CRC Handbook of Marine Mammal Medicine Second Edition 0839_frame_FM1 Page 2 Wednesday, May 23, 2001 10:38 AM Cover: In 1988, this marine mammal quilt was designed and constructed by scores of artists, needlework experts, and quilters to honor the efforts of The Marine Mammal Center (TMMC), in Sausalito, California. The quilt incorporates the designs of artists Richard Ellis, Pieter Folkens, Larry Foster, Dugald Stermer, and 25 others. The quilt travels on display, and to date has been exhibited at the California Academy of Sciences, the Monterey Bay Aquarium, and TMMC. This cover is in honor of the more than 800 volunteers who work at TMMC and for our contributors, reviewers, and editors. Thank you! CRC Handbook of Marine Mammal Medicine Second Edition Edited by Leslie A. Dierauf and Frances M. D. Gulland CRC Press Boca Raton London New York Washington, D.C. 0839_frame_FM1 Page 4 Tuesday, April 9, 2002 1:34 PM Senior Editor: John Sulzycki Production Manager: Carol Whitehead Marketing Manager: Carolyn Spence Illustrations in Chapters 9 and 19 are © Sentiel A. Rommel. Library of Congress Cataloging-in-Publication Data CRC Handbook of marine mammal medicine / edited by Leslie A. Dierauf and Frances M.D. Gulland.--2nd ed. p. cm. Includes bibliographical references and index. ISBN 0-8493-0839-9 (alk. paper) 1. Marine mammals--Diseases--Handbooks, manuals, etc. 2. Marine mammals--Health--Handbooks, manuals, etc. 3. Veterinary medicine--Handbooks, manuals etc. 4. Wildlife rehabilitation--Handbooks, manuals etc. I. Title: Handbook of marine mammal medicine. II. Dierauf, Leslie A., 1948- III. Gulland, Frances M. D. SP997.5.M35 C73 2001 636.9′5--dc21 2001025211 CIP This book contains information obtained from authentic and highly regarded sources. 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Visit the CRC Press Web site at www.crcpress.com © 2001 by CRC Press LLC No claim to original U.S. Government works International Standard Book Number 0-8493-0839-9 Library of Congress Card Number 2001025211 Printed in the United States of America 2 3 4 5 6 7 8 9 0 Printed on acid-free paper 0839_frame_FM1 Page 5 Tuesday, May 22, 2001 2:42 PM Dedication This book is dedicated to Dr. Nancy Foster— A whole generation of veterinarians for whom you, as a scientist, were our mentor, our inspiration, and our motivation in our pursuit of marine science, policy, and marine mammal medicine, thank you. We miss you. Thank you, Joe— for caring for Nancy for caring for the animals, and for being a leader for us in the field of marine mammal medicine. 0839_frame_FM1 Page 6 Tuesday, May 22, 2001 2:42 PM 0839_frame_FM1 Page 7 Tuesday, May 22, 2001 2:42 PM Preface Read not to contradict and confute, nor to believe and take for granted, nor to find talk and discourse, but to weigh and consider. —Francis Bacon, 1625 It has been more than 10 years since the first edition of the Handbook of Marine Mammal Medicine was published; during that time, the book has sold consistently (almost 2000 copies worldwide). Since its publication in 1990, there has been an exponential growth of experience and published literature addressing marine mammal medicine. Marine mammals have captured the imagination of not only the public, but also the scientific community. Despite this increase in information, much remains to be learned about the medicine of marine mammals. We hope that by sharing what is known to date, veterinarians will be encouraged to explore the unknown, and share this new information in the future. The meaning of the phrase “marine mammal medicine” has greatly expanded, and the contents of this second edition attempt to reflect this. As we enter the new millennium, veterinarians are not only involved in diagnosis and treatment of disease, but also in the bigger picture, including marine mammals as sentinels of ocean health, animal well-being, marine mammal strandings and unusual mortality events, legislation governing marine mammal health and population trends, and tagging and tracking of rehabilitated and released animals. To care for marine mammals effectively, veterinarians also need to understand their anatomy, physiology, and behavior. As the field develops, we must encourage new members of the profession and be able to advise students on careers in the field of marine mammal medicine. We hope our vision of what marine mammal medicine is in the 21st century becomes yours. With 66 contributors, and almost 100 reviewers, all working together to help craft 45 scientifically based chapters, we believe the contents of this textbook are light-years ahead of the topics presented in the first edition of the Handbook of Marine Mammal Medicine. For these extraordinary efforts, we wish to offer our utmost thanks to everyone involved. We appreciate the time taken away from their work to share their knowledge and experience with others. With all the reference books, journals, e-mails, and Web sites each author investigated, this second edition is an explosion of new information. We apologize for any current medical literature on marine mammals we may have inadvertently overlooked in this effort. Almost every year since 1995, CRC Press, the publisher of the first edition of the handbook, has contacted one of the editors (Dierauf ) asking if she “would be interested in publishing a second edition?” And almost every year since 1995, due to time constraints, more than full-time commitments elsewhere, and the fact that her current efforts are directed toward habitat protection for threatened and endangered species (U.S. Fish and Wildlife Service, Albuquerque, NM) and environmental education (co-founder and chair of the Alliance of Veterinarians for the Environment, Nashville, TN), she has emphatically and succinctly said “no.” Except, for early fall, 1999, when she hesitated . . . said she had to make a few phone calls, and would call back. 0839_frame_FM1 Page 8 Tuesday, May 22, 2001 2:42 PM The phone calls were to the now coeditor (Gulland) and, although she too had been asked previously and declined, she too hesitated. It really was time, almost the 10-year anniversary of the first edition; there was so much new information, so many new scientists entering the field, and an amazing array of students calling for help in advancing future careers in marine mammal medicine. We agreed that it was indeed time, if we could persuade fellow colleagues to join us in this effort. To our great surprise and wonder, considering how pressed for time everyone is these days, more than 90% of the scientists we called enthusiastically agreed to participate. We called CRC in October 1999, and said, “yes.” We are proud of our authors and our publisher for bringing this second edition to publication promptly to ensure that the information presented is as up to date and future oriented as is possible in this age of information. The first edition of this book limited its scope to U.S. and Canadian issues and species. This edition tries harder to address international concerns and the worldwide practice of marine mammal medicine. We chose to write the text in (no, not English — sorry Frances!) American (phrases, spelling) for consistency with the first edition. Both metric and American measurements are provided, and there is a conversion table in the appendix. In the references at the end of each chapter, we include abstracts from conference proceedings (many of which can be found on the International Association for Aquatic Animal Medicine, or IAAAM, CD-ROM; see Chapters 7 and 8 for ordering information), as well as peer-reviewed books and journals. This is to provide the reader with as much current information as possible; the reader is encouraged to seek peer-reviewed journal articles by the same authors as their pieces are published. We have Web information from reputable sources within the context of each chapter (in bold), information from veterinary and marine scientists through personal communications (pers. comm.), unpublished data (unpubl. data), cross-referencing that refers to pertinent information in other chapters (see Chapter …), and an extensive index. The chapters in this second edition have been peer-reviewed. Yet, despite this peer-reviewed information, the editors still wish to emphasize that, in the practice of marine mammal medicine, nothing—not Web information, not journal information, not e-mail information—substitutes for talking to your peers and colleagues prior to performing a new procedure, or administering a pharmaceutical to a marine mammal. Nothing beats a healthy exchange of questions, answers, and experiences to assist in decision making. Again, we wish to thank everyone we have worked with over the past year (authors, coauthors, editors, peer-reviewers, colleagues) for giving us their unending support, for responding to our unceasing phone calls and e-mails, and for helping us maintain our enthusiasm. We thank Raymond Tarpley, David St. Aubin, Shannon Atkinson, and Bill Amos for wonderful lastminute rescues. We offer special thanks to the staff and volunteers at The Marine Mammal Center, in Sausalito, CA (we quietly refer to these Editorial and Literary Volunteers as our “elves”) for their consistent, constant, and voluntary efforts on behalf of this production. In particular, we thank Rebecca Duerr, Danielle Duggan, Denise Greig, Michelle Lander, Gayle Love, Alana Phillips, Kathryn Zagzebski, Kelly Alman, Amber Clutton-Brock, and Tanya Zabka. Thanks are due to Andy Draper for ensuring polar bears were not left out in the cold, and to both Andy Draper and Jim Hurley for keeping our spirits up. We could not have done this without the help of every one of you. Leslie A. Dierauf Frances M. D. Gulland 0839_frame_FM1 Page 9 Tuesday, May 22, 2001 2:42 PM Editors Leslie A. Dierauf, V.M.D, is a wildlife veterinarian and conservation biologist with 17 years of clinical veterinary practice experience, specializing in marine mammal and small animal emergency medicine. She currently works with the U.S. Fish and Wildlife Service (Service), primarily on habitat conservation planning efforts for all types of threatened and endangered species in Texas, Arizona, New Mexico, and Oklahoma. Her primary focus is forming partnerships between the federal government and the private sector/citizenry. Prior to joining the Service, she worked as a scientific advisor on committee staff for the U.S. House of Representatives in Washington, D.C. In 1998, Dr. Dierauf was honored by the profession of veterinary medicine with the American Veterinary Medical Association’s National Animal Welfare Award. She also served as an American Association for the Advancement of Science Congressional Science Fellow. Dr. Dierauf currently sits on the Marine Ecosystem Health Program Advisory Board, a research and science policy effort located on Orcas Island, WA, and associated with the University of California, Davis, Wildlife Health Center. She also served 8 years on the American Veterinary Medical Association’s Environmental Affairs Committee, and 8 years on the National Marine Fisheries Service’s Working Group on Marine Mammal Unusual Mortality Events. She is the co-founder and chair of the Board of the Alliance of Veterinarians for the Enviornment. Dr. Dierauf is a member of the International Association for Aquatic Animal Medicine, the Alliance of Veterinarians for the Environment, the Society for Conservation Biology, and the American Veterinary Medical Association. She lives in Santa Fe, NM, with Jim Hurley, her partner of 22 years, and their three dogs. Frances M. D. Gulland, Vet. M.B., M.R.C.V.S., Ph.D., is a veterinarian interested in the role of disease in wildlife conservation. She obtained her veterinary degree from the University of Cambridge (England) in 1984 and her Ph.D., also from the University of Cambridge (Zoology Department) in 1991. Dr. Gulland worked at the Zoological Society of London as House Surgeon and later as Fellow in Wildlife Diseases, before moving to California in 1994. Dr. Gulland was introduced to marine mammals by her father, John A. Gulland, but became involved in their medicine when she started to work at The Marine Mammal Center (TMMC), Sausalito, CA, in 1994. As Director of Veterinary Services at TMMC, Dr. Gulland is involved in marine mammal strandings, rehabilitation, and disease investigation. She learns about marine mammal medicine on a daily basis from the animals and people around her. Dr. Gulland currently serves as a scientific advisor to the Oiled Wildlife Care Network in California and the Marine Mammal Commission, and is a member of the Working Group on Marine Mammal Unusual Mortality Events, the International Association for Aquatic Animal Medicine, the Wildlife Disease Association, and the Society for Marine Mammalogy. 0839_frame_FM1 Page 10 Tuesday, May 22, 2001 2:42 PM 0839_frame_FM1 Page 11 Wednesday, May 23, 2001 10:39 AM Contributors Brian M. Aldridge B.V.Sc., Ph.D., A.C.V.I.M. Department of Pathology, Microbiology, and Immunology School of Veterinary Medicine University of California Davis, California William Amos, Ph.D. Department of Zoology University of Cambridge Cambridge, England Brad F. Andrews SeaWorld of Florida Orlando, Florida Jim Antrim SeaWorld of California San Diego, California Kristen D. Arkush, Ph.D. Bodega Marine Laboratory University of California Bodega Bay, California Shannon K. C. Atkinson, Ph.D. Alaska SeaLife Center and University of Alaska Seward, Alaska Cathy A. Beck, M.S. U.S. Geological Survey Florida Caribbean Science Center Sirenia Project Gainesville, Florida Robert K. Bonde, Ph.D. U.S. Geological Survey Florida Caribbean Science Center Sirenia Project Gainesville, Florida Gregory D. Bossart, V.M.D., Ph.D. Division of Marine Mammal Research and Conservation Harbor Branch Oceanographic Institution Fort Pierce, Florida Michael Brent Briggs, D.V.M. Brookfield Zoo Brookfield, Illinois Fiona Brook, Ph.D., R.D.M.S., D.C.R. Department of Optometry and Radiography The Hong Kong Polytechnic University Hung Hom, Kowloon, Hong Kong John D. Buck, Ph.D. Mote Marine Laboratory Sarasota, Florida Daniel F. Cowan, M.D. Department of Pathology University of Texas Medical Branch Galveston, Texas Murray D. Dailey, Ph.D. The Marine Mammal Center Marin Headlands Sausalito, California Leslie M. Dalton, D.V.M. SeaWorld of Texas San Antonio, Texas Leslie A. Dierauf, V.M.D. Alliance of Veterinarians for the Environment Santa Fe, New Mexico Samuel R. Dover, D.V.M. Santa Barbara Zoological Garden Santa Barbara, California 0839_frame_FM1 Page 12 Tuesday, May 22, 2001 2:42 PM Deborah A. Duffield, Ph.D. Department of Biology Portland State University Portland, Oregon J. Lawrence Dunn, V.M.D. Department of Research and Veterinary Medicine Mystic Aquarium Mystic, Connecticut Ruth Y. Ewing, D.V.M. National Marine Fisheries Service South East Florida Science Center Miami, Florida Salvatore Frasca, Jr., V.M.D., Ph.D. Department of Pathobiology University of Connecticut Storrs, Connecticut Laurie J. Gage, D.V.M. Six Flags MarineWorld Vallejo, California Edward V. Gaynor, D.V.M. SeaWorld of Florida Orlando, Florida Scott Gearhart, D.V.M. SeaWorld of Florida Orlando, Florida Leah L. Greer, D.V.M. Department of Comparative Medicine College of Veterinary Medicine University of Tennessee Knoxville, Tennessee Frances M. D. Gulland, Vet. M.B., M.R.C.V.S., Ph.D. The Marine Mammal Center Marin Headlands Sausalito, California Martin Haulena, M.Sc., D.V.M. The Marine Mammal Center Marin Headlands Sausalito, California Robert Bruce Heath, D.V.M., M.Sc., Dipl. A.C.V.A. Fort Collins, Colorado Aleta A. Hohn National Marine Fisheries Service Beaufort Laboratory Beaufort, North Carolina Carol House, Ph.D. Cutchogue, New York James A. House, D.V.M., Ph.D. Cutchogue, New York Eric D. Jensen, D.V.M. U.S. Navy Marine Mammal Program San Diego, California Suzanne Kennedy-Stoskopf, D.V.M., Ph.D., Dipl. A.C.Z.M. North Carolina State University Raleigh, North Carolina Donald P. King, Ph.D. Department of Pathology, Microbiology and Immunology School of Veterinary Medicine University of California Davis, California Michelle E. Lander, M.Sc. The Marine Mammal Center Marin Headlands Sausalito, California Lynn W. Lefebvre, Ph.D. U.S. Geological Survey Florida Caribbean Science Center Sirenia Project Gainesville, Florida Linda J. Lowenstine, D.V.M., Ph.D., Dipl. A.C.V.P. Department of Pathology, Microbiology, and Immunology School of Veterinary Medicine University of California Davis, California 0839_frame_FM1 Page 13 Tuesday, May 22, 2001 2:42 PM James F. McBain, D.V.M. SeaWorld of California San Diego, California Ted Y. Mashima, D.V.M., Dipl. A.C.Z.M. Center for Government and Corporate Veterinary Medicine University of Maryland Baltimore, Maryland Debra Lee Miller, D.V.M., Ph.D. Division of Comparative Pathology University of Miami School of Medicine Miami, Florida Michael J. Murray, D.V.M. Monterey Bay Aquarium Monterey, California Daniel K. Odell, Ph.D. SeaWorld of Florida Orlando, Florida Todd M. O’Hara, D.V.M., Ph.D. North Slope Borough Department of Wildlife Management Barrow, Alaska Thomas J. O’Shea, M.S., Ph.D. U.S. Geological Survey Midcontinent Ecological Science Center Fort Collins, Colorado Michelle Lynn Reddy SAIC Maritime Services San Diego, California Sentiel A. Rommel, Ph.D. Eckerd College Florida Marine Research Institute Marine Mammal Pathobiology Laboratory St. Petersburg, Florida Teri K. Rowles, D.V.M., Ph.D. Office of Protected Resources National Marine Fisheries Service Silver Spring, Maryland David J. St. Aubin, Ph.D. Mystic Aquarium Mystic, Connecticut Sara L. Shapiro Florida Fish and Wildlife Conservation Commission Florida Marine Research Institute St. Petersburg, Florida Terry R. Spraker, D.V.M., Ph.D., Dipl. A.C.V.P. Diagnostic Laboratory College of Veterinary Medicine Colorado State University Fort Collins, Colorado Michael K. Stoskopf, D.V.M., Ph.D., Dipl. A.C.Z.M. Environmental Medicine Consortium College of Veterinary Medicine North Carolina State University Raleigh, North Carolina Thomas H. Reidarson, D.V.M., Dipl. A.C.Z.M. SeaWorld of California San Diego, California Jeffrey L. Stott, Ph.D. Department of Pathology, Microbiology, and Immunology School of Veterinary Medicine University of California Davis, California Michael G. Rinaldi, D.V.M. Department of Pathology University of Texas Health Science Center San Antonio, Texas Jay C. Sweeney, V.M.D. Dolphin Quest San Diego, California Todd R. Robeck, D.V.M., Ph.D SeaWorld of Texas San Antonio, Texas Forrest I. Townsend, Jr., D.V.M. Bayside Hospital for Animals Fort Walton Beach, Florida 0839_frame_FM1 Page 14 Tuesday, May 22, 2001 2:42 PM Pamela Tuomi, D.V.M. Alaska SeaLife Center Seward, Alaska William Van Bonn, D.V.M. U.S. Navy Marine Mammal Program San Diego, California Frances M. Van Dolah, Ph.D. National Ocean Services Charleston, South Carolina Michael T. Walsh, D.V.M. SeaWorld of Florida Orlando, Florida Andrew J. Westgate, Ph.D. Duke Marine Laboratory Beaufort, North Carolina Janet Whaley, D.V.M. Office of Protected Resources National Marine Fisheries Service Silver Spring, Maryland Scott Willens, D.V.M. North Carolina State University Raleigh, North Carolina Graham A. J. Worthy, Ph.D. Department of Biology University of Central Florida Orlando, Florida Nina M. Young, M.S. Center for Marine Conservation Washington, D.C. 0839_frame_FM1 Page 15 Tuesday, May 22, 2001 2:42 PM Contents Section I 1 Emerging Pathways in Marine Mammal Medicine Marine Mammals as Sentinels of Ocean Health Michelle Lynn Reddy, Leslie A. Dierauf, and Frances M. D. Gulland Introduction ................................................................................................3 Sentinels......................................................................................................3 Ecosystem Changes Detected by Sentinels..............................................4 Marine Mammals as Sentinels..................................................................5 Conclusion ..................................................................................................9 Acknowledgments ......................................................................................9 References ...................................................................................................9 2 Emerging and Resurging Diseases Debra Lee Miller, Ruth Y. Ewing, and Gregory D. Bossart Introduction ..............................................................................................15 Cetaceans ..................................................................................................16 Pinnipeds...................................................................................................19 Manatees ...................................................................................................22 Sea Otters..................................................................................................23 Polar Bears ................................................................................................24 Conclusion ................................................................................................24 Acknowledgments ....................................................................................25 References .................................................................................................25 3 Florida Manatees: Perspectives on Populations, Pain, and Protection Thomas J. O’Shea, Lynn W. Lefebvre, and Cathy A. Beck Introduction ..............................................................................................31 Maiming of Manatees in Collisions with Boats ....................................33 A Primer on Manatee Population Biology: Accounting for the Confusion and Uncertainty.....................................................36 Estimation of Population Size and Trend.....................................36 Carcass Counts, Mortality, and Survival......................................39 0839_frame_FM1 Page 16 Tuesday, May 22, 2001 2:42 PM Population Models..........................................................................40 Uncertainties on Population Status: A Red Herring? ...........................40 References .................................................................................................42 4 Marine Mammal Stranding Networks Frances M. D. Gulland, Leslie A. Dierauf, and Teri K. Rowles Introduction ..............................................................................................45 Objectives of Stranding Networks ..........................................................45 Stranding Networks Worldwide ..............................................................46 Acknowledgments ....................................................................................66 References .................................................................................................66 5 Marine Mammal Unusual Mortality Events Leslie A. Dierauf and Frances M. D. Gulland Introduction ..............................................................................................69 MMUME Responses in the United States .............................................70 The U.S. National Contingency Plan ...........................................71 Expert Working Group on MMUMEs...........................................71 The MMUME Response.................................................................74 MMUME Fund................................................................................76 Lessons Learned........................................................................................77 The Cooperative Response ............................................................77 The Process .....................................................................................78 UMMME Fund................................................................................78 Results Accrued from Title IV of the MMPA........................................78 How Can You Help? ................................................................................79 Conclusion ................................................................................................79 Acknowledgments ....................................................................................79 References .................................................................................................79 6 Mass Strandings of Cetaceans Michael T. Walsh, Ruth Y. Ewing, Daniel K. Odell, and Gregory D. Bossart Introduction ..............................................................................................83 Theories to Explain Mass Strandings .....................................................83 Current Investigations into Mass Strandings ........................................86 Evaluation of a Mass Stranding ..............................................................87 Management of a Mass Stranding...........................................................88 Disposition of Animals in a Mass Stranding .........................................92 Euthanasia .......................................................................................94 Return to the Sea ...........................................................................94 Survival of Treated Whales............................................................94 0839_frame_FM1 Page 17 Tuesday, May 22, 2001 2:42 PM Conclusion ................................................................................................94 Acknowledgments ....................................................................................95 References .................................................................................................95 7 Careers in Marine Mammal Medicine Leslie A. Dierauf, Salvatore Frasca, Jr., and Ted Y. Mashima Introduction ..............................................................................................97 Full-Time Employment..................................................................97 Part-Time Employment..................................................................98 Personality Traits and Other Tools...............................................98 Summary .........................................................................................99 The Six-Step Method for Landing That Perfect JobWorking with Marine Mammals ........................................................................99 1. The First Step—Taking a Personal Self-Assessment ...............99 2. The Second Step—Categorizing Your Unique Skills, Strategies, and Approaches ......................................................100 3. The Third Step—Planning for Action and Timing................102 4. The Fourth Step—Making Choices ........................................102 5. The Fifth Step—Preparing for the Interview..........................103 6. The Sixth Step—Starting Your New Job ................................106 Accessing Resources ..............................................................................107 Internships and Residencies ........................................................107 Matched Internships.........................................................107 Matched Residencies ........................................................108 Other Internships..............................................................108 Graduate Degree Programs ..........................................................109 Other Related Programs...............................................................110 Advanced Training Programs.......................................................111 Fellowships ...................................................................................112 Scientific Societies and Membership Organizations .................112 Recommendations and Conclusions.....................................................113 Acknowledgments ..................................................................................114 References ...............................................................................................114 8 The Electronic Whale Leslie A. Dierauf Introduction ............................................................................................117 Using Your Head on the Web................................................................117 Reference Databases...............................................................................118 General Biomedical and Veterinary Medical Sites ....................118 Model Web Sites and Evidence-Based Medicine ........................119 0839_frame_FM1 Page 18 Tuesday, May 22, 2001 2:42 PM Marine Mammal–Related Listserves...........................................120 Other Internet Discussion and Marine Mammal Information Lists ......................................................................121 Online Marine Mammal Journals and Textbooks .....................121 Fellowships, Foundations, and Grants........................................122 Fellowships........................................................................122 Foundations .......................................................................123 Grants ................................................................................123 Federal Government Listings ......................................................123 Miscellaneous Electronic Resources ...........................................123 Meetings and Proceedings on CD-ROM.....................................125 Electronic Addresses for Other Chapters in This Book ............125 Disclaimer...............................................................................................126 Conclusions ............................................................................................126 References ...............................................................................................126 Section II 9 Anatomy and Physiology of Marine Mammals Gross and Microscopic Anatomy Sentiel A. Rommel and Linda J. Lowenstine Introduction ............................................................................................129 External Features....................................................................................138 Sea Lions .......................................................................................138 Manatees .......................................................................................138 Seals...............................................................................................139 Dolphins ........................................................................................139 Microanatomy of the Integument.........................................................139 The Superficial Skeletal Muscles..........................................................141 The Diaphragm as a Separator of the Body Cavities ..........................142 Gross Anatomy of Structures Cranial to the Diaphragm ...................142 Heart and Pericardium .................................................................142 Pleura and Lungs ..........................................................................143 Mediastinum .................................................................................143 Thymus .........................................................................................143 Thyroids ........................................................................................143 Parathyroids ..................................................................................144 Larynx............................................................................................144 Caval Sphincter ............................................................................144 Microscopic Anatomy of Structures Cranial to the Diaphragm ........144 Respiratory System.......................................................................144 Thymus .........................................................................................145 Thyroids ........................................................................................145 Parathyroids ..................................................................................145 0839_frame_FM1 Page 19 Tuesday, May 22, 2001 2:42 PM Gross Anatomy of Structures Caudal to the Diaphragm ...................145 Liver...............................................................................................145 Digestive System ..........................................................................145 Urinary Tract ................................................................................147 Genital Tract.................................................................................147 Adrenal Glands .............................................................................148 Microscopic Anatomy of Structures Caudal to the Diaphragm.........148 Liver...............................................................................................148 Digestive System ..........................................................................148 Urinary Tract ................................................................................149 Genital Tract.................................................................................149 Adrenals ........................................................................................150 Lymphoid and Hematopoietic Systems ......................................150 Nervous System .....................................................................................150 Circulatory Structures ...........................................................................151 The Potential for Thermal Insult to Reproductive Organs ................152 Skeleton ..................................................................................................153 Ribs................................................................................................155 Sternum.........................................................................................155 Postthoracic Vertebrae .................................................................156 Sacral Vertebrae ............................................................................156 Chevron Bones..............................................................................156 Pectoral Limb Complex ...............................................................156 Pelvic Limb Complex...................................................................157 Sexual Dimorphisms ....................................................................157 Bone Marrow.................................................................................158 Acknowledgments ..................................................................................158 References ...............................................................................................158 10 Endocrinology David J. St. Aubin Introduction ............................................................................................165 Sample Collection and Handling ..........................................................166 Blood..............................................................................................166 Saliva .............................................................................................166 Feces ..............................................................................................166 Urine..............................................................................................166 Tissues...........................................................................................167 Pineal Gland ...........................................................................................167 Hypothalamus–Pituitary........................................................................169 Thyroid Gland ........................................................................................169 Adrenal Gland ........................................................................................177 0839_frame_FM1 Page 20 Tuesday, May 22, 2001 2:42 PM Osmoregulatory Hormones ...................................................................182 Vasopressin....................................................................................183 Renin–Angiotensin System..........................................................185 Atrial Natriuretic Peptide............................................................185 Endocrine Pancreas ................................................................................185 Future Studies.........................................................................................186 Acknowledgments ..................................................................................187 References ...............................................................................................187 11 Reproduction Todd R. Robeck, Shannon K. C. Atkinson, and Fiona Brook Introduction ............................................................................................193 Physiology of Reproduction...................................................................193 Pinniped Reproduction ..........................................................................195 Female Pinniped Reproduction ...................................................195 Reproductive Cycle ..........................................................195 Estrous Cycle ....................................................................196 Pregnancy and Pseudopregnancy .....................................197 Embryonic Diapause and Reactivation ...........................198 Implantation......................................................................198 Pregnancy Diagnosis.........................................................199 Induction of Parturition ...................................................199 Lactation............................................................................200 Milk Collection ................................................................200 Male Pinniped Reproduction .......................................................200 Anatomy ............................................................................200 Sexual Maturity ................................................................201 Seasonality ........................................................................201 Contraception and Control of Aggression ..................................202 Females ..............................................................................202 Males .................................................................................202 Reproductive Abnormalities in Pinnipeds .................................203 Cetacean Reproduction..........................................................................204 Female Cetacean Reproduction...................................................204 Reproductive Maturity .....................................................204 Bottlenose Dolphin ...........................................204 White-Sided Dolphin ........................................204 Killer Whale.......................................................204 False Killer Whale .............................................205 Beluga.................................................................205 Reproductive Cycle ..........................................................205 Bottlenose Dolphin ...........................................205 White-Sided Dolphin ........................................205 0839_frame_FM1 Page 21 Tuesday, May 22, 2001 2:42 PM Killer Whale.......................................................206 False Killer Whale .............................................206 Beluga.................................................................206 Estrous Cycle and Ovarian Physiology ...........................206 Bottlenose Dolphin ...........................................206 Killer Whale.......................................................208 False Killer Whale .............................................208 Suckling (Lactational) Suppression of Estrus .................209 Corpora Albicantia and Asymmetry of Ovulation ........210 Pseudopregnancy...............................................................210 Pregnancy ..........................................................................211 Bottlenose Dolphin ...........................................211 Killer Whale.......................................................211 Beluga.................................................................212 Pregnancy Diagnosis.........................................................212 Parturition .........................................................................212 Stages of Parturition .........................................212 Induction of Parturition ...................................212 Male Cetacean Reproduction ......................................................215 Sexual Maturity ................................................................215 Bottlenose Dolphin ...........................................215 White-Sided Dolphin ........................................215 Killer Whale.......................................................215 Beluga.................................................................216 Seasonality ........................................................................216 Bottlenose Dolphin ...........................................216 White-Sided Dolphin ........................................216 Killer Whale.......................................................217 False Killer Whale .............................................217 Beluga.................................................................217 Contraception and Control of Aggression ..................................217 Females ..............................................................................217 Males .................................................................................218 Reproductive Abnormalities in Cetaceans.................................218 Artificial Insemination.................................................................219 Semen Collection and Storage.........................................219 Manipulation and Control of Ovulation.........................221 Induction of Ovulation .....................................221 Synchronization of Ovulation..........................222 Insemination Techniques .................................................223 Future Applications ..........................................................224 Acknowledgments ..................................................................................225 References ...............................................................................................226 0839_frame_FM1 Page 22 Wednesday, April 10, 2002 8:17 AM 12 Immunology Donald P. King, Brian M. Aldridge, Suzanne Kennedy-Stoskopf, and Jeffrey L. Stott Introduction ............................................................................................237 Overview of the Immune System.........................................................238 Innate Immunity and the Inflammatory Response ...................238 Adaptive Immune Response ........................................................238 Cytokines ......................................................................................239 Immunodiagnostics ................................................................................240 Inflammation ................................................................................240 Cellular Immunity .......................................................................241 Functional Immune Testing ........................................................242 In Vitro ..............................................................................242 In Vivo ...............................................................................242 Humoral Immunity ......................................................................243 Measurement of Pathogen-Specific Antibodies (Serodiagnostics) ......243 Serum/Virus Neutralization Test ................................................244 Precipitation/Agglutination Techniques.....................................244 Enzyme-Linked Immunosorbent Assay ......................................245 Total Immunoglobulin .................................................................245 Clinical Approach to Suspected Marine Mammal Immunological Disorders...................................................................246 Conclusion ..............................................................................................248 Acknowledgments ..................................................................................248 References ...............................................................................................248 13 Stress and Marine Mammals David J. St. Aubin and Leslie A. Dierauf Introduction ............................................................................................253 Stressors ..................................................................................................253 Stress Response and Regulation............................................................254 Neurological Factors ....................................................................255 Endocrine Factors .........................................................................256 Catecholamines.................................................................256 Glucocorticoids.................................................................256 Mineralocorticoids............................................................260 Thyroid Hormones ...........................................................260 Other Hormones ...............................................................261 Immunological Factors.................................................................261 Indicators of Acute and Chronic Stress................................................262 Acute Response ............................................................................262 Chronic Response.........................................................................263 Future Research......................................................................................264 0839_frame_FM1 Page 23 Tuesday, May 22, 2001 2:42 PM Conclusion ..............................................................................................265 Acknowledgments ..................................................................................265 References ...............................................................................................265 14 Genetic Analyses Deborah A. Duffield and William Amos Introduction ............................................................................................271 Genetic Techniques ...............................................................................271 DNA Sequencing ..........................................................................271 “Tandem Repeats” and DNA Fingerprinting .............................272 Genetic Analyses Applied to Stranded Marine Mammals..................272 Species Identification ...................................................................273 Population Identification .............................................................273 Social Organization ......................................................................274 Genetic Analysis Applied to Captive Maintenance and Breeding Programs ..............................................................................275 Paternity Testing ..........................................................................275 Hybrid Detection..........................................................................276 Sampling .................................................................................................277 Conclusion ..............................................................................................278 Acknowledgments ..................................................................................278 References ...............................................................................................278 Section III Infectious Diseases of Marine Mammals 15 Viral Diseases Suzanne Kennedy-Stoskopf Introduction ............................................................................................285 Virus Isolation—An Overview ..............................................................285 Poxviruses ...............................................................................................286 Host Range....................................................................................286 Clinical Signs................................................................................287 Therapy .........................................................................................287 Pathology.......................................................................................287 Diagnosis .......................................................................................288 Differentials ..................................................................................288 Epidemiology ................................................................................289 Public Health Significance...........................................................289 Papillomaviruses ....................................................................................289 Host Range....................................................................................289 Clinical Signs................................................................................290 0839_frame_FM1 Page 24 Tuesday, May 22, 2001 2:42 PM Therapy .........................................................................................290 Pathology.......................................................................................290 Diagnosis .......................................................................................290 Differentials ..................................................................................290 Epidemiology ................................................................................291 Public Health Significance...........................................................291 Adenoviruses ..........................................................................................291 Host Range....................................................................................291 Clinical Signs................................................................................291 Therapy .........................................................................................291 Pathology.......................................................................................292 Diagnosis .......................................................................................292 Epidemiology ................................................................................292 Public Health Significance...........................................................292 Herpesviruses..........................................................................................292 Host Range....................................................................................292 Virology .........................................................................................293 Clinical Signs................................................................................293 Therapy .........................................................................................294 Pathology.......................................................................................294 Diagnosis .......................................................................................294 Differentials ..................................................................................295 Epidemiology ................................................................................295 Public Health Significance...........................................................295 Morbilliviruses .......................................................................................296 Host Range....................................................................................296 Virology .........................................................................................296 Clinical Signs................................................................................296 Therapy .........................................................................................297 Pathology.......................................................................................297 Diagnosis .......................................................................................297 Differentials ..................................................................................297 Epidemiology ................................................................................298 Public Health Significance...........................................................298 Influenza Viruses ....................................................................................298 Host Range....................................................................................298 Clinical Signs................................................................................298 Therapy .........................................................................................299 Pathology.......................................................................................299 Diagnosis .......................................................................................299 Differentials ..................................................................................299 Epidemiology ................................................................................299 Public Health Significance...........................................................300 0839_frame_FM1 Page 25 Tuesday, May 22, 2001 2:42 PM Caliciviruses (San Miguel Sea Lion Virus) ...........................................300 Host Range....................................................................................300 Clinical Signs................................................................................300 Therapy .........................................................................................300 Pathology.......................................................................................301 Diagnosis .......................................................................................301 Epidemiology ................................................................................301 Public Health Significance...........................................................302 Other Viruses..........................................................................................302 Hepadnavirus ................................................................................302 Coronavirus...................................................................................302 Retrovirus......................................................................................302 Rhabdoviruses...............................................................................303 Acknowledgments ..................................................................................303 References ...............................................................................................303 16 Bacterial Diseases of Cetaceans and Pinnipeds J. Lawrence Dunn, John D. Buck, and Todd R. Robeck Introduction ............................................................................................309 Microbial Sampling Techniques............................................................310 Specific Bacterial Diseases of Cetaceans and Pinnipeds .....................312 Septicemia.....................................................................................312 Brucellosis .....................................................................................312 Cetaceans ..........................................................................313 Pinnipeds ...........................................................................314 Vibriosis.........................................................................................314 Cetaceans ..........................................................................315 Pinnipeds ...........................................................................315 Pasteurellosis ................................................................................315 Cetaceans ..........................................................................315 Pinnipeds ...........................................................................315 Erysipelothrix................................................................................316 Cetaceans ..........................................................................316 Pinnipeds ...........................................................................318 Mycobacterial Disease .................................................................319 Cetaceans ..........................................................................319 Pinnipeds ...........................................................................319 Leptospirosis .................................................................................320 Pinnipeds ...........................................................................320 Nocardia ........................................................................................321 Cetaceans ..........................................................................322 Pinnipeds ...........................................................................325 0839_frame_FM1 Page 26 Tuesday, May 22, 2001 2:42 PM Miscellaneous Bacterial Disease ...........................................................325 Respiratory Disease ......................................................................325 Dermatological Disease ...............................................................326 Urogenital Disease .......................................................................327 Gastrointestinal Disease ..............................................................327 Conclusion ..............................................................................................328 Acknowledgments ..................................................................................328 References ...............................................................................................328 17 Mycotic Diseases Thomas H. Reidarson, James F. McBain, Leslie M. Dalton, and Michael G. Rinaldi Introduction ............................................................................................337 Mycotic Diseases....................................................................................337 Epidemiology of Fungi ...........................................................................338 Modes of Transmission ................................................................338 Mechanisms of Pathogenesis.......................................................338 Clinical Manifestations .........................................................................339 Clinical Diagnostic Features of the Fungi ...........................................340 Therapeutics ...........................................................................................349 Conclusion ..............................................................................................351 Acknowledgments ..................................................................................352 References ...............................................................................................352 18 Parasitic Diseases Murray D. Dailey Introduction ............................................................................................357 Removal and Fixation of Parasites for Identification..........................357 Treatment ...............................................................................................359 Parasites of Cetacea ...............................................................................359 Protozoa.........................................................................................359 Ciliates ..............................................................................359 Apicomplexans..................................................................359 Flagellates ..........................................................................360 Sarcodina ...........................................................................360 Helminths (Nematodes, Trematodes, Cestodes, Acanthocephalans)....................................................................361 Gastrointestinal Tract ......................................................361 Liver ...................................................................................365 Respiratory System, Sinuses, and Brain..........................365 Urogenital System ............................................................366 Connective Tissue ............................................................366 0839_frame_FM1 Page 27 Tuesday, May 22, 2001 2:42 PM Ectoparasites .................................................................................367 Parasites of Pinnipeds ............................................................................367 Protozoa.........................................................................................367 Apicomplexans..................................................................367 Flagellates ..........................................................................368 Helminths (Nematodes, Trematodes, Cestodes, Acanthocephalans)....................................................................369 Gastrointestinal Tract ......................................................369 Respiratory and Circulatory Systems .............................370 Liver, Biliary System, and Pancreas ................................372 Connective Tissue ............................................................372 Ectoparasites .................................................................................372 Parasites of Sirenia .................................................................................372 Protozoa—Apicomplexans ...........................................................372 Helminths (Nematodes, Trematodes) .........................................373 Parasites of Sea Otters ...........................................................................373 Protozoa—Apicomplexans ...........................................................373 Helminths (Nematodes, Trematodes, Cestodes, Acanthocephalans)....................................................................373 Parasites of Polar Bears ..........................................................................374 Acknowledgments ..................................................................................374 References ...............................................................................................374 Section IV Pathology of Marine Mammals 19 Clinical Pathology Gregory D. Bossart, Thomas H. Reidarson, Leslie A. Dierauf, and Deborah A. Duffield Introduction ............................................................................................383 Abnormalities and Artifacts..................................................................383 Blood Collection.....................................................................................384 Sampling Equipment and Processing ..........................................384 Blood Collection Sites..................................................................385 Cetaceans ..........................................................................385 Otariids ..............................................................................385 Phocids ..............................................................................385 Odobenids..........................................................................385 Manatees ...........................................................................387 Sea Otters ..........................................................................387 Polar Bears.........................................................................390 Hematology (CBC)..................................................................................390 Evaluation of Erythrocytes ....................................................................391 Indices ...........................................................................................391 0839_frame_FM1 Page 28 Tuesday, May 22, 2001 2:42 PM Anemia ..........................................................................................399 Classification of Anemia by RBC Indices ......................400 Normocytic, Normochromic ...........................400 Macrocytic, Hypochromic ................................400 Macrocytic, Normochromic .............................400 Microcytic, Normochromic, or Hypochromic ............................................401 Evaluation of Leukocytes ......................................................................401 Neutrophils or Heterophils..........................................................401 Eosinophils ....................................................................................401 Basophils .......................................................................................402 Monocytes and Lymphocytes ......................................................402 Leukocytes and Age .....................................................................402 Leukocytes and Disease ...............................................................403 Serum Analytes and Enzymes...............................................................403 Glucose, Lipids, and Pancreatic Enzymes ..................................403 Total Cholesterol and Triglycerides............................................404 Amylase, Lipase, and Trypsin-Like Immunoreactivity .............405 Markers of Hepatobiliary System Disorders ........................................406 Alanine Aminotransferase (ALT or SGPT) .................................406 Aspartate Aminotransferase (AST or SGOT) .............................407 Sorbitol Dehydrogenase (SDH) and Glutamate Dehydrogenase (GLDH) ...........................................................407 Lactate Dehydrogenase (LDH) .....................................................408 -Glutamyltransferase (GGT)......................................................408 Alkaline Phosphatase (ALP).........................................................409 Bilirubin ........................................................................................410 Bile Acids ......................................................................................411 Kidney-Associated Serum Analytes ......................................................411 Urea Nitrogen and Creatinine.....................................................411 Serum Proteins .......................................................................................413 Hematocrit and Total Plasma Protein ........................................413 Albumins and Globulins..............................................................414 Electrolytes .............................................................................................416 Sodium ..........................................................................................416 Potassium ......................................................................................416 Chloride.........................................................................................417 Total Carbon Dioxide...................................................................417 Calcium, Phosphorus, and Magnesium ......................................418 Calcium .............................................................................418 Phosphorus .......................................................................419 Magnesium ........................................................................419 Miscellaneous Serum Analytes .............................................................420 Uric Acid.......................................................................................420 Creatinine Phosphokinase ...........................................................420 0839_frame_FM1 Page 29 Wednesday, May 23, 2001 10:40 AM Hemostatic Parameters..........................................................................420 Blood Types ...................................................................................420 Screening for Hemostatic Disorders ...........................................420 Prothrombin Time and Partial Prothrombin Time ...................421 Markers of Inflammation.......................................................................422 Erythrocyte Sedimentation Rate .................................................422 Serum Iron ....................................................................................422 Bone Marrow Evaluation .......................................................................423 Urinalysis ................................................................................................423 Conclusion ..............................................................................................424 Clinical Cases.........................................................................................424 Cetaceans ......................................................................................424 CASE 1—Bottlenose Dolphin ............................................424 History ...............................................................424 Clinicopathological Findings............................424 Discussion .........................................................424 CASE 2—Bottlenose Dolphin ............................................424 History ...............................................................424 Clinicopathological Findings............................424 Treatment ..........................................................424 Progress ..............................................................424 Additional Clinicopathological Findings.........425 Further Treatment.............................................425 CASE 3—Bottlenose Dolphin ............................................425 History ...............................................................425 Clinicopathological Findings............................425 Treatment ..........................................................425 Discussion .........................................................425 CASE 4—Killer Whale........................................................425 History ...............................................................425 Diagnosis ...........................................................425 Treatment ..........................................................425 Discussion .........................................................426 CASE 5—Killer Whale........................................................426 History ...............................................................426 Clinicopathological Findings............................426 Discussion .........................................................426 CASE 6—Pacific White-Sided Dolphin .............................426 History ...............................................................426 Clinicopathological Findings............................426 Treatment ..........................................................426 Subsequent Clinicopathological Findings .......427 Additional Treatment .......................................427 0839_frame_FM1 Page 30 Tuesday, May 22, 2001 2:42 PM Diagnosis ...........................................................427 Discussion .........................................................427 Pinnipeds .......................................................................................427 CASE 1—Harbor Seal .........................................................427 History ...............................................................427 Clinicopathological Findings............................427 Treatment ..........................................................428 Post-Mortem Diagnosis ....................................428 Discussion .........................................................428 Manatees .......................................................................................428 CASE 1.................................................................................428 History ...............................................................428 Clinicopathological Findings............................428 Diagnosis ...........................................................428 Treatment ..........................................................428 Discussion .........................................................428 Sea Otters......................................................................................429 CASE 1.................................................................................429 History ...............................................................429 Clinicopathological Data..................................429 Radiographic Results ........................................429 Treatment ..........................................................429 Further Clinicopathological Data ....................429 Treatment ..........................................................429 Clinicopathological Data..................................429 Histopathological Diagnosis.............................429 Acknowledgments ..................................................................................430 References ...............................................................................................430 20 Cetacean Cytology Jay C. Sweeney and Michelle Lynn Reddy Introduction ............................................................................................437 Sample Collection ..................................................................................438 Collection of Respiratory Tract Samples....................................438 Collection of Gastric Samples.....................................................438 Collection of Fecal Samples ........................................................439 Collection of Urinary Tract Samples..........................................439 Collection of Aspirates from Masses ..........................................439 Slide Preparation ....................................................................................439 Examination of Specimens ....................................................................441 Determination of Cellular Concentration within Slide Preparation.......................................................................441 0839_frame_FM1 Page 31 Tuesday, May 22, 2001 2:42 PM Mucus............................................................................................441 Amorphous Material ....................................................................441 Interpretation ..........................................................................................441 Color..............................................................................................441 Epithelial Cells .............................................................................441 Leukocytes ....................................................................................442 Erythrocytes ..................................................................................442 Respiratory Tract....................................................................................442 Normal Findings...........................................................................442 Significant Findings......................................................................443 Stomach ..................................................................................................444 Normal Findings...........................................................................444 Significant Findings......................................................................444 Colon/Rectum ........................................................................................445 Normal Findings...........................................................................445 Significant Findings......................................................................445 Urinary Tract ..........................................................................................445 Normal Findings...........................................................................445 Significant Findings......................................................................446 Acknowledgments ..................................................................................446 References ...............................................................................................446 21 Gross Necropsy and Specimen Collection Protocols Teri K. Rowles, Frances M. Van Dolah, and Aleta A. Hohn Introduction ............................................................................................449 Necropsy Examinations and Specimen Collection .............................450 Carcass Condition Code ........................................................................453 Morphometrics .......................................................................................453 Morphometric Data Protocol...........................................453 Genetics ..................................................................................................453 Genetic Sample Protocol..................................................454 Stomach Contents..................................................................................454 Stomach Contents Protocol .............................................454 Age...........................................................................................................454 Age Protocol......................................................................456 Reproductive Status ...............................................................................456 Reproductive Status Protocol ..........................................457 Pathology—Gross Necropsy Examination............................................457 Human Interactions .....................................................................458 Histopathology .......................................................................................458 Histopathology Protocol...................................................459 Acoustic Pathology ................................................................................459 Acoustic Pathology Protocol............................................460 0839_frame_FM1 Page 32 Tuesday, May 22, 2001 2:42 PM Infectious Diseases.................................................................................460 Bacteriology...................................................................................460 Bacteriology Protocol........................................................460 Virology ...................................................................................................462 Virology Protocol ..............................................................462 Parasitology.............................................................................................462 Parasitology Protocol........................................................462 Non-Infectious Diseases ........................................................................464 Toxicology .....................................................................................464 Toxicology Protocol ..........................................................464 Harmful Algal Blooms ...........................................................................465 Harmful Algal Bloom Protocol ........................................467 Conclusions ............................................................................................467 Acknowledgments ..................................................................................467 References ...............................................................................................469 22 Toxicology Todd M. O’Hara and Thomas J. O’Shea Introduction ............................................................................................471 Classes of Toxicants...............................................................................477 Elements .................................................................................................478 Mercury .........................................................................................478 Cadmium ......................................................................................480 Lead ...............................................................................................481 Organotins.....................................................................................481 Other Elements.............................................................................482 Halogenated Organics ............................................................................482 Accumulation and Variability .....................................................482 Organochlorine Pesticides and Metabolites ...............................484 Polychlorinated Biphenyls ...........................................................485 Other Organohalogens .................................................................487 Effects of Organochlorines on Metabolism ................................488 Effects of Organochlorines on Reproduction and Endocrine Function ....................................................................................490 Effects of Organochlorines on Immunocompetence and Epizootics ...........................................................................491 Biotoxins .................................................................................................493 Brevetoxin .....................................................................................493 Paralytic Shellfish Poisoning .......................................................494 Domoic Acid.................................................................................495 Ciguatera .......................................................................................496 Oil............................................................................................................496 0839_frame_FM1 Page 33 Tuesday, May 22, 2001 2:42 PM Treatment and Diagnostic Procedures..................................................499 Dose Scaling..................................................................................499 Treatment......................................................................................499 Diagnosis .......................................................................................501 Acknowledgments ..................................................................................502 References ...............................................................................................502 23 Noninfectious Diseases Frances M. D. Gulland, Linda J. Lowenstine, and Terry R. Spraker Introduction ............................................................................................521 Congenital Defects.................................................................................521 Neoplasia ................................................................................................522 Trauma ....................................................................................................522 Intraspecific Trauma ....................................................................522 Interspecific Trauma ....................................................................528 Anthropogenic Trauma ................................................................530 Miscellaneous .........................................................................................531 Integumentary System .................................................................531 Musculoskeletal and Dental Systems.........................................532 Respiratory System.......................................................................533 Digestive System ..........................................................................533 Genitourinary System ..................................................................534 Endocrine System .........................................................................535 Cardiovascular System.................................................................535 Lymphoid System .........................................................................536 Nervous System and Special Senses ...........................................536 Acknowledgments ..................................................................................537 References ...............................................................................................537 Section V Diagnostic Imaging in Marine Mammals 24 Overview of Diagnostic Imaging William Van Bonn and Fiona Brook Introduction ............................................................................................551 Imaging Science......................................................................................551 From Human to Marine Mammal Diagnostic Imaging ......................552 Application of Diagnostic Imaging Techniques...................................554 Conclusion ..............................................................................................555 Acknowledgments ..................................................................................556 0839_frame_FM1 Page 34 Tuesday, May 22, 2001 2:42 PM 25 Radiology, Computed Tomography, and Magnetic Resonance Imaging William Van Bonn, Eric D. Jensen, and Fiona Brook Introduction ............................................................................................557 Indications ..............................................................................................561 Limitations .............................................................................................565 Technique................................................................................................568 Clinical Applications .............................................................................574 Dolphin .........................................................................................574 Normal Radiographic Anatomy.......................................574 Radiographic Pathology....................................................579 Pinniped ........................................................................................581 Normal Radiographic Anatomy.......................................581 Radiographic Pathology....................................................585 Computed Tomographic Anatomy .......................................................586 Magnetic Resonance Imaging Anatomy, Dolphin ...............................587 Acknowledgments ..................................................................................588 References ...............................................................................................590 26 Ultrasonography Fiona Brook, William Van Bonn, and Eric D. Jensen Introduction ............................................................................................593 Indications ..............................................................................................593 Limitations .............................................................................................594 Technique................................................................................................594 Equipment and Preparation .........................................................594 Image Orientation ........................................................................595 Clinical Applications .............................................................................596 Thoracic Imaging..........................................................................596 Heart and Mediastinum ...............................................................596 Lungs .............................................................................................597 Thoracic Lymph Nodes................................................................600 Abdominal Imaging ......................................................................601 Liver and Biliary System..............................................................601 Spleen ............................................................................................604 Pancreas.........................................................................................605 Gastrointestinal Tract ..................................................................605 Urinary Tract ................................................................................609 Reproductive Tract .......................................................................611 Males .................................................................................611 Females .............................................................................612 0839_frame_FM1 Page 35 Tuesday, May 22, 2001 2:42 PM Eyes................................................................................................616 Musculoskeletal System ..............................................................616 Body Condition.............................................................................618 Conclusion ..............................................................................................618 Acknowledgments ..................................................................................618 References ...............................................................................................618 27 Flexible and Rigid Endoscopy in Marine Mammals Samuel R. Dover and William Van Bonn Introduction ............................................................................................621 Indications ..............................................................................................622 Limitations .............................................................................................623 Equipment...............................................................................................624 Flexible Endoscopes......................................................................624 Rigid Telescopes ...........................................................................626 Light Sources ................................................................................626 Accessories and Instruments .......................................................627 Cameras.........................................................................................629 Video Monitors and Recorders ....................................................630 Clinical Applications in Cetaceans ......................................................630 Cetacean Gastroscopy ..................................................................630 Colonoscopy..................................................................................633 Respiratory Endoscopy .................................................................633 Urogenital .....................................................................................635 Clinical Applications in Other Marine Mammals ..............................635 Minimally Invasive Surgical Techniques .............................................636 Insufflation ....................................................................................636 Access............................................................................................637 Trocars and Cannulas...................................................................638 Closure ..........................................................................................639 Minimally Invasive Surgery in Cetaceans..................................640 Minimally Invasive Surgery in Other Marine Mammals..........640 Acknowledgments ..................................................................................641 References ...............................................................................................641 28 Thermal Imaging of Marine Mammals Michael T. Walsh and Edward V. Gaynor Introduction ............................................................................................643 Technique................................................................................................643 History ....................................................................................................644 Cameras ..................................................................................................645 0839_frame_FM1 Page 36 Tuesday, May 22, 2001 2:42 PM Clinical Applications .............................................................................645 Manatees .......................................................................................646 Pinnipeds .......................................................................................646 Cetaceans ......................................................................................647 Other Marine Mammal Species ..................................................649 Web Sites.......................................................................................650 Conclusion ..............................................................................................651 References ...............................................................................................651 Section VI Medical Management of Marine Mammals 29 Marine Mammal Anesthesia Martin Haulena and Robert Bruce Heath Introduction ............................................................................................655 Anesthetic Protocol................................................................................655 Preanesthetic Examination ..........................................................655 Choice of a Specific Anesthetic Protocol ...................................656 Monitoring Techniques..........................................................................656 Noninvasive Techniques..............................................................657 Invasive Techniques .....................................................................657 Support ....................................................................................................657 Cetaceans ................................................................................................657 Induction .......................................................................................657 Intubation......................................................................................660 Inhalation Anesthesia ..................................................................660 Monitoring ....................................................................................660 Support ..........................................................................................661 Emergencies ..................................................................................662 Otariids ...................................................................................................662 Induction .......................................................................................662 Intubation......................................................................................666 Inhalation Anesthesia ..................................................................667 Monitoring ....................................................................................668 Support ..........................................................................................668 Emergencies ..................................................................................669 Phocids ....................................................................................................670 Induction .......................................................................................670 Intubation......................................................................................674 Inhalation Anesthesia ..................................................................675 Monitoring ....................................................................................675 Support ..........................................................................................675 Emergencies ..................................................................................676 0839_frame_FM1 Page 37 Tuesday, May 22, 2001 2:42 PM Odobenids ...............................................................................................677 Induction .......................................................................................677 Intubation and Inhalation Anesthesia ........................................680 Monitoring ....................................................................................680 Support ..........................................................................................680 Emergencies ..................................................................................681 Sirenians..................................................................................................681 Sea Otters................................................................................................681 Induction .......................................................................................681 Intubation......................................................................................683 Inhalation Anesthesia ..................................................................683 Monitoring ....................................................................................683 Support ..........................................................................................683 Emergencies ..................................................................................684 Ursids ......................................................................................................684 Conclusion ..............................................................................................684 Acknowledgments ..................................................................................684 References ...............................................................................................684 30 Intensive Care Michael T. Walsh and Scott Gearhart Introduction ............................................................................................689 Records and Instructions .......................................................................689 Patient Evaluation..................................................................................689 Rehydration ............................................................................................690 Blood Transfusion...................................................................................692 Nutritional Therapy...............................................................................693 Hypoglycemia ...............................................................................693 Emaciation ....................................................................................693 Appetite Stimulants .....................................................................694 Respiratory Emergencies........................................................................695 Trauma ....................................................................................................695 Wound Management ..............................................................................696 Central Nervous System........................................................................696 Reproductive Emergencies.....................................................................697 Dystocia ........................................................................................697 Other Reproductive Emergencies................................................698 Antibiotics ..............................................................................................698 Analgesics ...............................................................................................699 Miscellaneous Therapeutic Agents.......................................................699 Support Equipment ................................................................................699 Conclusion ..............................................................................................700 References ...............................................................................................700 0839_frame_FM1 Page 38 Tuesday, May 22, 2001 2:42 PM 31 Pharmaceuticals and Formularies Michael K. Stoskopf, Scott Willens, and James F. McBain Introduction ............................................................................................703 Routes for Administering Drugs to Marine Mammals .......................704 Dose Scaling ...........................................................................................705 Drug Interactions ...................................................................................705 Cimetidine and Antacids .............................................................705 Tetracyclines .................................................................................706 Fluoroquinolones ..........................................................................706 Other Antibiotics .........................................................................707 Antifungals....................................................................................708 Antiparasitic Drugs ......................................................................708 Steroids..........................................................................................708 Diuretics........................................................................................708 Drug Dosages..........................................................................................709 Acknowledgments ..................................................................................722 References ...............................................................................................722 32 Euthanasia Leah L. Greer, Janet Whaley, and Teri K. Rowles Introduction ............................................................................................729 Stranded Animals ...................................................................................729 Display and Collection Animals...........................................................730 Methods of Euthanasia ..........................................................................730 Injectable Agents ....................................................................................731 Route of Administration..............................................................731 Barbiturates ...................................................................................732 Etorphine.......................................................................................732 T-61................................................................................................733 Paralytics .......................................................................................733 Inhalants .................................................................................................734 Physical Methods ...................................................................................734 Ballistics ........................................................................................734 Explosives......................................................................................736 Verification of Death..............................................................................736 Carcass Disposal.....................................................................................736 Acknowledgments ..................................................................................737 References ...............................................................................................737 0839_frame_FM1 Page 39 Tuesday, May 22, 2001 2:42 PM Section VII Marine Mammal Well-Being 33 U.S. Federal Legislation Governing Marine Mammals Nina M. Young and Sara L. Shapiro Federal Legislation and Regulations—Discussion ...............................741 Introduction ..................................................................................741 The Responsible Regulating Agencies ........................................742 The Endangered Species Act........................................................743 Listing, Critical Habitat, and Recovery Plans................744 Protection for Listed Species ...........................................744 Permits ..............................................................................745 Consultations ....................................................................745 Enforcement ......................................................................745 Implementation of the Convention on International Trade in Endangered Species of Wild Fauna and Flora ........................................................................746 The Marine Mammal Protection Act .........................................750 The MMPA Moratorium on Taking................................750 Exemptions and Permits for Incidental Take.................750 Reauthorizations of the MMPA.......................................753 Marine Mammal Strandings and Health ........................753 The Animal Welfare Act..............................................................755 The Law.............................................................................755 Licensing and Registration...............................................755 Research Facilities ............................................................755 AWA Enforcement ............................................................755 Regulations........................................................................756 Space Requirements .........................................................756 Overlap among the Agencies and the Various Laws .....757 The Lacey Act of 1901.................................................................758 The Fur Seal Act...........................................................................758 Conclusion ....................................................................................758 Definitions and Abbreviations Pertaining to U.S. Marine Mammal Legislation ................................................759 Contact Information.........................................................762 Marine Mammal Permits: Frequently Asked Questions (FAQs)........762 The Marine Mammal Stranding Networks ................................762 Scientific Research and Enhancement Permits .........................763 Public Display Permits ................................................................764 Other Permits ...............................................................................765 0839_frame_FM1 Page 40 Tuesday, May 22, 2001 2:42 PM Acknowledgments ..................................................................................765 References ...............................................................................................766 34 Public Health Daniel F. Cowan, Carol House, and James A. House Introduction ............................................................................................767 Viral Infections .......................................................................................768 Poxviruses .....................................................................................768 Calicivirus.....................................................................................768 Influenza........................................................................................769 Rabies ............................................................................................769 Bacterial Infections.................................................................................769 Vibrio spp. .....................................................................................769 Edwardsiella spp. .........................................................................770 Clostridium spp............................................................................770 Leptospira......................................................................................770 Streptococcus ................................................................................770 Brucella .........................................................................................771 Erysipelothrix rhusiopathiae .......................................................771 Mycobacterium spp......................................................................771 Coxiella burnetii ..........................................................................772 Other Mixed Infections................................................................772 Mycoplasma Infections ..........................................................................772 Fungal Infections ....................................................................................773 Protozoal Infections ...............................................................................773 Toxoplasma gondii .......................................................................773 Cryptosporidium spp. ..................................................................774 Giardia spp. ..................................................................................774 Potential for Transmission of Infectious Disease from Marine Mammals to Humans..................................................774 Acknowledgments ..................................................................................775 References ...............................................................................................775 35 Water Quality Kristen D. Arkush Introduction ............................................................................................779 Environmental Considerations..............................................................779 Space..............................................................................................780 System Water Source ...................................................................780 Temperature ..................................................................................780 0839_frame_FM1 Page 41 Tuesday, May 22, 2001 2:42 PM Lighting .........................................................................................781 Salinity and pH.............................................................................781 Filtration .................................................................................................781 Microorganisms (as Pathogens and/or Indicators of Water Quality) ......................................................................783 Mechanisms of Sterilization..................................................................784 Ozone ............................................................................................785 Conclusions ............................................................................................786 Acknowledgments ..................................................................................786 References ...............................................................................................787 36 Nutrition and Energetics Graham A. J. Worthy Introduction ............................................................................................791 Energy Requirements .............................................................................791 Metabolic Rate..............................................................................792 Thermoregulation.........................................................................794 Locomotion ...................................................................................796 Summary: Average Daily Metabolic Rate ..................................799 Water Requirements.....................................................................799 Fasting and Starvation..................................................................801 The Bioenergetic Scheme ......................................................................803 Maintenance Energy.....................................................................804 Production Energy ........................................................................804 Reproduction .....................................................................804 Molt ...................................................................................807 Heat Increment of Feeding ..........................................................807 Fecal and Urinary Energy Losses ................................................809 Calculation of Gross Energy Requirements ...............................810 Prey..........................................................................................................811 Species That Marine Mammals Consume in Captivity and in the Wild.........................................................................811 Seasonal Changes in Prey Composition .....................................813 Major Nutritional Disorders..................................................................813 Thiamine Deficiency....................................................................813 Hyponatremia ...............................................................................814 Vitamins A, D, and E ...................................................................815 Vitamin C......................................................................................816 Scombroid Poisoning....................................................................816 Conclusions ............................................................................................817 Acknowledgments ..................................................................................817 References ...............................................................................................817 0839_frame_FM1 Page 42 Tuesday, May 22, 2001 2:42 PM 37 Hand-Rearing and Artificial Milk Formulas Forrest I. Townsend, Jr. and Laurie J. Gage Introduction ............................................................................................829 Cetaceans ................................................................................................829 Formula .........................................................................................829 Delivery Methods and Techniques .............................................830 Feeding Frequency and Daily Requirements..............................830 Monitoring Neonates ...................................................................831 Weaning Procedures .....................................................................831 Other Practical Information ........................................................831 References and Suggested Further Reading ................................831 Pinnipeds.................................................................................................832 Harbor Seals ..................................................................................832 Formula .............................................................................832 Delivery Methods and Techniques..................................833 Feeding Frequency and Daily Requirements ..................833 Weaning Procedures..........................................................834 Other Practical Information.............................................834 References and Suggested Further Reading ....................834 Elephant Seals...............................................................................836 Formulas............................................................................836 Fish Mash ..........................................................836 Elephant Seal Formula......................................836 ESF 50–50 ..........................................................836 ESF 75–25...........................................................837 Feeding Frequency and Daily Requirements ..................837 Delivery Methods and Techniques..................................838 Weaning Procedures..........................................................838 Other Practical Information.............................................838 References and Suggested Further Reading ....................838 Sea Lions .................................................................................................839 Formula .........................................................................................839 Delivery Methods and Techniques .............................................839 Feeding Frequency and Daily Requirements..............................840 Weaning Procedures .....................................................................840 Other Practical Information ........................................................840 References and Suggested Further Reading ................................840 Walruses ..................................................................................................841 Formulas........................................................................................841 Beginning Formula............................................................841 Maintenance formula .......................................................841 Feeding Frequency and Daily Requirements..............................842 0839_frame_FM1 Page 43 Tuesday, May 22, 2001 2:42 PM Delivery Methods and Techniques .............................................842 Weaning Procedures .....................................................................842 Other Practical Information ........................................................842 References and Suggested Further Reading ................................842 Manatees .................................................................................................843 Formulas........................................................................................843 Miami Seaquarium Formula ............................................843 SeaWorld Formula ............................................................843 Delivery Methods and Techniques .............................................843 Feeding Frequency and Daily Requirements..............................844 Weaning Procedures .....................................................................844 Other Practical Information ........................................................844 References and Suggested Further Reading ................................845 Sea Otters................................................................................................845 Formula and Preparation .............................................................845 Delivery Methods and Techniques .............................................845 Feeding Frequency and Daily Requirements..............................846 Weaning Procedures .....................................................................846 Other Practical Information ........................................................846 References and Suggested Further Reading ................................847 Polar Bears ..............................................................................................847 Formulas........................................................................................847 Delivery Methods and Techniques .............................................848 Feeding Frequency and Daily Requirements..............................848 Weaning Process ...........................................................................848 Other Practical Information ........................................................848 References and Suggested Further Reading ................................848 Acknowledgments ..................................................................................849 38 Tagging and Tracking Michelle E. Lander, Andrew J. Westgate, Robert K. Bonde, and Michael J. Murray Introduction ............................................................................................851 Tracking Methodologies: A Brief Overview.........................................851 Pinnipeds.................................................................................................857 Cetaceans ................................................................................................862 Manatees .................................................................................................866 Sea Otters................................................................................................870 Polar Bears ..............................................................................................874 Conclusion ..............................................................................................874 Acknowledgments ..................................................................................874 References ...............................................................................................874 0839_frame_FM1 Page 44 Tuesday, May 22, 2001 2:42 PM 39 Marine Mammal Transport Jim Antrim and James F. McBain Introduction ............................................................................................881 Regulations .............................................................................................881 History of Marine Mammal Transport.................................................882 Cetaceans ......................................................................................882 Pinnipeds .......................................................................................888 Sea Otters......................................................................................888 Sirenians ........................................................................................889 Polar Bears.....................................................................................889 Additional Medical Considerations ......................................................889 Conclusion ..............................................................................................890 Acknowledgments ..................................................................................891 References ...............................................................................................891 Section VIII Specific Medicine and Husbandry of Marine Mammals 40 Cetacean Medicine James F. McBain Introduction ............................................................................................895 Philosophy ..............................................................................................895 Clinical Examination .............................................................................896 History...........................................................................................896 Visual Examination ......................................................................897 How Does the Animal Feel? .......................................................897 Buoyancy .......................................................................................897 Decreased Buoyancy .........................................................898 Increased Buoyancy ..........................................................898 Listing ................................................................................898 Social Behavior .............................................................................898 Hands-On Examination................................................................899 Urine Collection...........................................................................899 Stool Samples................................................................................899 Milk Samples ................................................................................899 Blowhole........................................................................................900 Additional Diagnostic Aids ...................................................................900 Body Weight ..................................................................................900 Ultrasonography ...........................................................................900 Radiography ..................................................................................900 Clinical Laboratory Tests.............................................................900 0839_frame_FM1 Page 45 Tuesday, May 22, 2001 2:42 PM Clinical Pathology..................................................................................901 Case Example: Pulmonary Disease .............................................901 Indicators of Inflammatory Disease ................................901 Therapeutics ...........................................................................................903 Surgery...........................................................................................903 Medical Therapy...........................................................................903 Oral Route.........................................................................903 Subcutaneous Route .........................................................904 Intramuscular Route.........................................................904 Intravenous Route ............................................................904 Topical Route ....................................................................904 Final Thoughts .......................................................................................905 Acknowledgments ..................................................................................905 References ...............................................................................................905 41 Seals and Sea Lions Frances M. D. Gulland, Martin Haulena, and Leslie A. Dierauf Introduction ............................................................................................907 Husbandry...............................................................................................907 Pools, Haul-Out Areas, and Enclosures ......................................907 Feeding ..........................................................................................908 Restraint..................................................................................................908 Physical Restraint.........................................................................908 Mechanical Restraint ...................................................................909 Chemical Restraint ......................................................................909 Physical Examination ............................................................................909 Diagnostic Techniques...........................................................................910 Blood Collection ...........................................................................910 Urine..............................................................................................910 Cerebrospinal Fluid ......................................................................911 Biopsies..........................................................................................911 Therapeutic Techniques ........................................................................911 Topical ...........................................................................................911 Oral................................................................................................911 Aerosol ..........................................................................................912 Subcutaneous ................................................................................912 Intramuscular................................................................................912 Intravenous ...................................................................................912 Intraosseous ..................................................................................912 Intraperitoneal ..............................................................................912 Diseases...................................................................................................913 Integumentary System .................................................................913 Musculoskeletal System ..............................................................915 0839_frame_FM1 Page 46 Tuesday, May 22, 2001 2:42 PM Digestive System ..........................................................................916 Respiratory System.......................................................................917 Cardiovascular ..............................................................................919 Urogenital System ........................................................................919 Endocrine System .........................................................................920 Eyes................................................................................................920 Nervous System............................................................................921 Acknowledgments ..................................................................................922 References ...............................................................................................922 42 Walruses Michael T. Walsh, Brad F. Andrews, and Jim Antrim Introduction ............................................................................................927 Biology.....................................................................................................927 Reproduction ..........................................................................................928 Diet..........................................................................................................929 Physical Examination ............................................................................929 Restraint..................................................................................................930 Manual ..........................................................................................930 Sedation and General Anesthesia................................................930 Specimen Collection and Diagnostic Techniques ...............................930 Medical Problems...................................................................................931 Dermatology .................................................................................931 Ophthalmology .............................................................................932 Tusk Infections and Trauma........................................................933 Foreign Bodies...............................................................................934 Intestinal Disease .........................................................................934 Miscellaneous Diseases................................................................935 Acknowledgments ..................................................................................935 References ...............................................................................................935 43 Manatees Gregory D. Bossart Introduction ............................................................................................939 Natural History ......................................................................................939 Anatomy, Physiology, and Behavior .....................................................941 Husbandry...............................................................................................942 Habitat Requirements ..................................................................942 Water Requirements.....................................................................942 Nutrition .......................................................................................943 Restraint, Handling, and Transport ............................................944 0839_frame_FM1 Page 47 Tuesday, May 22, 2001 2:42 PM Physical Examination...................................................................946 Diagnostic Techniques .................................................................946 Therapeutics .................................................................................948 Anesthesia .....................................................................................950 Environmental Diseases ........................................................................951 Brevetoxicosis ...............................................................................951 Cold Stress Syndrome ..................................................................951 Infectious Diseases.................................................................................952 Parasites ........................................................................................952 Miscellaneous Conditions .....................................................................953 Neoplasia.......................................................................................953 Neonatal Disease..........................................................................953 Human-Related Traumatic Injuries ............................................954 Acknowledgments ..................................................................................958 References ...............................................................................................958 44 Sea Otters Pamela Tuomi Introduction ............................................................................................961 History ....................................................................................................961 Classification ..........................................................................................962 Anatomy .................................................................................................963 Vision ......................................................................................................965 Social Organization ................................................................................965 Reproduction ..........................................................................................965 Causes of Mortality in Free-Living Otters ...........................................967 Feeding and Metabolism........................................................................967 Husbandry...............................................................................................969 Captive Nutrition...................................................................................971 Physical and Chemical Restraint..........................................................971 Clinical Examination .............................................................................973 Medical Abnormalities ..........................................................................974 Hypoglycemia ...............................................................................974 Hyperthermia................................................................................974 Hypothermia .................................................................................975 Loss of Coat Condition ................................................................975 Oil Exposure .................................................................................976 Abnormalities of Clinical Chemistry .........................................977 Gastroenteritis ..............................................................................978 Parasites ........................................................................................978 Miscellaneous Conditions ...........................................................979 Surgery ....................................................................................................979 Dentistry .................................................................................................980 0839_frame_FM1 Page 48 Tuesday, May 22, 2001 2:42 PM Preventive Medicine ..............................................................................980 Acknowledgments ..................................................................................980 References ...............................................................................................980 45 Polar Bears Michael Brent Briggs Introduction ............................................................................................989 Natural History and Physiology ...........................................................989 Nutrition .................................................................................................990 Nutrition of Juveniles, Early Pregnant, and Lactating Females..............................................................991 Infants............................................................................................992 Geriatrics.......................................................................................992 Reproduction ..........................................................................................992 Endocrinology .........................................................................................992 Reproductive Hormones ..............................................................992 Thyroid Hormones .......................................................................993 Housing ...................................................................................................993 Behavior ..................................................................................................994 Physical Examination ............................................................................994 Venipuncture ..........................................................................................995 Mechanical or Manual Restraint ..........................................................996 Anesthesia...............................................................................................996 Ketamine .......................................................................................997 Ketamine/Xylazine ......................................................................998 Tiletamine HCl and Zolazepam HCl .........................................998 Telazol/Medetomidine .................................................................999 Etorphine.......................................................................................999 Carfentanil ....................................................................................999 Fentanyl Citrate............................................................................999 Inhalation Agents .........................................................................999 Systemic Diseases ................................................................................1000 Developmental/Anomalous Diseases .......................................1000 Nutritional Diseases ..................................................................1000 Neoplasia.....................................................................................1000 Infectious Diseases .....................................................................1001 Viral Disease ...................................................................1001 Bacterial Disease.............................................................1001 Mycotic Disease..............................................................1001 Parasitic Disease .............................................................1002 Skin Disease................................................................................1002 Dental Disease............................................................................1003 Trauma ........................................................................................1003 Toxins ..........................................................................................1003 0839_frame_FM1 Page 49 Tuesday, May 22, 2001 2:42 PM Zoonoses ...............................................................................................1003 Acknowledgments ................................................................................1003 References .............................................................................................1004 Appendices Appendix A Conversions ...............................................................1011 Appendix B Abbreviations ............................................................1015 Appendix C Characteristics of Common Disinfectants ....1017 Index ...........................................................................................................1019 0839_frame_FM1 Page 50 Tuesday, May 22, 2001 2:42 PM 0839_frame_C01 Page 1 Tuesday, May 22, 2001 10:44 AM I Emerging Pathways in Marine Mammal Medicine 0839_frame_C01 Page 2 Tuesday, May 22, 2001 10:44 AM 0839_frame_C01 Page 3 Tuesday, May 22, 2001 10:44 AM 1 Marine Mammals as Sentinels of Ocean Health Michelle Lynn Reddy, Leslie A. Dierauf, and Frances M. D. Gulland Introduction It was January 1958, when Rachel Carson, a marine biologist who had been working with the U.S. Fish and Wildlife Service, received a letter from Olga Owens Huckins of Duxbury, Massachusetts. The letter told of birds dying after local applications of the pesticide DDT (dichlorodiphenyl trichloroethane) (Gore, 1994). DDT had already been known to have detrimental effects on birds (Robbins et al., 1951), and the evidence would continue to grow (Robinson, 1969; Faber and Hickey, 1973; Fry and Toone, 1981). More sensitive to the pesticides in their environment, the birds showed effects long before effects were seen in other wildlife species or in humans. Rachel Carson went on to write the landmark book Silent Spring (Carson, 1962), alerting the general public to the insidious effects of chemical pollutants. People were becoming better at understanding the importance of recognizing adverse reactions of wildlife to anthropogenic hazards in the environment. Carson’s local birds were sentinels of environmental changes that in time were shown to affect human health. However, these were not the first avian sentinels. At the turn of the 20th century, experiments by the Bureau of Mines showed that canaries taken into mines collapsed when exposed to carbon monoxide gas (the birds recovered when exposed to fresh air). Miners were able to avoid possible disaster by carrying caged canaries with them into mineshafts and tunnels. The birds alerted them to the presence of the deadly invisible gas (Burrell and Seibert, 1916). Sentinels The word sentinel has its origins in the Latin, sentire, which means to perceive or feel (Morris, 1975), and is now used to mean a person or animal who guards the group against surprise. The National Research Council (1991) defines an animal sentinel system as “a system in which data on animals exposed to contaminants in the environment are regularly and systematically collected and analyzed to identify potential health hazards to other animals or humans.” Sentinel systems provide knowledge needed to facilitate early responses to potentially hazardous conditions and to allow for more effective resource management. For such systems to be effective in controlling and preventing disease, they must be simple, sensitive, representative, 0-8493-0839-9/01/$0.00+$1.50 © 2001 by CRC Press LLC 3 0839_frame_C01 Page 4 Tuesday, May 22, 2001 10:44 AM 4 CRC Handbook of Marine Mammal Medicine and timely (CDC, 1988). Ideally, sentinels should detect changes prior to their effects becoming irreversible. Depending on what these systems are designed to monitor, animal sentinels can be wild or domestic, maintained in a laboratory or at a zoological park, and they can be terrestrial or marine (National Research Council, 1991). Animal species that are “charismatic megafauna”—such as whales and dolphins—make particularly good sentinels, because they have special public appeal and can be more effective at drawing societal attention and action to the plight of ecosystems. Invertebrates such as bivalves (clams, mussels, oysters) have been used widely as bioindicators of environmental contamination (Butler, 1973; Farrington et al., 1983). Bivalves are sedentary with relatively stable populations, so body burdens of contaminants reflect local conditions and can be used for long- and short-term pollution assessment. Additionally, they have a universal distribution that facilitates data comparison between many regions; they concentrate contaminants in their tissues; they have little or no detectable reactive enzyme systems to metabolize toxins, which makes assessment reasonably accurate; they are relatively tolerant of polluted conditions; and they are commercially available worldwide and thus have public health implications (Farrington et al., 1983; National Research Council, 1991). Vertebrates are also used as sentinels, and because they are at higher trophic levels than invertebrates, they are more likely to show the biomagnification effects of contaminants. Contaminant effects on sentinels, whether invertebrate or vertebrate, may occur at the suborganismal, organismal, or population level (Keith, 1996). Suborganismal effects include genotoxic effects, alterations in enzyme function, metallothionein induction, changes in thyroid function and retinol homeostasis, and hematological changes. Effects at the organismal level include pathological lesions, and alterations in development, growth, reproduction, and survival. Effects at the population level include alterations in abundance and distribution and changes in species assemblages (McCarthy and Shugart, 1990). Ecosystem Changes Detected by Sentinels Canaries are no longer used in mines; modern, technological carbon monoxide detection and monitoring devices have replaced them. Today the scope of environmental concern has expanded. The great number of humans inhabiting the Earth, in concert with their ever-increasing consumption and destruction of resources, places enormous pressures on the environment. By 2010, it is predicted that the Earth’s population will be 9.3 billion (Colborn et al., 1996). Yet we are far from understanding the effects of the alterations we are imposing on our environment. However, if data are carefully collected and analyzed from properly designed, implemented, and coordinated animal sentinel programs, we can make important inroads in detecting and mitigating some of the environmental threats we are inadvertently imposing upon ourselves. The effects of humans can be found in every ecosystem, whether it is deep in the dampest rain forest, high on the most frigid mountain top, or surrounded by the driest desert. However, the habitat that defines the planet Earth is the ocean, which covers 79% of the Earth’s surface. These effects may be direct, such as by the overharvesting of commercial fisheries, or indirect, through effects of runoff and global warming. Oceans facilitate the distribution of potentially toxic contaminants such as heavy metals and organochlorine (OC) chemicals. Comprising industrial chemicals such as polychlorinated biphenyls (PCBs) and chlorinated pesticides such as DDT, OCs tend to be stable and lipophilic. A group of experts attending a meeting on “Chemically Induced Alterations in Sexual Development: The Wildlife/Human Connection” concurred that “we are certain of the following: A large number of man-made chemicals that have been released into the environment … have the potential to disrupt the endocrine system of animals, including humans” (Colborn and Clement, 1992). 0839_frame_C01 Page 5 Tuesday, May 22, 2001 10:44 AM Marine Mammals as Sentinels of Ocean Health 5 In the sea, contaminants in runoff from urban, industrial, and agricultural activities intermix and bioaccumulate up the food chain, attaining the greatest concentrations in animals at the highest trophic levels, such as marine mammals. At an international workshop on marine mammals and persistent ocean contaminants in 1998, invited experts concluded that “there is good reason to be concerned that survival and reproduction in certain marine mammal populations may have been affected, and are being affected, by persistent contaminants, particularly OCs.” The workshop also concluded that there is a need for multidisciplinary studies on the significance of ocean contaminants in relation to the health and well-being of marine mammals (Marine Mammal Commission, 1998; see Chapter 22, Toxicology). Activities of humans and terrestrial animals also impact ocean health in other ways. Recently identified pathogens in marine mammals, such as Giardia lamblia, Sarcocystis neurona, Toxoplasma gondii, and antibiotic-resistant enteric bacteria, may all originate in waste from humans or their activities (Buergelt and Bonde, 1983; Olsen et al., 1997; Parveen et al., 1997; Johnson et al., 1998; LaPointe et al., 1999; Measures and Olsen, 1999). Runoff also increases nutrient load and availability, enhancing blooms of potentially toxic marine algae species such as Alexandrium spp. (produce saxitoxins), Gymnodinium breve (Ptychodiscus brevis) (produce brevitoxin), and Pseudonitzschia australis (produce domoic acid) (Geraci and Lounsbury, 1993; Smolowitz and Doucette, 1995; Scholin et al., 2000; see Chapter 2, Emerging and Resurging Diseases; Chapter 22, Toxicology). Whether such infectious agents and algal blooms are increasing in prevalence or are merely being detected more readily due to increasing awareness of ocean and marine mammal health issues is still subject of debate (Harvell et al., 1999). The ocean is also a sink for excess heat, and as such, it is an effective global thermostat (Carson, 1951). The National Oceanic and Atmospheric Administration (NOAA) National Climatic Data Center (NCDC) tracks land and sea temperature measurements. On its Web site (http://www.ncdc.noaa.gov/ol/climate/globalwarming.html), the NCDC reports that global surface temperatures have increased about 1°F (0.3 to 0.6°C) since the late 19th century, and about 0.5°F (0.2 to 0.3°C) over the past 40 years, which is the period with the most credible data. This warming trend is due to what is commonly known as the greenhouse gas effect—a result of industrial output of carbon dioxide, methane, and nitrous oxide that accumulates in the atmosphere and traps heat. Global climate change may alter animal abundance, distribution, and migration patterns, and has the potential to influence disease patterns worldwide (Aguilar and Raga, 1993; Daszak et al., 2000). Potential effects on cetaceans are reviewed by Burns (2000). Another form of pollution is noise pollution. Cetaceans have drawn attention to the increase in noise levels in the oceans (Richardson et al., 1995; National Research Council, 2000). Cetaceans use sound for a variety of purposes including foraging, communication, and navigation. It is feared that low-frequency, high-intensity noise generated by maritime shipping, polar icebreakers, offshore drilling, seismic surveys, oceanographic testing, and military use in the world’s oceans is a potentially serious problem for cetaceans, so there is a critical need for data on cetacean hearing for assessing the effects of such noise on these animals. Sound sources that have been developed for use in monitoring changes in ocean temperatures and detecting stealth submarines are currently hot topics. These sounds travel long distances, perhaps even masking sounds produced by marine mammals (National Research Council, 2000). Marine Mammals as Sentinels Holden (1972) was perhaps the first to formally propose the use of marine mammals as environmental sentinels. Marine mammals are good indicators of mid- to long-term changes in the environment, because many species have long life spans, feed at or near the top of the food chain, and have extensive fat stores (Aguilar and Borrell, 1994). Ironically, the blubber 0839_frame_C01 Page 6 Tuesday, May 22, 2001 10:44 AM 6 CRC Handbook of Marine Mammal Medicine that plays a crucial role in nutrition, buoyancy, and thermoregulation for these animals is an ideal repository for some contaminants. While the most inert and lipophilic of these contaminants may remain stored in the blubber until the animal dies, others may be metabolized, especially in times of physiological challenge such as illness, extreme temperature, nutritional compromise, or pregnancy and lactation (DeFreitas et al., 1969; McKenzie et al., 1997). The California sea lion (Zalophus californianus), harbor seal (Phoca vitulina), bottlenose dolphin (Tursiops truncatus), and beluga (Delphinapterus leucas) have been identified as model species for investigations into the effects of environmental contaminants on marine mammals (Marine Mammal Commission, 1998). The ecology and life histories of these animals are relatively well studied, they are relatively common thus more readily sampled, and they are well represented in facilities where breeding programs have been successful (Andrews et al., 1997). One way to more accurately ascertain contaminant effects on wild marine mammal populations is to use biomarkers in samples carefully collected from free-ranging animals (Peakall, 1992; Aguilar and Borrell, 1994). This is particularly true if samples are collected from representative members of populations that are the focus of long-term monitoring programs (Gaskin et al., 1982; Scott et al., 1990; Addison and Smith, 1998; Addison et al., 1998), especially when relevant biological data and health histories are available (Scott et al., 1990). However, regulations often prohibit collecting samples from young and their accompanying mothers in the wild, and there is no guarantee that any particular individual will be available for sampling. Additionally, data can be affected by variation in sample collection, handling, and processing, which can be difficult to control under field conditions. For example, when collecting blubber biopsies, it may be difficult to regulate the location and depth of the biopsy, both of which may affect results depending on the species (Aguilar and Borrell, 1994). In addition, because of the logistical difficulties and expenses involved in such operations, few are undertaken. Hunted marine mammals, such as the bowhead whale (Balaena mysticetus) harvested by the Inuit in Alaska, can also be sampled to yield information on ocean contaminants and marine mammal health (O’Hara et al., 1999). Because these animals are freshly dead and can be examined in detail, levels of contaminants can be correlated with histological changes in individual animals. Because bowhead whale populations have been well monitored, contaminant data from individuals yield insight into changes in reproduction and survival at the population level. Marine mammals have helped draw public attention to the current plight of fish stocks. For example, the western population of Steller sea lions (Eumetopias jubatus) has declined by more than 70% since the 1970s (Ferrero and Fritz, 2000), resulting in the addition of this species to the federal list of endangered species (National Marine Fisheries Service, 1992). The cause of the decline remains unclear and may be a combination of factors. Management actions have been implemented to reduce potential interactions between Steller sea lions and the Alaskan groundfish fishery (Ferrero and Fritz, 2000). However, it has been hypothesized that the large-scale harvesting of fish and whales that occurred from the 1950s through the early 1970s in the Bering Sea and Gulf of Alaska (National Research Council, 1996) may have altered the food web, allowing walleye pollock (Theragra chalcogramma) to become a dominant fish species (see Bowen, 1997). Pollock is an economically significant fish, as well as an important prey item for Steller sea lions (Lowry et al., 1989), so shortage in pollock stocks could significantly contribute to the decreasing numbers of these pinnipeds. Understanding the size composition of fishes eaten by a predator such as the Steller sea lion in relation to those of the commercial catch can lend much insight into marine mammal–fisheries interactions (Frost and Lowry, 1986). The Steller sea lion may thus prove to be an important sentinel for fish stocks in the Bering Sea. The exceptional hearing and sound production capabilities of cetaceans have long been recognized by scientists. Many species can hear sounds well outside the range of human hearing 0839_frame_C01 Page 7 Tuesday, May 22, 2001 10:44 AM Marine Mammals as Sentinels of Ocean Health 7 (Ridgway, 1997). Much has been learned about hearing in small cetacean species that are housed at marine mammal facilities. However, little or nothing is known about hearing in other cetacean species, such as the large baleen whales and some of the larger toothed whales such as beaked whales and the great sperm whale (Physeter catadon). Recently, intense sound from naval vessels has been implicated in several stranding events at various locations across the globe (Frantzis, 1998). Studies are currently under way to investigate the effects of anthropogenic noise on cetaceans (e.g., Au et al., 1999; Erbe and Farmer, 2000; Finneran et al., 2000; Schlundt et al., 2000). These studies will aid understanding of the effects of intense noise, which will contribute to the development of mitigation strategies ultimately to help find a balance between the basic needs of marine mammals and the important role the ocean plays in commerce, exploration, national defense, and travel. Stranded marine mammals are another source of information about the ocean environment (Geraci and Lounsbury, 1993; Gulland, 1999). Not only can they be sampled to quantify contaminant levels in tissues, but they can also alert researchers to diseases that are present in the more inaccessible wild animals that would be difficult to detect in random samplings of such populations. For example, 20% of sexually mature California sea lions that stranded and died along the northern coast of California showed neoplasia when examined post-mortem (Gulland et al., 1996). In comparison, only one case of neoplasia has been observed in California sea lions at rookeries on San Miguel Island, California, where more than 100,000 sea lions live (Spraker, pers. comm.). Study of neoplasia pathogenesis is more readily performed on stranded sea lions than on those in rookeries, and thus stranded animals essentially serve as sentinels for their wild conspecifics. Similarly, stranded belugas in the St. Lawrence estuary serve as sentinels of the health of the estuary. These whales have an unusually high prevalence of tumors and diseases for cetaceans, suggesting that this population is immunocompromised (Martineau et al., 1988; 1999; De Guise et al., 1994). These findings, coupled with the charismatic appeal of the beluga, have helped raise concern over contaminant levels in the St. Lawrence River and estuary. A number of infectious agents in marine mammals were first identified in stranded animals, after which their presence in the free-ranging population was confirmed. These include phocine distemper virus (PDV), which caused the death of over 18,000 harbor seals in Europe in 1988 (Osterhaus and Vedder, 1988), phocine herpes virus (PhHV1) isolated from stranded harbor seals in 1985 (Osterhaus et al., 1985), and Brucella in a variety of species (Ross et al., 1994; Garner et al., 1997) (see Chapter 15, Viral Diseases; Chapter 16, Bacterial Diseases). Live stranded animals offer an opportunity to monitor clinical signs that may result from changes in ocean health. For example, thorough examination of stranded, sick California sea lions resulted in the detection of domoic acid, a recently identified marine biotoxin, produced by the diatom Pseudonitzschia australis. The sea lions had consumed toxin-laden anchovies, and the domoic acid concentrated in the tissues of the sea lions caused muscle tremors, seizures, and death (Scholin et al., 2000) (see Chapter 2, Emerging and Resurging Diseases). In this case, the findings warned against human consumption of the anchovies, and increased monitoring of other seafood in the area. Stranded animals do not constitute an ideal sentinel system, as they do not represent the entire population (Aguilar and Borrell, 1994). In addition, samples of stranded animals are rarely age and sex structured, and biological data such as individual life histories, feeding habits, reproductive success, or disease progression are not typically available. Furthermore, contaminant levels in tissues collected from animals found dead may be significantly affected by decomposition of the samples (Borrell and Aguilar, 1990) (see Chapter 22, Toxicology). Marine mammals maintained at research and display facilities can be effective sentinels. The authors of the Marine Mammal Protection Act (MMPA), passed by Congress in 1972 (see Chapter 33, Legislation), understood the value of marine mammals in collections for conducting 0839_frame_C01 Page 8 Tuesday, May 22, 2001 10:44 AM 8 CRC Handbook of Marine Mammal Medicine research and raising environmental awareness. They specifically allowed for the collection of marine mammals, stating “(3) there is inadequate knowledge of the ecology and population dynamics of such marine mammals and of the factors which bear upon their ability to reproduce themselves successfully; (4) negotiations should be undertaken immediately to encourage the development of international arrangements for research on, and conservation of, all marine mammals” (MMPA sec. 2, p. 2). Reijnders (1988) stated, “Even more than before, marine mammals in captivity should be used to obtain a set of reference data to interpret values obtained from animals expected to be affected by contaminants.” There are many advantages to using animals under human care as sentinels. Longitudinal health data are available for long-term studies and may provide insight into transgenerational and long-term health trends. These animals are fed wild-caught fish that have naturally occurring levels and mixtures of contaminants. These contaminants can be identified and quantified to provide insight not only into the dietary exposure of the marine mammals, but also into ecosystem levels and distribution of OCs that may impact the seafood-consuming public. In addition, tissues and fluids, including storage (blubber) and circulating (blood) compartments, can be regularly and systematically collected using conditioned husbandry behaviors, whereby the animals cooperate in specimen collection. Biological data such as age, sex, nutritional state, and reproductive and health histories can be recorded and correlated with measured contaminant levels. Changes in blubber levels can be correlated with levels in blood. Studies can be designed to establish effective biomarkers for monitoring complex physiological functions, such as immune and neurological responses and effects on reproduction. Contaminant monitoring is currently ongoing in San Diego where a large collection of bottlenose dolphins is maintained by the U.S. Navy. The animals reside in netted enclosures in San Diego Bay, California, often work in the open ocean, and are fed a diet from known sources. Preliminary research has revealed that preprandially collected blood can be used to estimate blubber levels of contaminants using lipid-normalized levels of OCs found in blood (Reddy et al., 1998). Milk samples collected voluntarily (Kamolnick et al., 1994) from lactating females in this population showed that from day 94 to day 615 of lactation, lipid-normalized levels of PCB and DDE (dichlorodiphenyl dichloroethylene) decreased by 69 and 82%, respectively (Ridgway and Reddy, 1995). In addition, preliminary data showed that concentrations of several OC contaminants in maternal blubber correlated strongly with reproductive outcome in these animals (Reddy et al., 2000). This population may provide a useful benchmark for marine mammal OC studies. Marine mammals can also be temporarily collected for contaminant studies; two such studies have been conducted with groups of harbor seals (Reijnders, 1986; Brouwer et al., 1989; de Swart et al., 1994; 1996; Ross et al., 1995; 1996). In these studies, half of the animals were fed fish from a highly polluted source and the other half were fed fish from a lesspolluted source. Results showed that animals fed higher levels of contaminants had reduced levels of circulating thyroid hormone and vitamin A, suppressed immune responses, and reduced reproductive success. A comprehensive marine mammal sentinel system would best include data collected from many sources including stranded animals, wild populations, and animals in collections. To ensure data quality, and to facilitate comparison between studies, it is important to standardize sample collection and handling protocols and to maintain archived samples to study as new analytical methods and technologies are developed (Wise et al., 1993) (see Chapter 21, Necropsy; Chapter 22, Toxicology). Linking these studies with laboratory toxicity studies should provide valuable insight into natural exposure and potential risk assessment and management strategies (National Research Council, 1991; Ross, 2000). 0839_frame_C01 Page 9 Tuesday, May 22, 2001 10:44 AM Marine Mammals as Sentinels of Ocean Health 9 Conclusion Marine mammals are effective ambassadors for the ocean environment because of their great public appeal. O’Shea points out, “For the general public, marine mammals are one of the most conspicuous components of marine biological diversity. Any that come ashore dead or ill raise the levels of uneasiness about the health of our oceans” (Geraci and Lounsbury, 1993). Stenciled images of marine mammals on storm drains in coastal cities with reminders of “No dumping, we live downstream” support this sentiment. More than ever, it is imperative to use an interdisciplinary and interagency approach. Long-term monitoring of populations and toxicological and disease investigations are expensive, time-consuming, and complex. The collaborative expertise of specialists, including oceanographers, geographers, chemists, biologists, physicians, veterinarians, epidemiologists, and pathologists, is needed to understand the effects of ocean health on the health of marine mammals and potentially humans. Klamer et al. (1991) predicted, “If the increase in ocean PCB concentrations continues, it may ultimately result in the extinction of fish-eating marine mammals.” But there is still time. The ocean has not yet fallen silent in the fashion forewarned by Rachel Carson in Silent Spring (1962). The great mammals of the sea have much to tell us, if only we learn to listen. Acknowledgments The authors thank Gwen Griffith, Scott Newman, Andy Draper, and Donna Staples for reviewing this chapter. References Addison, R.F., and Smith, T.G., 1998, Trends in organochlorine residue concentrations in ringed seal (Phoca hispida) from Holman, Northwest Territories, 1972–91, Arctic, 51: 253–261. Addison, R.F., Stobo, W.T., and Zinck, M.E., 1998, Organochlorine residue concentrations in blubber of grey seal (Halichoerus grypus) from Sable Island, N.S. 1974–1994: Compilation of data and analysis of trends, Can. Data Rep. Fish. Aquat. Sci., 1043. Aguilar, A., and Borrell, A., 1994, Assessment of organochlorine pollutants in cetaceans by means of skin and hypodermic biopsies. Chapter 11, in Nondestructive Biomarkers in Vertebrates, Fossi, M.C., and Leonzio, C. (Eds.), Lewis Publishers, Boca Raton, FL, 245–267. 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Scholin, C.A., Gulland, F., Doucette, G.J., Benson, S., Busman, M., Chavez, F.P., Cordaro, J., DeLong, R., De Vogelaere, A., Harvey, J., Haulena, M., Lefebvre, K., Lipscomb, T., Loscutoff, S., Lowenstine, L.J., Marin III, R., Miller, P.E., McLellan, W.A., Moeller, P.D.R., Powell, C.L., Rowles, T., Silvagni, P., Silver, M., Spraker, T., Trainer, V., and Van Dolah, F.M., 2000, Mortality of sea lions along the central California coast linked to a toxic diatom bloom, Nature, 403: 80–84. 0839_frame_C01 Page 13 Tuesday, May 22, 2001 10:44 AM Marine Mammals as Sentinels of Ocean Health 13 Scott, M.D., Wells, R.S., and Irvine, A.F., 1990, A long-term study of bottlenose dolphins on the west coast of Florida, in The Bottlenose Dolphin, Leatherwood, S., and Reeves, R.R. (Eds.), Academic Press, San Diego, CA, 235–244. Smolowitz, R., and Doucette, G., 1995, The localization of saxitoxin and saxitoxin-producing bacteria in the siphons of butter clams, Saxidomus giganteus, Abstr., 26th Annual Proceedings of the International Association for Aquatic Animal Medicine, Mystic, CT, 66. Wise, S.A., Schantz, M.M., Koster, B.J., Demiralp, R., Mackey, E.A., Greenverg, T.T., Burow, M., Ostapczuk, P., and Lillestolen, T.I., 1993, Development of frozen whale blubber and liver reference materials for the measurement of organic and inorganic contaminants, Fresenius J. Anal. Chem., 345: 270–277. 0839_frame_C01 Page 14 Tuesday, May 22, 2001 10:44 AM 0839_frame_C02 Page 15 Tuesday, May 22, 2001 10:40 AM 2 Emerging and Resurging Diseases Debra Lee Miller, Ruth Y. Ewing, and Gregory D. Bossart Introduction Emerging and resurging diseases affect both plants and animals worldwide. Novel zoonotic diseases usually cause concern because of their potential impacts on human health, but other diseases that can cause significant morbidity or mortality are also of concern because of their potential conservation importance. They can be especially devastating to endangered species where population levels are critically low (Harwood and Hall, 1990). For the purposes of this chapter, emerging diseases are defined as those diseases that have not been identified previously, or are considered a novel threat to the currently afflicted species (Wilson, 1999), and the chapter concentrates on diseases that have emerged in the past decade. Here resurging diseases are defined as those that historically have been documented in the species currently affected, but were considered to be eradicated or to no longer pose a significant problem. Unfortunately, it is often difficult to correctly define a disease as emerging or resurging in free-ranging wildlife. It therefore may be more appropriate to label such diseases as presumptive emerging or resurging diseases, given the paucity of historical data and the lack of baseline reference values from which to draw conclusions one way or the other. Daszak et al. (2000) describe three ways that wildlife species are exposed to emerging diseases. First, diseases emerge among wildlife species as a result of spillover from domestic species. This route has become increasingly common as domestic species encroach upon wildlife habitat, resulting in increased contact between domestic and wild animals. The introduction of canine distemper virus (CDV) to seals is a prime example of spillover to the marine environment. Initially, the etiologies of phocine morbillivirus outbreaks occurring in the 1980s were characterized serologically as phocine distemper virus (PDV) 1 and PDV-2 (Ross et al., 1992). These two strains were antigenically distinct from CDV and from each other (Visser et al., 1990). Subsequently, molecular analysis of isolates from tissues of Baikal seals (Phoca sibirica) revealed a wild-type CDV (Visser et al., 1993; Mamaev et al., 1995). Transmission of this new strain is thought to be via aerosols from domestic or feral dogs (Lyons et al., 1993). Aerosol transmission of CDV from adjacent susceptible terrestrial species such as raccoons and foxes is also possible. A very recent outbreak of CVD in Caspian seals (P. caspica) is thought to be responsible for about 10,000 deaths (Kennedy et al., 2000). The second mode of disease emergence occurs as an unfortunate consequence of efforts to restock species for conservation purposes (Daszak et al., 2000). This practice has allowed the 0-8493-0839-9/01/$0.00+$1.50 © 2001 by CRC Press LLC 15 0839_frame_C02 Page 16 Tuesday, May 22, 2001 10:40 AM 16 CRC Handbook of Marine Mammal Medicine translocation of hosts and pathogenic organisms, facilitating the exposure of previously naive animals to new diseases. Examples in marine mammals are currently rare, although the spread of leptospirosis was described in harbor seals (P. vitulina) during rehabilitation, probably as a result of exposure to terrestrial mammals, such as skunks (Stamper et al., 1998). The difficulty in preventing spread of disease in the open ocean environment means that, once introduced, the consequences of a novel disease could be devastating. Finally, natural phenomena, such as weather patterns like El Niño, can have profound effects on species and may greatly enhance the proliferation and/or transport of pathogenic organisms (Fauquier et al., 1998; Hoegh-Guldberg, 1999). This third mode of disease emergence is especially relevant to marine wildlife, and may be a major cause of disease resurgence (Harvell et al., 1999). Whether they are emerging or resurging, the diseases that impact marine mammals today deserve close attention, since the results are often devastating and the etiologies complex. Epizootics often involve multiple disease entities, with a primary etiology often difficult or nearly impossible to determine. For example, morbillivirus infections, which had not been documented in pinnipeds or cetaceans prior to 1988, have resulted in at least six marine mammal epizootics, and were implicated in mass mortality of the fragile Mauritanian population of Mediterranean monk seals (Monachus monachus) (Osterhaus et al., 1997; Kennedy, 1998). However, some investigators attributed the primary etiology of the monk seal mortality event to a harmful algal bloom of Alexandrium spp. (Hernández et al., 1998), resulting in considerable debate (Harwood, 1998). To solve issues such as these, multidisciplinary teams of investigators are needed. Wildlife veterinarians and biologists are now embracing the challenge of identifying disease processes occurring in wildlife species, their etiologies, and the impact they have on individuals, populations, and the species as a whole. Advanced technologies, such as the polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), in situ hybridization, genetic sequencing, electron microscopy, and immunohistochemistry, have greatly enhanced our ability to identify disease etiologies. Similarly, advanced telemetry equipment has improved monitoring of free-ranging populations (see Chapter 38, Tagging and Tracking). Combining the laboratory-based identification of disease etiology with longterm population monitoring by field biologists is key to understanding diseases in wildlife. Given these tools, several diseases have recently been identified as either emerging or resurging in marine mammals. Cetaceans Viral, bacterial, and neoplastic diseases are among the most important emerging and resurging diseases of cetaceans (Table 1) (also see Chapter 15, Viral Diseases; Chapter 16, Bacterial Diseases; Chapter 18, Parasitic Diseases; and Chapter 23, Noninfectious Diseases). For example, in the last decade, morbilliviruses have emerged as significant pathogens of cetaceans and pinnipeds worldwide. The origin of these viruses is undetermined, and their pathogenesis and epidemiology are just unfolding. Nucleotide sequence analysis of viral RNA isolated from Atlantic bottlenose dolphins ( Tursiops truncatus) that died in the 1987–1988 Atlantic Coast and the 1993 Gulf of Mexico epizootics indicated that the porpoise morbillivirus (PMV) and dolphin morbillivirus (DMV) are not species specific (Taubenberger et al., 1996). The 1987–1988 Atlantic Coast epizootic was a mixed infection; animals were infected with either DMV or PMV, and some animals had dual infections with both viral types. Only PMV was detected in dead animals from the 1993 Gulf of Mexico epizootic and the 1994 Irish Coast harbor porpoise (Phocoena phocoena) die-off, and 0839_frame_C02 Page 17 Tuesday, May 22, 2001 10:40 AM 17 Emerging and Resurging Diseases TABLE 1 Identified Emerging and Resurging Diseases in Cetaceans Disease/Etiological Agent Papillomavirus Porpoise morbillivirus Dolphin morbillivirus Pilot whale morbillivirus Unknown type of morbillivirus, first in baleen whale Arbovirus (Togaviridae) encephalitis Hepadnaviral hepatitis Brucella spp. Host Species Orcinus orca (killer whale) Tursiops truncatus (Atlantic bottlenose dolphin) Phocoena phocoena (harbor porpoise) Lagenorhynchus obscurus (dusky dolphin) Phocoena spinipinnis (Burmeister’s porpoise) Tursiops truncatus (Pacific bottlenose dolphin) Tursiops truncatus (Atlantic bottlenose dolphin) Phocoena phocoena (harbor porpoise) Tursiops truncatus (Atlantic bottlenose dolphin) Stenella coeruleoalba (striped dolphin) Delphinus delphis (Pacific common dolphin) Delphinus delphis ponticus (Black Sea common dolphin) Globicephala melaena/melas (long-finned pilot whale) Balaenoptera physalus (fin whale) Orcinus orca (killer whale) Lagenorhynchus obliquidens (Pacific white-sided dolphin) Tursiops truncatus (Atlantic bottlenose dolphin) Tursiops truncatus (Atlantic bottlenose dolphin) Lagenorhynchus acutus (Atlantic white-sided dolphin) Stenella coeruleoalba (striped dolphin) Delphinus delphis (common dolphin) Phocoena phocoena (harbor porpoise) Orcinus orca (killer whale) Globicephala spp. (pilot whale) Balaenoptera acutorostrata (minke whale) Reference Bossart et al., 1997; 2000 Cassonnet et al., 1998 Van Bressem et al., 1999 Bossart and Ewing, unpublished data Barrett et al., 1993 Taubenberger et al., 1996 Domingo et al., 1990 Lipscomb et al., 1994 Taubenberger et al., 1996 Reidarson et al., 1998; Birkun et al., 1999 Taubenberger et al., 2000 Jauniaux et al., 1998 Bossart and Ewing, unpublished data Bossart et al., 1990; Bossart, unpublished data Foster et al., 1996 Clavareau et al., 1998 Miller et al., 1999 (Continued) 0839_frame_C02 Page 18 Tuesday, May 22, 2001 10:40 AM 18 CRC Handbook of Marine Mammal Medicine TABLE 1 Identified Emerging and Resurging Diseases in Cetaceans (continued) Disease/Etiological Agent Helicobacter spp. Lobomycosis Histoplasmosis Coccidioidomycosis Immunoblastic malignant lymphoma Oral squamous cell carcinoma Renal adenoma Pulmonary carcinoma Angiomatosis Host Species Balaenoptera physalus (fin whale) Balaenoptera borealis (sei whale) Lagenorhynchus acutus (Atlantic white-sided dolphin) Tursiops truncatus (Atlantic bottlenose dolphin) Tursiops truncatus (Atlantic bottlenose dolphin) Tursiops truncatus (Pacific bottlenose dolphin) Tursiops truncatus (Atlantic bottlenose dolphin) Stenella frontalis (Atlantic spotted dolphin) Stenella attenuata (pantropical spotted dolphin) Tursiops truncatus (Atlantic bottlenose dolphin) Tursiops truncatus (Atlantic bottlenose dolphin) Tursiops truncatus (Atlantic bottlenose dolphin) Tursiops truncatus (Atlantic bottlenose dolphin) Reference Fox et al., 2000 Haubold et al., 1998 Jensen et al., 1998 Reidarson et al., 1998 Bossart et al., 1997 Renner et al., 1999 Cowan and Turnbull, 1999 Ewing and MignucciGiannoni, in review Turnbull and Cowan, 1999 only DMV was recovered in the Mediterranean striped dolphin (Stenella coeruleoalba) epizootic. Taubenberger et al. (1996) proposed that cetacean morbilliviruses had actually been present in the western Atlantic prior to the European epizootics. Lipscomb et al. (1994) retrospectively examined histological specimens from the 1987–1988 Atlantic Coast epizootic for morbillivirus antigen; using immunocytochemical techniques, they detected morbillivirus antigen in 53% of the animals examined. Duignan et al. (1995a) found morbillivirus antibodies in 86% of two species of pilot whales (Globicephala melas and G. macrorhynchus) in the western Atlantic. They hypothesized that pilot whales were long-distance vectors during their trans-Atlantic migrations (Duignan et al., 1995b). Barrett et al. (1995) found that 93% of the long-finned pilot whales (G. melas) that mass-stranded between 1982 and 1993 were morbillivirus seropositive, providing further evidence that cetacean morbilliviruses are widespread, occurring in many cetacean species in the Atlantic. Interestingly, recent molecular findings of Taubenberger et al. (2000) suggest that the long-finned pilot whale is host to a different, novel type of cetacean morbillivirus, distinct from both PMV and DMV. Since the cetacean morbillivirus epizootics in Europe, the northwest Atlantic, and the Gulf of Mexico, there has been evidence of morbillivirus circulating through certain Pacific odontocete populations (Reidarson et al., 1998b; Van Bressem et al., 1998; Uchida et al., 1999). There are seropositive dusky dolphins (Lagenorhynchus obscurus), common dolphins (Delphinus delphis), and offshore bottlenose dolphins (T. truncatus) in the southeastern Pacific (Van Bressem et al., 1998). Common dolphins in the northeastern Pacific were seropositive and had viral RNA detected 0839_frame_C02 Page 19 Tuesday, May 22, 2001 10:40 AM Emerging and Resurging Diseases 19 by PCR, although they did not show clinical signs of disease (Reidarson et al., 1998b). Uchida et al. (1999) reported a striped dolphin with nonpurulent meningoencephalomyelitis that stranded in Miyazaki, Japan. Using immunocytochemical techniques, they applied monoclonal anti-CDV antibodies and detected positive immunoreactivity in degenerate and intact neurons, suggesting a spontaneous morbillivirus infection. Benign mucosal and cutaneous papillomas, and/or fibropapillomas, have been characterized macroscopically and microscopically in various cetacean species. A papillomavirus etiology has been implicated for lesions in killer whales (Orcinus orca), sperm whales (Physeter macrocephalus), belugas (Delphinapterus leucas), harbor porpoises, Burmeister’s porpoises (Phocoena spinipinnis), dusky dolphins, and the offshore stock of bottlenose dolphins (Lambertsen et al., 1987; De Guise et al., 1994; Van Bressem et al., 1996; 1999; Bossart et al., 2000). Strong supportive evidence includes transmission electron microscopy (TEM), immunocytochemistry, and DNA in situ hybridization. Papillomavirus DNA was recently amplified by PCR of DNA from warts on genital slits of Burmeister’s porpoises, dusky dolphins, and bottlenose dolphins retrieved from the Peruvian coast (Cassonnet et al., 1999). Although viral diseases have had the most dramatic effects on cetaceans in the last decade, bacterial diseases are also important emerging diseases in cetaceans. Brucellosis, an apparently novel infectious disease of marine mammals with both zoonotic and economic implications, was reported in various seals, porpoises, dolphins, and a river otter (Lontra canadensis) (Foster et al., 1996), and an aborted bottlenose dolphin (Miller et al., 1999). Interestingly, retrospective studies of banked serum from stranded pinnipeds and cetaceans from the coasts of England and Wales collected between 1989 to 1995 revealed that the first positive sample occurred as early as 1990 (Jepson et al., 1997) (see Chapter 16, Bacterial Diseases). Recently, a novel Helicobacter species was cultured from the gastric mucosa of stranded Atlantic white-sided dolphins (Lagenorhyncus acutus) and identified using PCR (Fox et al., 2000). By using 16s rRNA analysis, the isolates were determined to be a novel species. By using a Warthin–Starry stain, spirochete bacteria were observed associated with proliferative lymphoplasmocytic gastritis. These findings suggest that this novel Helicobacter species may have a role in the pathogenesis of dolphin gastritis and ulceration. Pinnipeds Toxins, neoplasia, and viral, bacterial, and parasitic diseases have all recently been identified as causing, or being associated with, significant morbidity or mortality in pinnipeds, especially in free-ranging populations (Table 2). Although the effects of morbilliviruses on pinnipeds have been dramatic, they will not be discussed further here (see Chapter 15, Viral Diseases). Domoic acid–induced morbidity and mortality may represent a resurging disease in eastern Pacific pinniped populations. Recent mortality of California sea lions (Zalophus californianus) along the central coast of California in 1998 and 2000 was attributed to harmful algal blooms (Gulland, 2000; Scholin et al., 2000). Domoic acid (DA) produced by the diatom Pseudonitzschia australis was detected in sea lion serum, urine, and feces, and in anchovy tissues (Lefebvre et al., 1999; Scholin et al., 2000). Demonstration of DA in the sea lion prey species suggests an oral route as the mode of toxin transmission. Histological examination of tissues revealed brain lesions characteristic of DA intoxication, including severe anterioventral hippocampal neuronal necrosis and marked neutrophil vacuolation within certain strata of the hippocampus and dentate gyri (Scholin et al., 2000). There have been documented cases of neurological dysfunction and mortality in sea lions, northern fur seals (Callorhinus ursinus), and dolphins (Gulland, 2000), which could have been associated with Pseudonitzschia blooms 0839_frame_C02 Page 20 Tuesday, May 22, 2001 10:40 AM 20 CRC Handbook of Marine Mammal Medicine TABLE 2 Identified Emerging and Resurging Diseases in Pinnipeds Disease/Etiological Agent Phocine herpesvirus-1 and -2 Phocine morbillivirus Canine distemper virus Monk seal morbillivirus-WA Monk seal morbillivirus-G Influenza B Coronavirus Brucella spp. Campylobacter-like bacterium Coxiella burnetii Mycobacterium spp. Host Species Phoca vitulina (harbor seal) Phoca vitulina (harbor seal) Pagophilus groenlandicus (harp seal) Cystophora cristata (hooded seal) Phoca hispida (ringed seal) Odobenus rosmarus rosmarus (Atlantic walrus) Halichoerus grypus (gray seal) Phoca sibirica (Baikal seal) Halichoerus grypus (gray seal) Phoca caspica (Caspian seal) Monachus monachus (Mediterranean monk seal) Monachus monachus (Mediterranean monk seal) Halichoerus grypus (gray seal) Phoca vitulina (harbor seal) Phoca vitulina (harbor seal) Phoca vitulina (harbor seal) Zalophus californianus (California sea lion) Odobenus rosmarus rosmarus (Atlantic walrus) Pagophilus groenlandicus (harp seal) Phoca hispida (ringed seal) Cystophora cristata (hooded seal) Halichoerus grypus (gray seal) Phocarctos hookeri (New Zealand sea lion) Phoca vitulina (harbor seal) Arctocephalus spp. (fur seal) Reference Gulland et al., 1997; Harder et al., 1996 De Koeijer et al., 1998 Duignan et al., 1994; 1997 Visser et al., 1993 Kennedy et al., 1990 Mamaev et al., 1995 Visser et al., 1993 Lyons et al., 1993; Forsyth et al., 1998; Kennedy et al., 2000 Osterhaus et al., 1998 Osterhaus et al., 1998 Osterhaus et al., 2000 Bossart and Schwartz, 1990 Forbes et al., 2000 Tryland et al., 1999 Foster et al., 1996 Baker, 1999 La Pointe et al., 1999 Hunter et al., 1998 0839_frame_C02 Page 21 Tuesday, May 22, 2001 10:40 AM 21 Emerging and Resurging Diseases TABLE 2 Identified Emerging and Resurging Diseases in Pinnipeds (continued) Disease/Etiological Agent Listeria ivanovii Sarcocystis neurona-like Giardia spp. Cryptosporidia spp. Contracaecum corderoi Ophthalmic condition Host Species Otaria byronia (southern sea lion) Arctocephalus australis (South American fur seal) Phoca vitulina (harbor seal) Phoca vitulina (harbor seal) Phoca hispida (ringed seal) Pagophilus groenlandicus (harp seal) Phoca vitulina (harbor seal) Halichoerus grypus (gray seal) Zalophus californianus (California sea lion) Zalophus californianus (California sea lion) Monachus schauinslandi (Hawaiian monk seal) Reference Bernardelli et al., 1996 Thornton et al., 1998 Lapointe et al., 1998 Olson et al., 1997 Measures and Olson, 1999 Deng et al., 2000 Deng et al., 2000 Fletcher et al., 1998 Banish and Gilmartin, 1992 that have occurred along the California coast over the past three decades (Walz et al., 1994). However, the DA-producing diatom P. australis did not receive much attention until a seabird mortality event occurred concurrently with a P. australis bloom in Monterey Bay, California, in 1991 (Work et al., 1993). The impacts of human and climatic activities on coastal seawater temperatures and quality may influence algal species diversity and abundance. Hernández et al. (1998) detected variable levels of numerous paralytic toxins, including decarbamoyl saxitoxin, neosaxitoxin, and gonyautoxin-1 in Mediterranean monk seal liver, kidney, skeletal muscle, and brain collected during a 1997 mortality event. The same toxins were detected in certain monk seal prey species, suggesting an available source of toxin and providing a strong indication that saxitoxins may have played a role in the monk seal mortality event. However, both the lethal toxin levels and the pharmacokinetics and baseline levels of saxitoxin in tissues of monk seals are unknown, making it difficult to interpret the toxin levels found in the animals from the 1997 epizootic (Harwood, 1998). Metastatic urogenital epithelial cell carcinomas have been reported in stranded California sea lions over the last 20 years (Gulland et al., 1996). The high prevalence of urogenital neoplasia in California sea lions suggests either a communicable infectious etiology or a common exposure to oncogenic environmental factors. Investigations of tumor etiopathogenesis have focused on the role of environmental chemical contaminants and viruses (Gulland et al., 1995; Buckles et al., 1999; Lipscomb et al., 2000). In examining cases of metastatic urogenital carcinoma, Lipscomb et al. (2000) described areas of intraepithelial neoplasia with cells containing eosinophilic intranuclear inclusion bodies. By using immunocytochemical techniques, these intranuclear inclusion bodies were shown to be positive for Epstein–Barr virus latent membrane protein. Additionally, herpesvirus-like particles were observed by TEM, and 0839_frame_C02 Page 22 Tuesday, May 22, 2001 10:40 AM 22 CRC Handbook of Marine Mammal Medicine amplification of DNA extracted from frozen tumor samples was positive for consensus regions of herpesvirus terminase and DNA polymerase genes. Additional nucleotide sequence data indicate that the herpesvirus detected is a member of the γ-herpesvirus family. The most significant emerging bacterial disease of pinnipeds is currently brucellosis (see Chapter 16, Bacterial Diseases). Brucella spp. have been isolated from harbor seals in the eastern Pacific (Garner et al., 1997b) and from ringed (Phoca hispida) and harp seals (Pagophilus groenlandicus) near the Magdalene Islands, Gulf of St. Lawrence (Forbes et al., 2000). These marine mammal isolates are genetically distinct from currently recognized terrestrial species of Brucella and are considered novel Brucella species (Jahans et al., 1997; Bricker et al., 2000). Serological surveys for antibodies to Brucella in various species, including hooded (Cystophora cristata), harp, and ringed seals, indicate that this Brucella species is well distributed in northern Atlantic marine mammal populations (Tryland et al., 1999). Other zoonotic organisms emerging as pathogens of marine mammals are Cryptosporidium and Giardia spp. Canadian researchers investigated the prevalence of Giardia spp. and Cryptosporidium spp. in marine mammals from the Canadian western Arctic region in 1994 and 1995 and on the eastern Canadian Coast in 1997 and 1998. Giardia spp. cysts were positively detected in feces by fluorescein isothiocyanate (FITC)-labeled monoclonal antibody (Olson et al., 1997; Measures and Olson, 1999). Along the eastern coast, Giardia spp. occurred at a prevalence of 25% in gray (Halichoerus grypus) and harbor seals from the Gulf of St. Lawrence and the St. Lawrence estuary (Measures and Olson, 1999). Adult harp seals, sampled near the Magdalene Islands, Gulf of St. Lawrence, had the highest prevalence of Giardia cysts, at 50%. All pups less than 1 year of age were negative for cysts. In the western Arctic region, specifically the Holman region of the Northwest Territories, there was a 20% prevalence of Giardia in ringed seals (Olson et al., 1997). Incidentally, belugas sampled from both sites, and a northern bottlenose whale (Hyperoodon ampullatus) sampled from eastern Canada, were negative for Giardia spp. (Olson et al., 1997; Measures and Olson, 1999). Deng et al. (2000) investigated the prevalence of Cryptosporidium spp. as well as Giardia spp. in Pacific harbor seals, northern elephant seals (Mirounga angustirostris), and California sea lions from the northern California coast. They detected Cryptosporidium spp. oocysts in three California sea lions, one of which also had Giardia spp. cysts. Oocysts were then isolated and purified for PCR characterization: C. parvum and G. duodenalis were identified based on genetic characterization and morphological and immunological findings. Another protozoan, Sarcocystis spp., has been recognized as an important cause of mortality in adult Pacific harbor seals along the central California coastline (La Pointe et al., 1998). Microscopically, every case presented with marked to severe cerebellar nonsuppurative meningoencephalitis associated with S. neurona–like protozoa (La Pointe et al., 1998; Chechowitz et al., 1999). This protozoal parasite was isolated from the brain tissue from one harbor seal, and investigations are currently under way to further characterize it genetically and serologically. A helminth of emerging importance to pinnipeds is the nematode Contracaecum corderoi. From January 1992 through December 1997, C. corderoi induced gastrointestinal perforations with associated peritonitis in stranded California sea lions along the central California coast (Fletcher et al., 1998). At that time, C. corderoi had only been reported in southern fur seals (Arctocephalus australis) (Dailey and Brownell, 1972). Manatees Currently, mortality associated with toxic algal blooms is the resurging disease with the most impact on manatees (see Chapter 22, Toxicology). From early March to late April 1996, at least 150 manatees died in an unprecedented epizootic along approximately 80 miles of the 0839_frame_C02 Page 23 Wednesday, May 23, 2001 10:41 AM Emerging and Resurging Diseases 23 southwest coast of Florida (U.S. Marine Mammal Commission Annual Report to Congress, 1996). Brevetoxicosis was a primary component (Bossart et al., 1998). Grossly, severe nasopharyngeal, pulmonary, hepatic, renal, and cerebral congestion was present in all cases. Staining with interleukin-1β-converting enzyme was positive for brevetoxin in lymphocytes and macrophages in the lung, liver, and in secondary lymphoid tissues. Retrospective immunohistochemical staining of manatee tissues from an epizootic in 1982 (O’Shea, 1991) revealed widespread brevetoxin, suggesting brevetoxicosis as a component of, and the likely primary etiology for, epizootics in 1982 and 1996. As for many marine mammal species, cutaneous viral papillomatosis is an emerging disease in the Florida manatee (Trichechus manatus latirostris). Ewing et al. (1997) first reported suspected viral cutaneous papillomatosis in a captive West Indian manatee (T. manatus); diagnosis was made by light and transmission electron microscopy, which showed 45 to 50 nm spherical to hexagonal papillomavirus-like viral particles in dense arrays and smaller aggregates. Sea Otters Parasites are emerging as a major cause of disease in the California sea otter (Enhydra lutris). Acanthocephalan parasites have long been identified as a cause of mortality in California sea otters, but in recent years the prevalence and intensity of infection appear to be increasing (Thomas and Cole, 1996). Mortality is due to peritonitis following migration of the parasites from the intestine. In a retrospective study of beached sea otters, Dailey and Mayer (1999) noted that young male otters are more frequently affected by acanthocephalans than are other animals in the population. Acanthocephalans, primarily Polymorphus spp. and Corynosoma spp., are acquired by consumption of crabs (Emerita spp. and Blepharipoda spp.) that serve as intermediate hosts for the parasites, but are not the preferred food of most otters. Dailey and Mayer (1999) hypothesize that young animals are more susceptible to infection by these parasites because of their lack of feeding experience and low social status, which leads to the foraging of less desirable food sources. Protozoans also pose a threat to sea otters. Researchers at the National Wildlife Health Center, Madison, WI, have been conducting necropsies on the threatened southern sea otter since 1992. Over the last 8 years, protozoal encephalitis was present in 8.5% of the otters received for necropsy (Thomas and Cole, 1996). Recently, Sarcocystis neurona–like protozoans and Toxoplasma gondii have been associated with encephalomyelitis and meningoencephalitis, respectively, in southern sea otters (Chechowitz et al., 1999; Rosonke et al., 1999; Cole et al., 2000). Merozoites have also been seen in skeletal muscle at multiple anatomical locations (Rosonke et al., 1999). Lindsay et al. (2000) described mostly minimal cerebral inflammation in animals examined, with only two cases showing severe fulminant meningoencephalitic sarcocystosis. They subsequently isolated protozoal merozoites from the brain of an otter with neurological disease, which were characterized as S. neurona by PCR. In general, the classic terrestrial life cycle for Sarcocystis includes an herbivore, as an intermediate host, and a carnivore or omnivore as a definitive host, but the mode of transmission to sea otters is still unclear. Another protozoan, T. gondii, has been isolated from southern sea otters, and was infective in subsequent passages through mice (Cole et al., 2000). All isolates characterized were genetically distinct, but of the same type II strain. The majority of human and pig toxoplasmosis cases are also due to the type II strain (Howe et al., 1997; Mondragon et al., 1998). It is unclear, in southern sea otters, whether the high incidence of the type II strain is due to high regional prevalence, an increased strain pathogenicity, and/or a high rate of infection. The majority of animals infected did not have severe inflammatory changes, but all presented with at least mild 0839_frame_C02 Page 24 Tuesday, May 22, 2001 10:40 AM 24 CRC Handbook of Marine Mammal Medicine meningoencephalitis. Sea otters may be infected through ingestion of the oocyst stage, either directly from the water or by consuming filter-feeding invertebrates. Environmental contamination by feral and domestic cat populations, either directly or due to human disposal of cat feces to the municipal water supplies, might play a significant role in epidemiology of sea otter toxoplasmosis (Cole et al., 2000). Recent outbreaks of toxoplasmosis in humans resulting from inadequately treated municipal water supplies favor the latter hypothesis (Bowie et al., 1997; Isaac-Renton et al., 1998). Polar Bears There are few novel diseases reported in polar bears (Ursus maritimus). Fatal hepatic sarcocystosis was recently reported in two polar bears from a zoo in Anchorage, Alaska (Garner et al., 1997a). The protozoa were considered to be Sarcocystis spp. based on morphology and immunohistochemistry. The point source of infection was not identified; however, fecal contamination by birds or through food fish were suspected routes. There is serological evidence that morbillivirus is endemic in the free-ranging polar bear populations of the Bering, Chukchi, and east Siberian Seas, although epidemics of disease have not been reported (Follmann et al., 1996). Conclusion Frequency and severity of reported emerging and resurging diseases are increasing (Harvell et al., 1999). The increase may be due, in part, to improved observation and record keeping following opportunistic examinations, increased numbers of necropsies performed by pathologists rather than by biologists, and multidisciplinary investigations of recent mortality epizootics. Stranded animals, fishery by-catch, subsistence-harvested animals, and animals caught for research purposes are being more closely examined by veterinarians and pathologists. Additionally, a variety of novel technologies have enhanced identification of pathogens and toxins, so that agents may be detected in small or decomposing tissue samples. Thus, it is difficult to determine whether there is a true increase in diseases in marine mammals or merely an improvement in technology and effort. The development of long-term monitoring programs is needed to establish the significance of emerging and resurging diseases. These programs need to be transboundary, to encompass the entire migratory route of a marine mammal and the factors affecting it, and multidisciplinary. Understanding the pathogenesis of a disease, as well as its etiology and epidemiology, is paramount to understanding the potential effects of emerging and resurging diseases on a population. Accompanying the problems posed by these newly recognized infectious agents are the complications associated with the emergence of pathogen antimicrobial resistance (PAR), which has been recognized in various individual marine mammal cases (Johnson et al., 1998). Frequent use and abuse of antibiotics within both human and veterinary medicine, as well as within the agricultural industry, combined with the contamination of the environment with resistant bacteria through raw sewage spills, municipal water dumping, and agricultural and storm/flood runoff, may have important effects on marine bacteria. Care must be taken when determining the impact of resurging and emerging diseases to distinguish between diseases that were present previously but not identified and those that were truly not present. It will also be important to distinguish between primary and secondary diseases, and for secondary diseases, to determine the possible underlying causes for morbidity and mortality. Data collection and baseline life history information are key to elucidating the answers to these questions, although there are often limitations on acquiring this information, such as public indifference, limiting management policies, and inadequate funding. Regardless 0839_frame_C02 Page 25 Tuesday, May 22, 2001 10:40 AM Emerging and Resurging Diseases 25 of these factors, routine and systematic sampling of animals in research, free-ranging, and captive environments must be implemented, and samples should be processed in three categories. First, samples from clinically normal animals should be analyzed to obtain normal values to use for comparisons. Second, samples from clinically normal and ill animals should be subjected to testing with currently available tests. Finally, a subsample of all collected samples should be archived for future analysis; this may prove to be the most valuable component of all. Information on disease mechanisms, pathogenesis, epidemiology, ecology, and biology can be acquired most efficiently and accurately through collaborative, international, and interdisciplinary baseline research and epizootic investigations. The authors hope that these will continue to develop, so that the role of diseases in marine mammal health and conservation can be understood. Acknowledgments The authors thank Julia Zaias, Rosandra Manduca, Ailsa Hall, and Kirsten Gilardi for their reviews and editorial comments on this chapter, as well as all those who provided updated information on emerging and resurging diseases in marine mammals. 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Walz, P.M., Garrison, D.L., Graham, W.M., Cattey, M.A., Tjeerdema, R.S., and Silver, M.W., 1994, Domoic acid-producing diatom blooms in Monterey Bay, California: 1991–1993, Nat. Toxins, 2: 271–279. Wilson, M.E., 1999, Emerging infections and disease emergence, Emerging Infect. Dis., 5: 308–309. Work, T.M., Barr, B., Beale, A.M., Fritz, L., Quilliam, L.A., and Wright., J.L.C., 1993, Epidemiology of domoic acid poisoning in brown pelicans (Pelecanus occidentalis) and Brandt’s cormorants (Phalacrocorax penicillatus) in California, J. Zoo Wildl. Med., 24: 54–62. 0839_frame_C03 Page 31 Tuesday, May 22, 2001 10:40 AM 3 Florida Manatees: Perspectives on Populations, Pain, and Protection Thomas J. O’Shea, Lynn W. Lefebvre, and Cathy A. Beck Introduction The Florida manatee (Trichechus manatus latirostris) has been the subject of intensive research for over 25 years, using both stranding and field ecology approaches. Mandated by specific state and federal legislation, the objectives of this research have been rooted in the desire to improve manatee management for conservation of populations. Although there have been a number of different management issues that have confronted conservation efforts, the most overwhelming and persistent has been the direct mortality of manatees from accidental collisions with boats. One of the world’s most thorough and long-standing marine mammal carcass recovery and necropsy programs has clearly demonstrated that deaths of manatees from this one anthropogenic source is undisputedly a chronic, major, and growing problem (see, for example, Beck et al., 1982; O’Shea et al., 1985; Ackerman et al., 1995; Wright et al., 1995). Straightforward management solutions to this problem have been proposed, but only slowly achieved. These solutions involve a legislatively mandated policy to implement and enforce speed limits on boats in areas known to be used by manatees. To a lesser degree, solutions also involve creating sanctuaries where no boat traffic is allowed. The simple rationale is that at reduced speeds, the force of impact will be less deadly, and manatees will be more able to avoid slower boats; additionally, accidental collisions with boats cannot occur in sanctuaries where boats are excluded. Resistance to these management tools can be substantial, and some arguments against them center around incomplete knowledge of manatee population trends. However, such arguments ignore the troubling issues raised by the widespread maiming and pain inflicted on individual manatees that are struck by boats (Figure 1), escape death, and are thus not included among carcass count statistics. This overview has three related objectives. First, it provides simple documentation, descriptive summaries, and anecdotal accounts that demonstrate the extent to which maiming, and likely pain and suffering, occur in wild manatees as a result of strikes by boats. The chapter calls attention to the issues wounding raises for policy makers and managers involved with implementing boat speed zones, particularly in regard to existing laws and emerging ethical points 0-8493-0839-9/01/$0.00+$1.50 © 2001 by CRC Press LLC 31 D B FIGURE 1 Boat-inflicted wounds on wild, living Florida manatees. (A) Multiple lacerations on dorsal tail fluke. (Photo credit: J. Reid, U.S. Geological Survey.) (B) Trunk and tail stock of adult female with completely amputated fluke. (Photo credit: T. O’Shea, U.S. Geological Survey.) (C) Lacerations of the head. (Photo credit: R. Bonde, U.S. Geological Survey.) (D) Healed severe dorsal and lateral propeller wounds. (Photo credit: K. Curtin.) C A 0839_frame_C03 Page 32 Tuesday, May 22, 2001 10:40 AM 32 0839_frame_C03 Page 33 Tuesday, May 22, 2001 10:40 AM Florida Manatees: Perspectives on Populations, Pain, and Protection 33 of view. The authors suggest that considerations related to wounding should also be embraced in developing boat speed zone and sanctuary decisions, and that this issue adds a strong dimension that can override debate about manatee population trends. The strength of the science behind the latter is often misunderstood, leading to unnecessary controversy. Therefore, the second major objective is to provide a simple primer on concepts and uncertainties in manatee population biology for manatee veterinarians, rehabilitators, and biomedical specialists. Although these specialists may have little training in population ecology, they are on the front lines in manatee rescue and treatment efforts, and are often asked by the media to comment on questions related to manatee population trends. This primer is generally restricted to review of information in the published literature or widely accessible management documents. Finally, the authors submit their viewpoint that issues surrounding uncertainty in manatee population biology may be “red herrings” that detract from implementation of management actions. As humanity enters an era of growing ethical concerns for animal welfare, the degree of maiming and injury to manatees by boats will become unacceptable. Indeed, long-standing statutes that have been overdue in their application are cited to justify manatee speed zones and sanctuaries. Maiming of Manatees in Collisions with Boats Clearly, many manatees are hit by boats, suffer pain and wounding, but survive. One of the first references to manatees being struck by boat propellers was made in the early 1940s, while by the late 1940s, biologists were using propeller scar patterns on living manatees in the wild to identify them as individuals (see historical summary in O’Shea, 1988). Although popular accounts stating that all Florida manatees bear scars from collisions with boats are not true, most carcasses examined bear scars from previous strikes (Wright et al., 1995), and a very large number of scarred manatees exist. A photoidentification system and database of scarred manatees currently maintained by the U.S. Geological Survey Sirenia Project in Gainesville, Florida (Beck and Reid, 1995) contains only individuals with distinct scars, the vast majority of which appear to have been inflicted by propeller blades or skegs (keels). This database now documents 1184 living individuals scarred from collisions with boats. Most of these manatees (1153, or 97%) have more than one scar pattern, indicating multiple strikes by boats. The severity of mutilations for some of these individuals can be astounding. These include long-term survivors with completely severed tails, major tail mutilations, and multiple disfiguring dorsal lacerations (Figures 1 and 2). These injuries not only cause gruesome wounds, but may also impact population processes by reducing calf production (and survival) in wounded females. Anecdotal observations also speak to the likely pain and repeated suffering endured by some of these individuals. For example, during fieldwork by the senior author (O’Shea) at Blue Spring and the surrounding St. Johns River, Florida, in the 1980s, known individual manatees were re-identified while snorkeling, and tracked by radiotelemetry. During snorkeling, a few individuals of known age allowed close approach, such that past scar patterns could be counted (including less-conspicuous wounds covered by gray pigmented tissue or algae). Adults with evidence of up to 19 separate hit patterns (some with multiple cuts in a single pattern) were recorded in field notes. Many individuals were struck relatively early in life (manatees can live up to 59 years) (Marmontel et al., 1996). Ages of eight individual manatees examined underwater in February 1985, and the corresponding number of strike patterns (in parentheses) by age were as follows: age 3 (12), age 3 (6), age 4 (12), age 5 (9), age 5 (11), age 6 (19), age 7 (14), and age 8 (7). In 1983, one small calf was observed with a severe dorsal mutilation trailing a decomposing piece of dermis and muscle as it continued to accompany and nurse from its mother. This individual was again severely hit in 1984, and by age 2 its dorsum was grossly 0839_frame_C03 Page 34 Tuesday, May 22, 2001 10:40 AM 34 Handbook of Marine Mammal Medicine A B C D FIGURE 2 Underwater photographs of severe healed dorsal and tail wounds on wild, living manatees from widely separated areas in Florida. Dorsal (A) and lateral (B) mutilations of two manatees at Crystal River in northwestern peninsular Florida, where in recent decades a variety of population data suggest increasing population trends, yet severe maiming remains evident. Similar wounds (C, D) on two manatees from the southeastern Atlantic Coast, where population data do not suggest recent population increases. (Photo credits: J. Reid, U.S. Geological Survey.) 0839_frame_C03 Page 35 Tuesday, May 22, 2001 10:40 AM Florida Manatees: Perspectives on Populations, Pain, and Protection 35 FIGURE 3 Underwater photograph of right dorsolateral area of a 2.5-year-old wild juvenile Florida manatee struck multiple times since birth in the St. John’s River system near Blue Spring. Note the compound fracture of the rib emerging just above and to the right of the center of the photograph. Population data suggest increasing trends at this site, yet severe maiming remains evident. (Photo credit: T. O’Shea, U.S. Geological Survey.) deformed and included a large protruding rib fragment visible in 1985 (Figure 3). While snorkeling close to this individual on January 16, 1985, patterns of 12 separate strikes by boats were counted. Despite such severe wounding, this individual remained alive in the year 2000. Carcasses examined at necropsy also often bear healed scars of multiple past strikes by boats; one extreme case, recently noted by the Florida Marine Research Institute, had evidence of more than 50 past collisions (Powell, pers. comm.). Traumatic injuries as a result of strikes by boats are also a major concern for manatee care and rehabilitation facilities (see Chapter 43, Manatees). Records maintained by the Sirenia Project since the late 1970s document rescue and rehabilitation attempts for 109 cases (69 of which died) directly linked to boat strike injuries, accounting for about 20 to 30% of the annual number of manatee rescues. The incidence of wounding by boats in Florida manatees is probably unparalleled in any marine mammal population in the world. Seals and sea lions recovered along the California coast from 1986 through 1999, for example, showed boat propeller damage in only 0.1% of 6196 live stranded individuals of six species (Goldstein et al., 1999). There is a growing sentiment in large segments of the U.S. and European public for animal welfare, animal well-being, and animal rights. One recent poll cited by Dennis (1997) found that two thirds of 1004 Americans queried by the Associated Press agreed with the statement, “An animal’s right to live free from suffering should be just as important as a person’s right to be free from suffering.” Despite modern philosophical debates on animal rights in relation to such topics as dietary use or biomedical experimentation, the inflicting of pain on animals has long been considered against most moral and ethical tenets of Western society, particularly when pain is inflicted carelessly and needlessly. Indeed, existing laws at both the state and federal levels with relevance to Florida manatees clearly reflect these tenets (Table 1), yet these laws are seldom brought to bear on the issues involving boat speed policies in Florida. The number one objective of the Florida Manatee Recovery Plan is “1. Identify and minimize causes of manatee injury and mortality” (U.S. Fish and Wildlife Service, 1996, p. 46), but the focus and debate to date has largely been on mortality only. This is due to population implications. 0839_frame_C03 Page 36 Tuesday, May 22, 2001 10:40 AM 36 Handbook of Marine Mammal Medicine TABLE 1 Florida Statutes and Federal Laws Pertaining to Injury and Wounding of Florida Manatees Florida Statutes, Title XLVI, Crimes, Chapter 828, Section 828.12 (1) “A person who unnecessarily overloads, overdrives, torments, deprives of necessary sustenance or shelter, or unnecessarily mutilates, or kills any animal, or causes the same to be done, or carries in or upon any vehicle, or otherwise, any animal in a cruel and inhumane manner, is guilty of a misdemeanor of the first degree, punishable as provided in s. 775.082 or by a fine of not more than $5,000, or both.” Florida Statutes, Title XXVIII, Natural Resources; Conservation, Reclamation, and Use, Chapter 370, Section 370.12 (2) (“Florida Manatee Sanctuary Act”) “(d)…it is unlawful for any person at any time, by any means, or in any manner intentionally or negligently to annoy, molest, harass, or disturb or attempt to molest, harass, or disturb any manatee; injure or harm or attempt to injure or harm any manatee; capture or collect or attempt to capture or collect any manatee; pursue, hunt, wound, or kill or attempt to pursue, hunt, wound, or kill any manatee; …(e) Any gun, net, trap, spear, harpoon, boat of any kind … used in violation of any provision of paragraph (d) may be forfeited upon conviction.” U.S. Marine Mammal Protection Act of 1972 (16 U.S.C. 1362, 16 U.S.C. 1372) Sec. 3. (4) “The term ‘humane’ in the context of the taking of a marine mammal means that method of taking which involves the least possible degree of pain and suffering practicable to the mammal involved.” Sec 3. (13) “The term ‘take’ means to harass, hunt, capture, or kill, or attempt to hunt, capture, or kill any marine mammal.” Sec. 102. (a) “…it is unlawful for any person or vessel or other conveyance to take any marine mammal in waters or on lands under the jurisdiction of the United States;…” U.S. Endangered Species Act of 1973 (16 U.S.C. 1531) Sec. 3 (18) “The term ‘take’ means to harass, harm, pursue, hunt, shoot, wound, kill, trap, capture, or collect, or attempt to engage in any such conduct.” Sec. 9 (a) (1) “… it is unlawful for any person subject to the jurisdiction of the United States to … (B) take any such species within the United Sates or the territorial seas of the United States.” Emphasis in italics added by authors (see also Chapter 33, Legislation). A Primer on Manatee Population Biology: Accounting for the Confusion and Uncertainty Three related facets of Florida manatee population biology have resulted in confusing interpretations of the status of the subspecies: the estimation of population size (and thus trends in size), carcass counts (and their relationships with death and survival rates), and population modeling. These are discussed below along with their implications for manatee protection policies. Estimation of Population Size and Trend There have been many studies in which manatee sightings from aircraft have been tallied (see summaries in Beeler and O’Shea, 1988; Ackerman, 1995). However, there are no estimates or confidence intervals for the size of the Florida manatee population that have been derived by reliable, statistically based, population-estimation techniques. This is not well understood by the public or by all individuals involved in manatee management, policy, or nonecological research programs. Nonetheless, this problem is clearly stated in the fundamental management document for the species, the Florida Manatee Recovery Plan: “Scientists have been unable to develop a useful means of estimating or monitoring trends in size of the overall manatee populations in the southeastern United States” (U.S. Fish and Wildlife Service, 1996, p. 9). In an ideal situation, biologists can determine sizes of animal or plant populations by conducting 0839_frame_C03 Page 37 Tuesday, May 22, 2001 10:40 AM Florida Manatees: Perspectives on Populations, Pain, and Protection 37 a census. A census is a complete count of individuals within a specified area and time period (Thompson et al., 1998). A survey, in contrast, is an incomplete count. With the exception of a few places where manatees may aggregate in clear shallow water, not all manatees can be seen from aircraft because of water turbidity, depth, surface conditions, variable times spent submerged, and other considerations. These and other factors affecting detectability of manatees in aerial surveys have been reviewed in detail by Lefebvre et al. (1995). Population estimation procedures for cetaceans and dugongs (Dugong dugon), in contrast, are based on sampling procedures that can be applied over broad, open areas. Survey techniques applied to these species allow adjustment for detectability and, thus, unlike Florida manatee surveys carried out along narrow stretches of coastline, yield unbiased estimates given certain sampling assumptions. These techniques generally involve forms of distance sampling (Buckland et al., 1993) or fixed-width transects that include methods to estimate correction factors for biases affecting detectability (Marsh, 1995). Differences between the reliability of results obtained by censuses or by sampling procedures that provide unbiased estimates, vs. simple count surveys, are often not appreciated by nonspecialists. Results obtained during typical manatee surveys yield unadjusted partial counts. These results are of value in providing information on where concentrations of manatees occur, likely relative abundance in various areas, and seasonal shifts in foci of abundance. However, the results do not provide good population estimates, nor can they reliably measure trends in populations. The counts are index values not calibrated by some known, empirically established, sampling relationship with the true numbers present. Index methods for estimating population trends in animals are flawed, because counts obtained are convolutions affected by numerous variables other than actual trends in populations—all of these variables can affect counts by altering detection probabilities in complex and unknown ways. These variables may also change with time, and their net effects on the index may not be linearly related to actual population size, obscuring the ability to understand true trends in populations. Attempts to standardize methods (e.g., air flight speed, altitude, time of day) and to adjust indices for some factors known to influence counts (e.g., temperature covariates in surveys at refugia) are important and have been followed in carrying out and interpreting results of manatee surveys. However, standardization of counting protocols does not compensate for the potentially large number of unknown or uncontrolled sources of variability in detectability (Thompson et al., 1998). Wildlife population specialists well grounded in sampling theory consider index monitoring as “an assessment protocol that collects data that usually represent at best a rough guess at population trends (and at worst may lead to an incorrect conclusion)” (Thompson et al., 1998). Thus over the years, manatee biologists have carried out numerous attempts to refine survey techniques as much as possible. These include attempts to test more sophisticated statistical approaches and to account for bias (Packard et al., 1985; 1986; Lefebvre and Kochman, 1991; Miller et al., 1998), as well as adjusting counts at aggregation sites for temperature and other covariates (Garrott et al., 1994; 1995; Ackerman, 1995; Craig et al., 1997). Nonetheless, an appropriate method for estimating the size of the entire manatee population in Florida has remained elusive. Despite these caveats, many biologists consider index approaches useful as opposed to the alternative of doing nothing (Fowler and Siniff, 1992). Thus, various aerial counts have been made in Florida since 1967, and the results from these numerous efforts have provided a longterm historical record. This large body of work (for review, see Ackerman, 1995) has led to the perception by nonspecialists that actual population size and trend are being monitored. Because it is likely that most manatees in Florida visit warm water sources, where they may occur in large numbers during periods of especially cold weather, surveys have been made at most of these places at such times each winter since the 1970s. During the initial years of such efforts, the most consistent high number obtained while circling these sites was considered a “minimum 0839_frame_C03 Page 38 Tuesday, May 22, 2001 10:40 AM 38 Handbook of Marine Mammal Medicine estimate” for numbers of manatees using that aggregation site, and the practice has been to sum these for each winter aggregation site and provide a “minimum estimate” for the size of the manatee population in Florida. These efforts did not consider manatees not counted, manatees tallied twice or more, manatees that may have moved between aggregation sites in short periods between high counts on different days, or manatees that were outside of the intensive survey areas. These “minimum estimates” are misnomers in that they are entirely different from the terminology used by population biologists for true population estimates based on sampling theory. The “minimum estimate” in 1978 was “at least 800–1000 manatees,” and in 1985 a summation of high counts made under unusually good conditions at aggregation sites was about 1200 manatees (see review by O’Shea, 1988). Confusion was further engendered when in 1990 the Florida legislature mandated “an impartial scientific benchmark census of the manatee population to be conducted annually” (Florida Statute 370.12.5a), despite recognition by scientists that a valid census was infeasible. In response, however, state resource agencies and cooperators have carried out intense synoptic surveys at simultaneous or nearly simultaneous times each year during winter. These surveys cover all known aggregation sites and most intervening areas, typically covering all areas in 1 or 2 days (Ackerman, 1995). Results of these index surveys are what are commonly, but incorrectly, cited as population estimates for Florida manatees. The first such survey in 1991 resulted in a count of 1268 manatees; a second survey 3 to 4 weeks later yielded a count of 1465. A year later the count was 1856. In January 1996, 2274 manatees were seen, and in the next month a count of 2639 was made. The most recent counts during two synoptic surveys in winter 1999–2000 were 1629, followed by 2222 10 days later. The wide variability in these numbers (differences of hundreds of animals within days or weeks, and a near doubling in 5 years) illustrates the unreliability of such counts as population estimates. This unreliability was further underscored when at least 150 manatees died during a red tide in southwestern Florida in early 1996 (Bossart et al., 1998), but the synoptic survey count for the west coast of Florida in January 1997 remained similar to that in 1996, prior to the die-off. Although over a 20- to 25-year period, counts have increased, perhaps reflecting an increase in the actual population in some of the regions surveyed over some segments of this time, the relationships between any of these numbers and the true population size remain unknown. Count data collected over multiple years from specific locations have also been analyzed for trends over time (Garrott et al., 1994; Ackerman, 1995; Craig et al., 1997). Conclusions about potential trends at specific sites may be stronger when they stem from more than one kind of data set. This can include combining inferences from counts, modeling population growth rates from survival and reproduction data (see below), examining carcass count data (see below), and weighing auxiliary information, such as habitat quality and factors promoting or reducing likelihood of survival, reproduction, or migration. This would provide a weight-of-evidence approach to aid policy makers and managers, based on a greater amount of information than count indices. Positive trends were observed in counts from the 1970s to early 1990s at Blue Spring (based on individual identification rather than aerial survey) and Crystal River, highly protected winter aggregation sites (Ackerman, 1995). Eberhardt and O’Shea (1995) showed that manatees at these two areas also had high population growth rates based on modeling of reproduction and survival data (but lower than rates of increase in counts, which were also influenced by immigration). Index counts adjusted for temperature and other covariates at several important power plant aggregation sites on the Atlantic Coast showed an increasing trend over 15 winters (ending in 1991–1992), whereas indices at one aggregation site in southwestern Florida (near Fort Myers) showed no trend (Garrott et al., 1994); previous analyses based on a 9-year period were also conducted by Garrott et al. (1995). This led to guarded speculation that manatee population trends on the Atlantic Coast may also have been 0839_frame_C03 Page 39 Tuesday, May 22, 2001 10:40 AM Florida Manatees: Perspectives on Populations, Pain, and Protection 39 increasing concomitant with increases in the adjusted index. However, the trend computed for adjusted counts from sites on the Atlantic Coast was too high to be compatible with the low to zero population growth estimates based on survival and reproduction data (Eberhardt and O’Shea, 1995). This seemingly conflicting information was recently clarified by a reanalysis of the counts at power plants using modifications to the statistical approach. The new analysis showed that an increasing trend in this adjusted index was only likely over the first third of the 15-winter data set, but that for the rest of the period the counts had not increased (Eberhardt et al., 1999). Craig et al. (1997) used a Bayesian approach (involving data-based hierarchial modeling to account for effects likely due to observation variables, movements among sites, and population trend) to reanalyze aerial survey data for the Atlantic Coast aggregation sites between 1982 and 1992. Although this analysis indicated possible population growth in the 1980s, it also concluded that trends leveled off or decreased during the early 1990s. Thus, unlike data for manatees at the Crystal River and Blue Spring sites, the weight of evidence from the late 1970s to early 1990s shows no suggestion of a continued increase in index counts of manatees on the Atlantic Coast or at Fort Myers (which together encompass a much larger geographic segment of the distribution than Blue Spring and Crystal River). Unfortunately, there are as yet no updated published analyses on which to base any trend conclusions for count indices in these areas for the full decade of the 1990s (although such work is in progress) and no comparable data for manatees in an extensive area encompassing the coastal Everglades. Carcass Counts, Mortality, and Survival Each year, authorities release details on the annual total number of Florida manatee carcasses recovered and their causes of death. This provides very valuable data for management in revealing sources, locations, and times of anthropogenic mortality (those most amenable to management), as well as a wealth of pathological and anatomical biological information. Carcass counts are growing, particularly in very recent years, and collision with boats remains the major identifiable cause of death. In 1995, 184 manatees were found dead in Florida and adjacent states, with 39 killed by boats, whereas by 1999 a total of 272 carcasses were recovered, with 83 killed by boats. During the first 5 months of 2000, the number of carcasses shown to be due to boat strikes was on a record pace (see Chapter 43, Manatees). Unfortunately, these carcass counts are often misunderstood as true mortality data, in the population biologist’s sense of number of deaths per unit of population (mortality as a rate). These are not mortality rate data, because the actual population size is unknown. Furthermore, carcass counts themselves are also index values, and dividing the existing “estimates” by carcass counts to obtain death rates would result in further complex convolutions (one uncalibrated index divided by another). There is no reliable knowledge of the numbers of carcasses that go undiscovered or how discovery varies spatially, seasonally, or temporally. As the number of people using Florida’s coasts continues to grow, for example, the probability of discovery and reporting is likely to increase, as is the likelihood of human-associated death. Mortality can be computed as a rate from the distribution of ages at death, using anatomical age estimation approaches on carcasses (Marmontel et al., 1997), but this requires statistical assumptions that are not always amenable to verification. However, there have been recent advances in obtaining unbiased estimates of survival rates in manatees that utilize methods based on solid statistical inference that are completely independent of carcass counts or aerial survey index data. Mortality can also be estimated from these methods (100 − % survival = % mortality). These advances are based on sight–resight models, which ironically capitalize on scarring of living manatees as markers of individual distinctiveness (O’Shea and Langtimm, 1995; Langtimm et al., 1998). These methods have not yet been applied statewide, but efforts are 0839_frame_C03 Page 40 Tuesday, May 22, 2001 10:40 AM 40 Handbook of Marine Mammal Medicine under way to increase regional coverage. Results obtained thus far for manatees in three important regions of Florida (the Big Bend coast encompassing Crystal River, the St. John’s River encompassing Blue Spring, and the Atlantic Coast), have been compatible with regional count indices and population growth models for these areas. Survival rate estimation cannot provide instant appraisals relative to status of the population for the most recent past year because of calculation requirements. This is a drawback for media and policy makers, who may prefer more immediate data even when scientifically less valuable. Population Models Population models employ mathematical relationships based on survival and reproduction rates to calculate population growth and trends in growth. Two sets of models of manatee population dynamics have been published. A deterministic model using classical mathematical approaches and various computational procedures with data on reproduction and survival of living, identifiable manatees suggests a maximum growth rate of about 7% per year (not including emigration or immigration) (Eberhardt and O’Shea, 1995). This maximum was based on the winter aggregation at Crystal River (an area with substantial protection), as studied from the late 1970s to early 1990s, and did not require estimates of population size. The analysis showed that the chief factor affecting potential for population growth is survival of adults. Low adult survival on the Atlantic Coast (a larger region with less protection) suggested very slow or no population growth over a similar period. This modeling shows the value of using survival and reproduction data obtained from photoidentification studies of living manatees to compute population growth rates with confidence intervals, information which can be used to infer long-term trends in the absence of reliable population size estimates. However, collection of similar data has been initiated only recently for other areas of the state (notably from Tampa Bay to the Caloosahatchee River beginning in the mid-1990s), and none is available over much of the remaining areas used by manatees in southwestern Florida. Population viability analysis (PVA) is a stochastic modeling approach, which varies potential scenarios impinging on reproduction and survival over long periods, and predicts responses in population growth. A PVA was carried out based on age-specific mortality rates computed from the age distribution of manatees found dead throughout Florida from 1979 through 1992 (Marmontel et al., 1997). This method of computing survival rests on certain assumptions that were not fully testable; yet, results point out the importance of adult survival to population persistence. Given population sizes that may reflect current abundance, the PVA showed that if adult mortality as estimated for the study period were reduced by a modest amount (e.g., from about 11 to 9%), as might be accomplished by management actions such as effective boat speed regulations, the Florida manatee population would likely remain viable for many years. Slight increases in adult mortality (a likely consequence of inadequate protection) would result in extinction over the long term. Given that the number of boats registered in Florida has increased from about 440,000 in 1975 to about 800,000 today, it is probably safe to accept the PVA-based conclusion that decreased adult survival and eventual extinction is a likely future outcome for Florida manatees, unless policies to protect them are aggressively implemented. Uncertainties on Population Status: A Red Herring? Arguments against designation of boat speed zones to protect manatees sometimes point to uncertainties about trends in population size as reasons to delay implementation of these regulations. However, the above review shows that the basis for statewide population size “estimates” of any kind is scientifically weak and unsuitable for computing trends, and that 0839_frame_C03 Page 41 Tuesday, May 22, 2001 10:40 AM Florida Manatees: Perspectives on Populations, Pain, and Protection 41 the weight of evidence suggesting population increases over the last two decades is strong only for two aggregation areas. Furthermore, new population analyses, based on more recent (since 1992) information, are not yet available in the peer-reviewed literature, but these will be fundamental to management decisions that are more relevant to the contemporary situation. Thus, population-based arguments against mandated actions to reduce collisions between manatees and boats have no solid footing. The increases in boat numbers and collision-caused carcass counts suggest a continuing problem, and this is underscored by the widespread evidence of pain and mutilation. There are several additional points often missed in discussions about manatee protection that render counterarguments about manatee population trend misleading and irrelevant. First, a variety of different kinds of population dynamics information is not available for much of the state, and a weight-of-evidence approach to evaluating population trend is currently impossible for these areas. Precaution dictates a conservative policy in favor of protection, in the absence of quality data. Manatees remain listed as endangered under the U.S. Endangered Species Act and are protected by the Florida Manatee Sanctuary Act of 1978 and the U.S. Marine Mammal Protection Act of 1972 (see Chapter 33, Legislation). Indeed, when protection efforts under these mandates become effective, populations will begin making slow increases. It should be remembered that when increasing trends become apparent, they are not equivalent to population recovery, but only a signal of movement toward recovery. Failure to implement or maintain protection measures simply because trends might be increasing (a position that is unsupported by published analysis of data from most of the state) would only slow progress toward full recovery. It would be poor and purely reactive management to take actions only when unequivocal evidence of decline exists. Second, the laws mandating boat speed zones for manatee protection do not link policy implementation to manatee population trend. The Florida Manatee Sanctuary Act (Florida Statutes, Title XXVIII, Section 370.12 (2)(f)) instead states: “In order to protect manatees or sea cows from harmful collisions with motorboats or from harassment, the Fish and Wildlife Conservation Commission shall adopt rules under Chapter 120…regulating the operation and speed of motorboat traffic, only where manatee sightings are frequent and it can generally be assumed, based on available scientific information, that they inhabit these areas on a regular or continuous basis.” Thus implementation of boat speed zones is directed to protect manatees from harm, not from death only, and is aimed at areas where manatees are abundant, not necessarily at areas where populations are declining. Likewise, sanctuaries have been designated in the headwaters of the Crystal River to minimize harassment by swimmers, as well as to reduce the risk of boat–manatee collisions (O’Shea 1995; Buckingham et al., 1999). Growing concern about the effects of human harassment of manatees resulted in a “Manatee Harassment Round Table Discussion” in October 1999, sponsored by the Florida Fish and Wildlife Conservation Commission. This discussion addressed the desirability of discouraging direct physical contact between people and manatees. While all would agree that the sublethal wounding of manatees by boats represents a far higher degree of harassment than any imposed by contact with humans, the issue of boating harassment, separate from boat-caused manatee deaths, has yet to receive much attention. Finally, unlike aspects of aerial count data, the overwhelming documentation of gruesome wounding of manatees leaves no room for denial. Minimization of this injury is explicit in the Recovery Plan, several state statutes, and federal laws, and implicit in our society’s ethical and moral standards and the direction of current trends in those standards. Thus, the little that can be said with reasonable scientific certainty about manatee population size and trend may be essentially irrelevant to implementation of boat speed zones and sanctuaries, the key management tools for addressing the primary and long-standing issue facing manatee conservation and protection efforts in Florida. 0839_frame_C03 Page 42 Tuesday, May 22, 2001 10:40 AM 42 Handbook of Marine Mammal Medicine References Ackerman, B.B., 1995, Aerial surveys of manatees: A summary and progress report, in Population Biology of the Florida Manatee, O’Shea, T.J., Ackerman, B.B., and Percival, H.F. (Eds.), U.S. Department of Interior, National Biological Service, Washington, D.C., Information and Technology Report No. 1: 13–33. Ackerman, B.B., Wright, S.D., Bonde, R.K., Beck, C.A., and Banowetz, D.J., 1995, Trends and patterns in mortality of manatees in Florida, 1974–1992, in Population Biology of the Florida Manatee, O’Shea, T.J., Ackerman, B.B., and Percival, H.F. (Eds.), U.S. Department of Interior, National Biological Service, Washington, D.C., Information and Technology Report No. 1: 223–258. Beck, C.A., and Reid, J.P., 1995, An automated photo-identification catalog for studies of the life history of the Florida manatee, in Population Biology of the Florida Manatee, O’Shea, T.J., Ackerman, B.B., and Percival, H.F. (Eds.), U.S. Department of Interior, National Biological Service, Washington, D.C., Information and Technology Report No. 1: 120–134. Beck, C.A., Bonde, R.K., and Rathbun, G.B., 1982, Analyses of propeller wounds on manatees in Florida, J. Wildl. Manage., 46: 531–535. Beeler, I.E., and O’Shea, T.J., 1988, Distribution and mortality of the West Indian manatee (Trichechus manatus) in the southeastern United States: A compilation and review of recent information, National Technical Information Service Publication PB88-207980/AS, Springfield, VA, two volumes, 613 pp. Bossart, G.D., Baden, D.G., Ewing, R.Y., Roberts, B., and Wright, S.D., 1998, Brevetoxicosis in manatees (Trichechus manatus latirostris) from the 1996 epizootic: Gross, histologic, and immunohistochemical features, Toxicol. Pathol., 26: 276–282. Buckingham, C.A., Lefebvre, L.W., Schaefer, J.M., and Kochman, H.I., 1999, Manatee response to boating activity in a thermal refuge, Wildl. Soc. Bull., 27: 514–522. Buckland, S.T., Anderson, D.R., Burnham, K.P., and Laake, J.L., 1993, Distance Sampling: Estimating Abundance of Biological Populations, Chapman & Hall, London, 446 pp. Craig, B.A., Newton, M.A., Garrott, R.A., Reynolds III, J.E., and Wilcox, J.R., 1997, Analysis of aerial survey data on Florida manatee using Markov chain Monte Carlo, Biometrics, 53: 524–541. Dennis, J.U., 1997, Morally relevant differences between animals and human beings justifying the use of animals in biomedical research, J. Am. Vet. Med. Assoc., 210: 612–618. Eberhardt, L.L., and O’Shea, T.J., 1995, Integration of manatee life-history data and population modeling, in Population Biology of the Florida Manatee, O’Shea, T.J., Ackerman, B.B., and Percival, H.F. (Eds.), U.S. Department of Interior, National Biological Service, Washington, D.C., Information and Technology Report No. 1: 269–279. Eberhardt, L.L., Garrott, R.A., and Becker, B.L., 1999, Using trend indices for endangered species, Mar. Mammal Sci., 15: 766–785. Fowler, C.W., and Siniff, D.B., 1992, Determining population status and the use of biological indices in the management of marine mammals, in Wildlife 2001: Populations, McCullough, D.R., and Barrett, R.H. (Eds.), Elsevier Applied Science, London, 1025–1037. Garrott, R.A., Ackerman, B.B., Cary, J.R., Heisey, D.M., Reynolds, J.E., Rose, P.M., and Wilcox, J.R., 1994, Trends in counts of Florida manatees at winter aggregation sites, J. Wildl. Manage., 58: 642–654. Garrott, R.A., Ackerman, B.B., Cary, J.R., Heisey, D.M., Reynolds, J.E., and Wilcox, J.R., 1995, Assessment of trends in sizes of manatee populations at several Florida aggregation sites, in Population Biology of the Florida Manatee, O’Shea, T.J., Ackerman, B.B., and Percival, H.F. (Eds.), U.S. Department of Interior, National Biological Service, Washington, D.C., Information and Technology Report No. 1: 34–55. Goldstein, T., Johnson, S.P., Phillips, A.V., Hanni, K.D., Fauquier, D.A., and Gulland, F.M.D., 1999, Human-related injuries observed in live-stranded pinnipeds along the central California coast 1986–1998, Aquat. Mammals, 25: 43–51. 0839_frame_C03 Page 43 Tuesday, May 22, 2001 10:40 AM Florida Manatees: Perspectives on Populations, Pain, and Protection 43 Langtimm, C.A., O’Shea, T.J., Pradel, R., and Beck, C.A., 1998, Estimates of annual survival probabilities for adult Florida manatees (Trichechus manatus latirostris), Ecology, 79: 981–997. Lefebvre, L.W., and Kochman, H.I., 1991, An evaluation of aerial survey replicate count methodology to determine trends in manatee abundance, Wildl. Soc. Bull., 19: 289–309. Lefebvre, L.W., Ackerman, B.B., Portier, K.M., and Pollock, K.H., 1995, Aerial survey as a technique for estimating trends in manatee population size—problems and prospects, in Population Biology of the Florida Manatee, O’Shea, T.J., Ackerman, B.B., and Percival, H.F. (Eds.), U.S. Department of Interior, National Biological Service, Washington, D.C., Information and Technology Report No. 1: 63–74. Marmontel, M., O’Shea, T.J., Kochman, H.I., and Humphrey, S.R., 1996, Age determination in manatees using growth-layer-group counts in bone, Mar. Mammal Sci., 54: 88. Marmontel, M., Humphrey, S.R., and O’Shea, T.J., 1997, Population viability analysis of the Florida manatee, 1976–1992, Conserv. Biol., 11: 467–481. Marsh, H., 1995, Fixed-width aerial transects for determining dugong population sizes and distribution patterns, in Population Biology of the Florida Manatee, O’Shea, T.J., Ackerman, B.B., and Percival, H.F. (Eds.), U.S. Department of Interior, National Biological Service, Washington, D.C., Information and Technology Report No. 1: 56–62. Miller, K.E., Ackerman, B.B., Lefebvre, L.W., and Clifton, K.B., 1998, An evaluation of strip-transect aerial survey methods for monitoring manatee populations in Florida, Wildl. Soc. Bull., 26: 561–570. O’Shea, T.J., 1988, The past, present, and future of manatees in the southeastern United States: Realities, misunderstandings, and enigmas, in Proceedings of the Third Southeastern Nongame and Endangered Wildlife Symposium, Odom, R.R., Riddleberger, K.A., and Ozier, J.C. (Eds.), Georgia Department of Natural Resources, Social Circle, GA, 184–204. O’Shea, T.J., 1995, Waterborne recreation and the Florida manatee, in Wildlife and Recreationists: Coexistence through Management and Research, Knight, R.L. and Gutzwiller, K. (Eds.), Island Press, Washington, D.C., 297–311. O’Shea, T.J., and Langtimm, C.A., 1995, Estimation of survival of adult Florida manatees in the Crystal River, at Blue Spring, and on the Atlantic Coast, in Population Biology of the Florida Manatee, O’Shea, T.J., Ackerman, B.B., and Percival, H.F. (Eds.), U.S. Department of Interior, National Biological Service, Washington, D.C., Information and Technology Report No. 1: 194–222. O’Shea, T.J., Beck, C.A., Bonde, R.K., Kochman, H.I., and Odell, D.K., 1985, An analysis of manatee mortality patterns in Florida, 1976–1981, J. Wildl. Manage., 49: 1–11. Packard, J.M., Summers, R.C., and Barnes, L.B., 1985, Variation of visibility bias during aerial surveys of manatees, J. Wildl. Manage., 49: 347–351. Packard, J.M., Siniff, D.B., and Cornell, J.A., 1986, Use of replicate counts to improve indices of trends in manatee abundance, Wildl. Soc. Bull., 14: 265–275. Thompson, W.L., White, G.C., and Gowan, C., 1998, Monitoring Vertebrate Populations, Academic Press, New York, 365 pp. U.S. Fish and Wildlife Service, 1996, Florida Manatee Recovery Plan, 2nd revision, U.S. Fish and Wildlife Service, Atlanta, GA, 160 pp. Wright, S.D., Ackerman, B.B., Bonde, R.K., Beck, C.A., and Banowetz, D.J., 1995, Analysis of watercraftrelated mortality of manatees in Florida, 1979–1991, in Population Biology of the Florida Manatee, O’Shea, T.J., Ackerman, B.B., and Percival, H.F. (Eds.), U.S. Department of Interior, National Biological Service, Washington, D.C., Information and Technology Report No. 1: 259–268. 0839_frame_C03 Page 44 Tuesday, May 22, 2001 10:40 AM 0839_frame_C04.fm Page 45 Tuesday, May 22, 2001 10:41 AM 4 Marine Mammal Stranding Networks Frances M. D. Gulland, Leslie A. Dierauf, and Teri K. Rowles Introduction Stranding networks are organizations that have developed to coordinate responses to stranded marine mammals. A stranded marine mammal has been defined in the United States as “Any dead marine mammal on a beach or floating nearshore; any live cetacean on a beach or in water so shallow that it is unable to free itself and resume normal activity; any live pinniped which is unable or unwilling to leave the shore because of injury or poor health” (Wilkinson, 1991). Although some causes of strandings have been identified, the majority remain enigmatic (Geraci, 1978; Geraci et al., 1999). The public concern for the welfare of stranded marine mammals, combined with the need to coordinate and maximize the information that can be obtained from these animals, are the forces behind stranding networks. This chapter describes the aims of stranding networks and reviews the history and structure of such networks worldwide. Objectives of Stranding Networks The goal of stranding networks is to maximize specimen and data collection pertinent to the natural history, ecology, and health of stranded marine mammals and, in some areas, to provide a humane response for a stranded marine mammal (Geraci and Lounsbury, 1993). This information is important, because most of what is known about the life history and ecology of marine mammal species that are rarely observed in the wild has been learned from stranded animals (Geraci and St. Aubin, 1979; Wilkinson and Worthy, 1999). Changes in stranding numbers may also act as early warnings for issues of management importance, such as boat strike and entanglement of marine mammals (Seagers et al., 1986). Although one of the aims of stranding networks is to rehabilitate and release live stranded animals, the importance of this activity to marine mammal conservation is contentious (St. Aubin et al., 1996; Wilkinson and Worthy, 1999). It is still unclear how likely a rehabilitated and released individual is to survive, as efforts at postrelease tracking to date have focused on limited individuals because of the expense involved (see Chapter 38, Tagging and Tracking). It is also argued that the least-fit members of a population are more likely to strand, so that rehabilitating and releasing these individuals may interfere with natural selection (Wilkinson and Worthy, 1999). Furthermore, translocation of animals may enhance spread of diseases (St. Aubin et al., 0-8493-0839-9/01/$0.00+$1.50 © 2001 by CRC Press LLC 45 0839_frame_C04.fm Page 46 Tuesday, May 22, 2001 10:41 AM 46 CRC Handbook of Marine Mammal Medicine 1996; Daszak et al., 2000). To counter these arguments, examination of stranded animals during rehabilitation has allowed detection of a variety of novel infectious agents and disease processes that would have been difficult to detect in dead stranded animals, which are often too decomposed for diagnostic purposes. There is also little doubt that the general public is concerned about the welfare of live stranded marine mammals. The public attention given to animals in rehabilitation offers great opportunity for education on factors affecting marine mammal populations. In addition, some argue that there is an obligation to attempt to rehabilitate animals that strand as a result of direct anthropogenic effects, such as oil spills and entanglement in marine debris. The number of animals released after rehabilitation is usually negligible compared with the total free-living population, so the contribution to conservation by rehabilitating live stranded animals may thus be more indirect, through public exposure, involvement, and education, and through scientific research, rather than as numerical additions to wild populations. Collection of data and specimens from dead stranded animals is less controversial, but protocols still need to be established in many countries and/or regions to ensure validity of the data collected, maximum use of the information, and the willing cooperation between parties involved in a stranding network. Stranding Networks Worldwide The degree of stranding network development varies worldwide, depending on funding availability, degree of public interest, extent of cooperation among federal, academic, and welfare organizations, facilities available, the number of strandings per year, and the duration of the existence of the network (Wilkinson and Worthy, 1999). In collecting information on stranding networks to compile this chapter, the most consistent concern of people contacted worldwide was the lack of funding. Contacts and brief descriptions of stranding networks are summarized in Table 1. A section on history is included, as developing networks may benefit from the experience of others. TABLE 1 Examples of Stranding Networks Worldwide ARGENTINA Buenos Aires City and Province H. Castello Marine Mammal Laboratory Museo Argentino de Ciencias Naturales Avda. Angel Gallardo 470 1406 Buenos Aires E-mail: [email protected] D. A. Albareda Acuario de Buenos Aires Avda. Las Heras 4155 Buenos Aires E-mail: [email protected] J. Loureiro Fundación Mundo Marino Avda.X s/n Casilla de Correo n°6 7105 San Clemente del Tuyú Buenos Aires Province E-mail: [email protected] R. Bastida and D. Rodriguez Universidad Nacional de Mar del Plata Depto de Ciencias Marinas Deán Funes 3350, 7600 Mar del Plata Buenos Aires Province E-mail: [email protected] 0839_frame_C04.fm Page 47 Tuesday, May 22, 2001 10:41 AM 47 Marine Mammal Stranding Networks TABLE 1 Examples of Stranding Networks Worldwide (continued) Río Negro Province R. González Instituto de Biología Marina y Pesquera Alte, Storni Casilla de Correo 104 8520 San Antonio Oeste Rio Negro Fax: 54-2934-421002 E-mail: [email protected] Chubut Province E. A. Crespo and S. N. Pedraza Marine Mammal Laboratory Centro Nacional Patagónico Blvd. Brown s/n 9120 Puerto Madryn, Chubut Fax: 54-2965-451543 E-mail: [email protected] [email protected] Tierra del Fuego Province N. Goodall and A. Schiavini Marine Mammal Laboratory Centro Austral de Investigaciones Científicas Casilla de Correo N° 92 9410 Ushuaia Tierra del Fuego E-mail: [email protected] [email protected] Structure Dead animals are examined and sampled for ecological studies, including age, structure, reproduction, feeding habits, genetics, virology, pollution, and parasitology. Live animals are taken to facilities (usually aquaria) for rehabilitation and monitoring of health status, where blood samples for routine health and serological tests are taken from live animals; federal and provincial laws regulate these institutions. Notes and Further Reading In Argentina there is no official stranding network, but there are several governmental and nongovernmental institutions concerned about stranding and health status of marine mammals. The Argentinean shoreline is so extensive that there are not enough groups to monitor it, but there is good communication between the research groups that work in the field. A stranding network has been in operation in Peninsula Valdéz since 1994, aimed at obtaining samples from stranded right whales; the Whale Conservation Institute collaborates with A. Carribero in this work. AUSTRALIA (Network varies by state) Queensland Michael Short Queensland Parks and Wildlife Service PO Box 2066 Cairns QLD 4870 Fax: 07-40523043 E-mail: [email protected] Tasmania Nigel Brothers Wildlife Management Officer Kerrin Jeffrey Nature Conservation Branch GPO Box 44A Hobart, Tasmania 7001 Fax: 0362-333477 E-mail: [email protected] Antarctic Wildlife Research Unit School of Zoology University of Tasmania GPO Box 252-05 Hobart, Tasmania 7001 E-mail: [email protected] Structure The Queensland Parks and Wildlife Service (QPWS) and the Great Barrier Reef Marine Park Authority work together to coordinate responses to strandings using the Incident Control Management System (ICMS). Most of the responses are performed by QPWS for logistical reasons. Strandings are reported on a hotline telephone number, which is diverted to a responder in the area with a mobile telephone. An e-mail listserve is used to (Continued) 0839_frame_C04.fm Page 48 Tuesday, May 22, 2001 10:41 AM 48 CRC Handbook of Marine Mammal Medicine TABLE 1 Examples of Stranding Networks Worldwide (continued) inform all network members of the status of a response. Live animals are transported to Sea World of the Gold Coast for rehabilitation. Dead animals are examined, samples banked for toxicology and genetics, and histology samples submitted to state laboratories. Jurisdiction over all marine mammals in Tasmanian waters and on the coastline falls to the Marine Unit of the Department of Primary Industries, Water and Environment (DPIWE, formerly the Parks and Wildlife Service) of Tasmania. Detailed necropsies are conducted on all cetaceans, and samples collected for morphology, pathology, toxicology, parasitology, reproductive, dietary, and aging investigations. All responses to strandings are conducted by volunteers trained to follow standard necropsy and sample collection procedures (Geraci and Lounsbury, 1993), and who are registered members of the Wildcare Organization. Samples from strandings are maintained and disseminated by the Tasmanian Museum and Art Gallery, and tracked by a database linked with that of DPIWE. History Concern over the status of dugongs initiated a formal stranding network in Queensland 3 years ago. Although dugongs remain the priority, the network now also responds to other marine mammals and turtles. The Antarctic Wildlife Research Unit (AWRU) began investigating cetacean stranding events in 1992, in response to strandings in Tasmania. The long-term goals of the unit were to gain a greater understanding of the biology and ecology of cetacean species in Tasmanian waters. It aimed to maximize the amount of scientific information collected from strandings, and build up a database of baseline data on these species. In 1996, the unit attended the first national stranding workshop coordinated by the then Australian National Parks and Wildlife Service (NPWS)—now Department of Primary Industries, Water and Environment (DPIWE)— providing protocols for the necropsy of and sample collection from stranded cetaceans. In 1998, due to the shift in priorities and goals of the NPWS, all strandings became the responsibility of the DPIWE. AWRU shifted its focus to the study of Globicephala melas, Physeter macrocephalus, and the Kogiidae, with federal funding received in 1997. Notes and Further Reading The response varies with species, dugongs being a priority, then endangered species. 90% of strandings are dead. Training courses are held regularly on the ICMS, stranding response, and sample collection. Tasmania has a relatively high number of strandings compared with other states in Australia. Although financial resources are limited, DPIWE seeks sponsorship for rescue equipment and training, and recently developed a flotation pontoon suitable for a 40-ton animal through sponsorship by the Australian Geographical Society. The Scientific Committee on Antarctic Research discourages the release of seals after being in captivity, especially to sub-Antarctic islands and the Antarctic continent. All pinniped releases must be approved by the relevant state agency, and require that a pre-release health assessment be performed. BELGIUM Administrative Coordination Management Unit of the North Sea Mathematical Models 3e en 23e Linieregimentsplein B-8400 Ostend Fax: 32-059704935 E-mail: [email protected] Scientific Coordination University of Liege Laboratory of Oceanology Sart Tilman B6 4000 Liege Fax: 32-43663325 E-mail: [email protected] T. Jauniaux Sart Tilman B43 4000 Liege Fax: 32-43663325/4065 E-mail: [email protected] Technical Coordination Jan Tavernier Royal Belgian Institute of Natural Sciences Rue Vautier, 29 1040 Brussels Fax: 32-026464433 0839_frame_C04.fm Page 49 Tuesday, May 22, 2001 10:41 AM 49 Marine Mammal Stranding Networks TABLE 1 Examples of Stranding Networks Worldwide (continued) Structure Dead animals are necropsied and sampled for histopathology, parasitology, bacteriology, virology, and toxicology. The post-mortem examinations are performed according to the proceedings of the European Cetacean Society (ECS) Workshop on Cetacean Pathology (Kuiken and Hartmann, 1993) and to the proceedings of the workshop on sperm whale strandings in the North Sea (Jauniaux et al., 1999). The Marine Animals Research & Intervention Network (MARIN) also assists in marine mammal rescues. Live stranded animals are transported to rehabilitation centers (Harderwijk Delphinarium, the Netherlands for cetaceans and National Sea Life Blankenberge, Belgium for seals). History MARIN determines the cause of death of marine mammals and seabirds stranded along the Belgian coast and has performed toxicological analyses on collected samples since 1989. In 1994, MARIN expanded southward to France, in association with the “Centre de Recherche sur les Mammifères Marins,” La Rochelle. Collaboration also exists between MARIN and Naturalis, the National Museum of Natural History, Leiden, the Netherlands. Notes and Further Reading Kuiken, T., and Hartmann, M.G., 1993, Proceedings of the First European Cetacean Society Workshop on Cetacean Pathology: Dissection Techniques and Tissue Sampling, Leiden, the Netherlands, 13–14 September 1991, ECS Newsl. 17: 1–39. Jauniaux, T., Garcia Hartmann, M., and Coignoul, F., 1999, Post-mortem examination and tissue sampling of sperm whales Physeter macrocephalus, in Proceedings of Workshop: Sperm Whales Strandings in the North Sea—The Event, the Action, the Aftermath. Web sites: http://www.ulg.ac.be/fmv/anp.htm www.mumm.ac.be BRAZIL Southern Coast I. B. Moreno, P. H. Ott, and D. Danilewicz Grupo de Estudos de Mamiferos Aquaticos do Rio Grande do Sul (GEMARS) Rua Felipe Neri, 382 conj. 203 90440-150 Porto Alegre, RS Fax: 55-51267-1667 E-mail: [email protected] Southeastern Coast Salvatore Siciliano Museo Nacional/UFRJ Dept. de Vertebrados, Setor de Mamiferos São Cristovao 20940-040 Rio de Janeiro, RJ Fax: 55-21568-1314 ext. 213 E-mail: [email protected] Northeastern Coast Regis P. de Lima and Cristiano L. Parente Centro Mamíferos Aquáticos/IBAMA Estrada do Forte Orange, s/n° Caixa Postal 01 Ilha de Itamaracá PE 53900-000 E-mail: [email protected] M. Cristina Pinedo Lab. Mamíferos Marinhos e Tartarugas Marinhas Dept. Oceanografia–FURG CP 474, Rio Grande–RS 96201-900 E-mail: [email protected] Also: [email protected] J. Laílson-Brito, Jr., B. Fragoso, A. de Freitas Azevedo Universidade do Estado do Rio de Janeiro Dept. de Oceanografia Projeto MAQUA Av. São Francisco Xavier 524 sala 4018E 20550-013 Rio de Janeiro, RJ E-mail: [email protected] Humpback Whale Project Marcia Engel Praia do Quitongo, s/n° CEP-45900-000 Caravelas, Bahia E-mail: [email protected] [email protected] http://www.criaativa. com.br/jubarte (Continued) 0839_frame_C04.fm Page 50 Tuesday, May 22, 2001 10:41 AM 50 CRC Handbook of Marine Mammal Medicine TABLE 1 Examples of Stranding Networks Worldwide (continued) Marcos César de Oliveira Santos Projeto Atlantis—LABMAR Instituto de Biociências Dept. de Ecologia Geral Universidade de São Paulo Cidade Universitária São Paulo, SP E-mail: [email protected] Structure At present, there is no centralized reporting system, but there are approximately ten research groups monitoring strandings along the Brazilian coast. Stranding data are collected by separate research groups that deploy their own individual monitoring programs. Many data are collected through collaborations with media, fishermen, and the public. Although studies of marine mammals were concentrated along the south–southeastern coast, there have been recent efforts to increase efforts on the northeastern coast. Most research groups will collect stranded marine mammals, although there is no specific national legislation. Most groups are at least partially funded by research grants from the Brazilian government, but some rely only on funds from nongovernmental organizations. History Although there is no centralized database, a large proportion of the Brazilian coastline has been monitored for marine mammal strandings over the last 10 years by a number of different organizations. In some areas (south and southeast), efforts of the different groups have overlapped at some time, whereas in the north and northeast regions long stretches of coastline are not monitored. The oldest program has been maintained by Dr. M. Cristina Pinedo (FURG) since 1976 for the coast of Rio Grande do Sul state. The monitoring program surveys 120 km of beach to the north and south of the city of Rio Grande (29°20′S to 33°45′S) every 2 weeks, and the whole coastline bimonthly. The National Center for Research, Conservation and Management of Aquatic Mammals–Aquatic Mammals Center was officially created in 1998, although it had been operating previously as the “Centro Peixe-Boi” (Manatee Center) for the rehabilitation of marine manatees. Notes and Further Reading A first draft structure for a Northeastern Coast Stranding Network is under consideration by IBAMA, the Federal Environmental Agency (IBAMA/CMA Relatório No. 007-99). When effective, this network will be coordinated by the Centro Mamíferos Aquáticos/IBAMA, and operated by several organizations, including Grupo de Estudos de Cetáceos do Ceará (GECC), Centro Golfinho Rotador/Fernando de Noronha, Programa de Estudos de Animais Marinhos (PREAMAR/Bahia), and Universidade Federal do Rio Grande do Norte (UFRN/Natal). IBAMA/CMA, 1999, Relatório do primeiro workshop sobre Rede de Encalhe de Mamíferos Aquáticos do Nordeste-REMANE. IBAMA/CMA Relatório No. 007 99, 35 pp. Pizzorno, J.L.A., Laílson-Brito, J. Jr., Dorneles, P.R., Azevedo, A. de F., and Gurgel, I.M.G. do N., 1998, Review of strandings and additional information on humpback whales, Megaptera novaeangliae, in Rio de Janeiro, southeastern Brazilian coast (1981–1997), Rep. Int. Whales Comm., 48: 443–446. Lodi, L., and Barreto, A., 1998, Legal actions taken in Brazil for the conservation of cetaceans, J. Int. Wildl. Law Policy, 1: 403–411. There is a marine mammal discussion group on the Web, contactable via Drs. Laílson-Brito and B. Fragoso. 0839_frame_C04.fm Page 51 Tuesday, May 22, 2001 10:41 AM 51 Marine Mammal Stranding Networks TABLE 1 Examples of Stranding Networks Worldwide (continued) CANADA East Coast Jerry Conway Marine Mammal Advisor Department of Fisheries and Oceans P.O Box 1035, Dartmouth Nova Scotia, B2Y 4T3 E-mail: [email protected] West Coast Ed Lochbaum Department of Fisheries and Oceans 3225 Stephenson Point Nanaimo, British Columbia V6B 5G3 E-mail: [email protected] Structure All responses to strandings are under the auspices of, and require licensing by, the Department of Fisheries and Oceans (DFO). In Nova Scotia, strandings can be reported by calling 1(800) 668-6868. The Nova Scotia Network has focused primarily on removing stranded marine mammals from where they are found and returning them to the water, as there are no holding facilities. Post-mortem examinations are performed, and samples and skeletons obtained and stored for further research. History A volunteer group in British Columbia, The Marine Mammal Research group, has attempted to serve as a stranding network for about 15 years, but is not very active currently. The Nova Scotia Stranding Network has existed for about 8 years. It has experienced a high turnover and has encountered difficulties at times primarily because the volunteers are university students and move on. After a couple of years of relative inactivity, it is re-grouping. Notes and Further Reading St. Lawrence beluga strandings have been well studied by Dr. Martineau and co-workers (see Chapter 22, Toxicology; Chapter 23, Noninfectious Diseases). The Nova Scotia Stranding Network has been associated with the rescue and recovery work carried out by East Coast Ecosystems with the northern right whale in the Bay of Fundy. CARIBBEAN Nathalie Ward Eastern Caribbean Cetacean Network Box 5, Bequia St. Vincent and the Grenadines West Indies or P.O. Box 573 Woods Hole, MA 02543, USA Fax: 508-548-3317 E-mail: [email protected] Structure The Eastern Caribbean Cetacean Network (ECCN) is a regional, volunteer network that records sightings and strandings of marine mammals in the eastern Caribbean. The ECCN is a research affiliate of the Smithsonian (Continued) 0839_frame_C04.fm Page 52 Tuesday, May 22, 2001 10:41 AM 52 CRC Handbook of Marine Mammal Medicine TABLE 1 Examples of Stranding Networks Worldwide (continued) Institute’s Marine Mammal Laboratory in Washington, D.C., and is sponsored by the United Nations Environment Program. It offers educational programs and workshops for children and adults, and training sessions for field identification and stranding protocols. Funding is provided by a number of nonprofit conservation organizations. The ECCN does not currently have a formal rescue or rehabilitation program nor a specimen collection. History The ECCN was founded in 1990 as a grassroots effort to identify whale and dolphin species of the eastern Caribbean. From 1990 to 1997, the facility was housed at the Museum of Antigua and Barbuda. As of June 1998, ECCN outreach programs have been housed in Bequia, St. Vincent and the Grenadines. The ECCN was founded by Nathalie Ward in response to the paucity of information available on cetaceans in the region. Notes and Further Reading The ECCN educational tools include a Field Guide to Whales and Dolphins of the Caribbean, available from Gecko Productions, Inc., P.O. Box 573, Woods Hole, MA 02543, U.S.A. CROATIA Dra s̆ ko Holcer Croatian Natural History Museum Department of Zoology Demetrova 1 HR-10000 Zagreb Fax: 385-1-4851644 E-mail: [email protected] Caterina Maria Fortuna Adriatic Dolphin Project Tethys Research Institute HR-51551 Veli Lo s̆ inj E-mail: [email protected] Structure The network includes the Ministry of Agriculture and Forestry through its connection with fishermen (primarily Fishing Inspectorate), the Ministry of Maritime Affairs through harbor masters’ offices, the Ministry of Internal Affairs through the Marine Police, and the Ministry of Defense through the National Center for Information and Alert. The ministries inform their offices of the project, and ask them to forward all information to the Croatian Natural History Museum (CNHM). Upon receipt of information on stranded animals, a team from the CNHM or the national stranding center goes to the site. Depending upon the animal’s condition, the team may collect the animal and transport it to Zagreb for post-mortem examination, or do a basic field examination, including species identification, measurements, collection of tissues and other samples (teeth, stomach contents), and determination of cause of death if possible. History In 1994, the Nature Protection Law was adopted under which a Special Act (Rule Book on Protection of Certain Mammalian Species, Mammalia) listing all protected species was issued in 1995. In this, bottlenose (Tursiops truncatus) and common dolphins (Delphinus delphis) were listed as protected species, but the Act extended legal protection to all other cetacean species that may be found in the Croatian part of the Adriatic Sea. Special Act (Rule Book on Compensation Fees for Damage Caused by Unlawful Actions on Protected Animal Species) was issued in 1996 by the same authority. Fines for deliberate killing or for actions that may cause damage or disturbance to cetaceans were set. The CNHM, in conjunction with the Adriatic Dolphin Project, tried to organize a stranding network at the national level in 1997. Notes and Further Reading In the first years, the network worked because of the enthusiasm of people involved, but lack of funding has stopped it almost entirely. Occasional reports are still forwarded to the CNHM, and depending on personal judgment, some stranded animals are collected. Information on strandings and carcasses is also occasionally collected by the veterinary faculty in Zagreb. 0839_frame_C04.fm Page 53 Tuesday, May 22, 2001 10:41 AM 53 Marine Mammal Stranding Networks TABLE 1 Examples of Stranding Networks Worldwide (continued) DENMARK Nature and Wildlife Section National Forest and Nature Agency Ålholtvej 1 DK-6840 Oksbøl Fax: 45-76541046 E-mail: [email protected] Fisheries and Maritime Museum Tarphagevej 2 DK-6710 Esbjerg V Fax: 45-76122010 Web site: http://www.fimus.dk Zoologisk Museum Universitetsparken 15 DK 2100 Copenhagen Ø Fax: 45-35321010 Web site: http://www.zmuc.dk Structure Since 1993, the network has been run cooperatively by the National Forest and Nature Agency, the Fisheries and Maritime Museum in Esbjerg, and the Zoological Museum of the University of Copenhagen. Stranding events are reported either directly to the museums or through the regional forest districts. All cetacean strandings are recorded and all specimens other than harbor porpoises are examined. A standard autopsy is performed on all suitable animals. Harbor porpoises are only collected within the framework of special projects. A record of available data and specimens for research are kept by the two museums, and a special tissue bank is associated with the network. A list of samples will be made available as a read-only database on the forthcoming Web site of the network. History In 1885, upon an inquiry by the Zoological Museum, the Danish Ministry of Interior Affairs set up a notification procedure for its rescue service officers, receiver of wrecks, and other local representatives who by telegraph were to report strandings of “unusual sea animals” to the museum. Although the museum received frequent reports, the prime scope of this network was to obtain rare specimens, not to record all strandings, nor to provide the basis for analyses and management. The more common species therefore remained unrecorded. This procedure lasted until about 1980, when the Zoological Museum and the Fisheries and Maritime Museum initiated a formal stranding network, aiming to collect as much information and as many specimens as possible. This network has been improved several times since, most recently with the launching of a contingency plan in 1993, involving the forest districts of the National Forest and Nature Agency. Notes and Further Reading A comprehensive review of Danish whale strandings was published in 1995 by Kinze covering the period 1575 to 1991. The first report covering the period 1992 to 1997 was published in 1998 (Kinze et al., 1998). Kinze, C.C., 1995, Danish whale records 1575–1991 (Mammalia, Cetacea), Review of whale specimens stranded, directly or incidentally caught along the Danish coasts, Steenstrupia, 21: 155–196. Kinze, C.C., Tougaard, S., and Baagøe, H.J., 1998, Danske hvalfund i perioden 1992–1997 [Danish whale records (strandings and incidental catches) for the period 1992–1997], Flora Fauna, 104: 41–53. [In Danish with English summary.] FRANCE Centre de Recherche sur les Mammifères Marins (CRMM) Institut de la Mer et du Littoral Port des Minimes 17000 La Rochelle Fax: 33-(0)-546449945 E-mail: [email protected] (Continued) 0839_frame_C04.fm Page 54 Tuesday, May 22, 2001 10:41 AM 54 CRC Handbook of Marine Mammal Medicine TABLE 1 Examples of Stranding Networks Worldwide (continued) Structure Strandings along the entire coastline are reported to authorities, which contact the local field operators authorized by the French Environment Office. These field operators are volunteers trained to respond to dead marine mammal strandings. When fresh, but dead, animals are dissected and samples collected for current or future studies (aging, stomach content analysis, ecotoxicology, genetics, reproductive biology, microbiology, parasitology, and pathology). For live stranded cetaceans, specialized personnel organize the rescue, or request euthanasia of the animal if its condition is too poor. Live stranded seals are taken to Océanopolis, Brest, or CRMM, La Rochelle, for rehabilitation. History The French stranding network was set up in 1971. All reported strandings are recorded in a database managed by the CRMM in La Rochelle. To date, over 8500 strandings have been recorded. Until 2000, administration of the network was funded mainly by the city of La Rochelle. It works thanks to the good-will, time, and funds of nonprofit organizations and authorized volunteers. Notes and Further Reading The CRMM produces annual reports on French marine mammal strandings. From 1990 to 1999, a mean of 460 cetaceans (4.5% of which were alive) and 40 seals (60% of which were alive) were recorded each year. There is a high rate of fisheries by-catch of small cetaceans, especially in winter. GERMANY Dr. Ursula Siebert Forschungs- und Technologiezentrum Westküste Hafentoern D 25761 Büsum Fax: 49-0-4834604199 E-mail: [email protected] H. Benke Director, Deutsches Museum für Meereskunde und Fischerei Katharinenberg 14–20 D 18439 Stralsund M. Stede Staatliches Veterinäriantersuchungsamt für Fische und Fischwaren Schleuenstrasse D 27472 Cuxhaven Structure Live stranded seals are taken to the Seal Station Friedrichskoog, and live stranded small cetaceans to the Delfinarium Harderwijk, the Netherlands, for rehabilitation. By-caught or stranded carcasses are taken to the Westcoast Research and Technology Center, University of Kiel for examination. If transportation cannot be organized in a few hours, carcasses are stored in one of the 21 freezers distributed along the coast of the North and Baltic Seas. Post-mortem examinations are performed according to Kuiken and Hartmann (1993). Depending upon the state of preservation and findings at necropsy, samples for histology, bacteriology, virology, parasitology, serology, and toxicology may be collected. Additional investigations include age determination, reproductive biology, genetics, stomach content analysis, and skeleton archiving. History The major harbor seal die-off of 1988–1989 in northern Europe led to the development of a well-functioning stranding network for marine mammals. Notes and Further Reading The majority of strandings of marine mammals in German waters occur along the coast of Schleswig–Holstein (100 to 150 cetaceans, 350 to 450 seals per year). Kuiken, T., and Hartmann, M. G., 1993, Proceedings of the First European Cetacean Society Workshop on Cetacean Pathology: Dissection Techniques and Tissue Sampling, Leiden, the Netherlands, 13–14 September 1991, ECS Newsl., 17: 1–39. 0839_frame_C04.fm Page 55 Tuesday, May 22, 2001 10:41 AM 55 Marine Mammal Stranding Networks TABLE 1 Examples of Stranding Networks Worldwide (continued) GREECE Dr. Alexandros Frantzis Institute of Marine Biological Resources National Centre for Marine Research Agios Kosmas GR-166 04 Hellenikon Fax: 301-9811713 E-mail: [email protected] Structure Whenever port authorities are informed of a cetacean stranding in their area of responsibility, they inform the National Centre for Marine Research (NCMR) via a stranding report. However, this does not always happen, nor are the port police always aware of stranded cetaceans. Stranding reports may contain information on the place, date, time, number of animals, their total length, plus other measurements, species, sex, cause of death, comments, and possibly photographs. Due to lack of specific knowledge and experience in most cases, all information provided by nonspecialized persons is considered suspect, except the fact that a stranding did occur. When a stranding is unusual (e.g., mass strandings) or seems to have a particular value (rare cetacean species), additional information is gathered by contacting people who saw the stranded cetacean, searching for photographic documents, and/or going to the site. Reports are retained for further analysis only when accompanied by photographs that allow species identification, or when a good description is accompanied by a precise total length. History Occasional efforts to record cetacean strandings in Greece began in the late 1980s. However, the formal start of a network came at the end of 1991, when morbillivirus infection of Mediterranean striped dolphins reached the Hellenic Seas, and the increasing number of stranded animals became disturbing. The NCMR and the Hellenic Society for the Study and Protection of the Monk Seal (HSSPMS) took the initiative to inform portpolice authorities formally about the necessity of gathering stranding data and samples. A special stranding and sighting form was prepared and distributed to competent authorities all along the Greek coasts. Two years later, the HSSPMS ceased its cetological activity and a new nongovernmental organization, “Delphis” (Hellenic Cetacean Research and Conservation Society), started to receive stranding data (simultaneously with NCMR), and responded to cetacean strandings whenever possible. Some additional data were given to Greenpeace by its supporters. No formal stranding network yet exists in Greece. Notes and Further Reading Greece has the longest coastline of all the Mediterranean countries (more than 16,000 km) and almost 10,000 islands and islets, including many small uninhabited ones. Due to these particular geographic characteristics, Greek coasts (which are often inaccessible by land) are very difficult to monitor. However, the main reasons no formal and appropriate cetacean stranding network exists in Greece are lack of dedicated funds and, to a lesser degree, lack of a national coordinating authority. Even so, the incomplete stranding data gathered during the last 7 years have contributed significantly to our knowledge of cetaceans in Greece and the Mediterranean Sea. HONG KONG Coordinator: Dr. Thomas Jefferson Fax: 858-278-3473 E-mail: [email protected] Local contacts: Samuel Hung, Mientje Torey, and Lawman Law MP 852-91990847 Contact within HKAFCD: Dick Choi E-mail: [email protected] Structure The network is funded by the Hong Kong Government Agriculture, Fisheries and Conservation Department (AFCD) and assisted by a local oceanarium, Ocean Park Corporation, for veterinary support/expertise. (Continued) 0839_frame_C04.fm Page 56 Tuesday, May 22, 2001 10:41 AM 56 CRC Handbook of Marine Mammal Medicine TABLE 1 Examples of Stranding Networks Worldwide (continued) History Hong Kong, China SAR, formally established a cetacean stranding network in 1994, although limited data have been collected since 1973. Notes and Further Reading Parsons, E.C.M., and Jefferson, T. A., 2000, Post-mortem investigations on stranded dolphins and porpoises from Hong Kong waters, J. Wildl. Dis., 36: 342–357. ISRAEL Oz Goffman Israeli Marine Mammal Research & Assistance Center (IMMRAC) Fax: 972-52692477 E-mail: [email protected] Structure IMMRAC is in the Naval High School, in Mikhmoret, in the center of the Mediterranean coast of Israel. IMMRAC has three main interests: research, increasing public awareness, and rescue and rehabilitation. Academic support comes from the Leon Recenati Institute for Maritime Studies at the Haifa University. The rescue team consists of 30 volunteers, 3 of whom are veterinarians, and conducts simulation exercises twice a month. The personnel are divided into three teams according to the different geographic regions: north, center, and south. Necropsies are performed to establish the cause of death, with all data analyzed by Mia Roditi. IMMRAC is willing to offer assistance to neighboring countries if requested. History IMMRAC was established by a number of individuals that dedicated their free time and efforts to protecting and researching marine mammals along the coasts of Israel. Previously there had been no data on marine mammals in this region. IMMRAC conducted the first dolphin population surveys in the eastern Mediterranean, the Gulfs of Suez and Eilat, using information from trawler boats, and later from Navy vessels and diving boats. Recently, IMMRAC received, as a donation from “Tnuva,” Israel’s largest dairy producer, a research and rescue boat, which will enable daily population surveys to be performed. The IMMRAC volunteers began collecting bodies of beached dolphins in their private cars, sometimes assisted by government authorities. Notes and Further Reading IMMRAC activities led to the following findings: In 1995 Orit Barnea showed that the long snouted spinner dolphin (Stenella longirostris) lives in the Gulf of Eilat. This is the northernmost habitat for this Indian Ocean population. The rough toothed dolphin (Steno bredanensis) is found in the waters along the Israeli Mediterranean coastline, and is probably a rare but permanent resident. ITALY Marco Borri, Coordinatore Centro Studi Cetacei (CSC) Museo Zoologico “La Specola” via Romana 17 50125 Firenze Fax: 39-(0)55-225325 E-mail: [email protected] 0839_frame_C04.fm Page 57 Tuesday, May 22, 2001 10:41 AM 57 Marine Mammal Stranding Networks TABLE 1 Examples of Stranding Networks Worldwide (continued) Structure A nationwide marine mammal stranding network is managed by the CSC of the Società Italiana di Scienze Naturali, based at the Civic Natural History Museum in Milan (Borri et al., 1997). Information on the stranding event is relayed from the stranding location, mostly by personnel from the Coast Guard, to a centralized answering service in Milan, provided at no cost by the insurance company Europe Assistance SpA. From there, the appropriate CSC correspondent from one of the 18 zones, into which the 8000 km of Italian coastline is subdivided, is alerted, and the appropriate intervention performed. CSC also coordinates research projects using samples obtained from the stranding program. History The CSC was created within the Milan Public Museum of Natural History with operational guidance from the Italian Society of Natural Sciences in 1985 at the first national conference on cetaceans in Riccione. CSC is recognized by Ministero delle Risorse Agricole, Alimentari e Forestali (Ministry of Agricultural, Food and Forest Resources) and is authorized by Ministero dell’Agricoltura e Foreste (Ministry of Agriculture and Forests) (CITES Office) and by Ministero dell’Ambiente (Ministry of Environment) (Service for the Conservation of Nature). One of the initial goals of CSC, whose aim is to unite researchers and institutions in Italy concerned with cetaceans, was to create “Progetto Spiaggiamenti” (a stranding project). This project, based upon similar projects in other countries, created a national network for the reporting and response to stranded cetaceans in 1986. In 1990, a second project was added, addressing the special needs of live stranded cetaceans. Notes and Further Reading Results of the network activities are published yearly in the Society’s proceedings (Atti della Società Italiana di Scienze Naturali). In 1986 through 1997, 2288 cetacean strandings were recorded. Of the 1724 identified species, 1054 (61.1%) were striped dolphins, 347 (20.1%) bottlenose dolphins, 99 (5.7%) sperm whales, 83 (4.8%) Risso’s dolphins, 40 (2.3%) fin whales, 40 (2.3%) long-finned pilot whales, 39 (2.3%) Cuvier’s beaked whales, with shortbeaked common dolphins, minke whales, false killer whales, and one dwarf sperm whale accounting for the remaining 1.4%. Borri, M., Cagnolaro, L., Podestà, M., and Ranieri, T., 1997, I1 Centro Studi Cetacei: dieci anni di attività (1986–1995), Natura (Milan), 88(1): 1–93. Cornaglia, E., Rebora, L., Gili, C., and Di Guardo, G., 2000, Histopathological and immunohistochemical studies on cetaceans found stranded on the coast of Italy between 1990 and 1997, J. Vet. Med., 47: 129–142. JAPAN T. K. Yamada National Science Museum 3-23-1 Hyakunin-cho Shinjuku-ku, Tokyo 164 E-mail: [email protected] Structure Local governments, aquaria, museums, research institutes, universities, and volunteers are loosely cooperating on stranding responses. The National Science Museum and Institute of Cetacean Research are responding mostly to dead strandings, the aquaria to live. There are about 100 to 200 strandings per year, of which 50 to 80 individuals are investigated to some extent. In 1999, about 50 necropsies were performed. Biological investigations, morphological research, and contaminant surveys have been conducted. (Continued) 0839_frame_C04.fm Page 58 Tuesday, May 22, 2001 10:41 AM 58 CRC Handbook of Marine Mammal Medicine TABLE 1 Examples of Stranding Networks Worldwide (continued) History The first symposium on marine mammal strandings was held in 1997 by the National Science Museum, the University of Tokyo, the Japanese Association of Zoos and Aquariums, the Institute of Cetacean Research, and the Sea of Japan Cetology Research Group. Further activities to save live strandings and to investigate dead strandings were decided upon. Training seminars have been held annually since then at the National Science Museum. Notes and Further Reading Traditionally, cetaceans have been heavily hunted for human consumption. MALDIVES H. Whitewaves Marine Research Centre Malé Republic of Maldives Fax: 960-322509/326558 E-mail: [email protected] Structure The Maldives is a country of some 1200 tiny coral islands, set upon a string of atolls, in the central Indian Ocean. Since mid-2000, an official strandings reporting scheme has been in place. Of the 1200 islands, some 200 are inhabited. Each inhabited island has a government office and government-appointed island chief. The Marine Research Centre (MRC) has sent recording forms to each island office, with instructions on how to report every marine mammal stranding. The scheme is inexpensive and is funded from the MRC budget. The main aim of the scheme is to obtain basic biological information about cetaceans in the Maldives. History Before early 2000 there was no marine mammal stranding network in the Maldives. Reports of cetacean strandings were occasionally sent to the MRC, in the capital Malé, and information on other strandings was collected by MRC staff during field trips. Notes and Further Reading Most stranded cetaceans are found floating dead at sea by fishermen. Nearly all those that wash up on islands or reefs appear to be dead at the time of stranding. There are only two known instances of live strandings to date. This, combined with the geography of the country (numerous small islands and reefs spread over a vast area of ocean, with consequent transport and communication difficulties), means that a network focusing on the welfare of live stranded marine mammals is unlikely to develop in the foreseeable future. Anderson R.C., A. Shaan, and Z. Waheed, 1999, Records of cetacean “strandings” in the Maldives, J. S. Asian Nat. Hist., 4: 187–202. MALTA Dr. A.Vella Department of Biology University of Malta Msida, MSD 06 Fax: 356-32903049 E-mail: [email protected] Structure The Director of the Environment Protection Department (EPD) is responsible for responding to strandings, and will send an inspector to the site to ensure that protocols are followed. The entities authorized to respond to a cetacean stranding are the Commissioner of Police, the Director of the Veterinary Services of Malta, field 0839_frame_C04.fm Page 59 Tuesday, May 22, 2001 10:41 AM 59 Marine Mammal Stranding Networks TABLE 1 Examples of Stranding Networks Worldwide (continued) cetacean researchers from the University of Malta, representatives of local NGOs, and the media. For dead cetacean strandings, the animal is measured, photographed, and a post-mortem examination undertaken. Legal proceedings may be undertaken if there is indication of human interaction. Specimens for further studies or for educational displays are taken to the University of Malta. For live cetacean strandings, the Director of the Veterinary Services determines the plan of action. The dolphinarium, Marineland, assists by making specialized equipment, a large treatment tank, and veterinary advice available. Fondazione Cetacea (Italy) is also willing to assist. History A cetacean stranding protocol was issued officially in March 1999, by the director of the EPD. Notes and Further Reading This protocol has been running smoothly since its establishment in March 1999. It is hoped that it will promote the proper handling of cetacean strandings. In the past, this was not the case, due to lack of available advice for inexperienced personnel. MEXICO Baja California Dr. Lorenzo Rojas-Bracho Programa Nacional de Investigación y Conservación de Mamíferos Marinos (PNICMM) c/o CICESE Ensnenada, Baja California, Tel. (6)174 50 50 al 53 ext 22115 Carribean Maria del Carmen Garcia: Parque Nacional Isla Contoy Subdirectora Tel (98) 497525 (98) 494021 Blvd Kukulkan km 4.8 ZH Cancún Q. Roo CP 77500 Gulf of Mexico Diana Madeleine AntochiwAlonzo Red de Varamientos de Yucatàn, A.C. Calle 53-E No. 232 entre 44 y 46 Fracc. Francisco de Montejo C.P. 97 200 Mérida Yucatán Tel. (9) 946 55 58 Tel./Fax. (9) 927 36 18 http://www.revay.org.mx E-mail: [email protected] Pacific Hector Pérez-Cortés CRIP/INP Km. 1 Caretera Pichilingue – La Paz La Paz 23020 E-mail: [email protected] Structure The SOMEMMA (Mexican Society for Marine Mammalogy–Sociedad Mexicana de Mastozoologia Marina) organizes and coordinates all the groups interested in stranding response by maintaining a strandings database and assisting with obtaining permits from the National Institute of Ecology (INE) and Procuraduria Federal de Proteccion al Ambiente (PROFEPA). In Ensenada, Baja California, a new way of organizing stranding response efforts is being attempted. All people interested in strandings in the Ensenada–Tijuana corridor (NGOs, university, research institutes, individuals, and INE) were contacted, and representatives met with PROFEPA. Delegates created the subcommittee for strandings attention, an organization with government representation. (Continued) 0839_frame_C04.fm Page 60 Tuesday, May 22, 2001 10:41 AM 60 CRC Handbook of Marine Mammal Medicine TABLE 1 Examples of Stranding Networks Worldwide (continued) Quintana Roo is the state that faces the Caribbean Sea, where the first stranding network on the east coast of Mexico was established in 1987. This group has concentrated mainly on manatees. History For over a decade, research groups have responded to marine mammal strandings, mainly in the southern state of Baja California Sur, where there is the highest density of marine mammalogists. Initially, each researcher worked independently, but efforts to coordinate responses are developing. A few years ago, in the northern State of Baja California, a group of students and researchers formed an NGO that focuses on marine mammal strandings, primarily California sea lions, in the Ensenada–Tijuana area. In the mid-1990s, the Attorneys General Office for the Environment (PROFEPA) was created, with almost every state in Mexico having a PROFEPA office. PROFEPA addresses any issue that affects the environment. It does not respond to strandings, but to be able to attend strandings, one must have its authorization and permits from the INE. Both PROFEPA and INE have created a number of subcommittees consisting of members of local communities to address environmental issues, from illegal fishing to pollution. Notes and Further Reading No government funding for these efforts exists, nor is there any possibility of financial support in the foreseeable future. Except for the states of Campeche and Tamaulipas, NGOs are currently attending strandings on the coasts of Veracruz, Tabasco, and Yucatán. Most of these groups formed in the last 3 to 4 years. Students mostly constitute these groups. Funding is extremely low and comes from contributions by the members. Some receive in-kind support from their local universities and aquaria. More recently, a national stranding e-mail correspondence group was created to discuss strategies and to exchange experiences. This information was kindly provided by SOMEMMA. THE NETHERLANDS Dr. Chris Smeenk National Museum of Natural History P.O. Box 9517 2300 RA Leiden Fax: 31-1-5687666 E-mail: [email protected] Structure The stranding network involves many official authorities and volunteers. It is coordinated by the National Museum of Natural History, Leiden. Stranding records are published in Lutra, the journal of the Dutch Mammal Society (Smeenk, 1995). Dead cetaceans are collected by or for the museum; most of them are frozen. A post-mortem on all suitable animals is carried out by a team of veterinarians and zoologists. Standard samples are taken for histopathology, bacteriology, virology, life-history, toxicology, and dietary studies (Kuiken and Hartmann, 1993). Live stranded animals are taken to the Marine Mammal Park at Harderwijk and to Zeehondencreche Pieterbuen. History Data and material from stranded cetaceans have been collected since about 1914. Archives and databases of strandings are kept in the National Museum of Natural History, Leiden. For some large species, records date back to the 16th century (Smeenk, 1997). Skeletal material and samples are deposited in the Leiden museum; other important osteological collections are in the Zoological Museum of Amsterdam University and in the Natural History Museum in Rotterdam. 0839_frame_C04.fm Page 61 Tuesday, May 22, 2001 10:41 AM 61 Marine Mammal Stranding Networks TABLE 1 Examples of Stranding Networks Worldwide (continued) Notes and Further Reading Addink, M.J., and Smeenk, C., 1999, The harbour porpoise Phocoena phocoena in Dutch coastal waters: Analysis of stranding records for the period 1920–1994, Lutra, 41: 55–80. Kuiken, T., and Hartmann, M.G., 1993, Proceedings of the First European Cetacean Society Workshop on Cetacean Pathology: Dissection Techniques and Tissue Sampling, Leiden, the Netherlands, 13–14 September 1991, ECS Newsl., 17: 1–39. Smeenk, C., 1995, Strandingen van Cetacea op de Nederlandse kust in 1990, 1991 en 1992, Lutra, 38: 90–104. Smeenk, C., 1997, Strandings of sperm whales Physeter macrocephalus in the North Sea: History and patterns, Bull. Inst. R. Sci. Nat. Belg. Biol., 67 Suppl.: 15–28. NEW ZEALAND Coordinator Anton van Helden Marine Mammals Collection Manager Museum of New Zealand Te Papa Tongarewa P.O. Box 467, Wellington Fax: 06443817310 E-mail: [email protected] Pathologist Pádraig Duignan New Zealand Wildlife Health Centre I.V.A.B.S. Massey University Palmerston North Fax: 006463505714 E-mail: [email protected] Department of Conservation Rob Suisted 58 Tory Street, Wellington E-mail: [email protected] Genetics Dr. Scott Baker School of Biological Sciences University of Auckland Auckland E-mail: [email protected] Volunteer Groups Project Jonah P.O. Box 8376 Symonds Street Auckland Fax: 064-95215425 Marine Watch Jim Lilley 59 Clydesdale St Linwood, Christchurch Structure The Department of Conservation (DOC) administers the Marine Mammal Protection Act of 1978, which provides for the conservation, protection, and management of marine mammals. Among other roles, DOC is responsible for dealing with beached and stranded cetaceans and pinnipeds. Cetaceans that can be refloated are saved with the help of volunteer groups. Those that die are examined by a pathologist to determine cause of death. Samples are archived at Massey University for diagnostic tests, toxicology, and genetics. The marine mammals collection manager at the Museum of New Zealand Te Papa Tongarewa maintains a database of all cetacean strandings as well as collecting, storing, and maintaining an extensive skeletal collection. A database of cetacean genetics is maintained at the University of Auckland. History The New Zealand Stranding Network was established as a collaboration among the Museum of New Zealand, the Department of Conservation, universities, and Maori interest groups. Notes and Further Reading New Zealand has a large number of cetacean strandings with an average of 80 incidents per year representing as many as 38 species (an average of 19 species each year). In addition, stranded pinnipeds include New Zealand fur seals, subantarctic fur seals, leopard seals, and, less commonly, New Zealand sea lions and southern elephant seals, with historic records of crabeater seals. Web: http://www.massey.ac.nz Web: http://www.doc.govt.nz (Continued) 0839_frame_C04.fm Page 62 Tuesday, May 22, 2001 10:41 AM 62 CRC Handbook of Marine Mammal Medicine TABLE 1 Examples of Stranding Networks Worldwide (continued) PERU CEPEC Department of Veterinary Research Jorge Chavez 302, Pucusana Lima 20 E-mail: [email protected] Centro Peruano de Estudios Cetologicos (CEPEC) Museo de la Fauna Marina Jorge Chavez 101, Pucusana Lima 20 E-mail: [email protected] Structure No official marine mammal stranding network exists in Peru, but specimens are collected opportunistically by a variety of individuals and institutions, including CEPEC. Fresh or live cetacean strandings typically are utilized by locals. History CEPEC is a private institute founded in 1985 for research on the distribution, biology, pathology, and management issues of cetaceans in developing countries, with particular emphasis on the Southeast Pacific. SPAIN Valencia Region Fax: 34-963864372 E-mail: [email protected] Murcia Region Tel: 34-968526817 and 34-689788515 Catalonia Region Fax: 34-937525710 E-mail: [email protected] Andalusia Region Fax: 34-952229287 E-mail: [email protected] Balearic Islands Tel: 34-971675125 Galician Region Cemma Tel./Fax: 34-981360804 E-mail: [email protected] Euskadi Region Ambar E-mail: [email protected] Cantabria Region Fax: 34-942281068 Canary Islands M. Andre Fax: 34-928451141 E-mail: [email protected] Asturias Region Cepesma E-mail: [email protected] Structure Each coastal regional government, of which there are five in the Mediterranean, four in the Atlantic, and one in the Canary Islands, has a coordinator. Coordinators collaborate with the Spanish Cetacean Society, funded by the Spanish Ministry of Environment, to establish standard protocols and methods for sightings, strandings, and rehabilitation of cetaceans and sea turtles in Spanish waters. In the Canary Islands, there is no official stranding network, but the veterinary school (Marine Mammal Conservation Research Unit, Veterinary School, University of Las Palmas de Gran Canaria) has responded to 85% of cetacean strandings in the Canary Islands. There are no pinniped strandings. Once a year, a complete report on all island strandings is sent to the government of each island. 0839_frame_C04.fm Page 63 Tuesday, May 22, 2001 10:41 AM 63 Marine Mammal Stranding Networks TABLE 1 Examples of Stranding Networks Worldwide (continued) SWEDEN Mats Olsson Swedish Museum of Natural History Contaminant Research Group Box 50007 SE 104 05 Stockholm Fax: 46-8 5195 4256 E-mail: [email protected] Structure Seals found dead in fishing gear or stranded within the Baltic have been sent to the Swedish Museum of Natural History in Stockholm. Collection is by the public, the police, and the Swedish Coast Guard. The animals are examined to determine cause of death or health status. The health studies are part of the Swedish Environmental Monitoring Program run by the Swedish Environmental Protection Agency (EPA). Simultaneous annual censuses of the three seal populations are carried out by the Swedish Museum of Natural History, also funded by the Swedish EPA. History The Swedish program for stranded seals has existed since the 1970s. Notes and Further Reading Olsson, M., Andersson, Ö., Bergman, Å., Blomkvist, G., Frank, A., and Rappe, C., 1992, Contaminants and diseases in seals from Swedish waters, Ambio, 21: 561–562. UKRAINE (and Bulgaria and Georgia) Dr. Alexei Birkun E-mail: [email protected] Structure This network that includes three countries is coordinated by the BREMA laboratory in Simferopol, Crimea, and includes 6 specialists and 30 to 40 volunteers (students, school children, fishermen, officers of the Ukrainian Fish Protection Service, coastal border guards). There is no financial support for the network at present. History A cetacean stranding network has been working in the Crimea (Ukraine, Black Sea region) since 1989. In 1997, the network was extended into Bulgaria and Georgia. Notes and Further Reading Birkun, A., Jr., Stanenis, A., and Tomakhin, M., 1994, Action plan for rescue, rehabilitation and reintroduction of wild sick and traumatized Black Sea cetaceans. European research on cetaceans, 8, in Proc. 8th Annual Conf. Eur. Cetacean Soc., Montpellier, France, 2–5 March 1994, Lugano, 237 pp. Krivokhizhin, S.V., and Birkun, A.A., 1999, Strandings of cetaceans along the coasts of the Crimean peninsula in 1989–1996, European research on cetaceans, 12, in Proc. 12th Annual Conf. Eur. Cetacean Soc., Monaco, 20–24 January 1998, European Cetacean Society, Valencia, 59–62. Birkun, A., Jr., Kuiken, T., Krivokhizhin, S., Haines, D.M., Osterhaus, A.D.M.E., van de Bildt, M.W., Joiris, C.R., and Siebert, U., 1999, Epizootic of morbilliviral disease in common dolphins (Delphinus delphis ponticus) from the Black Sea, Vet. Rec., 144: 85–92. (Continued) 0839_frame_C04.fm Page 64 Tuesday, May 22, 2001 10:41 AM 64 CRC Handbook of Marine Mammal Medicine TABLE 1 Examples of Stranding Networks Worldwide (continued) UNITED KINGDOM Institute of Zoology Regent’s Park London, NW1 4RY Fax: 0207 586 1457 E-mail: [email protected] The Natural History Museum Cromwell Road London, SW7 5BD Fax: 020 7942 5433 Wildlife Unit SAC Veterinary Science Division (Inverness) Drummondhill Stratherrick Road Inverness, IV2 4JZ Fax: 1463-711103 E-mail: [email protected] British Divers Marine Life Rescue 39 Ingham Road, Gillingham Kent, ME7 1SB Tel./Fax: 01634-281680 E-mail: 101375,[email protected] RSPCA Headquarters Wildlife Department Causeway Horsham West Sussex RH12 1HG http://www.rspca.org.uk Scottish SPCA 603 Queensferry Road Edinburgh, EH4 6EA Fax: 0131 339 4777 Structure Coordination of pathological investigations of strandings in England and Wales has been conducted by the Institute of Zoology (Zoological Society of London) in collaboration with the Natural History Museum, London, since 1990. The Scottish Agricultural College Inverness has coordinated all strandings research investigations within Scotland since 1992. Post-mortem examinations are performed according to Kuiken and Hartmann (1993). Live strandings are reported to the Royal Society for the Protection of Animals (RSPCA) in England and Wales (24-hour hotline: 0870 5555999). In Scotland, the Scottish Society for the Protection of Animals (SSPCA) has several local emergency phone numbers. Inspectors from both organizations routinely attend such events. Live seals are taken to seal rehabilitation centers throughout the U.K., when deemed necessary. Live stranded cetaceans are typically attended by veterinarians, members of British Divers Marine Life Rescue (BDMLR), and other rescue groups who have an extensive network of trained volunteers throughout the U.K. There are currently no appropriate facilities for cetacean rehabilitation within the U.K. History Since 1913, the Natural History Museum in London has collected data on cetacean strandings within the U.K. In 1990, 2 years after a major epizootic of phocine distemper occurred in harbor seals in northern Europe, the U.K. Department of the Environment decided to partially fund a systematic and collaborative program of marine mammal strandings research within the U.K. This research is currently ongoing. The main goals of this new strandings research, apart from investigating any future marine mammal mass mortalities, were systematically to investigate the diseases, causes of death, and potential relationships between exposure to contaminants and health status in marine mammals in U.K. waters. A centralized U.K. database for pathological and other data resulting from the strandings projects and national marine mammal tissue archives were also established. Although originally established to investigate both cetacean and pinniped strandings in U.K. waters, the U.K. strandings program has been heavily biased toward cetaceans in recent years to comply with a number of international cetacean conservation agreements to which the U.K. is a signatory. Notes and Further Reading Approximately 200 cetaceans (mainly harbor porpoises and common dolphins) and 300 pinnipeds (mainly gray seals and common seals) typically strand within the U.K. each year. A number of key collaborating organizations, such as the Veterinary Investigation Unit, Truro, the Centre for Environment, Fisheries and Aquaculture Science; Sea Mammal Research Unit; University College Cork, Ireland; University of Aberdeen; Institute of Animal Health, Pirbright; and the Natural History Museum of Scotland, are involved in many aspects of the strandings research. 0839_frame_C04.fm Page 65 Tuesday, May 22, 2001 10:41 AM 65 Marine Mammal Stranding Networks TABLE 1 Examples of Stranding Networks Worldwide (continued) Kuiken, T., and Hartmann, M.G., 1993, Proceedings of the First European Cetacean Society Workshop on Cetacean Pathology: Dissection Techniques and Tissue Sampling, Leiden, the Netherlands, 13–14 September 1991, ECS Newsl., 17: 1–39. UNITED STATES OF AMERICA http: //www.nmfs.noaa.gov/prot_res/PR2/Health_am_stranding_Response_program/mmhsrp.html Cetaceans, Seals, Sea Lions, Sea Turtles: Alaska NMFS Alaska Region P.O. Box 21668 Juneau, AK 99802-1668 Tel: (907) 586-7235 Fax: (907) 586-7249 Washington and Oregon NMFS Northwest Region 7600 Sand Point Way, N.E. Bldg. 1 Seattle, WA 98115-0070 Tel: (206) 526-6733 Fax: (206) 526-6736 Maine to Virginia NMFS Northeast Region One Blackburn Drive Gloucester, MA 01930-2298 Tel: (508) 495-2090 North Carolina to Texas, Puerto Rico, U.S. Virgin Islands NMFS Southeast Region 9721 Executive Center Drive St. Petersburg, FL 33716 Tel: (305) 361-4586 Sea Otters: U.S. Fish and Wildlife Service 2493 Portola Road, Suite B Ventura, CA 93003 Tel: (805) 644-1766 Manatees: Endangered Species Division U.S. Fish and Wildlife Service 75 Spring Street, S.W. Atlanta, GA 30303 Tel: (404) 679-7096 California and Hawaii NMFS Southwest Region 501 West Ocean Boulevard Suite 4200 Long Beach, CA 90802 Tel: (562) 980-4017 Polar Bears, Walrus, Sea Otters in Alaska: U.S. Fish and Wildlife Service 1011 East Tudor Road Anchorage, AK 99503-6199 Tel: (907) 786-3800 Structure Jurisdiction over cetaceans and seals and sea lions falls to the National Marine Fisheries Service (NMFS), while the U.S. Fish and Wildlife Service has jurisdiction over walrus, sea otters, and polar bears. The National Stranding Network is divided into five regions: Northwest, Southwest, Northeast, Southeast, and Alaska. Although officially part of the Southwest Region, all stranding responses in Hawaii are coordinated by the NMFS Pacific Area Protected Species Program Coordinator. Network members consist of a wide range of organizations and individuals, including government agencies, academic institutions, research institutions, rehabilitation facilities, aquaria, and interested individuals. Activities of members are coordinated by the NMFS regional coordinator. Training is available for network volunteers, primarily through a field guide (Geraci and Lounsbury, 1993), but also through newsletters and workshops. All participants are required to submit monthly stranding reports to their regional offices on which Level A, B, and C data are recorded. Level A data are minimum data to be collected at any stranding event and reported to the national office (exact location, date, initial species identification, number of animals involved, sex, length, evidence of human interaction, and condition of the animals). Level B data are basic life-history and specific event data (weather, carcass orientation, animals and human activities in area, collection of parts for age determination). Level C data are results of careful internal and external examination of animals involved, including specimen collection and preservation (Geraci and Lounsbury, 1993). Members do not receive direct funding from NMFS for stranding responses, except under special circumstances. History In 1972, the increased federal protection of marine mammals resulting from the passage of the Marine Mammal Protection Act (MMPA), combined with increased public awareness and compassion for marine mammals, highlighted a need for an organized response to marine mammal strandings beyond the Smithsonian Institution’s list of strandings. In 1977, the first Marine Mammal Stranding Workshop was held. The shortterm goals established at this workshop were to provide for a national network coordinator; to establish and (Continued) 0839_frame_C04.fm Page 66 Tuesday, May 22, 2001 10:41 AM 66 CRC Handbook of Marine Mammal Medicine TABLE 1 Examples of Stranding Networks Worldwide (continued) evaluate regional reporting and notification systems; to establish standard protocols for euthanasia, transport, release, specimen requests, and disposal of stranded marine mammals; to describe clearly and periodically evaluate data collection; and to develop and maintain up-to-date inventories of all interested parties and network-authorized institutions. The long-term goals of this workshop were to develop procedures that would minimize possible threats to human health, minimize pain and suffering of live stranded animals, derive maximum scientific and educational benefits, and result in collection of normal baseline data. In 1981, regional offices and methods for network participation and reporting were established. By 1987, there was sufficient new information from strandings and enough need to standardize collection protocols that a second Marine Mammal Stranding Workshop was held. In 1991, a national stranding coordinator was appointed to define national stranding policy, standardize network operations, and enhance and support capabilities of network members. In 1992, the stranding networks were recognized within the MMPA with the addition of Title IV, the Marine Mammal Health and Stranding Response Act (Public Law 102–687). Notes and Further Reading If an unusual increase in stranding numbers occurs, a protocol for response described by Wilkinson (1996) occurs (see Chapter 5, Unusual Mortality Events). An interagency National Marine Mammal Tissue Bank and Quality Assurance Program held at the National Institute of Standards and Technology in Gaithersburg, MD was established to collect and archive tissues from marine mammals that can be used for retrospective analysis of contaminant levels. Geraci, J.R., and Lounsbury, V., 1993, Marine Mammals Ashore: A Field Guide for Stranding, Texas A&M University Sea Grant College Program, Galveston, 305 pp. St. Aubin, D.J., Geraci, J.R., and Lounsbury, V.J., 1996, Rescue, rehabilitation and release of marine mammals: An analysis of current views and practices, Proceedings of a workshop held in Des Plaines, Illinois, 3–5 December 1991, NOAA Technical Memorandum, NMFS-OPR-8, 65 pp. Wilkinson, D., and Worthy, G., 1999, Marine mammal stranding networks, in Conservation and Management of Marine Mammals, Twiss, J.R., and Reeves, R.R. (Eds.), Smithsonian Institution Press, Washington, D.C., 396–411. Wilkinson, D.M., 1991, Report to Assistant Administrator for Fisheries: Program review of the marine mammal strandings networks, U.S. Department of Commerce, NOAA, National Marine Fisheries Service, Silver Spring, MD, 171 pp. Wilkinson, D.M., 1996, National Contingency Plan for Response to Unusual Marine Mammal Mortality Events, Technical Memorandum NMFS-OPR-9, U.S. Department of Commerce, NOAA, NMFS, Silver Spring, MD, 118 pp. Acknowledgments The authors thank K. Acevedo, M. Addink, D. Albareda, M. Andre, A. Barreto, J. Barnett, A. Birkun, M. Borri, N. Brothers, J. Conway, E. A. Crespo, E. Degollada, P. Duignan, K. Evans, D. Holcer, A. Frantzis, O. Goffman, T. Jauniaux, T. Jefferson, K. Jeffrey, P. Jepson, R. Kinoshita, C. Kinze, N. LeBoeuf, G. Notabartollo di Sciara, M. Olsson, E. Poncelet, J. A. Raga, B. Reid, L. Rojas, K. Rose, V. Ruoppolo, M. Short, S. Siciliano, U. Siebert, C. Smeenk, K. Soto, K. Van Waerebeek, N. Ward, A.Vella, and T. Yamada, for providing information on stranding networks, and Ailsa Hall for reviewing this chapter. References Daszak, P., Cunningham, A.A., and Hyatt, A.D., 2000, Emerging infectious diseases of wildlife—Threats to biodiversity and human health, Science, 287: 443–449. Geraci, J.R., 1978, The enigma of marine mammal strandings, Oceanus, 21: 38–47. 0839_frame_C04.fm Page 67 Tuesday, May 22, 2001 10:41 AM Marine Mammal Stranding Networks 67 Geraci, J.R., and Lounsbury, V., 1993, Marine Mammals Ashore: A Field Guide for Strandings, Texas A&M University Sea Grant College Program, Galveston, 305 pp. Geraci, J.R., and St. Aubin, D.J., 1979, Biology of marine mammals: Insights through strandings, Final Report MMC-77/13 to the U.S. Marine Mammal Commission, Washington, D.C., available from National Technical Information Service, Springfield, VA, PB-293 890, 343 pp. Geraci, J.R., Harwood, J., and Lounsbury, V.J., 1999, Marine mammal die-offs. Causes, investigations and issues, in Conservation and Management of Marine Mammals, Twiss, J.R., and Reeves, R.R. (Eds.), Smithsonian Institution Press, Washington, D.C., 367–395. St. Aubin, D.J., Geraci, J.R., and Lounsbury, V.J., 1996, Rescue, rehabilitation and release of marine mammals: An analysis of current views and practices, Proceedings of a workshop held in Des Plaines, Illinois, December 3–5, 1991, NOAA Technical Memorandum, NMFS-OPR-8, 65 pp. Seagers, D.J., Lecky, J.H., Slawson, J.J., and Sheridan Stone, H., 1986, Evaluation of the California Marine Mammal Stranding Network as a management tool based on record for 1983 and 1984, Administrative Report SWR-86-5, NMFS Southwest Region, Terminal Island, CA, 34 pp. Wilkinson, D.M., 1991, Report to Assistant Administrator for Fisheries: Program Review of the Marine Mammal Strandings Networks, U.S. Department of Commerce, NOAA, National Marine Fisheries Service, Silver Spring, MD, 171 pp. Wilkinson, D., and Worthy, G., 1999, Marine Mammal Stranding Networks, in Conservation and Management of Marine Mammals, Twiss, J.R., and Reeves, R.R. (Eds.) Smithsonian Institution Press, Washington, D.C., 396–411. 0839_frame_C04.fm Page 68 Tuesday, May 22, 2001 10:41 AM 0839_frame_C05.fm Page 69 Tuesday, May 22, 2001 10:41 AM 5 Marine Mammal Unusual Mortality Events Leslie A. Dierauf and Frances M. D. Gulland Introduction The stranding of large numbers of marine mammals always commands a great deal of public, media, and scientific curiosity. Although these events occur with greater frequency along certain coastlines, they can occur worldwide, posing questions about their causes and potential effects on human health. Many animals stranding at one time is referred to as a mass stranding (see Chapter 6, Mass Strandings). When many animals strand over an extended period of time or in an unusual fashion, this is referred to as a Marine Mammal Unusual Mortality Event (MMUME). Providing humane care for the animals in such strandings, and determining the cause of such events are challenging tasks. Although identifying the immediate cause of such events is difficult, identifying predisposing factors and determining the effects of the event on the population dynamics and genetics of the remaining marine mammal population can be even more demanding (Harwood and Hall, 1990; Harwood, 1998; Baker, 1999). Causes of recent marine mammal die-offs and their investigations have recently been reviewed by Geraci et al. (1999). Although many investigations have been successful, each has its own set of complications and complexities and teaches different lessons (Geraci et al., 1999). As more reports are produced following investigations of MMUMEs, future responses will improve. To facilitate responses, and to maximize the chances for identifying the causes of unusual mortality events and their effects on marine mammal populations, a number of countries have developed contingency plans. In the United States, three specific events triggered the need for interested parties to develop a legal framework and subsequent law that addressed MMUMEs. The first was the Exxon Valdez oil spill in Prince William Sound, Alaska, in 1989 (Loughlin, 1994). The second was a stranding of 14 endangered humpback whales (Megaptera novaeangliae) off Cape Cod, Massachusetts in 1987 (Geraci et al., 1989), and the third was a bottlenose dolphin (Tursiops truncatus) die-off along the Atlantic seaboard between 1987 and 1988 (Geraci, 1989). In the 1st Session of the 102nd Congress, Congressman Walter Jones of North Carolina, who was Chairman of the Committee on Merchant Marine and Fisheries in the U.S. House of Representatives, introduced a bill called the “Marine Mammal Health and Stranding Act.” By late July 1992, the bill had passed out of committee, and a similar bill was moving through the Senate. On November 4, 1992, the Marine 0-8493-0839-9/01/$0.00+$1.50 © 2001 by CRC Press LLC 69 0839_frame_C05.fm Page 70 Tuesday, May 22, 2001 10:41 AM 70 CRC Handbook of Marine Mammal Medicine Mammal Health and Stranding Response Act was signed into law by the President, and became Title IV of the Marine Mammal Protection Act (MMPA) (see Chapter 33, Legislation). In 1988, the dramatic phocine distemper epizootic that killed over 18,000 harbor seals (Phoca vitulina) in Europe raised awareness of the need for contingency plans to investigate marine mammal die-offs, and for long-term monitoring of strandings (Heide-Jorgensen et al., 1992; Thompson and Hall, 1993). In 1989, the Department of the Environment in the United Kingdom established a national program to investigate marine mammal mortalities in the United Kingdom and to coordinate responses. The sudden death of about 100 adult Hooker’s sea lions and over 1600 pups (Phocarctos hookeri) in the remote Auckland Islands off the southern tip of New Zealand in 1998 highlighted the need for preexisting sampling protocols and response plans. Although these have subsequently been developed, the lack of such plans at the time contributed to the difficulty in determining the predisposing factors that triggered the event (Baker, 1999). The Oxford English Dictionary defines the word contingency as a future event or circumstance where there is uncertainty of occurrence. Contingency plans are thus designed to guide responses during unusual events. These plans are imperative during MMUMEs, as such events are often sudden in onset, require early sampling to determine cause, are large scale, expensive to investigate, and command high public and media attention. This chapter reviews MMUMEs and the contingency plans in place to improve responses in the United States; Chapter 6 discusses mass strandings. MMUME Responses in the United States To clarify protocols for response in the United States, strandings and MMUMEs have been clearly defined by law. A stranding (see Chapter 4, Stranding Networks; Chapter 6, Mass Strandings) is: • One or more marine mammals in the wild, and • Dead on the beach or in the waters of the United States, or • Alive and on the beach or shore, and —Either unable to return to the water, or —Although able to return to the water, is in need of medical attention, or —Unable to return to the water under its/their own power or without assistance. Examples of stranding events are the regular and recurrent false killer whale (Pseudorca crassidens) mass strandings in Florida; the gray whale (Eschrichtius robustus) that becomes disoriented and caught up in a freshwater river; or the premature harbor seal (Phoca vitulina) pup that is abandoned by its mother. These are potential marine mammal mortalities, but they are not unusual. A MMUME is a stranding, but that stranding must: • Be unexpected; • Involve a significant die-off of any marine mammal population; and • Demand an immediate response. Events deemed MMUMEs generally are caused by such things as geophysical catastrophic events, chemical spills, pollutant or contaminant discharges, biotoxins, microbial or parasitic 0839_frame_C05.fm Page 71 Tuesday, May 22, 2001 10:41 AM Marine Mammal Unusual Mortality Events 71 infections, and/or any other emergency affecting marine mammals in the wild. Recent examples of MMUMEs include the 1989 Exxon Valdez oil spill and sea otters (Enhydra lutris) in Alaska; the 1996 brevetoxicosis event in manatees (Trichechus manatus) off the west coast of Florida; and the 1998 domoic acid event in California sea lions (Zalophus californianus) along the California coast (Table 1). The U.S. National Contingency Plan The United States has developed a contingency plan to respond to MMUMEs as mandated by Title IV of the Marine Mammal Protection Act. The purposes of Title IV are the following: 1. To bring together individuals with “knowledge and experience in marine science, marine mammal science, marine mammal veterinary and husbandry practices, and marine conservation, including stranding network participants”; 2. To establish a marine mammal health and stranding program and to set up a process within that program to facilitate the collection and dissemination of marine mammal health and health trend data, on marine mammal populations in the wild; 3. To help gather, collate, and correlate data on marine mammal health and marine mammal populations with data on physical, chemical, and biological environmental parameters, such as water sampling data from the Environmental Protection Agency (EPA), microbiological testing from the National Centers for Disease Control (CDC), weather data from the National Oceanic and Atmospheric Administration (NOAA), degree of habitat degradation, human disturbance, or food availability from the U.S. Fish and Wildlife Service (FWS); and 4. To provide coordinated and effective responses to unusual mortality events by establishing a mandated and timely process in which to act (MMPA, Title IV). In addition, the processes within Title IV are designed to provide stranding network participants and marine mammal medical and conservation scientists with easily available broadbased data and reference materials. These reference materials are meant to be sufficient to help them better understand the connections between marine mammal health and the habitats upon which they depend for survival, as well as serve as general overall indicators of the health of our coastal and marine environs. The purpose of the MMUME National Contingency Plan is to outline actions that should be taken to protect public health and welfare; investigate and identify the cause of a mortality event, to minimize or mitigate the effects of a mortality event on the affected population, to provide for the rehabilitation of individual animals, and to determine the impact of a mortality event on the affected population. The FWS also has written an Oil Spill Response Contingency Plan (for wildlife in general), which is distributed through its Contaminants Program (USFWS, 1995). Expert Working Group on MMUMEs Title IV established a decision-making body of scientific experts, called the Working Group on Marine Mammal Unusual Mortality Events (WGMMUME). The WGMMUME operates year round and meets once a year to coordinate efforts and apprise members of ongoing or past events. The group is composed of 12 experts from the fields of marine science, marine mammal science, marine mammal veterinary and husbandry practices, and marine conservation, including stranding network participants. A staff person from the National Marine Fisheries Service (NMFS) serves as executive director of the working group, and every 2 years, the working group chooses a chair from among its 12 members. Additional staff from the NMFS, the Marine Mammal Commission (MMC), and the FWS, and past members of the WGMMUME are welcome to a Common dolphins (Delphinus delphis) 1995 California sea lions (Zalophus californianus) Mediterranean monk seals (Monachus monachus) 100 >150 28 Bottlenose dolphins (Tursiops truncatus) c 6 Right whales (Eubalaena glacialis) b ∼150 Manatees (Trichechus manatus) 10 >200 220 2528 59 No. of Animals FL panhandle, then MS, then AL, then LA Mauritania in Africa (western Sahara, southwest of Spain) North-central CA coast Western North Atlantic SW Coast of FL Gulf of California (Sea of Cortez) Mexico Monterey Harbor, CA Gulf Coast, TX Coast of CA Gulf Coast, TX Location Dx: Saxitoxin from dinoflagellate, Alexandrium, and/ or morbillivirus Dx: Leptospirosis Unk; possibly red tide intoxication Dx: Brevetoxin from the dinoflagellate (Gymnodinium breve) TDx: Ship strike and U.S. Navy underwater explosions Unk TDx: 18/25 dead dolphins exhibited morbillivirus TDx: Cyanide poisoning Dx: Morbillivirus epizootic TDx: El Niño Diagnosis WG+, NOSC, leptospirosis occurs in California sea lions about every 4 years Emaciated pups and juveniles, WG+, NOSC, NCP NOSC, NCP: in average year, fewer than 80 bottlenose dolphins strand here WG+; dead seabirds, too; cyanide found in dolphin liver and lung samples; source never identified WG+, NOSC, necropsies and testing for environmental contaminants negative WG+, OSC, IDST, R, toxic algal bloom; death via inhalation and ingestion report filed; 12% of 2/96 total manatee count WG+, NOSC, December to March, during winter calving season; 3 calves and 3 adults; skull fractures; abrupt deaths; eardrum ruptures WG+, NOSC, generally three or fewer strand in each of these areas; red tides and oyster bed closures WG; prior to event, total population only ~500 animals WG+, OSC, NCP Notes Gulland et al., 1996 Osterhaus et al., 1997; Harwood, 1998; Hernandez et al., 1998 Bossart et al., 1998 Lipscomb et al., 1996 Lipscomb et al., 1996; Colbert et al., 1999 Reference 72 1997 1996 Sea otters (Enhydra lutris) Bottlenose dolphins (Tursiops truncatus) California sea lions (Zalophus californianus) Bottlenose dolphins (Tursiops truncatus) 1992 1994 Species Year TABLE 1 Marine Mammal Unusual Mortality Events since 1992 0839_frame_C05.fm Page 72 Tuesday, May 22, 2001 10:41 AM CRC Handbook of Marine Mammal Medicine West coast of North America (Bering Sea to Baja Mexico) FL panhandle, in and near St. Joseph and St. Andrews Bays Mid-Atlantic Coast (MA to NC) Point Reyes, 20 miles north of San Francisco, CA Central CA coast Dx: Brevetoxin from dinoflagellate (Gymnodinium breve) Dx: Numerous causes, including decreased food availability, fisheries interactions, entanglement Unk Unk; 3 of 85 were confirmed with sarcocystis meningitis Dx: Domoic acid intoxication from diatom (Pseudonitzschia australis) WG+, NOSC, NCP, emaciation suggestive of nutritional disorder; variable chlorinated hydrocarbon levels WG−, large numbers of dead fish, birds, and sea turtles, as well WG+, OSC, NCP, IDST, R, diatom cell counts reached 200,000/l; ingestion of sardines/anchovies; neurological signs, including seizures WG−, NCP, emaciated subadults WG+, NOSC Gulland, 2000; Scholin et al., 2000 Source: Table constructed from Marine Mammal Commission reports, 1992–1999. Key: = contingency plan; CP NCP = no contingency plan; WG+ = WGMMUME decides it is a UME, requiring a response; WG− = WGMMUME decides it is not a UME, is within the normal range IDST = interdisciplinary scientific team participated in UME diagnostics; of variation for this particular species; R = scientific report written and filed/published in the scientific literature; WG+ = not a U.S. event; Dx = diagnosis made; OSC = on-site coordinator designated; TDx = tentative diagnosis only; NOSC = no on-site coordinator designated; Unk = cause unknown. a For mass die-offs prior to 1992, see Twiss and Reeves (1999, p. 376). b The northern right whale is the most endangered marine mammal in U.S. waters, and the most endangered large whale in the world, with only about 300 animals left in the population. c The Mediterranean monk seal is highly endangered. 87 Bottlenose dolphins (Tursiops truncatus) 216 (11 of them were alive; 55 carcasses were fresh) 273 Harbor porpoises (Phocoena phocoena) 1999 70 85 Gray whales (Eschrichtius robustus) California sea lions (Zalophus californianus) 1998 Pacific harbor seals (Phoca vitulina) 0839_frame_C05.fm Page 73 Tuesday, May 22, 2001 10:41 AM Marine Mammal Unusual Mortality Events 73 0839_frame_C05.fm Page 74 Tuesday, May 22, 2001 10:41 AM 74 CRC Handbook of Marine Mammal Medicine attend the annual meetings. Member terms are 3 years, with no person being allowed to serve more than two terms. Every 3 years, a third of the members rotates off, and new members are selected. The charges of the WGMMUME as mandated in Title IV are the following: • To determine whether or not a MMUME is occurring, • To determine after a MMUME has begun, when response to that MMUME is no longer necessary, and • To help develop a contingency plan for responding to MMUMEs. The MMUME Response Details of the response to MMUMEs are given in the National Contingency Plan for Response to Marine Mammal Unusual Mortality Events (Wilkinson, 1996). To respond to a MMUME efficiently and effectively, there are several crucial elements that must be in place and operating: 1. A functional stranding network, with primary responders observing stranded marine mammals and reporting them to their regional stranding coordinator. The responders must provide precise information on the geographic location and approximate number and species of marine mammals involved. Each animal reported should have Level A data collected (Chapter 4, Stranding Networks; Chapter 21, Necropsy). 2. A regional coordinator, a national coordinator (from either the NMFS or the FWS, depending on the primary species involved in the UME), and a working group on MMUMEs, all of which work together according to the established plan. 3. A blueprint, plan, and protocols for animal rescue, rehabilitation and release, euthanasia, sample collection, referral laboratories to analyze collected samples, and long-term habitat and species protection. 4. Commitment and funding from the federal government to initiate a rapid response and to conduct complete investigations. The response to a MMUME is shown in Figure 1. Each step of this process is essential for an effective response to proceed. Rapid and accurate information from each member of the stranding network to the regional stranding coordinator is the trigger for the process to begin. There are then two critical time constraints built into the MMUME response. First, the MMUME national coordinator is required to contact as many members of the working group as possible within 24 hours of a regional stranding coordinator contacting the NMFS. Second, members of the working group must call the MMUME national coordinator back immediately. Title IV does allow some flexibility if, at the request of any working group member, the MMUME coordinator needs to gather additional information on numbers, species, sexes, ages, and/or specific conditions associated with the MMUME to aid in decision making. Theoretically, the law states that each person in the working group within a maximum of 24 hours of obtaining the data needed must decide independently whether or not a MMUME is occurring and must register that decision with the MMUME coordinator. Once a majority of the working group has registered a yes or no vote, the MMUME coordinator announces whether (majority voted yes) or not (majority voted no) a MMUME is taking place. There are seven questions each expert working group member must ask: 1. Compared to historical records, is there a marked increase in the number of strandings of this species? 2. Are these marine mammals stranding at a time of year when historically strandings are unusual? 3. Are the increased strandings occurring in a localized area or over a wide geographic range, or is the event spreading geographically over time? 4. Is the species, age, or sex composition in the stranded animals different from what occurs normally in that geographic area or at that time of year? 0839_frame_C05.fm Page 75 Tuesday, May 22, 2001 10:41 AM 75 Marine Mammal Unusual Mortality Events 5. Are stranded animals exhibiting similar and/or unusual pathological changes or changes in general body condition from what is seen normally? 6. Are there animals alive in the area(s) where mortalities are occurring, and, if so, are they exhibiting any aberrant behaviors? 7. Does the stranding involve a critically endangered species? Then, by law, unless time is needed to gather additional information as requested by any member of the working group, determination of whether or not an MMUME is occurring must timeline Has the Regional Stranding Coordinator called the NMFS National MMUME Coordinator? 0 hours YES NO Process Stops Has the NMFS National MMUME Coordinator called all the Members of the Working Group? 24 hours YES NO Contact NMFS Again Has the NMFS National MMUME Coordinator received calls back from Working Group Members to be able to make a decision whether a MMUME is occurring? YES NO Contact Working Group Again Is a MMUME occurring? YES 48 hours NO Process Stops Regional Stranding Coordinator, Continues to Watch, and Keeps Regular Contact with NMFS MMUME Coordinator MMUME National Coordinator informs Regional Stranding Coordinator a MMUME is occurring MMUME National Coordinator through Secretary of Commerce designates On-Site Coordinator MMUME National Coordinator transfers responsibility for action to the On-Site Coordinator On-Site Coordinator makes immediate recommendations to the Regional Stranding Coordinator on how best to proceed with response activities On-Site Coordinator takes over response, following the Contingency Plan to the best of his/her abilities, utilizing professional judgment, and assembles response team and plan On-Site Coordinator or his/her designee remains on site at MMUME coordinating the response FIGURE 1 Flowchart and timing of response to MMUME in the United States. 0839_frame_C05.fm Page 76 Tuesday, May 22, 2001 10:41 AM 76 CRC Handbook of Marine Mammal Medicine On-Site Coordinator Live/Dead Animal Rescue Response Legal Counsel and National MMUME Coordinator Live/Dead Animal Research Response Activities Command Operations and Administrative Response FIGURE 2 Coordinated team response interactions during a MMUME. (Adapted from the U.S. National Contingency Plan.) take place within 48 hours of a regional stranding coordinator contacting the NMFS about a possible event. If the working group believes a MMUME is indeed occurring, an appropriately qualified onsite coordinator (OSC) is immediately designated to mobilize and manage the national response to the event. Depending on the species involved and the location of the MMUME, the OSC will be either a NMFS or a FWS regional director or an individual designated by that regional director. Because the OSC is responsible for directing the response, the individual must have strong management and leadership capabilities, highly effective communication skills, the capacity to make decisions with minimal use of intermediaries, the ability to access information and expertise including interagency expertise, and a familiarity with the contingency plan and the stranding network. The OSC is also responsible for preparing a report containing results of scientific investigations and recommendations for subsequent monitoring and/or management activities. The coordination of team efforts once an on-site coordinator has been designated for a MMUME is shown in Figure 2. Through the National Contingency Plan, adequate funding, personnel for the team, and logistical support, such as ships, aircraft, and other heavy equipment, are made available to carry out an efficient and effective response, whether the marine mammal involved in the MMUME is under NMFS or FWS jurisdiction (see Chapter 33, Legislation). MMUME Fund Title IV established an interest-bearing account in the Federal Treasury called the “Marine Mammal Unusual Mortality Event Fund” to be used exclusively for costs associated with preparing for and responding to MMUMEs, which remains available until expended. Monies provided to the fund come from multiple sources, including Congressional appropriations, special funds appropriated to the Secretary of Commerce, and monies received by the U.S. government in the form of public or private gifts, devises, and/or bequests. The acceptance and solicitation of donations into a fund such as this is highly unusual in the federal government, but allowable and anticipated under Title IV of the MMPA. 0839_frame_C05.fm Page 77 Tuesday, May 22, 2001 10:41 AM Marine Mammal Unusual Mortality Events 77 Anyone wishing to donate funds to the MMUME Fund is asked to contact the NMFS or the chair of the working group. Donations can be sent directly to NMFS, 1335 East-West Highway, Silver Spring, MD 20910, with a notation attached that the money is to be used “exclusively for marine mammal MMUME through the MMUME Fund.” If every person reading this chapter sent just $5 each, the fund would grow incrementally and be able to support the important tasks and responses needed to continue to make the MMUME program successful. Although the fund is coordinated by the NMFS, it is available for response to any MMUME, including those under NMFS and FWS jurisdiction. Lessons Learned The Cooperative Response Stranding network participants are highly vigilant in alerting federal officials whenever there is even an inkling of a MMUME. Scientists and stranding network participants give maximum effort in reacting to MMUMEs and in providing tissues and samples for furthering knowledge of MMUMEs in general and of individual MMUMEs in particular. Facilities must, as new volunteers arrive to assist, make all stranding network volunteers aware of national plans and needs. Participants must understand their reporting obligations and the importance of Level A data (see Chapter 4, Stranding Networks; Chapter 21, Necropsy). All original members of the working group have now been replaced through attrition, and the working group under the directorship of its chair continues to be highly productive, developing standardized protocols, assisting with developing new contingency plans and revising existing plans, and devising strategies to increase funding for MMUME responses. Plans are being developed for MMUMEs that recur, such as leptospirosis, El Niño events, and domoic acid toxicity in California sea lions off the West Coast of the United States. Interdisciplinary scientific and logistical teamwork is important to obtain diagnoses. In the last few years, each MMUME in the United States and elsewhere has garnered a response from a multitude of players in the scientific community, a kind of collaborative response rarely seen in the past. Federal, state, regional, stranding network, and private agencies and individuals participate, as do many academic institutions. The scientific and gray literature associated with MMUMEs now is written by multiple scientific contributors. Interagency cooperation has improved. The NMFS, the U.S. Geological Survey, the EPA, and the FWS met in October 1998 and decided to create an interagency working group to address the uncertainties and unknowns regarding contaminant levels that are being detected in marine mammals. Although the NMFS, the FWS, and the EPA do not yet work seamlessly together, there has been noticeable improvement since Title IV of the MMPA came into existence. Around the world, national contingency plans to respond to unusual mortality events in marine mammals are under development or under discussion. The UN Environment Program (UNEP) has an action plan for marine mammals worldwide. Although lack of funding at any particular time can hinder the magnitude of a response anywhere at any time, it is the unending support of the volunteers in stranding networks worldwide that makes the response possible and successful. The WGMMUME has assisted the NMFS and the FWS in developing and releasing a series of contingencies plans, including the National Contingency Plan for Response to Marine Mammal Unusual Mortality Events (Wilkinson, 1996), and the Contingency Plan for Catastrophic Rescue and Mortality Events for the Florida Manatee and Marine Mammals (Geraci and Lounsbury, 1997). In addition, the NMFS is working on a new contingency plan for the Hawaiian monk seal. 0839_frame_C05.fm Page 78 Tuesday, May 22, 2001 10:41 AM 78 CRC Handbook of Marine Mammal Medicine The Process In the United States, not all stranding network members or participants are aware of the MMUME law or process, or of the existence of a national contingency plan. Communication between the federal agencies and the working group must be as rapid as possible, as does the response of the working group. Members of the working group must make their individual decisions about whether or not a MMUME is occurring within 48 hours, so the response time is effective. Single, local response teams at the stranding network level cannot be left to respond on their own to huge, time-consuming MMUMEs without the aid of personnel or funding from the federal government. UMMME Fund The NMFS and the FWS always are concerned about funding constraints in trying to implement their programs relating to marine mammals. Funding is important because it supports the following efforts: • Communication, helping staff, who at times can feel overburdened with excessive workloads; • Baseline data collection and collation, including information on stranding rates, disease, and environmental contaminants for use in securing diagnoses of MMUME causes; • MMUME sample/tissue data collection, archiving, and analysis; and • Rapidity of the response to MMUME. A 1994 Congressional amendment to the MMPA allows monies from the MMUME Fund to be used for care and maintenance of marine mammals seized by NMFS or FWS agents when the level of care the animals are receiving is inadequate. This seizure is important to marine mammal well-being, but is not a MMUME, and original Congressional intent was never to use the fund for such purposes. The intent was always to use the fund for wild marine mammals and not for animals held in captivity at aquaria, zoos, or other U.S. facilities (U.S. House of Representatives, 1992). Thus, it is extremely important when making donations to the MMUME Fund that the NMFS be instructed that the money is to be used “exclusively for marine mammal UME.” Results Accrued from Title IV of the MMPA There has been definite improvement in the collection quantity and quality of marine mammal disease data. More final diagnoses have been made since passage of Title IV, although the predisposing factors often remain unclear. It is the authors’ hope that in the future there will be more integration of baseline health, population parameters, and ecosystem changes with investigations of MMUMEs. This will help determine whether or not there are real long-term alterations occurring in ocean health, as suggested by Harvell and co-workers (1999), rather than simply improvements in detection and reporting. Relative to a response to unusual stranding events prior to 1992, there is now a coordinated effort, with much interaction among federal, state, regional, and local participants. Funding for MMUME responses and tissue analyses, as well as database establishment and maintenance, is critical. The more people who know about MMUMEs and Title IV, and the more people who have a passion for marine mammal and ecosystem health, the more people there will be to lobby Congress and their individual Senators and Representatives to ensure that annual appropriations are provided for the program. Private donations and gifts are welcome also. 0839_frame_C05.fm Page 79 Tuesday, May 22, 2001 10:41 AM Marine Mammal Unusual Mortality Events 79 How Can You Help? Volunteer with local stranding networks on a regular basis. Understand the plans and legislation in place to facilitate responses to dead marine mammals (see Chapter 4, Stranding Networks; Chapter 6, Mass Strandings; Chapter 33, Legislation). Donate supplies and funds to support local efforts. Help tackle logistical problems facing stranding network participants during investigations. Assist with administrative and communication tasks, as well as with the more attractive jobs working directly with the animals. Send gifts and donations to the national fund for MMUMEs. Tell everyone you know about MMUMEs and how we can learn more from responding quickly to them and working together to determine and explain the causes of MMUMEs. In your research endeavors, keep marine mammal health and well-being in the forefront, developing rapid, sensitive, and specific tests for diagnosing disease and finding new and effective ways to treat marine mammals found alive during MMUMEs. Always consider factors beyond conventional clinical medicine when dealing with wild animals—environmental changes, population dynamics, and genetics. Conclusion Unusual mortality events and other marine mammal strandings are effective learning tools for diagnosing factors affecting the health of marine mammal populations. If a marine mammal is still alive or freshly dead, tissues can be collected, using a standardized set of methodologies for quality-controlled analysis. The results may lead to an explanation of what caused the individual or group of marine mammals to strand. Even more importantly, placing these data in a national, accessible database will allow information from one event to be compared with that from another. All of this information can be compared with reference materials taken from nonstranding marine mammals in the wild. Such carefully planned procedures will provide the most insightful evidence for determining why marine mammals strand, how MMUMEs occur, and when these events are harmful to marine mammal populations and the ecosystems upon which they depend. Marine ecosystems worldwide are being negatively impacted by multiple factors, and they need immediate attention. Only by concentrating everyone’s attention on marine mammals and the habitats in which they live, will we be able to continue to be fascinated and mesmerized by healthy marine mammals in the wild for generations to come. Acknowledgments The authors thank Mona Haebler and Tom O’Shea for their reviews of this chapter. Both have served on the WGMMUME, as have the authors. References Baker, A., 1999, Unusual mortality of the New Zealand sea lion Phocarctos hookeri, Auckland Islands, January–February 1998, Report of a workshop held 8–9 June 1998, Wellington, NZ, and a contingency plan for future events, New Zealand Department of Conservation, 84 pp. Bossart, G.D., Baden, D.G., Ewing, R.Y., Roberts, B., and Wright, S., 1998, Brevetoxicosis in manatees (Trichechus manatus latirostris) from the 1996 epizootic: Gross, histologic and immunohistologic features, Toxicol. Pathol., 26: 276–282. Colbert, A.A., Scott, G.I., Fulton, M.H., Wirth, E.F., Daugomah, J.W., Key, P.B., Strozier, E.D., and Galloway, S.B., 1999, Investigation of unusual mortalities of bottlenose dolphins along the midTexas coastal bay ecosystem during 1992, NOAA Technical Report NMFS 147, U.S. Department of Commerce, Seattle, Washington, 23 pp. 0839_frame_C05.fm Page 80 Tuesday, May 22, 2001 10:41 AM 80 CRC Handbook of Marine Mammal Medicine Costas, E., and Lopez-Rodas, V., 1998, Paralytic phycotoxins in monk seal mass mortality, Vet. Rec., 142: 643–644. Geraci, J. R., 1989, Clinical investigation of the 1987–1988 mass mortality of bottlenose dolphins along the U.S. central and south Atlantic coast, Final Report, U.S. Marine Mammal Commission, Washington, D.C., 63 pp. Geraci, J.R., and Lounsbury, V.J., 1997, Contingency plan for catastrophic manatee rescue and mortality events, Florida Department of Environmental Protection, Florida Marine Research Institute, Contract Report MR 199, 136 pp. Geraci, J.R., Anderson, D.M., Timperi, R.J., St. Aubin, D.J., Early, G.A., Prescott, J.H., and Mayo, C.A., 1989, Humpback whales (Megaptera novaeangliae) fatally poisoned by dinoflagellate toxin, Can. J. Fish. Aquat. Sci., 46: 1895–1898. Geraci, J.R., Harwood, J., and Lounsbury, V.J., 1999, Marine mammal die-offs. Causes, investigations and issues, in Conservation and Management of Marine Mammals, Twiss, J.R., and Reeves, R.R. (Eds.), Smithsonian Institution Press, Washington, D.C., 367–396. Gulland, F., 2000, Domoic acid toxicity in California sea lions (Zalophus californianus) stranded along the central California coast, May–October 1998, NOAA Technical Memorandum, NMFS-OPR, 17, 45 pp. Gulland, F.M.D., Koski, M., Lowenstine, L.J., Colagrass, A., Morgan, L., and Spraker, T., 1996, Leptospirosis in California sea lions (Zalophus californianus) stranded along the central California coast, 1981–1994, J. Wildl. Dis., 32: 572–580. Harvell, C.D., Kim, K., Burkholder, J., Colwell, R.R., Epstein, P.R., Grimes, J., Hofmann, E.E., Lipp, E.K., Osterhaus, A.D.M.E., Overstreet, R., Porter, J.W., Smith, G.W., and Vasta, G.R., 1999, Emerging marine diseases—climate links and anthropogenic factors, Science, 285: 1505–1510. Harwood, J., 1998, What killed the monk seals? Nature, 393: 17–18. Harwood, J., and Hall, A., 1990, Mass mortality in marine mammals: Its implications for population dynamics and genetics, Trends Ecol. Evol., 5: 254–257. Heide-Jorgensen, M.P., Harkonen, T., Dietz, R., and Thompson, P.M., 1992, Retrospective of the 1988 European seal epizootic, Dis. Aquat. Organisms, 13: 37–62. Hernandez, M., Robinson, I., Aguilar, A., Gonzalez, L.M., Lopez-Jurado, L.F., Reyero, M.I., Cacho, E., Franco, J., Lopez-Rodas, V., and Costas, E., 1998, Did algal toxins cause monk seal mortality? Nature, 393: 28–29. Lipscomb, T.P., Kennedy, S., Moffett, D., Krafft, A., Klaunberg, B.A., Lichy, J.H., Regan, G.T., Worthy, G.A.J., and Taubenberger, J.K., 1996, Morbilliviral epizootic in bottlenose dolphins of the Gulf of Mexico, J. Vet. Diagn. Invest., 8, 283–290. Lipscomb, T.P., Schulman, Y.D. Moffett, D., and Kennedy, S., 1994, Morbilliviral disease in Atlantic bottlenose dolphins (Tursiops truncatus) from 1987–1988 epizootic, J. Wildl. Dis., 30: 567–571. Loughlin, T.R. (Ed.), 1994, Marine Mammals and the Exxon Valdez, Academic Press, San Diego, CA, 395 pp. MMC, Marine Mammal Commission, 1992–1999, Annual Reports to Congress, Bethesda, MD, available January of each following year. MMPA, Title IV, Marine Mammal Protection Act of 1972, as amended, 1995, 16 USC 1421 ff. Osterhaus, A., Groen, J., Neisters, H., Van de Bildt, M., Vedder, B.M.L., Vos, J., van Egmond, H., Sidi, B.A., and Barham, M.E.O., 1997, Morbillivirus in monk seal mass mortality, Nature, 388: 838–839. Scholin, C.A., Gulland, F., Doucette, G.J., Benson, S., Busman, M., Chavez, F.P., Cordaro, J., DeLong, R., De Vogelaere, A., Harvey, J., Haulena, M., Lefebvre, K., Lipscomb, T., Loscutoff, S., Lowenstine, L.J., Marin III, R., Miller, P.E., McLellan, W.A., Moeller, P.D.R., Powell, C.L., Rowles, T., Silvagni, P., Silver, M., Spraker, T., Trainer, V., and Van Dolah, F.M., 2000, Mortality of sea lions along the central California coast linked to a toxic diatom bloom, Nature, 403: 80–84. Thompson, P.M., and Hall, A.J., 1993, Seals and epizootics—what factors might affect the severity of mass mortalities? Mammal Rev., 23: 149–154. USFWS, U.S. Fish and Wildlife Service, 1995, Oil Spill Contingency Plan, 1995. 0839_frame_C05.fm Page 81 Tuesday, May 22, 2001 10:41 AM Marine Mammal Unusual Mortality Events 81 U.S. House of Representatives, Marine Mammal Health and Stranding Response Act, Committee Report, 1992, Report 102-758, July 30, 14 pp. Wilkinson, D.M., 1996, National Contingency Plan for Response to Unusual Marine Mammal Mortality Events, NOAA Technical Memorandum NMFS-OPR-9, 9/96, Silver Spring, MD. 0839_frame_C05.fm Page 82 Tuesday, May 22, 2001 10:41 AM 0839-frame_C06 Page 83 Tuesday, May 22, 2001 10:42 AM 6 Mass Strandings of Cetaceans Michael T. Walsh, Ruth Y. Ewing, Daniel K. Odell, and Gregory D. Bossart Introduction A mass stranding of cetaceans is an event in which two or more individuals of the same species, excluding a single cow–calf pair, beach within a given spatial and temporal reference (Wilkinson, 1991). A mass stranding event may span 1 or more days and range over miles of shoreline, bridging multiple counties, or sandbars and outlying keys. A variety of species have been affected; Odell (1987) listed 19 odontocete species known to mass-strand. Aristotle recorded sightings of stranded cetaceans 2300 years ago. Cetaceans continue to mass-strand, yet the causes of the majority of these events remain unclear. Mass strandings have received more attention as coastal human populations increase, making discovery of stranded animals more likely. Documentation of stranding events has improved over the last 70 years, the earliest organized attempts originating in England. These records have allowed reviews of such occurrences (Fraser, 1934; 1946; 1953; 1956; Geraci, 1978; Sergeant, 1982). Despite the attention mass strandings receive from the public and scientific community alike, they remain hard to manage, and the reasons for their occurrence remain hard to identify. Geraci et al. (1999) produced an excellent review of marine mammal die-offs, summarizing various etiologies of mass-stranding events. Table 1 lists a compilation of mass strandings, mostly from the Smithsonian marine mammal database and the Southeast United States (SEUS) marine mammal stranding network database, that have occurred along the East Coast of the United States within the past 12 years (1987 through 1999). Causes of most of these events are either unknown or ambiguous, theories being supported only by circumstantial evidence. Theories to Explain Mass Strandings As long as people have been aware of mass strandings, theories have been formulated to explain why marine mammals mass-strand on beaches (Dudok Van Heel, 1962; Geraci et al., 1976; Eaton, 1979; 1987; Geraci and St. Aubin, 1979; Odell et al., 1980; Best, 1982; Cordes, 1982; Wareke, 1983). Anecdotal theories for why whales strand include that these species whose ancestors were land mammals have an evolutionary memory compelling them back to land, that the animals are distressed and/or in pain and are committing suicide, and that they are avoiding drowning. Other more accredited theories include that sloping beaches give poor sonar reflection 0-8493-0839-9/01/$0.00+$1.50 © 2001 by CRC Press LLC 83 0839-frame_C06 Page 84 Tuesday, May 22, 2001 10:42 AM 84 CRC Handbook of Marine Mammal Medicine TABLE 1 Mass Strandings along the East Coast of the United States from 1987 through 1999 Species Year Month Day Number of Animals State Ref. P. crassidens D. delphis L. acutus K. breviceps L. acutus L. acutus S. bredanensis G. macrorhynchus T. truncatus D. delphis L. acutus L. acutus F. attenuata S. coeruleoalba P. crassidens K. breviceps L. acutus P. macrocephalus L. acutus G. melas G. griseus S. coeruleoalba G. macrorhynchus G. macrorhynchus S. bredanensis G. macrorhynchus G. melas G. melas G. melas G. melas G. melas G. macrorhynchus G. macrorhynchus G. macrorhynchus F. attenuata Z. cavirostris (?) F. attenuata L. acutus K. breviceps F. attenuata S. clymene G. melas T. truncatus D. delphis S. frontalis L. acutus S. attenuata G. macrorhynchus G. griseus K. breviceps D. delphis 1987 1987 1987 1987 1987 1987 1987 1987 1987 1988 1988 1988 1988 1989 1989 1989 1989 1990 1990 1990 1991 1991 1991 1991 1991 1991 1991 1991 1991 1991 1991 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1992 1993 1993 1993 1993 1993 1993 1993 1993 1 2 3 8 9 9 10 11 12 2 4 4 5 1 7 8 8 4 8 12 1 3 3 4 4 7 9 9 9 10 12 1 2 2 3 6 7 8 8 9 12 12 12 1 3 4 9 11 11 11 12 2 4 7 23 5 5 18 14 1 4 29 30 7 26 11 9 30 19 9 11 20 9 b 24 –30 b 11–20 24 b 21–22 9 10 29 8 24 30 10 15 30 25 3 27 31 4 6–10 12 13 1 15 6 6 3 20 21 20 6 5 3 3 20 10 3 29 3 5 3 3 4 3 3 3 4 5 9 53 3 b 4 /5 27 12 10 11 32 27 17 16 31 13 3 8 2 3 2 6 3 3 23 19 6 6 2 8 5 6 5 2 4 LA MA MA FL ME MA FL FL SC MA MA MA GA MA FL NC ME FL ME MA NC FL FL FL FL FL MA MA MA MA MA FL FL FL FL FL FL MA FL FL FL MA MA MA MS MA FL FL MA FL MA a a a a; b a a a; b a; b a a a a a; b a a; b a a a; b a a; c a; b a; b a; b a; b a; b a; b b; c c a; c a; c a; c a; b a; b a; b b a; b b a; c b b b a; c a a b c a; b b a a c 0839-frame_C06 Page 85 Tuesday, May 22, 2001 10:42 AM 85 Mass Strandings of Cetaceans TABLE 1 Mass Strandings along the East Coast of the United States from 1987 through 1999 (continued) Species G. macrorhynchus G. macrorhynchus D. delphis L. acutus L. hosei L. acutus K. breviceps L. acutus L. acutus G. macrorhynchus S. clymene G. macrorhynchus G. macrorhynchus G. macrorhynchus G. macrorhynchus F. attenuata K. breviceps S. attenuata G. macrorhynchus L. acutus L. acutus S. bredanensis D. delphis G. macrorhynchus G. macrorhynchus L. acutus D. delphis S. bredanensis M. europaeus G. melas S. bredanensis L. acutus G. macrorhynchus S. attenuata L. acutus S. bredanensis Z. cavirostris Year 1994 1994 1994 1994 1994 1994 1994 1994 1995 1995 1995 1995 1995 1995 1995 1995 1995 1996 1996 1997 1997 1997 1997 1998 1998 1998 1998 1998 1998 1998 1998 1999 1999 1999 1999 1999 1999 Month 2 b 2–3 3 3 7 10 11 12 1 3 6 7 8 8 9 9 12 1 5 5 8 12 11 1 1 1 1 2 8 11 12 3 5 8 8 8 10 Number of Animals Day b 17–24 b 26–24 5 14 13 9 5 30 4 24 15 1 15 21 15 16 11 16 31 28 12 14 16 3 12 c 29 /31 31 4 28–31 6 28 19 5 2 11 21 3 46 b 4/3 3 6 b 30/28 7 4 23 12 2 18 32 4 9 7 5 b 6/3 11 2 2 2 34 10 7 8 c 97/82 16 2 9 2 12 50 2 3 6 5 4 State Ref. FL NC MA MA FL MA NJ MA MA NC FL FL FL FL FL VI FL FL FL MA MA FL MA FL FL MA MA FL NC FL FL MA FL FL MA GA VI a; b a; b a; c a; c a; b a a a; c c b a; b a; b b a; b a; b a a; b a; b b a a a; b a; c a; b a; b a; c c b b b a; b c b b c b b Note: (?) indicates species uncertain in database record. Shaded individual species records have been considered to be from the same mass stranding event; however, they have been recorded as separate events within the referenced databases. a Refers to data within the Cetacean Distributional Database, Smithsonian Institute. b Refers to data in the SEUS marine mammal stranding network database. c Refers to data referenced in Wiley et al., in review. which misleads the animals ashore; that geomagnetic disturbances affect their ability to navigate geomagnetically; that acoustic navigation is lost as a result of parasitic destruction of the eighth cranial nerve; that coastlines are unfamiliar to the animals; that the animals strand as a result of geologic disturbances, such as earthquakes or underwater volcanoes; and that mass strandings involve pelagic species, which may have difficulty navigating in shallow waters. 0839-frame_C06 Page 86 Tuesday, May 22, 2001 10:42 AM 86 CRC Handbook of Marine Mammal Medicine It is likely that many species involved in mass strandings use geomagnetic cues to migrate (Kirschvink et al., 1985; Klinowska, 1985a,b). Klinowska (1985b) proposed geomagnetic disturbances as an explanation for live strandings in the United Kingdom. This theory is based on the study of coastline geomagnetic maps and the finding that correlations exist between stranding sites and relative intensity of the local geomagnetic fields. It is likely that this theory is a factor in explaining where animals strand, rather than why they strand, and it is certainly possible that a group of ill individuals will overlook other sensory modalities and ultimately follow geomagnetic or shoreline clues into a specific location. This may be a partial explanation for why certain beaches, such as on Cape Cod, Massachusetts, seem to experience repeated mass-stranding events. The loss of acoustic navigation ability (“sonar”) as a result of parasitic involvement may explain some mass strandings (Ridgway and Dailey, 1972). Parasites are common in wild species (see Chapter 18, Parasitic Diseases), and their presence in locations such as the middle or inner ear could lead to disorientation. Morimitsu et al. (1986) demonstrated eighth cranial nerve destruction induced by Nasitrema spp. at the junction with the inner ear in three cetacean species. However, there is some question about the validity of these conclusions, as it was stated in a subsequent publication that these specimens were not fresh, and freeze artifact may have affected the histological appearances of the tissues (Morimitsu et al., 1987). The lack of early evidence for specific viral or bacterial etiologies in some stranding events in the mid-1980s reawakened the discussion of the role of pod cohesion as a major factor in mass strandings. In 1986, during a mass stranding of false killer whales (Pseudorca crassidens) in the Florida Keys, the influence of social structure was plainly illustrated (Walsh et al., unpubl. data). After repeatedly stranding and being pushed back to sea by the public, a group of false killer whales eventually stranded in the Florida Keys (Odell et al., 1980). The group of 30 animals was spread over more than 12 miles along shallow waters and numerous islands. The effort to coordinate and relocate the surviving 16 animals to a central location resulted in the youngest and smallest animals being moved first to a small isolated bay. At first these five young animals were actively swimming and investigating the shallow bay. They appeared confused, but they were active. When one of the larger adult male animals was transported into the bay, he immediately beached himself on one edge of the shore. Each of the younger animals then lined up neatly beside him and did not move from his side. Whether the response was based on visual or auditory cues was unknown, but as each animal was added to the group, this response was repeated until all survivors were in one line. Current Investigations into Mass Strandings Investigations of mass-stranding events have evolved and continue to evolve as more standardized approaches are applied. For example, a mass stranding of Atlantic white-sided dolphins (Lagenorhynchus acutus) yielded valuable information on pathological conditions that were present, including parasite identification and numbers, along with other baseline life history data (Geraci and St. Aubin, 1977). In a subsequent mass-stranding investigation in 1986 involving shortfinned pilot whales (Globicephala macrorhynchus), clinical pathology was emphasized. Blood samples for complete blood counts (CBC) and serum chemistries were taken from all live animals to elucidate observed clinical symptoms of disease (Walsh et al., 1991). The diagnostic workups also included cultures of the respiratory, reproductive, and gastrointestinal systems. Serum was initially used for serological analyses for certain known domestic animal and marine mammal pathogens; however, serum subsamples were also archived for future retrospective analyses. At necropsy, samples were collected for histopathological and toxicological analyses, urinalysis, and various additional tissue cultures (Bossart et al., 1991). This investigation, while comprehensive, was limited by three factors: interest/disciplinary focus, response crew abilities, and finances. 0839-frame_C06 Page 87 Tuesday, May 22, 2001 10:42 AM 87 Mass Strandings of Cetaceans Although there was evidence of illness in individuals from these mass strandings, no specific etiology for the stranding event was identified. New issues have been raised as each incident is more thoroughly investigated. What infectious agents, such as viruses or bacteria, may be involved? What role do anthropogenic or naturally occurring biotoxins or contaminants play in mass strandings? What factors are primary; which are secondary? Could the original problem, which may have occurred weeks or months before, out at sea, be missed? Evaluation of a Mass Stranding One approach to evaluating a stranding event is given in Table 2. This approach includes assessments of environmental conditions and trends, the group of animals as a whole, and the individuals of that group. The environmental evaluation should list all potential factors, including: 1. Previous strandings at this site (historical perspective); 2. Geomagnetic maps (if available); 3. Topographic and bathymetric characteristics and anomalies (beach type, slope, presence of barrier islands, sandbars, landslides, volcanic eruptions, earthquakes); 4. Tide factors, sea surface temperature, salinity, fronts, currents, and other oceanographic factors; 5. Storms within the last few weeks; 6. Available local fishing data on local fishery changes; 7. Algal blooms; 8. Toxic material spills; 9. Acoustic events; and 10. Other species mortalities. Evaluation of animal groups should include: 1. Recognition that in some species of cetaceans there are strong social ties between group members, which may result in individuals blindly congregating around ill leaders or other ill individuals; thus, the species involved, and the leader (if possible) should be identified; 2. Group demographics (sex and age distribution); 3. The ratio of live to dead animals; 4. Cow–calf pairs; and 5. Evaluation of individuals involved. TABLE 2 Factors to Evaluate during a Mass Stranding Environmental Local Adverse weather: Storms Beach Topography Previous stranding history Current and tides Acoustic events: Land slides Volcanic eruption Underwater experiments Anthropogenic noise Cetacean Regional Group Individual Weather pattern shift: El Niño La Niña Foodborne toxins Food availability Harmful algal blooms Oil spill Pesticide runoff Social bonds: Leader illness Cow–calf pairs Breeding season: Pregnant females Infectious disease: Acute process Chronic disease Appearance Attitude Heart rate and character Respiratory rate and character Hematology and serum biochemistry 0839-frame_C06 Page 88 Tuesday, May 22, 2001 10:42 AM 88 CRC Handbook of Marine Mammal Medicine Management of a Mass Stranding Strandings are generally very complicated events. Proper management requires experienced, organized rescue efforts, including individuals trained in stabilizing live animals, rapidly diagnosing illnesses, and arranging for possible extended rehabilitation of ill animals. In some cases, controlling interference from untrained individuals is also a priority. To work in concert with local law authorities, such as the Marine Patrol or local police, members of the mass-stranding rescue team should make contact with the law enforcement officer in charge. A temporary plan (which may include aerial survey and observations) should be implemented to determine the number of animals involved, where they are located, the accessibility of the stranding location and to evaluate other pertinent circumstances (Figure 1). If the animals are spread over a large area, it may be advisable to consolidate the individual animals (weather permitting) into one location. If there is adequate help available, individuals are assigned to each animal to provide temporary first aid, including keeping the animal sternal to avoid inhalation of debris. Animals exposed to sunlight must be kept moist, cool, and shaded. Zinc oxide can be applied to briefly towel-dried skin, to help deflect sunlight and decrease sunburn. Pouring water over the animal’s body will also help keep the skin from drying and the animal from overheating. If towels are placed over the animal, they must be kept wet and not placed where they may occlude respiration. All individual animals should be identified with tape or tags (such as small spaghetti tags or roto tags) (see Chapter 38, Tagging and Tracking) placed in the dorsal fin to facilitate correlation between clinical and pathological data collection, as well as later identification should the animals be released and re-strand. Algorithms to aid in evaluations of individuals within the group are summarized in Figures 2 and 3. These flowchart approaches to individual evaluations involve on-site monitoring of Verification Site Evaluation Accessible Evaluate Group Inaccessible Return to Sea See Figure 2 FIGURE 1 Algorithm for initial mass stranding response. Euthanasia 0839-frame_C06 Page 89 Tuesday, May 22, 2001 10:42 AM 89 Mass Strandings of Cetaceans Verified Stranding Accessible All Alive Evaluation and Triage Alive and Dead Alive All Dead Dead Keep Sternal Heart Rate Respiratory Rate Physical Exam Blood Sample See Figure 3 Necropsy Field Data Level A Data Cetacean Data Other Data Measurements Photos Necropsy Tissues Cultures FIGURE 2 Algorithm for evaluation of animals that are accessible. health status and separation of affected individuals into groups, based on clinical findings, which include (1) those likely to survive; (2) those apparently stable, but showing obvious signs of illness; and (3) those unlikely to survive. Individual health monitoring needs to include heart rate, respiratory rate, and attitude. Heart rates can be monitored in a partially submerged animal by placing the hand on the area between the pectoral flippers, and feeling for the reverberations of the heart through the chest wall. For safety reasons, this procedure should not be attempted with struggling or very large animals. In totally beached animals, which are lying laterally (although some animals beach sternally), heart rate may be visualized by movement of the sternal area. In a mass stranding of 30 false killer whales in Florida, heart rates ranged from 60 to 150 beats per minute (bpm) (Walsh et al., unpubl. data). Normal heart rates of this species are approximately 60 to 100 bpm and respiratory rates are 8 to 18 breaths per 5 min. The animals that lived the longest were five animals with near normal heart and respiratory rates (Walsh et al., unpubl. data). In addition to physical information, blood samples should be taken from each individual before any treatments are given. Blood collection is discussed elsewhere (see Chapter 19, Clinical 0839-frame_C06 Page 90 Tuesday, May 22, 2001 10:42 AM 90 CRC Handbook of Marine Mammal Medicine Blood Analysis and Physical Exam Results Normal Abnormal Release Rehabilitation Rehabilitation Survival (>6 mo) Death Euthanasia Necropsy Field data Release Retain Radio-tag or Mark Tissues Cultures FIGURE 3 Algorithm for animal evaluation and disposition. Pathology). Care must be taken when sampling stranded cetaceans, because they are capable of inflicting injury with their flukes, especially to inexperienced volunteers. At a minimum, blood sample volume should be sufficient to include CBC, serum chemistries, and serum electrolyte levels; however, optimally, additional serum is required for additional diagnostic analyses and for archival purposes. It is often possible to have pertinent tests run on an emergency basis utilizing local hospitals and veterinary clinics close to stranding sites. Emergency clinical laboratory tests should include manual packed cell volume, refractometer-determined total protein, fibrinogen, white blood cell count, glucose, blood urea nitrogen, creatinine, calcium, and electrolytes. These tests can aid the on-site clinician and rescue crew making decisions regarding the disposition of the group. Any residual serum and EDTA plasma should be retrieved from the hospitals and/or veterinary clinics and archived for future analyses. Fibrinogen tests require special tubes containing sodium citrate, and need to be spun, plasmaseparated, and analyzed or frozen in plastic vials within 1 hour of sampling to ensure accuracy. If possible, a centrifuge should be available on site to allow serum or plasma separation as soon as possible. New handheld, portable analyzers are available to analyzed some electrolytes, chemistries, and blood gas parameters on site. Blood glucose monitors may also be helpful in evaluating animals. Biochemical and hematological abnormalities found in individuals of each stranding may vary widely. In the stranded false killer whales, pod abnormalities included hemoconcentration, leukopenia, elevated liver enzymes, hypernatremia, hyperchloremia, and hypocalcemia (Table 3). 0.2 0.2 0.3 0.1 0.3 0.2 0.4 — 1.0 0.3 0.3 0.4 0.3 0.2 111 88 96 135 122 115 131 — 128 122 154 99 138 280 26 92 74 113 25 166 53 — 74 25 58 139 — 65 6.4 6.3 5.5 5.3 5.7 6.4 7.5 — 6.9 5.7 5.9 4.5 6.5 7.2 TP g/dl 2.7 2.3 3.1 2.9 2.8 2.8 3.1 — 3.2 2.8 3.0 2.0 3.0 3.6 Alb g/dl 3.7 4.0 2.4 2.4 2.9 3.6 4.4 — 3.7 2.9 2.9 2.5 3.5 2.8 Glob g/dl 9 14 32 20 49 22 12 47 8 49 17 30 11 14 Amy U/l 239 166 — 240 440 317 — 317 250 208 76 — — — Lip U/l 106 106 363 269 159 66 108 56 158 159 201 479 160 242 AP U/l 112 59 3 15 80 40 9 60 105 80 30 38 33 15 ALT U/l 675 1490 423 279 1080 655 490 603 >2500 1080 382 740 830 110 AST U/l 20 21 — 27 27 29 — 30 16 28 19 — — 26 GGT U/l 787 281 155 104 498 984 677 331 1174 498 606 1205 535 60 CK U/l 2692 1089 567 380 1258 980 1517 1083 725 1258 725 1054 1546 382 LDH U/l 9.0 6.8 7.0 6.8 6.6 7.6 7.6 — 8.3 6.6 7.1 7.6 7.5 8.9 2.7 4.9 7.3 6.5 6.8 8.6 5.9 — 9.0 6.8 4.8 4.8 2.5 5.6 Ca Phos mg/dl mg/dl Notes: Glu = glucose, BUN = blood urea nitrogen, Cr = creatinine, Bili = bilirubin, Chol = cholesterol, Trig = triglycerides, TP = total protein, Alb = albumin, Glob = globulin, Amy = amylase, Lip = lipase, AP = alkaline phosphatase, ALT = alanine aminotransferase, AST = aspartate aminotransferase, GGT = gamma glutamyl transpeptidase, CK = creatine phosphokinase, LDH = lactic dehydrogenase, Ca = calcium, Phos = phosphorus, N = normal individual in captivity. 6.5 2.5 1.3 1.3 2.0 2.4 2.2 3.0 4.6 2.0 2.6 1.2 1.5 1.2 132 131 119 170 232 140 167 314 135 232 252 172 207 131 1 2 3 4 5 6 7 8 9 10 11 12 13 N 62 44 44 41 44 74 56 108 57 44 47 84 40 40 Glu BUN Cr Bili Chol Trig mg/dl mg/dl mg/dl mg/dl mg/dl mg/dl ID TABLE 3 Serum Chemistry Findings in a Mass Stranding of False Killer Whales (Pseudorca crassidens) 0839-frame_C06 Page 91 Tuesday, May 22, 2001 10:42 AM Mass Strandings of Cetaceans 91 0839-frame_C06 Page 92 Tuesday, May 22, 2001 10:42 AM 92 CRC Handbook of Marine Mammal Medicine Stranded short-finned pilot whales differed from the stranded false killer whales, in that no consistent biochemical or hematological abnormalities were present within the pod; however, individuals showed evidence of hemoconcentration, leukopenia, elevated serum creatinine, hyperbilirubinemia, hypocalcemia, and hypophosphatemia (Walsh et al., 1991). Similarly, in both strandings there was evidence of dehydration and stress that were supported by hemoconcentration and hyponatremia and by leukopenia, respectively. The hypocalcemia and hypophosphatemia were the result of unknown mechanisms but are not uncommon in stranded cetaceans, or subsequent to prolonged transport (Ewing, pers. comm.). Often, members of the pod have died by the time the rescue team intervenes. These animals should be necropsied to help determine what potential pathological processes are afflicting the pod. Sample collection is often difficult because of environmental conditions or logistics, but it is important that as thorough a necropsy as possible be performed (see Chapter 21, Necropsy). Table 4 illustrates the pathological findings for a group of stranded pilot whales from the Florida Keys in 1986 (Bossart et al., 1991). The pathological changes observed were diversified within the pod and even varied within individuals. The predominant findings were nonspecific gastrointestinal inflammation and degenerative changes. There was also marked lymphoid tissue depletion, suggesting chronic stress, immunosuppression, or cachexia (see Chapter 12, Immunology; Chapter 13, Stress). The histopathological changes were nonspecific although they were indicative of chronic progressive disease (Bossart et al., 1991). Based on blood work and necropsy results, it was evident that the animals involved in this stranding were not healthy at the time of intervention. Disposition of Animals in a Mass Stranding After all animals have been tagged for identification and blood has been collected for clinical laboratory analyses, the rescue team must decide on the disposition of the animals in the group (see Figure 3). Because illness may be a major factor by the time a pod of whales strands, choices of what to do with the group may be complicated. It is important to consider two points. If illness is a major factor, a wide range of illness severity may be manifested within the group. Some individuals may be critically ill, whereas others may be only slightly debilitated. Second, there may be a combination of other factors, in addition to the illness, that determines where the whales strand. Geomagnetic field differences may help determine where an ill group is more likely to strand. Local storms, currents, tides, bottom topography, and environmental oddities may be contributing factors. Hours or days after being pushed back out to sea, the same animal may not be leading the group, or environmental factors may have changed; as a result, the group may not re-strand, but instead go back to sea, perhaps to die, and valuable information may be lost. With prior knowledge of illness within the group, it may be inappropriate simply to turn the pod out to sea. The choices available to the rescue team are dependent upon the size of the pod, background of the rescue team, environmental conditions, and the availability of rehabilitation facilities. Each stranding should be viewed as an individual event, with the initial goal being to learn as much as possible about the primary factors involved. For example, on the northeast coast of Cape Cod Bay, Massachusetts, there is an area where mass strandings of pilot whales regularly occur (Geraci et al., 1999). Blood results and histopathological findings do not entirely incriminate illness as the major stranding factor. It is suspected that the local coastline and the rapid tide changes are the primary factors contributing to these strandings, although morbillivirus has been found associated with numerous strandings since 1982 (Geraci et al., 1999). a N +2(Pu) +2 +2(Pn) +1(Pt) +3(Pn) N +2(Pt) +5 +2(Pn) A (123 cm, M) B (144 cm, F) C (292 cm, M) D (323 cm, F) E (328 cm, F) F (330 cm, F) G (331 cm, F) H (350 cm, F) I (380 cm, F) J (440 cm, M) N +3 +5 N N N N +5 +5 N N +3 +3 N N N +1(Pn) +2 N +3 Pulmonary Inflammation Intestinal +2 +2 N +2 N +1 +1 +3 +4 N Cardiovascular N +3 +3 N +3 +3 N N N +3 Hepatic Degeneration +5 +4 NE NE +5 +5 +5 +5 +5 +5 Lymphoid Depletion +2 +3 NE NE +3 NE N N +5 +3 Adrenocortical Lipid Depletion Kidney: pyelitis, necrotizing, chronic–active, multifocal, moderate — Subcutis: cellulitis, necrotizing, chronic–active, multifocal, severe Skeletal muscle: myositis, necrotizing, chronic–active, severe Skin: dermatititis, ulcerative, chronic–active, multifocal, severe — Pancreas: pancreatitis, fibrosing, chronic, multifocal, moderate Pancreas: pancreatitis, necrotizing, chronic–active, multifocal, moderate to severe Tumor: uterus, fibroleiomyoma — — Other Source: Bossart, G.D., Walsh, M.T., Odell, D.K., Lynch, J.D., Buesse, D.O., Friday, B., and Young, W.G., 1991, Histopathologic findings of a mass stranding of pilot whales (Globicephala macrorhynchus), Proceedings Second Marine Mammal Stranding Workshop, NOAA Technical Report. b Grade ranges (+1 = mild; +3 = moderate; +5 = severe). Animal identification indicates straight-line length in centimeters from tip of rostrum to fluke notch and sex (M = male, F = female). N = No specific lesions present; P = Lesions associated with parasites (n = nematode, t = trematode, c = cestode, u = unknown); NE = Not examined. a Gastric Animal b ID (length, sex) TABLE 4 Graded Histopathological Findings in a Mass Stranding of Pilot Whales (Globicephala macrorhynchus) 0839-frame_C06 Page 93 Tuesday, May 22, 2001 10:42 AM Mass Strandings of Cetaceans 93 0839-frame_C06 Page 94 Tuesday, May 22, 2001 10:42 AM 94 CRC Handbook of Marine Mammal Medicine Euthanasia The realistic options facing a stranding response team must include the possibility of euthanasia. This procedure should never be implemented unless all other possibilities have been investigated and eliminated (see Chapter 32, Euthanasia). Return to the Sea Rescue groups around the world differ in their reactions to mass strandings, with some limiting their response solely to returning the animals to the water. This solution, which assumes that all is well with both the individuals and the group as a whole, has met with mixed success (Odell et al., 1980). On the west coast of Florida, it is common for cetaceans that strand to be pushed back into the water and to re-strand, each time with increased mortality. Occasionally, the whales are never seen again, so some assume this is the best way to handle the problem. In strandings where health and/or illness have been investigated, this cannot be the sole response. While certain rescue groups feel they are doing the best thing for the pod, they are not considering that they are sending many or all of the whales out of sight to die. It should also be considered that, if some of the animals are infected with a fatal infectious disease, returning these animals to sea may result in further spread of the pathogen. In addition, a great amount of valuable information that could help in future strandings is lost when animals are prematurely released back out to sea. Disease problems affecting these groups may not be discovered or documented. Miniaturization of tracking devices has allowed transmitters to be temporarily applied to cetaceans (see Chapter 38, Tagging and Tracking), which should be considered a possible approach to study the survival of animals returned to the sea. Survival of Treated Whales The approach to treatment of individuals from mass strandings is similar to that for any other marine mammal that is ill. Survival time of members of the two mass strandings mentioned earlier ranged from 2 days to 18 months. Because medical investigations into stranding events have been limited, it is not known what percentage of a pod of stranded whales may survive. It appears that the survival rate will be very low, with the chance of survival depending upon the stage of illness, the type of illness, and the adaptability and age of the individual. It must be assumed that survival of the pod will be low if members have already perished. A review of the treatments of nine stranded individuals that survived longer than 1 month indicated that most of these individuals continued to have recurrent bouts of illness. Premature release of these individuals may infect other healthy pods that would not have been exposed without human intervention. The recognition of the presence of infectious diseases in beached cetaceans has changed the approach to rehabilitation. Facilities with in-house collections that accept stranded animals put resident individuals at risk, unless all beached animals are placed in total isolation. Personnel working with beached animals must not have any contact with collection animals. Wet suits, food utensils, shower facilities, and handling equipment must be totally separate to eliminate vector transmission. Failure to implement full quarantine procedures can result in disaster (Bossart, 1995). Conclusion To date, investigations into the causes of cetacean mass strandings have improved with the increased involvement and cooperation of oceanaria, rehabilitation facilities, academic institutions, and federal agencies. Increased financial support has increased the return of information, but more must be done to ensure the thoroughness of each investigation. 0839-frame_C06 Page 95 Tuesday, May 22, 2001 10:42 AM Mass Strandings of Cetaceans 95 Although histopathology and limited serology are becoming more common, it remains important to synthesize these data with the environmental, natural history, clinical, bacterial, toxic, and viral components to yield a comprehensive final evaluation of each stranding event. As new diagnostic tests are developed, retrospective analyses of archived tissues and serum are critical. To accomplish this goal, laboratories designated as receiving hubs for this material must be identified. It may be helpful to partner with colleagues in other countries who are already accomplished in specialized fields. This will require development of research gateways to allow easier passage of research material between experts. It must be remembered that the initiating factor(s) of a stranding may have occurred days or weeks before the animals encountered land, so that some strandings may not be explainable, even if all possible information is gathered. Only ongoing detailed examinations of mass strandings will slowly lead to understanding of this phenomenon. Acknowledgments The authors thank the staff and participants in the Northeast and Southeastern U.S. Marine Mammal Stranding Networks, the National Marine Fisheries Service, Mote Marine Laboratory, Miami Seaquarium, and Dolphin Research Center for their involvement in the gathering of this information. They also thank Julia Zaias (University of Miami, Miami, FL) for editorial assistance, Teri Rowles for reviewing this chapter, and Jim Mead and the Marine Mammal Program at the Smithsonian Institution for their vigilance in the pursuit of information on cetaceans and for their compilation of information on mass strandings. References Best, P.B., 1982, Whales, why do they strand? Afr. Wildl., 36: 6. Bossart, G.D., 1995, Morbillivirus infection: Implications for oceanaria marine mammal stranding programs, in Proceedings of the International Association for Aquatic Animal Medicine, IAAAM CDROM Archives. Bossart, G.D., Walsh, M.T., Odell, D.K., Lynch, J.D., Buesse, D.O., Friday, R.B., and Young, W.G., 1991, Histopathologic findings of a mass stranding of pilot whales (Globicephala macrorhynchus), Proceedings Second Marine Mammal Stranding Workshop, NOAA Technical Report, 85–90. Bossart, G.D., Baden, D.G., Ewing, R.Y., Roberts, B., and Wright, S.D., 1998, Brevetoxicosis in manatees (Trichechus manatus latirostris) from the 1996 epizootic: Gross, histologic, and immunohistochemical features, Toxicol. Pathol., 26: 276–282. Cetacean Distributional Database, Marine Mammal Program, Smithsonian Institution, Washington, D.C. Cordes, D.O., 1982, The causes of whale strandings, N.Z. J. Med., 30: 21. Dudok Van Heel, W.H., 1962, Sound and cetacea, Neth. J. Sea Res., 1: 402. Eaton, R.L., 1979, Speculations on strandings as burial, suicide, and interspecies communication, Carnivora, 2: 24. Frantzis, A., 1998, Does acoustic testing strand whales? Nature, 392(6671): 29. Fraser, F.C., 1934, Report on cetacea stranded on the British coast from 1927–1932, Br. Mus. Nat. Hist., 11. Fraser, F.C., 1946, Report on cetacea stranded on the British coast from 1933–1937, Br. Mus. Nat. Hist., 12. Fraser, F.C., 1953, Report on cetacea stranded on the British coast from 1938–1947, Br. Mus. Nat. Hist., 13. Fraser, F.C., 1956, Report on cetacea stranded on the British coast from 1948–1956, Br. Mus. Nat. Hist., 14. Geraci, J.R., 1978, The enigma of marine mammal strandings, Oceanus, 21: 38–47. Geraci, J.R., 1989, Clinical investigation of the 1987–88 mass mortality of bottlenose dolphins along the U.S. central and south Atlantic coast, Final Report National Marine Fisheries Service, U.S. Navy (Office of Naval Research), and Marine Mammal Commission, 63 pp. 0839-frame_C06 Page 96 Tuesday, May 22, 2001 10:42 AM 96 CRC Handbook of Marine Mammal Medicine Geraci, J.R., and St. Aubin, D.J., 1977, Pathologic findings in a stranded herd of Atlantic white-sided dolphins, Lagenorhynchus acutus, in Proceedings of the International Association for Aquatic Animal Medicine, IAAAM CD-ROM Archive. Geraci, J.R., and St. Aubin, D.J., 1979, Biology of marine mammals: Insights through strandings, U.S. Marine Mammal Commission, Report Number MMC-77/13, Washington, D.C., PB-293, 890. Geraci, J.R., Testaverde, S.A., Staubin, D.S., and Loop, T.H., 1976, A mass stranding of the Atlantic white-sided dolphin (Lagenorhynchus acutus): A study into pathobiology and life history, U.S. Marine Mammal Commission, Report Number MMC 75/12, Washington, D.C., PB-289, 361. Geraci, J.R., Anderson, D.M., Timperi, R.J., St. Aubin, D.J., Early, G.A., Prescott, J.H., and Mayo, C.A., 1989, Humpback whales (Megaptera novaeangliae) fatally poisoned by dinoflagellate toxin, Can. J. Fish. Aquat. Sci., 46: 1895–1898. Geraci, J.R., Harwood, J., and Lounsbury, V.J., 1999, Marine mammal die-offs, in Conservation and Management of Marine Mammals, Smithsonian Institution Press, Washington, D.C., 367–395. Kennedy, S., Smyth, J.A., Cush, P.F., McCullough, S.J., Allan, G.M., and McQuaid, S., 1988, Viral distemper now found in porpoises, Nature, 336: 21. Kirschvink, J.L., Dizon, A.E., and Westphal, J.A., 1985, Evidence from strandings for geomagnetic sensitivity in cetaceans, J. Exp. Biol., 120: 1–24. Klinowska, M., 1985a, Interpretation of the U.K. cetacean strandings records, Rep. Int. Whaling Comm., 35: 459. Klinowska, M., 1985b, Cetacean live stranding sites relate to geomagnetic topography, Aquat. Mammals, 11: 2–32. Klinowska, M., 1985c, Cetacean live stranding date relate to geomagnetic disturbances, Aquat. Mammals, 11: 109–119. Mead, J., 1997, Pathobiology of cetacean strandings along the Atlantic coast, 1976–1977, in Proceedings of the International Association for Aquatic Animal Medicine, IAAAM CD-ROM Archive. Morimitsu, T., Nagai, T., Ida, M., Ishii, A., and Koono, M., 1986, Parasitogenic octavus neuropathy as a cause of mass stranding in odontoceti, J. Parasitol., 72: 469. Morimitsu, T., Nagai, T., Ida, M., Kawano, H., Naichuu, A., Koono, M., and Ishii, A., 1987, Mass stranding of odontoceti caused by parasitogenic eighth cranial neuropathy, J. Wildl. Dis., 23: 586–590. Odell, D.K., 1987, The mystery of marine mammal strandings, Cetus, 7: 2. Odell, D.K., Asper, E., Baucom, J., and Cornell, L., 1980, A recurrent mass stranding of false killer whales, Pseudorca crassidens, in Florida, Fish. Bull., 78: 171–177. Ridgway, S., and Dailey, M., 1972, Cerebral and cerebellar involvement of trematode parasites in dolphins and their possible role in stranding, J. Wildl. Dis., 8: 33–43. Sergeant, D.E., 1982, Mass strandings of toothed whales (Odontoceti) as a population phenomenon, Sci. Rep. Whale Res. Inst., 34: 1. Walsh, M.T., Beusse, D.O., Young, W.G., Lynch, J.D., Asper, E.D., and Odell, D.K., 1991, Medical findings in a mass stranding of pilot whales (Globicephala macrorhynchus) in Florida, Proceedings Second Marine Mammal Stranding Workshop, NOAA Technical Report 98, January, 75–83. Wareke, R., 1983, Whales, whale stranding—accident or design? Aust. Nat. Hist., 21: 4312. Wiley, D.N., Early, G., Mayo, C.A., and More, M.J., in review, The rescue and release of mass stranded cetaceans from beaches on Cape Cod, Massachusetts, USA: A review of some response action, Aquat. Mammals. Wilkinson, D.M., 1991, Report to the Assistant Administrator for Fisheries, in Program Review of the Marine Mammal Stranding Network, U.S. Department of Commerce, NOAA, NMFS, Silver Spring, MD, 171 pp. Wilkinson, D.M., 1996, National contingency plan for response to unusual marine mammal mortality events, U.S. Department of Commerce, NOAA Technical Memorandum, NMFS-OPR-9. 0839_frame_C07 Page 97 Tuesday, May 22, 2001 10:42 AM 7 Careers in Marine Mammal Medicine Leslie A. Dierauf, Salvatore Frasca, Jr., and Ted Y. Mashima Introduction All veterinarians working in the field of marine mammal medicine have many stories to tell about veterinary students and seasoned veterinarians with career changes in mind coming to them and asking for direction on where to find that perfect job in marine mammal medicine. One of the authors (S.F.) as director of education of the International Association for Aquatic Animal Medicine (IAAAM), for example, responds to an average of one to two e-mail inquiries per week from high school students, undergraduate and graduate students, veterinary students, or veterinary practitioners, regarding the availability of jobs in marine mammal medicine. Such a deceptively simple inquiry actually entails a long and complicated answer. Each individual career path represents a unique blend of what that person wants to do, what experience and training he or she brings to the pursuit, and what personal lifestyle choices that person wishes to honor (Dierauf, 1996). In 1994, the Society for Marine Mammalogy published a useful guide, which is available on the Web, that is the basis for some of the information in this chapter (Thomas and Odell, 1994). Other aspects come from the authors’ own personal searches for that “perfect job.” One may ask, “How can I have a great life, pursue my interest in marine mammals, and at the same time enthusiastically participate in this marvelous profession of veterinary medicine?” The choices really are very personal. Whether you are seeking a position in marine mammal clinical practice or marine mammal conservation and management, the opportunities available are varied and depend on your interests, skills, expertise, and abilities. One thing is certain: as a veterinarian with broad medical, scientific, and customer service expertise, you have excellent basic training in a variety of fields (Mashima, 1997), and can take your career in any direction that you wish. When you consider everything you are capable of doing, you will amaze yourself. One of the authors (L.A.D.) keeps this inspirational message on her desk, above a picture of a snow-covered, blue-skied mountain: “I am not in the habit of starting my day by thinking of things that I cannot get done!” Any one of the multitude of scientific, technical, and nontechnical topics/fields discussed in this textbook is a potential job opportunity for you. Full-Time Employment Full-time jobs in clinical veterinary medicine of marine mammals are rare, and primarily limited to display facilities, the military, and rehabilitation centers. Currently in the United States, the authors estimate there are fewer than three dozen veterinarians employed in the fulltime practice of marine mammal medicine; a number of these are employed in marine research. 0-8493-0839-9/01/$0.00+$1.50 © 2001 by CRC Press LLC 97 0839_frame_C07 Page 98 Tuesday, May 22, 2001 10:42 AM 98 CRC Handbook of Marine Mammal Medicine Even at the four Sea World facilities in the United States, where most “full-time” marine mammal veterinarians are employed, the caseload goes beyond marine mammals to birds, fish, and other marine organisms. Each year the selection criteria for positions in the field of marine mammal medicine become more stringent. In the opinion of one of the authors (S.F.), it is no longer a reasonable and wise career decision to consider yourself a viable candidate for these positions based solely on your classical veterinary education and degree. The competition for salaried positions and funding to perform clinically relevant research pertaining to marine mammals is intense. The viable candidate is someone who has developed skills in addition to formal veterinary training. These are skills in fields such as biomedical technology, computer science, population dynamics, public and environmental health, and conservation, which are complementary to formal veterinary medical training. Individuals with such skills often can improve their job opportunities, because they can present themselves as multifaceted professionals capable of multitasking at high levels and capable of filling more than one niche within the infrastructure. Part-Time Employment Now that the concerns for full-time employment have been addressed, there are a number of ways to work as a marine mammal veterinarian on a part-time basis, either as a volunteer or consultant, in a variety of state and federal agencies, nonprofit private organizations, environmental groups, or in academia. In addition to clinical jobs, there are positions in marine mammal medicine involving preventive medicine, pathology, epidemiology, management, policy making, and public education, outreach, and awareness. More often than not, developing an expertise in some associated field, such as epidemiology, pathology, or education, may be a principal route into the field of marine mammal health management (King, 1996; Marshall, 1998; Smith, 1998a,b). The concept of conservation medicine can be well applied to marine mammal medicine. This movement blends conservation biology with veterinary and human medicine, and it is gaining rapid recognition as an interdisciplinary, team-oriented science (Jacobson et al., 1995; Aguilar and Mikota, 1996; Deem et al., 1999; Meffe, 1999; Society for Conservation Biology, 2000). Conservation medicine in the marine context addresses the application of biomedical principles and technology to global issues of ecology and environmental health. It also encompasses a wide range of interests, ranging from collaborative research in marine mammal population status to the effects of changes in marine ecosystems on marine mammal health and disease; from conservation efforts to protect vital habitats to concerns over international public health; from the effects of ecotourism to policy-making and funding opportunities for protection of natural resources and marine environments. Thus, although conventional clinical jobs may be few and far between, there are a myriad of opportunities that involve marine mammal health interests. You may create many of these opportunities, as you apply your background in alternative ways (Environmental Careers Organization, 1993; Gerson, 1996; Doyle, 1999; National Wildlife Federation, 2000). Personality Traits and Other Tools Personality traits that lend themselves to exploration, risk-taking, and creativity are a plus in finding new career directions (Covey, 1990; Fassig, 1998; Johnson, 1998; Sylvester, 1998). Tools that come in handy are imagination, vigilance, practicality, patience, enthusiasm, and a willingness to dare to dream. These are the traits that lead to “making your own luck” (Wells, 1992). Luck is really the meeting of opportunity and preparation. 0839_frame_C07 Page 99 Tuesday, May 22, 2001 10:42 AM Careers in Marine Mammal Medicine 99 Another set of tools that is vital to veterinarians who wish to move outside the traditional practice setting consists of those skills learned outside the profession itself. Such skills include creative writing, editing, networking, computer science, leadership, and critical thinking. Additional training or experience in conservation biology, ecology, population biology, environmental science, foreign languages, and journalism can help in developing these skills. Oftentimes, the result is a global perspective with big-picture views of the world, its people, and cultures, an awareness of the effects marine mammals and other animals have on our world, and the effects our world has on them. In reality, the majority of marine mammal medical skills will also be learned outside of formal veterinary training (Dierauf, 1996). These personal development strategies, professional improvement opportunities, and global perspectives are not improvement strategies unique to the field of marine mammal medicine. Some veterinary colleges have recognized the importance of these personal skills and the role that veterinary medicine can play in the realm of world health. They have developed didactic and active learning experiences in such fields as international veterinary medicine and population biology that address global concerns and apply the veterinary medical degree in alternative ways. Summary Not everyone involved in marine mammal health is a veterinarian. Individuals who hold masters and doctorates in biomedical fields, such as molecular biology, cell biology, physiology, immunology, toxicology, neurobiology, ecology, and evolutionary biology, have contributed greatly to the advancement of marine mammal health over the past decades. Indeed, some of the most prolific and influential investigators in marine mammal biology have been nonveterinary professionals. The theme among all those individuals who have successfully developed careers in marine mammal health and medicine is excellence. Developing a reputation for excellence in some discipline and applying that excellence to the field of marine mammal health is the key to professional growth in this arena. In any case, this chapter is a generalized approach to identifying and seeking that “ideal” job, rather than an exacting formula for obtaining a position in marine mammal medicine. This chapter can be used as a guide, yet the decisions to be made are up to you alone. Use the suggestions in our “six-step method” as best suit your needs and desires for professional and personal development and fulfillment. The Six-Step Method for Landing That Perfect Job Working with Marine Mammals 1. The First Step—Taking a Personal Self-Assessment The field of marine mammal medicine and conservation may look enchanting, but is it really for you? Do you have the personal desires and lifestyle needs that will fit into this professional field? What are your work ethics and interests? Will a job in the field of marine mammalogy fit your current time frame? Are there any particular patterns that have emerged in your career choices to date (Buss, 1998)? We would be remiss if we did not tell you that the field of marine mammal medicine today is less than lucrative in terms of salary and advancement. To date, the majority of vacancies have occurred in aquaria, academic institutions, and federal/state government agencies, because there are only so many coastal areas in the United States and abroad upon which to base a career in marine mammal medicine. 0839_frame_C07 Page 100 Tuesday, May 22, 2001 10:42 AM 100 CRC Handbook of Marine Mammal Medicine First, you need to determine what exactly you are seeking with a position in the field of marine mammal medicine. Here are some questions to ask yourself, so that you have a clear picture of where you want to go in your professional career and what is most important to you in making any career change. We recommend that you not only read these questions, but also actually write down your answers to assist you as you move through the six-step process outlined in this chapter. Self-Assessment Questions Do you want to work part-time or full-time? Have you considered volunteering? Can you commit to an externship, internship, residency, or fellowship at this time? Is where you live important to you at this time? If so, where would you like to live? Do you have the means to live abroad, or are you planning to stay in the country where you currently reside? Do you have a family to support? If so, can you support your family in this career path? How motivated are you? Do you have the skills and training necessary for a position in this field? Do you have the time and resources available to take additional coursework or training? Does the position you are seeking fit your philosophy of life, lifestyle, and life goals? Are you ready to commit to a full-time job search, or are you peripherally interested at this time? Are you ready to commit to a job in a competitive field such as this? Have you paid enough attention to this field? Have you taken time to work with a veterinarian in an aquarium or a teaching institution to appreciate the commitment of hours and effort that are required to maintain a job position? Do you realize that in some of the marine mammal medicine positions, especially in field research or clinical practice, the hours can be long, erratic, and unpredictable? If they involve administrative duties, these can entail daily paperwork, writing, reporting, and supervising. Because many marine mammal positions require you to be out of doors, even regular tasks and chores can become onerous if performed under extreme climatic conditions, such as scorching sun, brutal rain, unending wind, and rough seas. We urge you to consider each of these questions and issues seriously. 2. The Second Step—Categorizing Your Unique Skills, Strategies, and Approaches These days it appears that businesses, organizations, and institutions are searching for employees who stand out in a crowd. Tom Peters (1999) calls it “hiring to talent.” He frames whom to hire by looking for special “projects, passion, provocation, partnerships, politics, professionalism and performance.” He said that once, in pouring over 200 applications for a single position, he made his first cut by looking at the applications and watching for something peculiar; in this case, it was a computer scientist who had been entered into Ripley’s Believe It or Not for creating and baking a 1-ton cookie! A good foundation in small animal medicine and surgery and critical care medicine may serve you well in your marine mammal pursuits and casework. Some marine mammal clinicians have expressed to us that they prefer to hire individuals who have strong small animal medicine backgrounds and/or have completed small animal internships or residencies. In fields such as marine mammal medicine and conservation, potential employees exhibiting imagination and creativity often stand out from the rest. We believe it is scientifically founded, innovative 0839_frame_C07 Page 101 Tuesday, May 22, 2001 10:42 AM Careers in Marine Mammal Medicine 101 thinking that will bring new marine mammal positions to the forefront, expanding the opportunities available for us all. Additional academic training and/or degrees can be helpful, as can short courses and continuing education in the field of marine mammal medicine. The ability to conduct self-motivated and self-motivating training and teaching and to participate in a volunteer capacity at facilities that cater to programs for advanced study and public involvement can add to your experiences in the field and build your professional skills and credentials. Volunteering in an organization, for no pay and hard work, can be an admission ticket to the world of paid employment, assuming you are productive and resourceful in choosing a particular role and how you focus on that role within the organization. For example, one of us (L.A.D.) came to be in charge of veterinary services (a paid position) at The Marine Mammal Center in Sausalito, California, by first volunteering every Sunday (over a year’s time) to set up a clinical laboratory and design a veterinary medical education course for the volunteers. However, in today’s economy, this may not be the most practical way individuals can acquire jobs in marine mammal health care. The advice often given by one of the authors (S.F.) regarding volunteerism is to strive to produce tangible results from your volunteer efforts and investments of time and expertise. This is especially true for students. Paid positions for veterinary students at display facilities or academic institutions are rare, and, when offered, the pay is often not commensurate with the effort. However, volunteer efforts may furnish opportunities to participate in clinical investigations or research projects that produce journal publications, conference presentations, or posters. Presenting your work at scientific conferences is an excellent way you can introduce yourself to large groups of potential employers or future collaborators. Some organizations, such as the IAAAM, encourage and support student presenters with competitions for student travel and conference presentation awards. On-the-job training, be it paid or unpaid, is always of value. Equal in importance to such active learning is discovering and committing to a mentor in the field (Harris, 1998). The mentor should be someone who can guide you and be an advocate for your career choices; someone who gives you an inside view of what the profession of marine mammal medicine and conservation is all about; someone who helps you build a base of contacts and networking individuals for future reference and support. All the authors have no doubts that the conscientious guidance and advice obtained from our mentors has been, and continues to be, integral to our career development. What tasks really fire you up? What tasks exhaust you? Richard Bolles (2000, p. 349) recommends that you make a list of all the things you enjoy doing with regard to work and play in general, and then categorize each item under one of these headings: “Skills with People,” “Skills with Things,” and “Skills with Information.” Bolles (2000, p. 79) also provides a list of 246 action verbs that describe a great variety of skills that, again, can be categorized under People, Things, and Data. How many of these action verbs relate to your skills and abilities? For example, are you a “people” person—Do you like mentoring, negotiating, instructing, supervising, persuading, speaking, serving, helping? Are you a “things” person—Do you like setting up things, working with precision instruments, operating technical devices, manipulating mechanical things, handling tools? Or are you a “data/information” person—Do you like collating, synthesizing, coordinating, analyzing, compiling, solving, computing, comparing? Once you have an idea of the variety of skills you have, write them down in order, beginning with the activities you enjoy doing the most. You will be surprised what clarity this simple exercise can bring to your marine mammal job search. This answers the question for you of “What do I want to do with marine mammals in my professional life?” 0839_frame_C07 Page 102 Tuesday, May 22, 2001 10:42 AM 102 CRC Handbook of Marine Mammal Medicine 3. The Third Step—Planning for Action and Timing The next step involves How to find the jobs that will give you the greatest satisfaction and opportunity to use your favorite skills. It is time to set some objectives and devise some options for planning your job-seeking strategy. Who knows you the best? It is likely to be your friends, family, peers, and colleagues. One of us (L.A.D.) “discovered” The Marine Mammal Center, when her friend, an art therapist, took her there during a summer afternoon outing. Ask the people who know you best to help you think of job opportunities and locate leads. With each lead, investigate the position and organization thoroughly to make certain each fits your current wants and needs. Use passive resources, such as telephone books, entertainment guides, the Internet, and on-line and hardcopy newsletters; sometimes these resources can trigger new job ideas, as well. Compare each job you come across with your prioritized list of skills and with your own strengths and weaknesses. Talk with anyone and everyone you meet who has the slightest connection to marine mammal medicine and health, to glean suggestions on other sources of information or other recommended organizations. Consider doing an elective “externship” that allows you to spend 4 to 6 weeks at a zoological park, aquarium, marine park, research facility, rehabilitation center, or government agency. After you find individuals who hold jobs you find attractive, ask them what they enjoy about their jobs, why they have kept their jobs, and how they obtained their jobs. Then make a list of the potential jobs and organizations and begin to investigate those people who are actually responsible for hiring to the kind of position you are seeking. Take a look at the section “Accessing Resources” at the end of this chapter, and the electronic job-hunting sources and ideas available in The Electronic Whale (Chapter 8), as well. In other words, it is just like school all over again; do your homework and you will succeed in gathering the information you need to make choices regarding the next phase of your professional career. 4. The Fourth Step—Making Choices The next step entails writing a job description for that perfect job, where you can use all your favorite skills, meet all your current lifestyle goals and objectives, and have some fun doing it. Try not to criticize or obstruct any ideas that might flow from your pen. Just keep writing, until you have on paper what your perfect job in the field of marine mammal medicine would be. This may seem like a fruitless, time-consuming exercise, but in reality it will truly clarify the direction you may want to take in choosing which positions to apply for, and then directing your career growth once you are in an organization. It will also insert some patience into your job search, recognizing that being in the right place at the right time may take time. You cannot really plan for the right time or the right place, but you can be prepared, and thereby recognize when the time and place are right. You will know. Now it is time to determine where you want to work. The best way to find where there are marine mammal medicine jobs is to network with people already in the marine mammal field. Choose one or more organizations you are interested in and start to nurture your networks. Find out what veterinarians or marine scientists already work there. Attend scientific marine mammal meetings, have coffee with these folks, get to know them, and, most importantly, let them begin to get to know you. Have patience, do not be overbearing, and make sure you ask the people you are networking with if they have time to talk with you. If they do not, ask them when (and where) would be more convenient. Be diplomatic and respectful of time in cultivating and maintaining your network. As another approach, if you are unable to make personal contact (although that is what these authors strongly recommend), pick up the telephone and call those facilities, organizations, 0839_frame_C07 Page 103 Tuesday, May 22, 2001 10:42 AM Careers in Marine Mammal Medicine 103 institutions, or laboratories that most interest you. Visit the companies/institutions of your choice. Ask to see their lists of job openings and general job descriptions. If there are no written job descriptions, come prepared to ask a set of preapplication questions of the people responsible for hiring in the organizations of your choice. In the case of academic positions within the laboratory of a primary investigator, make every attempt to schedule a meeting and a tour with the investigator. Tour the campus and examine the locations of buildings and facilities that are likely to be important resources for you. Assess whether the support facilities are truly convenient and accessible when you evaluate the opportunity as a whole. Again, take your time, be mild-mannered, and do not waste/hog the time of the people who work at the facilities of interest to you. Prepare and carry with you one or more résumés that speak to the particular type of job or organizational framework of interest. If you see a job description that appeals to you, ask who is in charge of hiring for that position. Get the correct spelling of the person’s name, his or her title or position, and telephone number. Bring professional stationery and envelopes with you. Insert a made-to-order résumé and list of references in an envelope, hand-write a short note to the appropriate person, insert it in the envelope, and write the person’s name, title, and division or organization on the envelope. Ask the personnel office or the office assistant to hand-deliver this note for you. If there is an application form for the position, fill it out thoughtfully. Be neat, organized, and concise, providing the exact information the application seeks, no more, no less. In your answers, “lead with the lead”; begin with a sentence that directly answers the question the application asks. Mail or hand-deliver the application on time (or even prior to the closing or due date—do not fax an application or supporting documents or e-mail information, unless that is what the application asks for). Include a cover letter that tells the hiring person that you are very interested in the position and that urges that person to inspect your application in detail and seriously consider you as a candidate. Be patient. All things come to those who wait. One of the authors (L.A.D.) decided in 1977 that she wanted to go into the field of marine mammal medicine. Not until 1979 did she take a hands-on marine mammal medicine workshop and meet her mentors. Not until 1980 was she hired into a paying job at The Marine Mammal Center; it took another 10 years (1990) to move into the marine mammal policy and conservation medicine arena. On a regular and consistent basis, make friendly calls to the people with whom you have been networking, so they know that you continue to remain interested. Finally, remember that the early bird catches the worm; be persistent, resourceful, and friendly in your efforts and contacts. 5. The Fifth Step—Preparing for the Interview The hope is that your networking, homework, legwork, and follow-up calls and letters have brought you the opportunity for an interview. Never walk into an interview or respond to a phone call for an interview until you have prepared and composed yourself. Do not appear desperate (even if you feel that way!) or too eager (even if you are ecstatic) when you are contacted. Be calm, cool, collected, polite, professional—and ready! In the phone call inviting you to an interview, make sure you ask what type of interview format will be used: in-person, by telephone, one-on-one, small panel, large panel, tour through a number of different offices for a series of interviews, on-the-job, real-life situations, or a combi-nation of these formats. There are a number of questions (Ryan, 2000) you may want to ask yourself and answer in writing prior to any interview opportunity. So, as soon as you have any hint that you 0839_frame_C07 Page 104 Tuesday, May 22, 2001 10:42 AM 104 CRC Handbook of Marine Mammal Medicine may be called for a telephone or in-person interview, begin preparing. Robin Ryan (2000) suggests that before you answer any preparatory questions, you first list as many as ten of your strongest traits. Then choose the five that most fit the job you have applied for, and rank those from 1 to 5. This is your five-point strategy. Consider these five points as your main answers to any of the questions posed here. Insert humor, enthusiasm, and anecdotes that demonstrate situations in which you successfully completed tasks related to the particular points you are presenting. Preparation is key; when someone tells you that you are lucky to be offered such an opportunity, be humble and recognize that you really do “make your own luck.” Tips for any interview (Dierauf, 1994): • The first 60 seconds of your interview are the most important; be prompt, neat in appearance, confident, and, above all, be prepared. Check your ego at the door. • Listen carefully to each question the interviewer asks you, pause, compose your thoughts, and then give an answer that is succinct, clear, and to the point. Use your five-point agenda whenever appropriate. Plan a number of different ways to deliver the same message. • Never take less than 20 or more than 90 seconds to answer a question. This ensures that the interviewer remains informed and energized by your presentations. • Remember that information and knowledge are power; the more you can absorb before your interview, the more smoothly the interview will proceed. Understand all aspects of any potential issue you may be asked to address. • If at all possible during the interview, do not discuss salary and benefits. This is a negotiation strategy you will want to work on if and when you are offered the job. This is just an interview. If the interviewer persists, ask what the salary range is for the position. Then deflect the question diplomatically by saying, “I believe the skills and experience I offer fit within that range,” or “That range is a bit lower than I had anticipated, but I am sure we can discuss that more fully at a later time, should you offer me this position.” • Have a rehearsed and practiced closing statement (60 seconds or less) to give yourself that final marketing sell before you exit the interview. During an interview, you can anticipate being asked a number of standard questions. For example, the first things on any interviewer’s mind, although he or she may not express them out loud, are these two: Can you and will you do the job? Will you fit into the philosophy and mission of this organization/institution? Work the answers to these often unasked questions into responses to actual questions, by talking about your current job and responsibilities, your commitment to your job, that you really find work enjoyable, and remember your five-point strategy. Assuming the person interviewing you is the person who will become your supervisor, answer in such a way that does not threaten that supervisor’s position in any way. You want to point out that you can complement his or her wishes and needs. Be sure that during the interview, if the interviewer is not clear or detailed enough, that you pleasantly ask for clarification or more detail. There are other common questions you should expect to be asked: Tell me about yourself (stick to your professional accomplishments, briefly summarizing your professional life over the past few years—keep it simple and short). Why should I hire you? 0839_frame_C07 Page 105 Tuesday, May 22, 2001 10:42 AM Careers in Marine Mammal Medicine 105 What makes you think that you have the qualifications for this position? Why do you want this job? What are the features of your current job that you like the most? The least? Why did you leave your last job? Why are you unemployed? Why are there time gaps in your work history? What are your strengths? Your weaknesses? All interviewers have their own reasons for asking the questions they do and in the manner they ask them. Prepare for unique questions or variations on them, such as: The Positive Approach—These are the interview questions that are the most enjoyable, where you can really shine, tell your stories and display your skills. Describe your current typical workday. Who was your favorite manager or supervisor and why? What do you know about this job and this organization? Name two or three things that are important to you in performing your job. What is the one thing you are proudest of in your (professional, not personal) life? What motivates you? What are you currently doing to improve yourself ? To you, what is the perfect job? The Negative Approach—Your responses to negative questions are best framed in a positive light. For example, take the question, give a brief answer, and then tell how you improved and/or learned from the situation, and how it made you grow and achieve greater success. Tell me about a time when you were criticized for poor performance. Describe a difficult co-worker. Tell me about one of your failures. How do you work under pressure? How do you handle stress? This job is a pressure-cooker. Can you handle it? Tell me something about your current boss that you dislike. Can you work odd hours, nights, weekends? Travel up to 20 days per month? How do you handle criticism? What was the most unpopular decision you ever made and what happened? What is the most difficult challenge you have ever faced (in your professional life)? If the interviewer chooses such a negative approach, seriously consider whether you really want to work with this person. Was it a game he or she was playing, or is that person, with whom you will presumably be working, truly a negative sort? Regardless of the interviewer’s style, anticipate some not-so-common questions, such as the following, that you will definitely want to consider, to avoid being surprised and unprepared in your responses: What is the most recent book you have read? Who is the president/CEO of this company? Tell me about a personal goal you want to achieve. May I contact your current employer? Also, be ready for any technical questions related to the scientific aspects of the job. The answers to the majority of these questions will be easy after all the homework and preparation you have done in the course of these first five steps. Be sure to write out your answers, so that you can review them prior to the actual interview. 0839_frame_C07 Page 106 Tuesday, May 22, 2001 10:42 AM 106 CRC Handbook of Marine Mammal Medicine Anticipate that, at some time during your interview, the interviewer may ask if you have any additional questions. Prior to entering the interview, determine which of the following questions are appropriate for the marine mammal medicine position you are seeking: What is your professional background? What motivates you? Can you describe what my day-to-day responsibilities will be in this position? With whom will I be working? Tell me a little bit about their backgrounds and skills. Can you explain the organizational structure here? Describe the atmosphere and politics in this office. What financial and support resources underlie the department/program in which I will be working? Since coming to this organization and your current position, what would you describe as your two greatest successes? What do you feel are your greatest strengths? Weaknesses? What are your short- and long-term visions for this organization/institution? Do you anticipate hiring/firing staff in the next 24 months? For what reasons? What are the strengths and weaknesses of this organization? What is your management style and your favorite type of employee? Give me examples of three challenges that you and I can work together to resolve. I would like you to speak with my references. May we look at my reference list together? Then close with what Ryan (2000) and Peters (1999) call the “Sixty Second Sell” or “Marketing the Brand YOU”—your own personal marketing ticket. Bring your interview back full circle by discussing what you do best, and how your enthusiasm and personality fit into, and complement, the mission and goals of the organization/institution, noting a few of your previous accomplishments that relate directly to the needs of the person hiring you and the job available. Be sure to tell the interviewer that, if you are hired, you intend to make a commitment to, and a difference in, the organization. Thank the interviewer, shake hands, smile, and calmly walk out. Go outside, sit down with pen and paper, and take notes about the interview and if you really believe you are a good fit for the job. Pat yourself on the back for a job well done. Follow up with a thank you note to the interviewer, and wait for the call. 6. The Sixth Step—Starting Your New Job In 1992, 24 scientists responded to a survey regarding career choices. From that survey, eight attributes important to any professional scientific career surfaced (Lebovsky, 1994): • • • • • • • • Be knowledgeable in the subject of science; Develop and practice good communication skills; Be enthusiastic in the presentation of science; Support and encourage students and pre-professionals; Respect the abilities of students and peers and listen carefully to them; Be willing to give time and effort to help students; Relate subject matter to real-life situations; and Have compassion for, and commitment to, your profession. How you communicate in your new career is very important. We are sure many of you already have excellent communication skills, and practice them every day, knowingly or unknowingly. Following is a basic list of communication tips one of us (L.A.D.) uses. These things are easy to do. The trick is to develop your own set of communication skills and practice 0839_frame_C07 Page 107 Tuesday, May 22, 2001 10:42 AM Careers in Marine Mammal Medicine 107 using them every day. They will serve you well in your interactions with peers, colleagues, and hiring personnel in the field of marine mammal medicine. Communications Basics • • • • • • • • • • • • • • Enhance and expand your oral and written communications (courses, practice, formal, informal). Train yourself to speak only after really listening and thinking. Do not let yourself get distracted when you are listening. Immerse yourself in a subject to learn it. Maintain a network of tried-and-true colleagues. Keep a positive attitude. Take nothing anyone says to you personally, even if it is so intended. Never take anything for granted. Steer away from viewing an issue as black or white, right or wrong. Take courses in teamwork, facilitation, mediation, and negotiation. Find a clear window of time (at least two 15-minutes periods) to think every day. Work at developing multiple options. Take risks; embracing risk is an exciting and energizing challenge. Have fun and keep your sense of humor. Accessing Resources Resources are what the majority of your efforts will revolve around as you plan your strategies and needs for a career in marine mammal medicine. First, we invite you to consider contacting marine mammal specialists who have contributed to this edition of the Handbook of Marine Mammal Medicine as sources of career information and ideas. In addition, the majority of programs, organizations, and other information sources listed below with their Web site addresses can provide greater detail, including contact information. The Electronic Whale (Chapter 8) provides further sources of electronic information. The following list of professional resources is not intended to be exhaustive. Opportunities listed below may change in terms of content, instructors, requirements, and/or dynamics. It is the responsibility of self-motivated individuals to investigate the current status of opportunities that interest them. Internships and Residencies Matched Internships Kansas State University, College of Veterinary Medicine, Manhattan, KS http://www.vet.ksu.edu The Ohio State University, College of Veterinary Medicine, Columbus, OH http://www.vet.ohio-state.edu University of Georgia, College of Veterinary Medicine, Athens, GA http://www.vet.uga.edu University of Michigan, College of Veterinary Medicine (also residencies), East Lansing, MI http://www.cvm.ms.edu 0839_frame_C07 Page 108 Tuesday, May 22, 2001 10:42 AM 108 CRC Handbook of Marine Mammal Medicine These matched internship programs concentrate to varying degrees on exotic, wildlife, and zoo animals in the format of a rotating 1-year internship through a veterinary teaching hospital. These programs are not aquatic specific, but each offers open rotations and vacation in which to accomplish aquatic studies. Each of these programs participates in the Veterinary Medical Intern-Resident Matching Program, administered through the American Association of Veterinary Clinicians. http://cvm.msu.edu/~judy/aavcl.htm Matched Residencies North Carolina State University, College of Veterinary Medicine, Raleigh, NC http://www.cvm.ncsu.edu University of California, Davis, School of Veterinary Medicine, Davis, CA http://www.vetmed.ucdavis.edu University of Florida, College of Veterinary Medicine, Gainesville, FL http://www.vetmed.ufl.edu University of Tennessee, College of Veterinary Medicine, Knoxville, TN http://web.utk.edu/~vetmed/default.html University of Wisconsin, School of Veterinary Medicine, Madison, WI http://www.vetmed.wisc.edu Each of these matched residency programs concentrates on exotic, wildlife, aquatic, and zoo animals in the context of a multiyear residency program through a veterinary teaching hospital and participates in the Veterinary Medical Intern-Resident Matching Program, administered through the American Association of Veterinary Clinicians. Individuals interested in residencies should contact the colleges offering such programs for admission requirements and application policies, and to introduce themselves to instructors. In addition, the dynamics of such programs may vary with regard to affiliations with regional aquariums and zoos. Other Internships Internships at aquaria or rehabilitation centers: Mystic Aquarium, Mystic, CT http://www.mysticaquarium.org National Aquarium at Baltimore, Baltimore, MD http://www.aqua.org New England Aquarium, Boston, MA http://www.neaq.org SeaWorld, San Diego, CA http://www.seaworld.com The Marine Mammal Center, Sausalito, CA http://www.tmmc.org These are veterinary internships, which are oriented to aquatic animal, for periods of 1 year or less, by arrangement, and are offered by institutions that are independent of the Veterinary Medical Intern-Resident Matching Program. The application policies and terms are determined by the admissions committee of each particular institution, and the content and experiences offered vary with the collection of animals being maintained, the research and veterinary services offered, and the affiliations established with other academic or research 0839_frame_C07 Page 109 Tuesday, May 22, 2001 10:42 AM Careers in Marine Mammal Medicine 109 institutions. It is advisable to contact these institutions directly to learn of their unique application policies. Internships at zoos with exposure to aquatic animal medicine: Birmingham Zoo, Birmingham, AL http://www.birminghamzoo.com Brookfield Zoo, Chicago, IL http://www.brookfieldzoo.org Columbus Zoo, Columbus, OH http://www.colszoo.org Louisville Zoological Gardens, Louisville, KY http://www.iglou.com/louzoo John G. Shedd Aquarium and Lincoln Park Zoo, Chicago, IL http://www.shednet.org and http://www.lpzoo.com Smithsonian National Zoological Park, Washington, D.C. http://natzoo.si.edu St. Louis Zoo, St. Louis, MO http://www.stlzoo.org These are veterinary internships offered by institutions independent of veterinary teaching hospitals, although most collaborate with regional research institutions and/or veterinary colleges. The conditions for application vary. It is advisable to contact these institutions directly to inquire about their programs. Internships affiliated with institutions or agencies: Alaska SeaLife Center, Seward, AK http://www.alaskasealife.org California Department of Fish and Game/UC Davis Wildlife Health Center, Davis, CA http://www.vetmed.ucdavis.edu/whc The Smithsonian Institution, Conservation and Research Center, Front Royal, VA http://www.si.edu/crc University of Alabama, Dauphin Island Sea Lab, Marine Sciences Program, Dauphin Island, AL http://www.disl.org The Wildlife Center of Virginia, Waynesboro, VA http://www.wildlifecenter.org Graduate Degree Programs Programs with aquatic and marine mammal emphasis (from departments outside veterinary schools) Department of Biology, San Francisco State University, San Francisco, CA http://www.sfsu.edu/~biology Department of Biology, University of Alaska Southeast, Juneau, AK http://www.jun.alaska.edu 0839_frame_C07 Page 110 Tuesday, May 22, 2001 10:42 AM 110 CRC Handbook of Marine Mammal Medicine Department of Pathobiology and Veterinary Sciences, University of Connecticut, Storrs, CT http://www.lib.uconn.edu/CANR/patho/index.html Department of Zoology, College of Biological Science, Guelph, Ontario, Canada http://www.uoguelph.ca/graduate studies Aquatic Pathobiology Center, Department of Pathology, School of Medicine, University of Maryland, Baltimore, MD http://som1.umaryland.edu/aquaticpath/ Aquatic Animal Disease Research and Diagnostic Center, School of Marine Science, The Virginia Institute of Marine Science, Gloucester Point, VA http://www.vims.edu/ These programs are graduate degree programs (i.e., Master’s and Ph.D.) offered by university departments or schools with faculty expertise in aquatic animal health. They are independent of veterinary teaching hospitals, although some, such as the Department of Pathobiology and Veterinary Sciences at the University of Connecticut, educate veterinarians in specialty training programs (e.g., veterinary anatomical pathology). The faculty of these programs determines the program offerings, and application policies vary according to the institution. This list of degree programs is not exhaustive; other programs are available and equally worthwhile. Interested individuals should investigate the course offerings and research opportunities at these and other institutions for programs that match their interests. Alternative sources of career opportunities include the Web sites, journal publications, and newsletters of following organizations: the American Association of Zoo Veterinarians, the Alliance of Veterinarians for the Environment, the American Veterinary Medical Association, the American Association of Zoos and Aquaria, the American Association of Wildlife Veterinarians, the Wildlife Disease Association, and the International Association for Aquatic Animal Medicine (see Chapter 8, The Electronic Whale). Other Related Programs American Veterinary Medical Association, Government Relations Division, Schaumburg, IL and Washington, D.C. http://www.avma.org Center for Coastal Studies, Provincetown, MA http://www.coastalstudies.org Center for Marine Conservation, Washington, D.C. http://www.cmc-ocean.org Center for Oceanic Research and Education, Essex, MA http://www.coreresearch.org Conference on Trade in Endangered Species, U.S. Fish and Wildlife Service, Washington, D.C. http://international.fws.gov Dolphin Internship Program, Honolulu, HI http://www.pacificwhale.org/internships Global Green, USA, Green Cross International, Washington, D.C. http://www.globalgreen.org Long Island University Southampton Campus College of Marine Science, Southampton, NY http://www.southampton.liu.edu 0839_frame_C07 Page 111 Tuesday, May 22, 2001 10:42 AM Careers in Marine Mammal Medicine 111 Moss Landing Marine Laboratories, Moss Landing, CA http://www.mlml.calstate.edu Oregon State University School of Oceanography, Newport, OR http://www.oce.orst.edu PAWS Wildlife Center, Lynnwood, WA http://www.paws.org/wildlife Scripps Research Institute, La Jolla, CA http:/www.scripps.edu SeaWorld, Orlando, FL; San Diego, CA; San Antonio, TX; Aurora, OH http://www.seaworld.com Stanford University Hopkins Marine Station of Behavior, Pacific Grove, CA http://www-marine.stanford.edu Texas A&M University, Galveston, TX http://www.marinebiology.edu University of Alaska College of Fisheries and Ocean Sciences, Fairbanks, AK http://www.uaf.edu University of Alaska Southeast Department of Marine Biology, Juneau, AK http://www.uas.alaska.edu University of California Long Marine Laboratory, Santa Cruz, CA http://www.ganesa.com/ecotopia/long.html University of Hawaii Marine Option Program, Honolulu, HI http://www.uhhmop.hawaii.edu University of Washington, College of Ocean and Fishery Sciences, Seattle, WA http://www.cofs.washington.edu Wildlife Conservation Society, Bronx, NY http://www.wcs.org Woods Hole Oceanographic Institute, Falmouth, MA http://www.whoi.edu Although less widely publicized and broader in scope than medicine alone, these programs relate to marine mammals, marine sciences, and marine research, policy, and/or environmental advocacy. Advanced Training Programs AQUAMED, An aquatic animal pathobiology course, sponsored by the Gulf States Consortium of Colleges of Veterinary Medicine at Auburn University, Mississippi State University, Louisiana State University, Texas A&M University, and the University of Florida; presented at the Louisiana State University School of Veterinary Medicine, Baton Rouge, LA http://www.vetmed.lsu.edu/aquamed.htm AQUAVET, A program in aquatic veterinary medicine, sponsored by the School of Veterinary Medicine at the University of Pennsylvania and the College of Veterinary Medicine at Cornell University; presented in collaboration with the Marine Biological Laboratory, the Northeast Fisheries Science 0839_frame_C07 Page 112 Tuesday, May 22, 2001 10:42 AM 112 CRC Handbook of Marine Mammal Medicine Center of the National Marine Fisheries Service, and Woods Hole Oceanographic Institute, Falmouth, MA http://zoo.vet.cornell.edu/public/aquavet/aquavet.htm ENVIROVET, An intensive short course in wildlife and ecosystem health in a developed country and an international development context, sponsored by the College of Veterinary Medicine, University of Illinois at Urbana-Champaign, IL http://www.cvm.uiuc.edu/vb/envirovet/ MARVET, An intensive short summer course in marine mammal medicine presented by Dr. Raymond Tarpley at Texas A&M [email protected] Fellowships American Association for the Advancement of Science, Washington, D.C. http://www.aaas.org American Veterinary Medical Association Congressional Science Fellowships, Washington, D.C. http://www.avma.org/avmf/csfapp.htm David H. Smith Conservation Research Fellowship Program http://consci.tnc.org/Smith.htm Harbor Branch Oceanographic Institute, Fort Pierce, FL http://www.hboi.edu International Oceanographic Foundation, Miami, FL http://www.rsmas.miami.edu/divs/mbf Sea Grant College Programs, Sea Grant Colleges and Universities nationwide (U.S.) search the web for Sea Grant College Fellowships Scientific Societies and Membership Organizations Alliance of Veterinarians for the Environment http://www.AVEweb.org American Association of Wildlife Veterinarians http://www.aawv.net American Association of Zoo Veterinarians http://www.worldzoo.org/aazv/aazv.htm American Cetacean Society http://www.acsonline.org American College of Zoological Medicine http://www.worldzoo.org/aczm American Veterinary Medical Association http://www.avma.org American Zoo and Aquarium Association http://www.aza.org European Association for Aquatic Mammals http://www.eaam.org 0839_frame_C07 Page 113 Tuesday, May 22, 2001 10:42 AM Careers in Marine Mammal Medicine 113 International Association for Aquatic Animal Medicine http://www.iaaam.org International Society for Ecosystem Health http://www.oac.uoguelph.ca/ISEH/index.htm National Sea Grant Program http://www.nsgo.seagrant.org Sarasota (FL) Dolphin Research Program http://www.mote.org/~rwells Society for Conservation Biology http://conbio.rice.edu/scb Student Conservation Association http://www.sca-inc.org The Society for Marine Mammalogy http://pegasus.cc.ucf.edu/~smm/about.htm Wildlife Conservation Society http://wildlifedisease.org Women’s Aquatic Network http://orgs.women.connect.com/WAN/welcome.html World Veterinary Association http://www.worldvet.org One additional Web site offers a large array of additional marine mammal Web resources: http://ourworld.compuserve.com/homepages/jaap/mmmain.htm Many of the resource organizations listed in this chapter maintain directories of their members by state to use for contact and networking purposes. They also produce newsletters and hold regular conferences and training workshops, which often involve roundtables on careers in marine mammal sciences and medicine (see Chapter 8, The Electronic Whale, for additional references related to marine mammal medicine). Recommendations and Conclusions Although this chapter offers no guarantees for finding a position in marine mammal medicine, if you follow the general recommendations, the six-step method, and access the information resources, as well as remember the six recommendations below, you will make your own luck and may actually find that perfect job in marine mammal medicine or conservation. • • • • • • Keep your eyes and ears open and keep networking. Be opportunistic. Find a mentor and work with that person as often as possible. Be patient. Maintain a public or professional presence. Be persistent. 0839_frame_C07 Page 114 Tuesday, May 22, 2001 10:42 AM 114 CRC Handbook of Marine Mammal Medicine Acknowledgments The authors thank Scott Newman and Gwen Griffith for peer-reviewing this chapter, and especially Jocelyn Catalla for her Web research and for her perspectives from the point of view of a student. In addition, the authors thank the members of the MarMam and Wildlife Health listserves for responding so enthusiastically to our listserve question: “What are your favorite marine mammal Web sites?” References Aguilar, R.F., and Mikota, S.K., 1996, To reach beyond: A North American perspective on conservation outreach, J. Zoo Wildl. Med., 27(3): 301–302. Bolles, R.N., 2000, What Color Is Your Parachute, 2000, Ten Speed Press, Berkeley, CA. Buss, D.D., 1998, Career development pathways in veterinary medicine, Convention notes, American Veterinary Medical Association, 135th Annual Convention, July 25–29: 114–115. Covey, S.R., 1990, Seven Habits of Highly Effective People, Covey Leadership Center, Provo, UT, 6 audiotapes. Deem, S.L., Cook, R.A., and Karesh, W.B., 1999, International opportunities in conservation medicine, Convention notes, American Veterinary Medical Association, 136th Annual Convention, July 10–14: 860–862. Dierauf, L.A., 1994, Potomac fever: I had it bad! in From the Lab to the Hill: Essays Celebrating 20 Years of Congressional Science and Engineering Fellows, Fainberg, A. (Ed.), American Association for the Advancement of Science, Washington, D.C., 31–35. Dierauf, L.A., 1996, The Career Changing Tool Kit, Connections Newsl. Alliance Vet. Environ., 1(1): 4–5. Doyle, K. (Ed.), 1999, The Complete Guide to Environmental Careers in the 21st Century, Island Press, Washington, D.C., 447 pp. Environmental Careers Organization, 1993, The New Complete Guide to Environmental Careers, Island Press, Washington, D.C., 364 pp. Fassig, S.M., 1998, Job-seeking skills, Convention notes, American Veterinary Medical Association, 136th Annual Convention, July 10–14: 753–755. Gerson, R., 1996, How to Create the Job You Want: Six Steps to a Fulfilling Career, Enrichment Enterprises, Austin, TX, 201 pp. Harris, J.M., 1998, Leo K. Bustad, DVM, Ph.D.: A veterinarian for all seasons, Convention notes, American Veterinary Medical Association, 136th Annual Convention, July 10–14: 449–450. Jacobson, S.K., Vaughan, E., and Miller, S.W., 1995, New directions in conservation biology: Graduate programs, Conserv. Biol., 9(1): 5–17. Johnson, S., 1998, Who Moved My Cheese? G.P. Putnam’s Sons, New York, 94 pp. King, L.J., 1996, Seven habits of highly effective globalized veterinarians, J. Vet. Med. Educ., Winter: 45. Lebovsky, A., 1994, The role of college and precollege science teachers in determining the education and career choices of Congressional fellows: A legacy of the class of 1990–1991, in From the Lab to the Hill: Essays Celebrating 20 Years of Congressional Science and Engineering Fellows, Fainberg, A. (Ed.), American Association for the Advancement of Science, Washington, D.C., 383–386. Marshall, K.E., 1998, Twenty laws of successful job hunting in the veterinary jungle, Convention notes, American Veterinary Medical Association, 136th Annual Convention, July 10–14: 758–760. Mashima, T.Y., 1997, Conservation and Environmental Career Opportunities, Connections Newsl. Alliance Vet. Environ., 2(1): 2–3. Meffe, G.K., 1999, Conservation medicine, Conserv. Biol., 13: 953–954. National Wildlife Federation, 2000, The 2000 Conservation Directory: A Guide to Worldwide Environmental Organizations, 45th ed., Washington, D.C., 544 pp. Peters, T., 1999, Reinventing Work: Fifty Ways to Transform Every Task into a Project That Matters, Alfred A. Knopf, New York, 28 pp. 0839_frame_C07 Page 115 Tuesday, May 22, 2001 10:42 AM Careers in Marine Mammal Medicine 115 Ryan, R., 2000, Sixty Seconds and You’re Hired, Penguin Books, New York, 175 pp. Smith, C.A., 1998a, How students and practitioners can prepare for international opportunities, Convention notes, American Veterinary Medical Association, 136th Annual Convention, July 10–14: 863–865. Smith, C.A., 1998b, Career Choices for Veterinarians: Beyond Private Practice, Smith Veterinary Services, Leavenworth, WA, 255 pp (see http://www.smithvet.com). Society for Conservation Biology, 2000, Symposium 7 on Conservation Medicine: The ecological context of health, 14th Annual SCB Meeting, Program and Abstracts, Missoula, MT, June 9–12: 102. Sylvester, N., 1998, Leadership skills for the new millennium: Interpersonal skills, Convention notes, American Veterinary Medical Association, 136th Annual Convention, July 10–14: 772–775. Thomas, J., and Odell, D., 1994, Strategies for pursuing a career in marine mammal science, Suppl. Mar. Mammal Sci., 10(2), April, The Society for Marine Mammalogy, Allen Press, Lawrence, KS, 14 pp. Wells, W.G., Jr., 1992, Working with Congress: A Practical Guide for Scientists and Engineers, American Association for the Advancement of Science, Washington, D.C., 153 pp. 0839_frame_C07 Page 116 Tuesday, May 22, 2001 10:42 AM 0839_frame_C08 Page 117 Tuesday, May 22, 2001 10:43 AM 8 The Electronic Whale Leslie A. Dierauf Introduction On January 1, 2000, an Alta Vista search engine Web search for “marine mammal medicine” yielded 1,196,440 matches! Just prior to sending the chapters for this textbook off to the publisher, a second search was conducted using the same search phrase and again on Alta Vista; this time we found 11,426,338 matches, a tenfold increase in sites in less than 1 year! We also asked a number of listserves what were their members’ favorite Web sites pertaining to marine mammal medicine; we received over 50 responses from people around the world, many of whose suggestions are noted in this chapter and in Chapter 7 (Careers). These kinds of numbers provide but a hint of the explosion of Internet-based information that is occurring. Accessing information and products on the Internet is the wave of the future, and the future is here today. Using Your Head on the Web Along with the World Wide Web to access information has come a tangle of difficulties. Reading materials on the Web really is no different from scientifically reviewing a potential paper for publication in a scientific journal. First, you must scrutinize the document and its authors to determine if the paper is even worthy of consideration. Then, using your best scientific judgment, you must decide if what you are reading is valid. The Web has no quality control per se; anyone in the world can represent him or herself as a marine mammal expert. Peer review is often lacking. Web writers span the spectrum from a leading expert in the field, who includes superb references and acknowledgments of peer reviewers, to someone with primarily an emotional interest in marine mammals, with minimal factual information and few to no scientific citations to back up assumptions or conclusions. We must each ensure that the marine mammal medicine and conservation information that comes online is accurate, scientifically based, and statistically valid. Since the public will have access to any scientific information online, electronic publications will need to be written in plain language, so that we, as veterinarians, communicate our scientific information to the public in an understandable and comprehensible fashion, just as if we were in an examination room trying to explain a disease process to a pet owner. Electronic information can be unbiased scientific results, or it can be advertisements for products, goods, or services of commercial ventures. Simply reading raw data can lead the information gatherer to misleading and incorrect conclusions. Accessing electronic information can be stressful. Try as we might, we expend more paper now in printing out the information we need than 0-8493-0839-9/01/$0.00+$1.50 © 2001 by CRC Press LLC 117 0839_frame_C08 Page 118 Tuesday, May 22, 2001 10:43 AM 118 CRC Handbook of Marine Mammal Medicine we did prior to the electronic age. Perhaps this too will change as time progresses. Perhaps in the future, Web sites of all kinds will have internal search methodologies that will allow a viewer to print specific sections of an article, and search more easily and quickly for specific detailed information, rather than getting ensnarled in the Web site. We look to a future in Internet technology where we all have the skills to know how best to frame a medical question, to use appropriate and accurate databases to access that information, to apply the answer to our marine mammal work, and, even more importantly, to be able to do this on-site in the field, just like a poolside rapid diagnostic test. So, you are urged to use your “sixth sense,” and if you have any doubts about what you are reading on a Web page, please be certain to check with known experts in the field before utilizing any potential diagnoses and the techniques and/or treatments the Web article recommends. Reference Databases General Biomedical and Veterinary Medical Sites Conducting searches of the scientific literature by traditional methods, such as a library search, can be time-consuming, tedious, and expensive. Once you find the article you need, if it is in the library at all, you then need to photocopy it and carry it home to read. With each passing year, however, online searchable scientific reference databases become more numerous, more helpful, and more easily browsed. Following is a list of those most applicable online reference sources for accessing biomedical, veterinary medical, and/or marine mammal medical literature. The University of Michigan School of Information and Library Studies manages a series of Internet resource guides covering a huge number of subjects, one of which is veterinary medicine: http://www.lib.umich.edu/chhome.html Michigan also has an electronic library that provides reliable access to scientific Internet resources. The site listed here allows you to enter the science and environment collection: http://mel.lib.mi.us The San Diego Library Consortium is a searchable database by author, subject, title, or biomedical subject, and links to other California state system universities, so it is quite complete. Access it at: http://circuit.sdsu.edu The U.S. Department of Agriculture, Food and Drug Administration maintains a database of biological collections on the Internet. The database covers specific subject matter and a large array of journals, which can be accessed: http://vm.cfscan.fda.gov/~frf/biologic.html ProMED is a scientific information request site, on which animal science papers can be located. Although designed for physicians, this site contains invaluable diagnostic and therapeutic information and, therefore, can be useful in marine mammal clinical practice: http://promed-windows.com Grateful Med and PubMed through the U.S. National Library of Medicine homepage is your entry to searches of Medline, standard medical vocabulary, public health, general medical, veterinary medical, and scientific literature abstracts, catalogs, databases, and disease research. These databases give you access to more than 20 billion scientific citations and abstracts, and cover French, Spanish, Portuguese, and Russian biomedical literature, in addition to English. The National Cancer Institute has similar online access to biomedical topics and literature. 0839_frame_C08 Page 119 Tuesday, May 22, 2001 10:43 AM The Electronic Whale 119 U.S. National Library of Medicine http://www.nlm.nih.gov Grateful Med http://igm.nlm.nih.gov/igm_intro/title.html PubMed http://www.nlm.nih.gov/pubs/factsheets/pubmed.html National Cancer Institute http://www-library.ncifcrf.gov SNOMED (Systemized Nomenclature of Human and Veterinary Medicine) is a conceptbased reference site related to record keeping, laboratory and clinical pathology system tracking, decision-support systems, disease registries, and more. It also identifies and defines veterinary medical standardized terminology, rather like a veterinary medical dictionary (Monti, 2000): http://www.snomed.org The U.S. Fish and Wildlife Service, National Conservation Training Center (NCTC) in Shepherdstown, West Virginia has an online conservation library. Articles, journals, and scientific literature related to a multitude of conservation issues can be searched by accessing: http://training.fws.gov/library NetVet is an ingenious site (also accessible through the AVMA Web site) developed in 1993 by a veterinarian now at Washington University in St. Louis, Missouri. The site contains a wealth of information about veterinary medical and animal resources available on the Internet; it references hundreds of veterinary and animal health–related Web sites through its Electronic Zoo, and is updated regularly. In 1995 alone, more than 650,000 computer users referenced this site. Within NetVet is a general reference site for writers, which includes dictionaries, encyclopedias, virtual libraries, and other valuable resources you may need if you are writing scientific or lay literature on marine mammals. Be sure to contact the NetVet site and have your new domains included on the Electronic Zoo list. The NetVet site can give your scientific publications excellent public and scientific exposure. American Veterinary Medical Association http://www.avma.org NetVet http://netvet.wustl.edu NetVet specific to Marine Mammal Information http://netvet.wustl.edu/marine.htm Model Web Sites and Evidence-Based Medicine The Health on the Net (HON) Foundation in Switzerland is a nonprofit organization intent on demonstrating the benefits of the Internet and related technologies to the fields of medicine and health care. Available in both English and French, HON includes Web site listings, journal articles, multimedia, and health news to provide integrated search results. The Organized Medical Networked Information (OMNI) is the self-described “United Kingdom’s gateway to high quality biomedical Internet resources.” OMNI relies on “unbiased, high quality, internetbased resources relevant to the medical, biomedical, and health communities.” These model Web sites insist that medical information on the Internet be peer-reviewed and “given [only] by medically trained and qualified professionals” (HON). Both sites welcome relevant resource 0839_frame_C08 Page 120 Tuesday, May 22, 2001 10:43 AM 120 CRC Handbook of Marine Mammal Medicine additions. The veterinary profession would do well to model its information for the Internet following the guidelines of these organizations and to utilize the opportunity to add its scientific works to these databases. Health on the Net http://www.hon.ch Organized Medical Networked Information http://omni.ac.uk Evidence-based medicine has a number of health and human medicine guidelines (AAFP, 1999), which the marine mammal medicine community would be wise to follow. For use in your scientific writing, the author recommends the following sites: University of Washington Library http://www.hslib.washington.edu/clinical/guidelines.html U.S. Government Guidelines http://www.guideline.gov Marine Mammal–Related Listserves One of the more rapid ways to gather information is through a listserve. A listserve is a mail system for creating, managing, and controlling electronic mailing lists of names and addresses. Messages, questions, answers, and announcements are sent to groups of people with similar interests. You can subscribe to and unsubscribe from a listserve as your time and commitment warrant. The two listserve sources marine mammal scientists use most commonly are MarMam and WildlifeHealth. To subscribe to the MarMam listserve, send an e-mail message to: [email protected] For the WildlifeHealth listserve, send an e-mail message to: [email protected] You can join these listserves by typing in the Web address, then in the body of the e-mail inserting “subscribe” “marmam” or “wildlifehealth” followed by “Yourfirstname Yourlastname” on the subject line, and sending it electronically. To post messages, use: [email protected] and [email protected] To contact the editors for MarMam, e-mail: [email protected] To contact WildlifeHealth within the Wildlife Information Network in the United Kingdom, e-mail: [email protected] MarMam—Marine Mammal Conservation and Discussion—list functions as an exchangeof-ideas location. The types of messages posted at MarMam range from requests for information to case studies to announcements of meetings and training opportunities to book reviews and journal abstracts. The WildlifeHealth listserve, originally set up through the National Wildlife Health Center (NWHC), which is a science center within the Biological Resources Division of the U.S. Geological Survey in Madison, Wisconsin, addresses wildlife health and 0839_frame_C08 Page 121 Tuesday, May 22, 2001 10:43 AM The Electronic Whale 121 facilitates the exchange of questions, answers, general information, case histories, and other concerns regarding wildlife health; any member of the listserve can post information, questions, answers, or concerns at the site. Both sites offer free access and unlimited use. Each site is archived, so past messages can be viewed and retrieved. Other Internet Discussion and Marine Mammal Information Lists There are currently at least four major information sites where e-mail discussion groups, chat rooms, announcements, and information lists can be registered, advertised, and accessed, including Lyris (Lyris Technologies, Inc., Berkeley, CA), Majordomo (Great Circle Associates, Mountain View, CA), LISTSERV (L-Soft, Landover, MD), and ListProc (Corporation for Research and Educational Networking (CREN, Washington, D.C.). Lyris http://www.lyris.net Majordomo http://www.greatcircle.com/majordomo [shareware] LISTSERV http://www.listserv.net CREN http://www.listproc.net [for UNIX users] List Identification http://tile.net/lists The sites listed here are excellent linkage points for marine mammal medicine and science sites. Dalhousie University http://is.dal.ca/~whitelab/links.htm Five Colleges Coastal & Marine Sciences http://www.science.smith.edu/departments/marine Marine Mammal Net http://marinemammal.net National Marine Mammal Laboratory http://nmml.afsc.noaa.gov/library/resources/resources.htm Whale Net http://whale.wheelock.edu Online Marine Mammal Journals and Textbooks In this age of electronic information, many veterinary medical journals, including marine mammal journals, are online, and textbooks are expected to be online soon. If you are an electronic textbook editor, ensure that your authors electronically submit their publications only through a quality-control gateway, and only after peer review. Materials with highquality electronic information will serve the public well, will improve accessibility, and will lead to lower costs for accessing information and greater opportunity for interacting electronically with colleagues regarding marine mammal medical information. This is already happening on a regular basis in the medical profession (BioMedicina, 1999) and at academic institutions. However, even in the medical profession, not enough physicians 0839_frame_C08 Page 122 Tuesday, May 22, 2001 10:43 AM 122 CRC Handbook of Marine Mammal Medicine have the skills and abilities that are required to frame diagnostic queries or clinical questions or to use the databases available to locate and apply the answers to the care of their patients. The author urges marine mammal veterinary medical specialists to participate in this arena of high-quality and quality-controlled electronic information. The journal Marine Mammal Science is available online, as are additional journal and textbook reference materials. Marine Mammal Science online http://pegasus.cc.ucf.edu/~smm/mms.htm Library of Michigan http://mel.lib.mi.us/science/auth.html National Council for Science and the Environment http://www.cnie.org/journal.htm Nova Southeastern University, Ocean Center Library http://www.nova.edu/cwis/oceanography/library.html San Diego State University http://circuit.sdsu.edu University of Buffalo Science and Engineering Library http://ublib.buffalo.edu/libraries/units/sel/collections/ejournal2.html#a University of Montreal Beluga Whale Info http://www.medvet.umontreal.ca/services/beluga/index_an.html U.S. Fish and Wildlife Service Literature Search http://training.fws.gov/library Fellowships, Foundations, and Grants Fellowships Congressional Science Fellowships are paid positions, sponsored by the American Veterinary Medical Foundation (AVMF) and the American Association for the Advancement of Science (AAAS). They are awarded competitively to scientists, who serve for 1 year in Washington, D.C., for either the U.S. House of Representatives or the U.S. Senate, acting as science advisors, researchers, and staff consultants to members of Congress or Congressional committees. An annual stipend is paid by the sponsoring association. AVMF http://www.avmf.org AAAS http://www.aaas.org There are 29 Sea Grant Colleges across the United States (associated with Land Grant Colleges) that offer Sea Grant Fellowships, where university scientists, educators, and outreach specialists are competitively chosen to work on Capitol Hill, on either House or Senate staff, in positions sponsored by Sea Grant, for as long as 1 year. Information on these fellowships can be accessed at: http://www.nsgo.seagrant.org 0839_frame_C08 Page 123 Tuesday, May 22, 2001 10:43 AM 123 The Electronic Whale Foundations The Foundation Directory, for many years available at libraries, is now available online, and has listings by state and subject matter for private foundations offering grants to nonprofit organizations for special projects and operating expenses. Find the directory at: http://www.fconline.fdncenter.org Grants The Grantsnet Web site is of great assistance in accessing grantors, as well as in providing tips on grant writing, career development, and foundation news. The site is accessed at: http://www.grantsnet.org Federal Government Listings Federal jobs listing: Federal Office of Personnel Management http://www.usajobs.opm.gov U.S. federal government listings: National Marine Fisheries Service, Silver Spring, MD http://www.nmfs.gov U.S. Agency for International Development, Washington, D.C. http://www.usaid.gov U.S. Department of Agriculture, Beltsville, MD http://www.usda.gov U.S. Department of the Interior, Washington, D.C. http://www.doi.gov U.S. Environmental Protection Agency, Washington, D.C. http://www.epa.gov U.S. Fish and Wildlife Service, Washington, D.C. http://www.fws.gov U.S. Geological Service (research arm of the Department of the Interior), Washington, D.C. http://www.usgs.gov National Park Service, Washington, D.C. http://www.nps.gov Federal listings abroad: Canadian Department of Fisheries and Oceans http://www.ncr.dfo.ca Miscellaneous Electronic Resources* A number of the organizations listed here also offer funds for research, as well as general veterinary and/or specific marine mammal medical information. Argus Clearinghouse http://www.clearinghouse.net/ * In alphabetical order; in the United States and abroad. For subject-oriented topics, including science 0839_frame_C08 Page 124 Tuesday, May 22, 2001 10:43 AM 124 CRC Handbook of Marine Mammal Medicine AVMA’s NOAH http://www.avma.org/network.html Network of Animal Health Cetacean Research Unit http://www.whalecenter.or The Whale Center of New England College of the Atlantic http://www.coa.edu/internships Marine mammal courses and internships Dalhousie University Whale Laboratory http://is.dal.ca/~whitelab/index.htm Publications, information, and programs Duke University Marine Mammal Laboratory http://www.env.duke.edu/marinelab/ marine.html Marine resources, biomedical information, and library Eckerd College Marine Mammal Courses http://www.eckerd.edu Marine academic courses and programs Institut Maurice Lamontagne http://www.qc.dfo-mpo.gc.ca/iml Canadian oceans and fisheries information (French and English) International Association for Bear Research http://www.bearbiology.com Specific scientific information on bears (including polar bears) International Biodiversity Measuring Course http://www.si.edu/simab/biomon.htm Standardized protocols for biodiversity monitoring International Marine Animal Trainers Association http://www.imata.org Marine mammal science and public display International Marine Mammal Association, Inc. http://www.imma.org Marine mammal conservation and news International Whaling Commission http://ourworld.compuserve.com/ homepages/iwcoffice International convention for regulation of whaling Ionian Dolphin Project http://www.tethys.org Tethys Research Institute (Italian and English) Manatee Awareness Coalition http://www.fmri.usf.edu/mammals.htm Protecting Florida’s marine resources Marine Mammal Careers (see also Chapter 7, Careers) http://www.seaworld.org/careers SeaWorld http://www.pegasus.cc.uct.edu/~smm Society for Marine Mammalogy http://www.rsmas.miami.edu/iof International Oceanographic Foundation Marine Mammal and Seabirds Course http://www.unb.ca/web/huntsman University of New Brunswick, Canada National Marine Educators Association http://www.marine-ed.org Marine education, science, and research 0839_frame_C08 Page 125 Tuesday, May 22, 2001 10:43 AM 125 The Electronic Whale National Marine Mammal Laboratory http://nmml.afsc.noaa.gov Marine mammal research in Northwest United States North Atlantic Marine Mammal Commission http://www.nammco.no Norway, Iceland, and Greenland marine mammal conservation and management North Pacific Marine Mammal Research Consortium http://www.marinemammal.org Bering Sea marine mammal research Polar Bears Alive http://www.polarbearsalive.org Polar bear and Arctic habitat information Seal Conservation Society http://www.greenchannel.com/tec Marine mammal welfare and conservation Universita degli Studi di Pavia http://www.unipv.it/cibra Marine mammal information (Italian and English) Whales on the Net http://whales.magna.com.au/home.html Cetacean information Wildlife Disease Association http://www.vpp.vet.uga.edu/wda Wildlife diseases, including marine mammals Meetings and Proceedings on CD-ROM The following association annual meetings have aquatic animal medicine sessions, and proceedings of each meeting are available on CD-ROM. American Veterinary Medical Association (each year in July) Environmental Affairs, Aquatic Medicine, Public Health Sessions http://www.avma.org North American Veterinary Conference (each year in February in Orlando, FL) Aquatic Medicine, Wildlife Health Sessions http://vetshow.com/navc International Association for Aquatic Animal Medicine (each year in May) Aquatic Animal Medicine http://www.iaaam.org Western States Veterinary Conference (each year in February in Las Vegas, NV) Aquatic Medicine, Wildlife Health Sessions http://www.wvc.org Electronic Addresses for Other Chapters in This Book Other pertinent Web sites specific to the scientific topics in each chapter of this book are noted in those chapters. You are directed to Chapter 7 (Careers) for information on continuing education opportunities in marine mammal medicine, as well as for a list of scientific societies and membership organizations related to marine mammal medicine. The Diagnostic Imaging Section of this book (Chapters 24 through 28) also contains a number of relevant technical Web site addresses. 0839_frame_C08 Page 126 Tuesday, May 22, 2001 10:43 AM 126 CRC Handbook of Marine Mammal Medicine Disclaimer Because the number of Web sites related to marine mammal medicine is growing exponentially, the author cannot take responsibility for the complete exactness of the Internet addresses in this chapter. Although Web access to each site mentioned in this chapter was accomplished multiple times, be advised that Web site addresses change. To access the information if Web site addresses do change, we have provided the full organizational name and brief subject contents for each item in this chapter in order for you to conduct a search for the particular item of interest through standard search engines on the Web. The author, in accessing Internet Web sites in preparation of this chapter, has attempted to weed out those sites that are not of apparent high quality and/or value. Conclusions One thing is certain, however. If you access the marine mammal medicine, conservation, and information sites included in this chapter, you will be better educated, not only in how to access the information, but also in how to read it with a critical eye and utilize it to your greatest advantage. The future of World Wide Web–based information systems is better designed Web sites, with consistency across veterinary medical information sites. In addition, the use of the Internet takes practice, just as any professional endeavor. The more you use the Web to access critical marine mammal resources and the more you attend seminars and continuing education sessions at conventions on accessing the Web, the better prepared you will be to manage and learn from the information you receive from the Internet. If we do this, along with our daily clinical practice and scientific reading, the marine mammals in our care will receive the best diagnostic and therapeutic approaches we can gather and implement. References AAFP (American Academy of Family Practice), 1999, Computer Zoo, AAFP, Annual Meeting, Orlando, FL, 13 pp. BioMedicina, 1999, Medicine on the Internet: Surgery and ophthalmology in the information age, BioMedicina, 2(6): 295–298. Monti, D.J., 2000, SNOMED browser latest in informatics, J. Am. Vet. Med. Assoc. 216(7): 1049. 0839_frame_C09 Page 127 Tuesday, May 22, 2001 10:43 AM II Anatomy and Physiology of Marine Mammals 0839_frame_C09 Page 128 Tuesday, May 22, 2001 10:43 AM 0839_frame_C09 Page 129 Tuesday, May 22, 2001 10:43 AM 9 Gross and Microscopic Anatomy Sentiel A. Rommel and Linda J. Lowenstine Introduction The California sea lion (Zalophus californianus) (Figure 1), Florida manatee (Trichechus manatus latirostris) (Figure 2), harbor seal (Phoca vitulina) (Figure 3), and bottlenose dolphin (Tursiops truncatus) (Figure 4) are used in this chapter to illustrate gross anatomy. These species were selected because of their availability and the knowledge base associated with them.* Gross anatomy of the sea otter (Enhydra lutra) is presented in Chapter 44 covering medical aspects of that species. Illustrations of the (A) external features, (B) superficial skeletal muscles, (C) relatively superficial viscera with skeletal landmarks, (D) circulation, body cavities, and some deeper viscera, and (E) skeleton are presented as five separate “layers” on the same page for each of the four species. These illustrations, based on dissections by one of the authors (S.A.R.), are of intact carcasses and thus help show the relative positions of organs in the live animals. The major lymph nodes are illustrated, but to simplify the illustrations, most are not labeled. The drawings represent size, shape, and position of organs in a healthy animal; the skeleton is accurately placed within the soft tissues and body outline. The scale of the drawings is the same for each species so that vertical lines can be used to compare features on all five; a photocopy onto a transparency would allow the reader to compare layers directly. Names of structures are labeled with three-letter abbreviations.** A brief figure legend helps the reader apply basic veterinary anatomical knowledge to the marine mammals illustrated. The style found in Miller’s Anatomy of the Dog (Evans, 1993) is followed as much as possible. Most technical terms follow the Illustrated Veterinary Anatomical Nomenclature by Schaller (1992). Recent comparative work on anatomy of marine mammals is found in Pabst et al. (1999), Rommel and Reynolds (2000; in press), and Reynolds et al. (in press). Older but valuable anatomical works include Murie (1872; 1874), Schulte (1916), Howell (1930), Fraser (1952), Slijper (1962), Green (1972), St. Pierre (1974), Bonde et al. (1983), King (1983), and Herbert (1987). *A set of illustrations of a mysticete would be valuable, but as space is limited and they are less likely to be under veterinary care, we chose an odoctocete; the skeletal anatomy of the right whale (Eubalaena glacialis) is compared with that of other marine mammals in Rommel and Reynolds (in press). **Abbreviations in the text use capital letters to refer to the label on the structure. The first letter refers to the layer (A being external features at the top and E the skeleton) followed by a hyphen and then the abbreviation of the structure. For example, D-HAR refers to the heart on layer D. 0-8493-0839-9/01/$0.00+$1.50 © 2001 by CRC Press LLC 129 0839_frame_C09 Page 130 Tuesday, May 22, 2001 10:43 AM 130 CRC Handbook of Marine Mammal Medicine FIGURE 1 Left lateral illustrations of a healthy California sea lion (Zalophus californianus). Based on dissections by S.A.R., with details and nomenclatures from the literature: Murie, 1874; Howell, 1930; English, 1976a. Thanks to Rebecca Duerr for many helpful suggestions. (© Copyright S. A. Rommel. Used with permission of the illustrator.) (Layer A) External features. The following abbreviations are used as labels: ANG = angle of mouth; ANS = anus; AXL = axilla, flipperpit; CAL = calcaneus, palpable bony feature; EAR = external auditory opening, ear; EYE = eye; INS = cranial insertion of the extremity; flipper, fin, and/or fluke; NAR = naris; OLC = olecranon, palpable bony feature; PAT = patella, palpable bony feature; PEC = pectoral limb, fore flipper; PEL = pelvic limb, hind flipper; PIN = pinna, external ear (as opposed to external ear opening); SCA = dorsal border of the scapula, palpable (sometimes grossly visible) bony feature; TAI = tail; UMB = umbilicus; UNG = unguis, finger and toe nails; U/G = urogenital opening; VIB = vibrissae. (Layer B) The superficial skeletal muscles. The layer of skeletal muscles just deep to the blubber and panniculus muscles. The following abbreviations are used as labels: ANS = anus; BIF = femoral biceps; BRC = brachiocephalic; DEL = deltoid; DIG = digastric; EAM = external auditory meatus; EXT = external oblique; FAS = fascia; F,S,B&P = fur, skin, blubber, and panniculus muscle (where present) cut along midline; GLU = gluteals; LAT = latissimus dorsi; MAM = mammary gland; MAS = masseter; PECp = deep (profound) pectoral; PECs = superficial pectoral; REC = rectus abdominis; SAL = salivary gland; SER = serratus; nipple; STC = sternocephalic; TFL = tensor fascia lata; TMP = temporalis; TRAc = trapezius, cervical portion; TRAt = trapezius, thoracic portion; TRI = triceps brachii; UMB = umbilicus. (Layer C) The superficial internal structures with “anatomical landmarks.” This perspective focuses on relatively superficial internal structures; the other important bony or soft “landmarks” are not necessarily visible from a left lateral view, but they are useful for orientation. The relative size of the lung represents partial inflation—full inflation would extend the lung margins to the distal tips of ribs. The female is illustrated because there is greater variation in uterine anatomy than in testicular and penile anatomy; note, however, that only the sea lion (of the illustrated species) is scrotal (actually the sea lion testes migrate into the scrotum in response to environmental temperature). The following abbreviations are used as labels (structures in midline are in type, those off-midline are in italics): ANS = anus; AXL lnn = axillary lymph nodes; BLD = urinary bladder; F,S&B = fur, skin, blubber (cut at midline); HAR =heart; HYO = hyoid apparatus; INT = intestines; ILC = lliac crest; KID = left kidney; LIV = liver; LUN = lung (note that the lung extends under the scapula); MAN = manubrium of the sternum; OVR = left ovary; PAN = pancreas; PAT = patella; PSC ln = prescapular lymph nodes; RAD = radius; REC = rectum; SAL = salivary glands; SCA = scapula; SIG ln = superficial inguinal lymph node; SPL = spleen; STM = stomach; TIB = tibia; TMP = temporalis; TRA = trachea; TYR = thyroid gland; TYM = thymus gland; ULN = ulna; VAG = vagina. (Layer D) A view slightly to the left of the midsagittal plane illustrating the circulation, body cavities, and selected organs. Note that the diaphragm separates the heart and lungs from the liver and other abdominal organs. The following abbreviations are used as labels (structures on the midline are in normal type, those off-midline are in italics): AAR = aortic arch; ADR = adrenal gland; ANS = anus; AOR = aorta; ARH = aortic hiatus; AXL = axillary artery; BIF = tracheobronchial bifurcation; BLD = urinary bladder; BRC = bronchus; BRN = brain; CAF = caval foramen; CAR = carotid artery; caMESa = caudal mesenteric artery; CEL = celiac artery; CRZ = crus of the diaphragm; crMESa = cranial mesenteric artery; CVC = vena cava, between diaphragm and heart; DIA = diaphragm, cut at midline, extends from crura dorsally to sternum ventrally; ESO = esophagus (to the left of the midline cranially, on the midline caudally); ESH = esophageal hiatus; F,S&B = fur, skin, blubber (cut at midline); HAR = heart; HYO = hyoid bones; KID = right kidney; LIV = liver, cut at midline; LUN = right lung between heart and diaphragm; MAN = manubrium of sternum; OVR = left ovary; PAN = pancreas; PUB = pubic symphysis; PULa = pulmonary artery, cut at hilus of lung; PULv = pulmonary vein, cut at hilus of lung; REC = rectum; REN = renal artery; SPL = spleen; STM = stomach; STR = sternum, sternabrae; TNG = tongue; TRA = trachea; TYM = thymus gland; TYR = thyroid gland; UMB = umbilicus; UTR = uterus; VAG = vagina; VRT- vertebral artery; XIP = xyphoid process of the sternum. (Layer E) The skeleton. Regions of the vertebral column (cervical, thoracic, lumbar, sacral, and caudal) are abbreviated (in lower case) as cer, tho, lum, sac, and cau, respectively, and are used as modifiers after an abbreviation in caps and a comma. If a specific vertebra is labeled, it will be represented by a capitalized first letter (for caudal, Ca will be used) and the vertebral number, i.e., first cervical = C1. The following abbreviations are used as labels: CAL = calcaneus; CAN = canine tooth (not present in cetaceans or manatees); DIG = digits; FEM = femur; FIB = fibula; HUM = humerus; HYO = hyoid bones; ILC = iliac crest of the pelvis; LRB = last, or caudalmost, rib; MAN = mandible; MNB = manubrium, the cranialmost bony part of the sternum; NSP = neural spine (spinous process), e.g., thoracic neural spines = NSP, tho; OLC = olecranon; ORB = orbit; PAT = patella; RAD = radius; SCA = scapula; STN = sternum, composed of individual sternabrae; SRB = sternal ribs, costal cartilages; TIB = tibia; TMF = temporal fossa; TPR = transverse process, e.g., TPR, C1 = transverse process of the first cervical vertebra; ULN = ulna; VBR = vertebral ribs; ZYG = zygomatic arch. 0839_frame_C09 Page 131 Tuesday, May 22, 2001 10:43 AM 131 Gross and Microscopic Anatomy OLC PIN SCA EAR PAT EYE ANS CAL TAI UNG NAR ANG VIB INS A U/G PEL INS U/G UMB AXL UNG PEC LAT TRAt EAM BRC SER FAS TRAc TMP F, S & B TFL GLU BIF ANS MAS DIG SAL STC B F, S, B & P MAM DEL EXT PECs PECp TRI LUN AXL Inn 1-3 HYO TMP UMB REC F, S & B SCA PSC Inn PAN KID ILC REC EYE ANS VAG SAL TYR C SIGIn TRA MAN HUM HAR TYM LIV SPL STM OVR INT PAT BLD TIB RAD ULN c Rommel 2000 ESO ESO AAR F, S & B BRN CAF CVC DIA CEL crMESa AOR ESH ESO LUN ARH BRC PAN CRZ ADR REN KID caMESa PUB TNG ANS HYO VAG TYR D UTR CAR TRA BLD VRT BIF MAN TMF OVR REC AXL TYM PULa PULv SPL HAR STR DIA XIP LIV STM UMB NSP tho NSP, cer VBR LRB SCA NSP, Ium ILC ORB NSP, cau CAL CAN MAN E ZYG HYO TPR, C1 TIB PAT MNB FIB FEM DIG HUM SRB OLC STN RAD ULN DIG 0839_frame_C09 Page 132 Tuesday, May 22, 2001 10:43 AM 132 CRC Handbook of Marine Mammal Medicine FIGURE 2 Left lateral illustrations of a healthy Florida manatee (Trichechus manatus latirostris). Based on dissections by S.A.R., with details and nomenclatures from the literature: Murie, 1872; Domning, 1977; 1978; Rommel and Reynolds, 2000. Thanks to D. Domning for suggestions on the muscle illustration. (© S. A. Rommel. Used with permission of the illustrator.) (Layer A) External features. The following abbreviations are used as labels: ANG = angle of mouth; ANS = anus; AXL = axilla; EAR = external auditory opening, ear; EYE = eye; FLK = fluke entire caudal extremity in manatees; flukes = entire caudal extremity in dugongs; INS = cranial insertion of the extremity, flipper and/or fluke; NAR = naris; OLC = olecranon, palpable bony feature; PEC = pectoral limb, flipper; PED = peduncle, base of tail, between anus and fluke; SCA = dorsal border of the scapula, palpable bony feature in emaciated individuals; UMB = umbilicus; UNG = unguis, fingernails; U/G = urogenital opening; VIB = vibrissae. (Layer B) The superficial skeletal muscles. The layer of skeletal muscles just deep to the blubber and panniculus muscles. The following abbreviations are used as labels: ANS = anus; CEP = cephalohumeralis; DEL = deltoid; EXT = external oblique; FAS = fascia; S,B&P = skin, blubber, and panniculus muscle (where present) cut along midline; IIN = internal intercostals; ILC = iliocostalis; ITT = intertransversarius; LAT = latissimus dorsi; LEN = levator nasolabialis; LON = longissimus; MAM = mammary gland, in axillary region, thus partly hidden under the flipper; MEN = mentalis; MND = mandibularis; PAN = panniculus, illustrated using dotted lines, is a robust and dominant superficial muscle; a layer of blubber is found on both the medial and lateral aspects of this muscle; REC = rectus abdominis; SLT = mammary slit, nipple; SPC = sphincter colli; SVL = sarcoccygeus ventralis lateralis; TER = teres major; TMP = temporalis; TRA = trapezius; TRI = triceps brachii; UMB = umbilicus, XIN = external intercostals. (Layer C) The superficial internal structures with “anatomical landmarks.” This perspective focuses on relatively superficial internal structures. Skeletal elements are included for reference, but not all are labeled. The left kidney (not visible from this vantage in the manatee) is illustrated. The relative size of the lung represents partial inflation. The following abbreviations are used as labels: ANS = anus; BLD = urinary bladder (dotted, not really visible in this view); BVB = brachial vascular bundle; CHV = chevrons, chevron bones; EYE = the eye (note how small it is); HAR = heart; HUM = humerus; INT = intestines; note the large diameter of the large intestines; KID = left kidney, not visible from this vantage in the manatee; LIV = liver; LUN = lung (note lung extends under scapula, and over heart); OVR = left ovary; PEL = pelvic vestige; RAD = radius; SAL = salivary gland; S&B = skin and blubber; SCA = scapula; SIG ln = superficial inguinal lymph node; S,B&P = skin, blubber, and panniculus muscle, cut at midline; STM = stomach; TMJ = temporomandibular joint; TYM = thymus gland; ULN = ulna; UMB = umbilical scar; UTR = uterine horn; VAG = vagina. (Layer D) A view slightly to the left of the midsagittal plane illustrates the circulation, body cavities, and selected organs. Note that the diaphragm of the manatee is unique and that the distribution of organs and the separation of thoracic structures from abdominal structures requires special consideration. The following abbreviations are used as labels (structures on the midline are in normal type, those off-midline are in italics): AAR = aortic arch; ADR = left adrenal gland; ANS = anus; AOR = aorta; AXL = axillary artery; BLD = urinary bladder; BRN = brain; BVB = brachial vascular bundle (cut); CAF = caval foramen; CAR = carotid artery; CDG = cardiac gland; CEL = celiac artery; CER = cervix; CHV = chevron bones; CRG = cardiac gland; CVB = caudal vascular bundle; DUO = duodenum; ESO = esophagus (to the left of the midline cranially, on the midline caudally); EXI = external iliac artery; HAR = heart; KID = right kidney; LIV = liver, cut at midline; OVR = right ovary; PAN = pancreas; PULa = pulmonary artery, cut at hilus of lung; PULv = pulmonary vein, cut at hilus of lung; REC = rectum; REN = renal artery; S&B = skin and blubber; SKM = skeletal muscle; SM&B = skin, muscle, and blubber (cut at midline); SPL = spleen; STM = stomach; STR = sternum; TNG = tongue; TRA = trachea; TRS = transverse septum; TYM = thymus gland; TYR = thyroid gland; UMB = umbilical scar; UTR = uterus; VAG = vagina. (Layer E) The skeleton. Regions of the vertebral column (cervical, thoracic, lumbar, sacral, and caudal), are abbreviated (in lowercase) as cer, tho, lum, sac, and cau, respectively, and are used as modifiers after an abbreviation in caps and a comma. If a specific vertebra is labeled, it will be represented by a capitalized first letter (for caudal, Ca will be used) and the vertebral number, i.e., first cervical = C1. The following abbreviations are used as labels: CHV = chevrons, chevron bones; DIG = digits, columns of finger bones; HUM = humerus; HYO = hyoid apparatus; HYP = hypapophysis, ventral midline vertebral process; LRB = last, or caudalmost, rib; LVR = last, or caudalmost, vertebra; MAN = mandible; NSP = neural spine (spinous process), e.g., thoracic neural spines = NSP, tho; OLC = olecranon; ORB = orbit; PEL = pelvic bone; RAD = radius; SCA = scapula; STN = sternum, if sternabrae are commonly fused; SBR = sternal ribs, costal cartilages; TMF = temporal fossa; TPR = transverse process, C1; ULN = ulna; VBR = vertebral ribs; XNR = external (bony) nares; XIP = xyphoid process, cartilaginous caudal extension of the sternum; ZYG = zygomatic process of the squamosal. 0839_frame_C09 Page 133 Tuesday, May 22, 2001 10:43 AM 133 Gross and Microscopic Anatomy OLC SCA FLK EAR PED EYE NAR INS VIB A ANS INS ANG AXL U/G UMB PEC UNG U/G ILC XIN IIN LAT LON TER S, B, & P TRA CEP ITT FAS TEM LEN SVL MEN SPC DEL MND ANS TRI MAM SLT B S, B, & P UMB REC S&B LUN KID (not visible) LUN LUN SCA PAN EXT UTR LIV OVR PEL TMJ S&B SAL EYE CHV HUM ANS TYM C BVB STM CRG HAR INT (lg) RAD INT (sml) UMB SIG In BLD INT (lg) S, B & P ULN PULa AXL AAR TRA PULv ESO AOR CRG AOR CEL ADR c Rommel 2000 SKM REN OVR EXI CVB TYR S&B BRN CAR CHV BVB TNG SKM TYM D ANS REC HAR STR CAF TRS LIV STM SPL DUO PAN UMB SM&B KID UTR BLD VAG CER NSP, tho SCA TPR, C1 NSP, lum NSP, cer LVR TPR, Ca1 HYO NSP, ca TMF ZYG XNR ORB CHV MAN HUM STN E PEL OLC RAD DIG ULN SBR LRB HYP VBR 0839_frame_C09 Page 134 Tuesday, May 22, 2001 10:43 AM 134 CRC Handbook of Marine Mammal Medicine FIGURE 3 Left lateral illustrations of a healthy harbor seal (Phoca vitulina). Based on dissections by S.A.R., with details and nomenclatures from the literature: Howell, 1930; Huber, 1934; Bryden, 1971; Tedman and Bryden, 1981; Rommel et al., 1998; Pabst et al., 1999. (© Copyright S. A. Rommel. Used with permission of the illustrator.) (Layer A) External features. The following abbreviations are used as labels: ANG = angle of mouth; ANS = anus; AXL = axilla; CAL = calcaneus, palpable bony feature; EAR = external auditory opening, ear; EYE = eye; INS = cranial insertion of the flipper; NAR = naris; OLC = olecranon, palpable bony feature; PAT = patella, palpable bony feature; PEC = pectoral limb, fore flipper; PEL = pelvic limb, hind flipper; SCA = dorsal border of the scapula, palpable bony feature; TAI = tail; UMB = umbilicus; UNG = unguis, finger and toe nails; U/G = urogenital opening; VIB = vibrissae. (Layer B) The superficial skeletal muscles. The layer of skeletal muscles just deep to the blubber and panniculus muscles. The following abbreviations are used as labels: ANS = anus; BIF = femoral biceps; BRC = brachiocephalic; DEL = deltoid; DIG = digastric; EAM = external auditory meatus; EXT = external oblique; FAS = fascia; F,S&B = fur, skin, blubber, and panniculus muscle (where present) cut along midline; GLU = gluteals; GRA = gracilis; LAT = latissimus dorsi; MAM = mammary gland; MAS = masseter; PAR lnn = parotid lymph nodes (ln for a single lymph node); PECa = ascending pectoral, extends over the patella and part of hind limb; PECs = superficial, pectoral; PECp = deep (profound) pectoral; REC = rectus abdominis; SAL = salivary gland; SEM = semitendinosus; SER = serratus; STC = sternocephalic; STH = sternohyoid; TFL = tensor fascia lata; TMP = temporalis; TRAc = trapezius, cervical portion; TRAt = trapezius, thoracic portion; TRI = triceps brachii; UMB = umbilicus. (Layer C) The superficial internal structures with “anatomical landmarks.” A view focused on relatively superficial internal structures visible from that perspective; the other important bony or soft “landmarks” are not necessarily visible from a left lateral view, but they are useful for orientation. The relative size of the lung represents partial inflation—full inflation would extend margins to distal tips of ribs. The following abbreviations are used as labels: ANS = anus; AXL = axillary lymph node; BLD = urinary bladder; EYE = eye; FEM = femur; FIB = fibula; HAR = heart; HUM = humerus; HYO = hyoid apparatus; INT = intestines; ILC = lliac crest; KID = left kidney; LIV = liver; LUN = lung; MAN = manubrium of the sternum; OLE = olecranon; OVR = left ovary; PAN = pancreas; PAT = patella; PRE = presternum, cranial sternal cartilage; PSC ln = prescapular lymph node; RAD = radius; REC = rectum; SAL = salivary glands; SIG ln = superficial inguinal lymph node; SCA = scapula; SPL = spleen; STM = stomach; TMJ = temporomandibular joint; TIB = tibia; TRA = trachea; TYR = thyroid gland; TYM = thymus gland; ULN = ulna; UMB = umbilical scar; UTR = left uterine horn; VAG = vagina; XIP = xiphoid. (Layer D) A view slightly to the left of the midsagittal plane illustrates the circulation, body cavities, and selected organs. Note that the diaphragm separates the heart and lungs from the liver and other abdominal organs. The following abbreviations are used as labels (structures on the midline are in normal type, those off-midline are in italics): AAR = aortic arch; ADR = left adrenal gland; ANS = anus; AOR = aorta; AXL = axillary artery; BCT = left brachiocephalic trunk; BRC = left bronchus as it enters the lung; BLD = urinary bladder; BRN = brain; CAF = caval foramen, with caval sphincter; CAR = carotid artery; CEL = celiac artery; CER = cervix; CVC = caudal vena cava; CRZ = left crus of the diaphragm; DIA = diaphragm, cut at midline, extends from crura dorsally to sternum ventrally; ESO = esophagus (to the left of the midline cranially, on the midline caudally); ESH = esophageal hiatus; EXI = external iliac artery; F,S&B = fur, skin, and blubber, plus panniculus where appropriate, cut on midline; HAR = heart; HPS = hepatic sinus within liver; KID = right kidney; LIV = liver, cut at midline; LUN = lung, right lung between heart and diaphragm; MAN = manubrium of sternum; caMESa = caudal mesenteric artery; crMESa = cranial mesenteric artery; OVR = ovary; PAN = pancreas; PUB = pubic symphysis; PULa = pulmonary artery, cut at hilus of lung; PULvv = pulmonary veins, cut at hilus of lung; REC = rectum; REN = renal artery; SKM = skeletal muscle; SPL = spleen; STM = stomach; STR = sternum made up of individual sternabrae; TNG = tongue; TRA = trachea; TYM = thymus gland; TYR = thyroid gland; UMB = umbilicus; UTR = uterus; VAG = vagina; XIP = xyphoid process of the sternum. (Layer E) The skeleton. Regions of the vertebral column (cervical, thoracic, lumbar, sacral, and caudal) are abbreviated (in lower case) as cer, tho, lum, sac, and cau, respectively, and are used as modifiers after an abbreviation in caps and a comma. If a specific vertebra is labeled, it will be represented by a capitalized first letter (for caudal, Ca will be used) and the vertebral number, i.e., first cervical = C1. The following abbreviations are used as labels: CAL = calcaneus; CAN = canine tooth; DIG = digits; FEM = femur; FIB = fibula; HUM = humerus; HYO = hyoid bones; ILC = iliac crest of the pelvis; LRB = last, or caudalmost, rib; LVR = last, or caudalmost, vertebra; MAN = mandible; MNB = manubrium, the cranialmost bony part of the sternum; NSP = neural spine (spinous process), e.g., thoracic neural spines = NSP, tho; OLC = olecranon; ORB = orbit; PAT = patella; PRS = presternum, cartilaginous extension of the sternum, particularly elongate in seals; PUB = pubic symphysis; RAD = radius; SCA = scapula; SBR = sternal ribs, costal cartilages; TIB = tibia; TMF = temporal fossa; TPR = transverse process, e.g., TPR, C1 = transverse process of the first cervical vertebra; ULN = ulna; VBR = vertebral ribs; XNR = external (bony) nares, nasal aperture of the skull; XIP = xyphoid process, cartilaginous caudal extension of the sternum; ZYG = zygomatic arch. 0839_frame_C09 Page 135 Tuesday, May 22, 2001 10:43 AM 135 Gross and Microscopic Anatomy AXL OLC EAR SCA EYE CAL TAI ANS U/G NAR VIB ANG A INS PEL UNG PAT U/G INS UNG PEC UMB FAS LAT F, S & B TFL TRI EAM TMP GLU BRC TRAt TRAc SAL BIF SEM ANS MAS DIG PAR Inn B STH GRA STC F, S & B DEL PECs SER PECa UMB F, S & B REC KID LUN HYO TMJ OVR OLE SAL ILC SCA PSC In EYE EXT MAM PECp FEM FIB REC ANS TYR C U/G TRA TIB PRE SIN In MAN BLD HUM TYM PAT AXL In RAD ULN XIP LIV SPL STM INT UMB PAN UTR HAR c Rommel 2000 CAR BRN ESO SKM VRT AAR PULa ESO BRC LUN ESH DIA CEL crMESa CAF AOR ADR CRZ KID REN caMESa EXI F, S & B REC ANS VAG TNG TYR D CER PUB TRA MAN BLD AXL TYM BCT STR PULvv HAR CVC DIA XIP HPS LIV STM VBR NSP, tho NSP, C2 ORB OVR F, S & B UTR LRB NSP, lum OLC TMF SPL UMB PAN ILC SCA NSP, cau CAL XNR LVR CAN MAN ZYG E HYO TPR,C1 PUB FIB PRS TIB MNB PAT HUM RAD FEM ULN XIP DIG SBR DIG 0839_frame_C09 Page 136 Wednesday, May 23, 2001 10:42 AM 136 CRC Handbook of Marine Mammal Medicine FIGURE 4 Left lateral illustrations of a healthy bottlenose dolphin (Tursiops truncatus). Based on dissections by S.A.R. with details and nomenclatures from the literature: Howell, 1930; Huber, 1934; Fraser, 1952; Slijper, 1962; Mead, 1975; Strickler, 1978; Klima et al., 1980; Pabst, 1990; Rommel et al., 1998; Pabst et al., 1999. Thanks to T. Yamada for suggestions on the muscle illustration. (© S. A. Rommel. Used with permission of the illustrator.) (Layer A) External features. The following abbreviations are used as labels: ANG = angle of mouth; ANS = anus; AXL = axilla; BLO = blowhole, external naris in dolphin; EAR = external auditory opening, ear; EYE = eye; FIN = dorsal fin; FLK = flukes (entire caudal extremity in cetaceans); INS = cranial insertion of the extremity; flipper, fin, and/or fluke; NOC = fluke notch in dugongs and in most cetaceans; PEC = pectoral limb, flipper; PED = peduncle, base of tail, between anus and flukes; MEL = melon; SCA = dorsal border of the scapula, palpable bony feature in emaciated dolphins; SNO = snout, cranial tip of upper jaw; UMB = umbilicus; U/G = urogenital opening. (Layer B) The superficial skeletal muscles. The layer of skeletal muscles just deep to the blubber and panniculus muscles. Note that the large muscles ventral to the dorsal fin are surrounded by a tough connective tissue sheath (Pabst, 1990). The following abbreviations are used as labels: ANS = anus; BLO = blowhole; DEL = deltoid; DIG = digastric; EAM = external auditory meatus; EPX = epaxial muscles, upstroke muscles; EXT = external oblique; HYP = hypaxialis; HPX = hypaxial muscles, downstroke muscles; ILI = iliocostalis; INT = internal oblique; ISC = oschium; ITTd = intertransversarius caudae dorsalis; ITTv = intertransversarius caudae ventralis; LAT = latissimus dorsi; LEV = levator ani; LON = longissimus; MAM = mammary gland; MAS = masseter; MUL = multifidus; PECp = deep (profound) pectoral; PSC ln = presacpular lymph node; REC = rectus abdominis; RHO = rhomboid; ROS = rostral muscles; S,B,&P = skin, blubber, and panniculus muscle (where present) cut along midline; SER = serratus; SLT = mammary slit, nipple; SPL = splenius; STE = sternohyoid; STM = sternomastoid; TER = teres major; TMP = temporalis; TRAd = trapezius dorsalis; TRAc = trapezius cranialis; TRI = triceps brachii; UMB = umbilicus. (Layer C) The superficial internal structures with “anatomical landmarks.” The relative size of the lung represents partial inflation—full inflation would extend margins to distal tips of ribs. The following abbreviations are used as labels: ANS = anus; BLD = urinary bladder; BLO = blowhole; EYE = eye; HAR = heart; HPX = hypaxial muscles; HUM = humerus; HYO = hyoid apparatus; INT = intestines; KID = left kidney; LIV = liver; LUN = lung (note that it extends beneath the scapula); MEL = melon; OVR = left ovary; PEL = pelvic vestige; PSC ln = prescapular lymph node; PUL ln = pulmonary lymph node, unique to cetaceans; RAD = radius; REC = rectum; ROS = rostral muscles, to manipulate the melon; SAC = lateral diverticulae, air sacs in dolphin; S&B = skin and blubber; SCA = scapula; SKM = skeletal muscle; SPL = spleen; STM = stomachs; TMJ = temporomandibular joint; TRA = trachea; TYR = thyroid gland; ULN = ulna; UMB = umbilical scar; UOP = uterovarian plexus; URE = ureter; UTR = uterine horn; VAG = vagina. (Layer D) A view slightly to the left of the midsagittal plane illustrates the circulation, body cavities, and selected organs. Note that the diaphragm separates the heart and lungs from the liver and other abdominal organs. The following abbreviations are used as labels (structures on the midline are in normal type, those off-midline are in italics): AAR = aortic arch; ADR = left adrenal gland; ANS = anus; AOR = aorta; AXL = axillary artery; BLD = urinary bladder; BLO = blowhole; BRC = bronchus; BRN = brain; CAR = carotid artery; CEL = celiac artery; CER = cervix; CRZ = left crus of the diaphragm; CVB = caudal vascular bundle; DIA = diaphragm, cut at midline, extends from crura dorsally to sternum ventrally; ESO = esophagus (to the left of the midline cranially, on the midline caudally); ESH = esophageal hiatus; EXI = external iliac artery; FINaa = arteries arrayed along the midline of the dorsal fin; FLKaa = arterial plexus on dorsal and ventral aspects of each fluke; HAR = heart; KID = right kidney; LAR = larynx or goosebeak; LIV = liver, cut at midline; MEL = melon; OVR = right ovary; PAN = pancreas (hidden behind first stomach); PMX = premaxillary sac; PULa = pulmonary artery, cut at hilus of lung; PULv = pulmonary vein, cut at hilus of lung; REC = rectum; REN = renal artery; S&B = skin and blubber, panniculus where appropriate cut at midline; SKM = skeletal muscle; SPL = spleen; STM1 = forestomach; STM2 = main stomach; STM3 = pyloric stomach; STR = sternum, sternabrae; TNG = tongue; TRA = trachea; TYM = thymus gland; TYR = thyroid gland; UMB = umbilicus; UOP = right uterovarian vascular plexus in dolphin; URE = right ureter; UTR = uterus; VAG = vagina. (Layer E) The skeleton. Regions of the vertebral column (cervical, thoracic, lumbar, sacral, and caudal), are abbreviated (in lower case) as cer, tho, lum, sac, and cau, respectively, and are used as modifiers after an abbreviation in caps and a comma. If a specific vertebra is labeled, it will be represented by a capitalized first letter (for caudal, Ca will be used) and the vertebral number, i.e., first cervical = C1. The following abbreviations are used as labels: CHV = chevrons, chevron bones; DIG = digits; HUM = humerus; HYO = hyoid apparatus; LRB = last, or caudalmost, rib; LVR = last, or caudalmost, vertebra; MAN = mandible; NSP = neural spine; e.g., thoracic neural spines = NSP, tho; OLC = olecranon; ORB = orbit; PEL = pelvic vestige; RAD = radius; SCA = scapula; STR = sternum; SBR = sternal ribs, costal ribs; TMF = temporal fossa; ULN = ulna; VBR = vertebral ribs; XNR = external (bony) nares, nasal aperture of the skull; ZYG = zygomatic arch. 0839_frame_C09 Page 137 Tuesday, May 22, 2001 10:43 AM 137 Gross and Microscopic Anatomy INS SCA FIN EAR BLO EYE PED MEL FLK SNO ANG INS INS A ANS U/G PEC UMB AXL PSC In SPL TRAc SEM TRAd LAT RHO MUL NOC U/G LON ILI EPX S&B EAM BLO MUL LON TEM ITTd ROS MAS DIG STE STM MAS DEL B PSC In SAC EYE TRI PECp INF TER SCA LUN LUN SER REC INT UMB EXT MAM SLT ANS ITTv ISC HYP HPX S, B & P SPL KID URE OVR BLO REC S&B MEL SKM ROS TMJ HYO TRA TYR HUM PEL VAG ANS RAD ULN C LIV PUL In HAR STM UMB UTR INT HPX S&B SKM BLD UOP REN CAR TRA BRN PMX ESO BRC AAR PULa CRZ PAN (hidden) CEL PULv ESH SKM SPL FINaa OVR UOP c Rommel 2000 AOR BLO EXI SKM MEL REC S&B CVB SKM TNG LAR TYR TYM AXL STR HAR D DIA LIV STM 2 STM 3 STM 1 CER UMB UTR ADR KID URE VAG ANS SKM S&B FLKaa BLD NSP, tho SCA NSP, C1&2 NSP, lum TMF XNR NSP, cau ORB MAN LVR ZYG HYO HUM PEL RAD E OLC STR ULN DIG SBR VBR LRB CHV 0839_frame_C09 Page 138 Tuesday, May 22, 2001 10:43 AM 138 CRC Handbook of Marine Mammal Medicine Included is a section on microanatomy to introduce the microanatomical peculiarities of marine mammals to pathologists and thus aid them in performing routine histopathological examination of marine mammal tissues. The microscopic appearance of organs and tissues is presented following the gross anatomical descriptions. This information has been gathered from the examination of tissues submitted to the University of California Veterinary Medical Teaching Hospital Pathology Service over the last 20 years. These tissues were acquired from stranded marine mammals, such as California sea lions, harbor seals, northern elephant seals (Mirounga angustirostris), southern sea otters (Enhydra lutris nereis), and a few small odontocetes and gray whales (Eschrichtius robustus). Anatomical observations from the literature are also included and referenced. Previous reviews of microanatomy include Simpson and Gardner (1972), Britt and Howard (1983), and Lowenstine and Osborne (1990). Histological recognition of organs and tissues from marine mammals poses little problem for individuals acquainted with the microanatomy of terrestrial mammals. The patterns of degenerative, inflammatory, and proliferative changes observed in marine mammal tissues are also similar to those observed in domestic mammalian species. Knowledge of specific microanatomy is necessary, however, for subtle changes to be recognized. External Features Consider the morphological features of the selected marine mammals. Streamlining and thermoregulation have caused changes in the appearance of marine mammals; these adaptations include the modification of appendages and other extremities for swimming, an increase in blubber for insulation, the development of axial locomotion, and the development of ascrotal testes (Pabst et al., 1999). Sea Lions The otariids (fur seals and sea lions), represented by the California sea lion, are also called eared seals because they have distinct pinnae (A-PIN) associated with their external ear openings (A-EAR). Like other pinnipeds, sea lions have robust vibrissae (A-VIB) on their snouts. Fur and/or blubber help streamline and insulate their bodies. Otariids (and walruses) can assume distinctly different postures on land by rotating their pelves to position their pelvic (or hind) flippers (A-PEL) under their bodies. Note the presence of nails (unguis; A-UNG) on the extremities. Eared seals propel themselves with their pectoral (or fore) flippers (A-PEC) when swimming. The adult males of the sexually dimorphic California sea lion (and most other otariids) are much larger than the females. The teeth of sea lions are often stained dark brown or black in the absence of significant dental calculus. As in other carnivora, the nasal turbinates are well developed (Mills and Christmas, 1990). Manatees The sirenians are represented by the Florida manatee. They lack hind limbs and have a dorsoventrally flattened fluke (A-FLK; note that it is flukes in cetaceans and dugongs and fluke in manatees). There is no dorsal fin, and the pectoral limbs or flippers are much more mobile than those of cetaceans—it is common to see manatees with their flippers folded across their chests or manipulating food into the mouth. The skin is rough and relatively thick and massive when compared with that of terrestrial mammals of the same body size. The skin is denser than water and contributes significantly to negative buoyancy (Nill et al., 2000). The vibrissae are robust but short (from wear), and the body hairs are fine but sparse, and give a nude 0839_frame_C09 Page 139 Tuesday, May 22, 2001 10:43 AM Gross and Microscopic Anatomy 139 appearance to the skin of the manatee. Although body hairs are sparse, they are uniquely innervated and might provide vibrational and other tactile sensations (Reep et al., 1999). The eyes (A-EYE) of manatees are small and, unlike the eyes of other mammals, close using a sphincter rather than distinct upper and lower eyelids. Seals The phocids, or earless seals (also called hair seals), are represented by the harbor seal. They have vibrissae similar to those of a dog. Their nares (A-NAR) are located at the dorsal aspects of their snouts. Phocid eyes are typically large (C-EYE) when compared with those of other marine mammals. Note that the appearance of phocids is generally the same, whether they are in the water or on land. Phocids commonly tuck their heads back against the thoraxes, making the neck look shorter than it really is, and they locomote in the water by lateral undulation of their pelvic flippers (A-PEL). Their flippers have long curved nails (A-UNG). Some phocids have multiple cusps on the caudal teeth, which in some species are quite complex and ornate. Dolphins The odontocetes are represented by the bottlenose dolphin. The cetaceans are characterized by the absence of pelvic limbs but are graced with large caudal structures called flukes (A-FLK). The melon (A-MEL) is a rostral fat pad that, together with elongated premaxillae and maxillae, gives the dolphin its “bottlenose.” The external nares are joined as a single respiratory opening at the blowhole (A-BLO), located at or near the apex of the skull. The externally smooth skin of dolphins has a thickened dermis, referred to as blubber. Some cetaceans also have dorsal fins (A-FIN), which are midline, nonmuscular, fleshy structures that may help stabilize them hydrodynamically. The keel of the peduncle (A-PED) provides streamlining and acts as a mechanical spring (Pabst et al., 1999). Cetaceans also have a pair of pectoral flippers that help them steer. Dolphins have facial hairs in utero but lose them at or near the time of birth (Brecht et al. 1997). Drawings contrasting features of the head and teeth of a representative porpoise and a representative dolphin appear in Reynolds et al. (1999). The unusual head of the sperm whale (Physeter macrocephalus) is described in detail by Cranford (1999). Dolphins have conical, pointed (when young and unworn) teeth. In contrast to dolphins, porpoises have flattened spade-shaped teeth and the lower, cranial margin of the melon extends all the way to the margin of the upper jaw or beak—there is no “bottle-shaped nose.” As dolphins age, their teeth wear down, as they are abraded by ingested material and each other; the name truncatus is derived from the truncated appearance of the teeth in the original specimen. The tongues of the bottlenose dolphin and some other odontocetes have elaborate cranial and lateral marginal papillae, which are important for nursing (Donaldson, 1977). Microanatomy of the Integument The cetacean integument differs significantly from that of terrestrial mammals in that there are no hair follicles (save for a few on the snouts of some species) and no sebaceous or apocrine glands (Greenwood et al., 1974; Ling, 1974). The thick epidermis is nonkeratinizing, lacks a granular layer, and is composed primarily of stratum spinosum (stratum intermedium) with deep rete pegs. The basal layer has continuous mitoses. Continuous desquamation caused by water friction may account for the absence of a keratinized stratum corneum and the continuous cell replication in the basal layer. The papillary dermis is extremely well vascularized (Elsner et al., 1974). The reticular dermis grades into the fat-filled panniculus adiposus, creating a fatty 0839_frame_C09 Page 140 Tuesday, May 22, 2001 10:43 AM 140 CRC Handbook of Marine Mammal Medicine layer referred to as the blubber layer. The blubber contains many collagen (fibrous) bundles and elastic fibers, and adipocytes are interspersed so that blubber thickness may not diminish significantly during catabolism of fat. The blubber layer is connected to the underlying musculature by loose connective tissue (subcutis). Pinnipeds, sea otters, and sirenians are haired (although hair density varies enormously from sea otters to walruses and sirenians), and therefore their skin is more similar to domestic mammals than is cetacean skin. The epidermis of these species is partially or entirely keratinizing. The stratum corneum is thickest on weight-bearing surfaces, such as the relatively glabrous ventral surfaces of fore and hind flippers, where the entire epidermis is quite thick. A stratum granulosum is present in phocids. Compound hair follicles consisting of a single guard hair follicle and several intermediate and underfur follicles are common, especially in fur seals and sea otters. Elephant seals, monk seals, and walruses, which lack underfur, all have simple hair follicles consisting of a single guard hair. Like terrestrial mammals, hair follicles of sea otters and pinnipeds are associated with well-developed sebaceous and apocrine (sweat) glands. Apocrine sweat glands are relatively large in the otariid seals, whereas the sebaceous glands are more prominent in the phocids. In densely haired regions of fur seals, the sweat glands enter the hair follicle above (distal) the sebaceous gland duct, but in sparsely haired species (such as the harp seal) and in sparsely haired areas of densely haired species, the pattern is reversed (Ling, 1974). Concentrations of glands vary with location on the animal, and patterns of gland distribution have not been fully described for all species. In some pinniped species, apocrine gland secretion may be more evolved for scent and olfactory communication than for thermoregulation (Greenwood et al., 1974). Hair follicles in all species are said to lack arrector pili muscles and have a fairly fixed angle relative to the skin surface. Vibrissae may be selectively heated by changes in blood flow (Mauck et al., 2000). The blubber layer is relatively thin in fur seals and sea otters; in these species, the pelage is presumed to provide primary insulation. The connective tissue in the pinniped dermis contains many elastic fibers. The reticular layer is thicker than the papillary layer. The lower portions of hair follicles extend into the deep reticular dermis and are often surrounded by adipose tissue in those species with a thick blubber layer. An interesting physiological phenomenon involving the marine mammal integument is the catastrophic cyclic molting that occurs in some phocids (Ling, 1974). Domestic mammals also tend to shed hair cyclically, but the stratum corneum is desquamated continuously, accompanied by continuous proliferation of the basal cell layer. In some phocids, basilar mitosis is seasonal, and the lipid-rich stratum corneum is parakeratotic and persists as a protective, presumably waterproof, sheet from one molt to the next. Prior to molt, a granular cell layer develops, and during molt, the surface epithelium is shed in great sheets along with the hair. In harp seals, this process is manifest grossly as small circular lesions that open and become confluent, leading to a drying-out and sloughing of the entire epidermal surface. Catastrophic molt has been best described histologically in the southern elephant seal (M. leonina) and is also evident in the northern elephant seal. Cyclic shedding or molt has also been seen in otariids but occurs more slowly, with shedding of the hair over several weeks or months. Mammary glands (B-MAM) are ventral, medial, and relatively caudal in most marine mammals, but they are axillary in sirenians. Cetaceans and some phocids have a single pair of nipples (B-SLT), but otariids and polar bears have two pairs of nipples. In cetaceans, the nipples are within mammary slits located lateral to the urogenital opening (note that some male cetaceans have distinct mammary slits). Detailed anatomy of the phocid mammary gland is described by Bryden and Tedman (1974) and Tedman and Bryden (1981). 0839_frame_C09 Page 141 Tuesday, May 22, 2001 10:43 AM Gross and Microscopic Anatomy 141 The Superficial Skeletal Muscles The skeletal muscles that are encountered when the skin, blubber,* and panniculus muscles are removed are illustrated in layer B of each figure. Note that the panniculus (B-PAN) is represented as dotted lines in the manatee because it is such a robust muscle, bordered on its lateral and medial aspects by “blubber.” The skeletal muscle of most marine mammals is very dark red, almost black, because of the relatively high myoglobin concentration. The design of the musculoskeletal system profoundly influences any mammal’s power output because it affects both thrust and propulsive efficiency (Pabst et al., 1999). Thrust forces depend on muscle morphology and the mechanical design of the skeletal system. The propulsive efficiency of the animal depends on the size, shape, position, and behavior of the appendage(s) used to produce thrust. Terrestrial mammals usually use their appendicular musculoskeletal system to swim using the proverbial dog paddle—alternate strokes of the forelimbs (and sometimes hind limbs). Pinnipeds use their more-derived appendicular musculoskeletal systems to swim. Unlike the other marine mammals, the fully aquatic sirenians and cetaceans swim using only their vertebral or axial musculoskeletal systems. Thus, in mammals that use their appendicular musculoskeletal systems to swim, two morphological “solutions” to increase thrust production are observed (Pabst et al., 1999). Proximal locomotor muscles tend to have large cross-sectional areas and so would have the potential to generate large in-forces. Proximal limb bones (i.e., humerus and femur) tend to be shorter than more distal bones (i.e., radius, ulna, tibia, and fibula), which increases the mechanical advantage of the lever system. The short proximal limb bones have an added hydromechanical benefit. These bones tend to be partially or completely enveloped in the body, which helps reduce drag on the appendage and increased body streamlining (Tarasoff, 1972; English, 1977; King, 1983). Contrast the distribution of muscle mass in the four species. Note that adaptations to each locomotory specialization have enlarged or reduced the corresponding muscles found in terrestrial mammals. Contrast the massiveness of the pectoral muscles (B-PEC) of the sea lion with those in the seal. The triceps (B-TRI) and deltoids (B-DEL) are also enlarged in both pinnipeds to increase thrust, and the olecranons (C,E-OLC) of both the seal and sea lion are enlarged to increase the mechanical advantage of these muscles. Note that the harbor seal has a unique component of the pectoral—an ascending pectoral muscle (B-PECa)—that extends over the humerus (also described for another phocid, the southern elephant seal; see Bryden, 1971). A dramatic change in thickness of the abdominal wall muscles (B-INT, EXT) occurs in young seals as they make the transition from a more terrestrial to a more aquatic lifestyle. Cetaceans and sirenians use their axial musculoskeletal systems to swim. Epaxial muscles (B-EPX) bend the vertebral column dorsally in upstroke; hypaxial muscles (B-HPX) and abdominal muscles bend the vertebral column ventrally in downstroke. Because there is no “recovery” phase, efficiency is increased. These muscles generate thrust forces that are delivered to the fluid medium via their flukes (Domning, 1977; 1978; Strickler, 1980; Pabst, 1990). The elongated neural spines (E-NSP) and transverse processes (E-TPR) of cetaceans also increase the mechanical advantage of the axial-muscle lever system, relative to that system in terrestrial mammals. By inserting far from the point of rotation, the lever arm-in is increased and, thus, force output is increased. A novel interaction between the tendons of the epaxial muscles and a connective tissue sheath that envelops those muscles also increases the work output of the axial musculoskeletal system in cetaceans (Pabst, 1993; Pabst et al., 1999). The *The term blubber is used differently in different species. In sea lions, seals, and manatees, it is subcutaneous fat in one or two layers, and resembles that found in terrestrial mammals. Blubber in cetaceans is fat—“inflated” dermis (Pabst et al., 1999). 0839_frame_C09 Page 142 Tuesday, May 22, 2001 10:43 AM 142 CRC Handbook of Marine Mammal Medicine sirenian axial skeleton does not display elongated processes, which would increase the lever arm-in for dorsoventral flexion. Instead, the lumbar and cranialmost caudal vertebrae have elongated transverse processes (Domning, 1977; 1978). The Diaphragm as a Separator of the Body Cavities The orientation of the diaphragm (C,D-DIA) in most marine mammals is very similar to the orientation of the diaphragm in the dog. Visualizing size, shape, and extent of the diaphragm will help one visualize the dynamics of respiration and diving. The diaphragm lies in a transverse plane and provides a musculotendinous sheet to separate the major organs of the digestive, excretory, and reproductive systems (all typically caudal to the diaphragm) from the heart with its major vessels; the lungs (C-LUN) and associated vessels and airways; the thyroid (C,D-THY), thymus (C,D-TYM), and a variety of lymph nodes, all located cranial to the diaphragm. The diaphragm is generally confluent with the transverse septum, so it attaches medially at its ventral extremity to the sternum. Although the diaphragm acts as a separator between the heart and lungs and the other organs of the body, the diaphragm is traversed by nerves and other structures, such as the aorta (D-AOR) (crossing in a dorsal and central position), the vena cava (D-CVC) (crossing more ventrally than the aorta, and often slightly left of the midline, although appearing to approximate the center of the liver), and the esophagus (D-EOS) (crossing slightly right of the midline, at roughly a midhorizontal level). This transverse orientation exists in most marine mammals, although the orientation of the diaphragm may be slightly diagonal, with the ventral portion more cranial than the dorsal portion. The West Indian manatee’s diaphragm differs from this general pattern of orientation and attachment. The manatee diaphragm and the transverse septum (D-TRS) are separate, with the latter occupying approximately the “typical” position of the diaphragm, and the diaphragm itself occupying a horizontal plane extending virtually the entire length of the body cavity. This apparently unique orientation presumably relates to buoyancy control (Rommel and Reynolds, 2000). There are two separate hemidiaphragms in the manatee. The central tendons firmly attach to hypapophyses (E-HYP) on the ventral aspects of the thoracic vertebrae, thereby producing the two pleural cavities. Gross Anatomy of Structures Cranial to the Diaphragm Heart and Pericardium The pericardium is a fluid-filled sac surrounding the heart; in manatees, it often contains more fluid than is found in the typical mammal or in other marine mammals. The heart occupies a ventral position in the thorax (immediately dorsal to the sternum; D-STR). The heart lies immediately cranial to the central portion of the diaphragm (D-DIA; or the transverse septum in the manatee, D-TRS). In some species, the lungs (D-LUN) may embrace the caudal aspect of the heart, separating the caudal aspect of the heart from the diaphragm. As in all other mammals, marine mammal hearts have four chambers, separate routes for pulmonary and systemic circulation, and the usual arrangements of great vessels (venae cavae, D-CVC; aorta, D-AOR; coronary arteries; pulmonary arteries, PULaa; pulmonary veins, PULvv). Many marine mammal hearts are flattened from front to back (ventral to dorsal), are relatively squat from top to bottom, and have a rounded apex, giving them a shape quite different from the hearts of most terrestrial mammals (Drabek, 1975). Most pinnipeds and some cetaceans also have a distinctive dilatation of the aortic arch (Drabek, 1977). Cardiac fat occurs, but is rapidly lost in debilitated animals. 0839_frame_C09 Page 143 Tuesday, May 22, 2001 10:43 AM Gross and Microscopic Anatomy 143 Pleura and Lungs The pleural cavities and lungs (C-LUN) are generally found dorsal and lateral to the heart; in the manatee, the lungs are unusual in that they extend virtually the length of the body cavity and remain dorsal to the heart (Rommel and Reynolds, 2000). Lungs of some marine mammals (cetaceans and sirenians) are unlobed. The cranial ventral portion of the left lung in the bottlenose dolphin and other small odontocetes is very thin, almost veil-like, where it overlies the heart. Lobation in the pinnipeds is generally similar to that in the dog, that is, two lobes on the left (the cranial lobe has cranial and caudal parts) and three (including the accessory lobe) on the right. Reduction of lobation occurs in some phocids (Boyd, 1975; King, 1983). The terminal airways in all marine mammals are reinforced with either cartilage or muscle (Pabst et al., 1999). Apical (tracheal) bronchi are present in dolphins. In otariids, it is important to note that the bifurcation (D-BIF) of the trachea into the main-stem bronchi takes place at the thoracic inlet, not at the pulmonary hilus as is the case in phocids and cetaceans (McGrath et al., 1981; Nakakuki, 1993a,b; Wessels and Chase, 1998). The lungs of cetaceans are grossly smooth, but those of many pinnipeds are divided into distinct lobules in the ventral fields. Interestingly, sea otter lungs have distinct interlobular septa. The size of marine mammal lungs depends upon each species’ diving proficiency. Marine mammals that make deep and prolonged dives (e.g., elephant seals) tend to have smaller lungs than expected (based on allometric relationships), whereas shallow divers (e.g., sea otters) tend to have larger than expected lungs (Pabst et al., 1999). Mediastinum The mediastinum is an artifact of the downward expansion of the lungs on either side of the heart in the typical mammal (Romer and Parsons, 1977); thus, the traditional definition of the mammalian mediastinum does not apply to manatees. The positions of the aortic hiatus, caval foramen (D-CAF), and esophageal hiatus (D-ESH) are unusual because of the configuration of the diaphragm. The manatee mediastinum (see manatee, layer D) is the midline region dorsal to where the pericardium attaches to the heart and ventral to the diaphragm, cranial to the transverse septum up to approximately the level of the first cervical vertebra. This is essentially what constitutes the cranial mediastinum of other mammals. The thyroid, thymus, tracheobronchial lymph nodes, and the tracheobronchial bifurcation are in the region defined as mediastinal in the manatee (Rommel and Reynolds, 2000). The mediastinum is thin and generally complete in the pinnipeds. Thymus The thymus (C,D-TYM), which typically is relatively larger in young than in old individuals of any species, is found on the cranial aspect of the pericardium (sometimes extending caudally to embrace almost the entire heart) and may extend into the neck in otariids, the bottlenose dolphin (Cowan and Smith, 1999), and some other species. Thyroids The thyroid glands (C,D-TYR) of the bottlenose dolphin and the manatee are located in the cranial part of the mediastinum along either side of the distal part of the trachea (C,D-TRA), prior to its bifurcation (D-BIF) into the bronchi. The paired, large, oval, dark-brown thyroid glands of pinnipeds, however, lie along the trachea just caudal to the larynx outside of the thoracic inlet (similar to the position in dogs). 0839_frame_C09 Page 144 Tuesday, May 22, 2001 10:43 AM 144 CRC Handbook of Marine Mammal Medicine Parathyroids The parathyroid glands have been described in small cetaceans, and their location relative to the thyroid gland varies among species examined to date (Hayakawa et al., 1998). In Risso’s dolphins (Grampus griseus) they are dorsal to the thyroids or embedded within them, whereas in bottlenose dolphins they are on the surface of the thyroids and in the connective tissue surrounding the dorsal side of the thyroids. Little is known about the parathyroids of pinnipeds and sirenians. Larynx The cetacean respiratory system has undergone several modifications that are associated with the production of sound. Immediately ventral and lateral to the blowhole (B,C,DBLO) are small sacs or lateral diverticulae (C-SAC). Medial to the diverticulae are the paired internal nares that extend on the cranial aspect of the braincase (D-BRN). The larynx (C-LAR), a spout-shaped structure referred to as the goosebeak, is composed of an elongated epiglottis and corniculate cartilage (Reidenberg and Laitman, 1987). The goosebeak extends through a small opening in the esophagus (supported laterally by an enlarged thyroid cartilage) into the relatively vertical narial passage; food can pass to either side of the goosebeak. Cetaceans have a robust hyoid apparatus (C,E-HYO) to support movements of the larynx. A palatopharyngeal sphincter muscle can keep the goosebeak firmly sealed (Pabst et al., 1999). For a detailed description of sound-producing anatomy, see Cranford et al. (1996). Caval Sphincter One additional structure that is associated with the circulatory system, located on the cranial aspect of the diaphragm in seals and sea lions, is a feature atypical in mammals. This is the muscular caval sphincter (D-CAS), which can regulate the flow of oxygenated* blood in the large venous hepatic sinus (D-HPS) to the heart during dives (Elsner, 1969). Microscopic Anatomy of Structures Cranial to the Diaphragm Respiratory System In cetaceans and otariids, cartilage extends around small bronchioles to the periphery of the lungs. In most phocids, cartilage is present around bronchi and bronchioles (Tarasoff and Kooyman, 1973; Boshier, 1974; Boyd, 1975). Bronchial glands are especially numerous in largercaliber bronchi and bronchioles of phocids. The configuration of terminal airways branching into alveoli varies among marine mammals, but, in general, respiratory ducts with small alveolar sacs make up the functional parenchyma. Myoelastic sphincters are present in the terminal bronchioles, presumably as an adaptation to diving (Boshier, 1974; Wessels and Chase, 1998). The number of alveolar duct units per lobule varies with species. The interalveolar septa have double rows of capillaries in most cetaceans and some otariids (e.g., in Steller but not California sea lions) but a single row of capillaries in phocids. *In diving mammals with abundant arteriovenous anastomoses (shunts between arteries and veins before capillary beds), one can find high blood pressure and highly oxygenated blood in veins. One such venous reservoir of oxygenated venous blood is the hepatic sinus of seals (King, 1983). 0839_frame_C09 Page 145 Tuesday, May 22, 2001 10:43 AM Gross and Microscopic Anatomy 145 Thymus The thymus of marine mammals is composed of lobules, each with a distinct lymphocyte-rich cortex and a less cellular medulla. In many stranded immature marine mammals, there is profound thymic atrophy, with lymphoid depletion, and mineralization and keratinization of Hasell’s corpuscles. Thyroids The thyroids of neonatal California sea lions, harbor seals, and elephant seals have plump cuboidal epithelium and little colloid (Little, 1991; Schumacher et al., 1993). In adults of the former two species, the epithelium also remains cuboidal, and the follicles remain fairly uniform in size. The thyroids of cetaceans are often distinctly lobulated, and the follicles of both young and adults are often small and lined with cuboidal epithelium similar to that of pinnipeds (Harrison, 1969b). Parathyroids The parathyroids of Risso’s dolphins are divided into lobules by connective tissue, and have parenchymal cells consisting of chief cells with intracellular lipid droplets (Hayakawa et al., 1998). Gross Anatomy of Structures Caudal to the Diaphragm Easy-to-find landmarks caudal to the diaphragm include a massive liver (C,D-LIV) and the various components of the gastrointestinal (GI) tract. The gonads and most other parts of the reproductive tracts are found only after the removal of the GI tract, except in a pregnant uterus. Liver Typically, the liver is located immediately caudal to the diaphragm. It is a large, brownish, multilobed organ that tends to have most of its volume or mass positioned to the left of the body midline. Marine mammal livers are generally not too different from those of other mammals, although the manatee liver is a little more to the right and dorsal than are the livers of most other mammals. The number of lobes and the fissures in the lobes may vary, particularly in the sea lion’s liver, in which deep fissures give the lobes a deeply scalloped appearance. Bile may be stored in a gall bladder (often greenish in color) located ventrally, between lobes of the liver, although some mammals (e.g., cetaceans, horses, and rats) lack a gall bladder. Bile enters the duodenum (D-DUO) to facilitate chemical digestion of fats. Digestive System Most of the volume of the cavity caudal to the diaphragm (the abdominal cavity) is occupied by the various components of the GI tract: the stomach, the small intestine (C-INTsml; duodenum, jejunum, ileum), and the large intestine (C-INTlg; cecum, colon, and rectum; C,D-REC). A strong sphincter marks the distal end of the stomach (the pylorus) before it connects with the small intestine (duodenal ampulla in cetaceans and sirenians). The separation between jejunum and ileum of the small intestine is difficult to distinguish grossly, although the two sections differ microscopically. The junction of the small and large intestines may be marked by the presence of a midgut cecum (homologous to the human appendix). The cecum is absent in most toothed whales, but present in some baleen whales (not the bowhead whale), vestigial but present in pinnipeds, and 0839_frame_C09 Page 146 Tuesday, May 22, 2001 10:43 AM 146 CRC Handbook of Marine Mammal Medicine absent in sea otters. In manatees, the cecum is large, globular, and has two blind pouches called cecal horns. The large intestine, as its name implies, has a larger diameter than the small intestine in some marine mammals. In the sea lion, seal, and dolphin there is little difference in gross appearance between the small and large intestines. The cecum of sea lions and seals is about a meter from the anus, whereas the small intestines are about 20 times as long; in adult manatees, both the large and small intestines may approach or even exceed 20 m (Reynolds and Rommel, 1996). The proportions and functions of these components reflect feeding habits and trophic levels of the different marine mammals. Accessory organs of digestion include the salivary glands (C-SAL; absent in cetaceans, present in pinnipeds, very large in the manatee), pancreas (D-PAN), and liver. The pancreas is sometimes a little difficult to locate, because it can be a rather diffuse organ and decomposes rapidly; however, a clue to its location is its proximity to the initial part of the duodenum into which pancreatic enzymes flow (Erasmus and Van Aswegen, 1997). Another organ that is structurally, but not functionally, associated with the GI tract is the spleen (D-SPL), which is suspended by a ligament, generally from the greater curvature of the stomach in simple-stomached species, or from the first stomach in cetaceans). It is usually on the right side, but may have its greatest extent along the left side of the body. The spleen is usually a single organ, but in some species (mainly cetaceans), accessory spleens (occasionally referred to as hemal lymph nodes) may accompany it. It varies considerably in size among species; in manatees and cetaceans it is relatively small, but the spleen is relatively massive in some deep-diving pinnipeds (Zapol et al., 1979; Ponganis et al., 1992), where it acts to store red blood cells temporarily. The length and mass of the GI tract may be very impressive and create three-dimensional relationships that can be complex. Tough connective tissue sheets called mesenteries suspend the organs from the dorsal part of the abdominal cavity, and shorter connective tissue bands (ligaments*) hold organs close to one another in predictable arrangements (e.g., the spleen is almost always found along the greater curvature of the stomach and is connected to the stomach by the gastrosplenic ligament). Numerous lymph nodes and fat are also suspended in the mesenteries. The GI tracts of pinnipeds and other marine mammal carnivores follow the general patterns outlined above, although the intestines can be very long in some species (Schumacher et al., 1995; Stewardson et al., 1999). Cetaceans, however, have some unique specializations (Gaskin, 1978). In these animals, there are three or more compartments to the stomach, depending on the species. Functionally, the multiple compartments of cetacean stomachs correspond well to regions of the single stomach of most other mammals. Most cetaceans have three compartments; the first, called the forestomach (D-STM1; essentially an enlargement of the esophagus), is muscular and very distensible; it acts much like a bird crop (i.e., as a receiving chamber). The second (D-STM2), or glandular compartment, is the primary site of chemical breakdown among the stomach compartments; it contains the same types of enzymes and hydrochloric acid that characterize the “typical” mammalian stomach. Finally, the “U-shaped” third compartment, or pyloric stomach (D-STM3), ends in a strong sphincteric muscle that regulates flow of digesta into the duodenum of the small intestine. The initial part of the cetacean duodenum is expanded into a small saclike ampulla (occasionally mistaken for a fourth stomach). *Ligament has several meanings in anatomy: a musculoskeletal element (e.g., the anterior cuciate ligament), a vestige of a fetal artery or vein (e.g., the round ligament of the bladder), the margin of a fold in a mesentery (e.g., broad ligament), and a serosal fold between organs (e.g., the gastrolienal ligament). Note: In human terminology anterior and posterior are used; in comparative and veterinary terminology cranial and caudal are used when relating to the head and tail, respectively. 0839_frame_C09 Page 147 Tuesday, May 22, 2001 10:43 AM Gross and Microscopic Anatomy 147 Among the marine mammals, sirenians have the most remarkable development of the GI tract. Sirenians are herbivores and hindgut digesters (similar to horses and elephants), so the large intestine (specifically the colon) is extremely enlarged, enabling it to act as a fermentation vat (see Marsh et al., 1977; Reynolds and Rommel, 1996). The sirenian stomach is single chambered and has a prominent accessory secretory gland (the cardiac gland) extending prominently from the greater curvature. The duodenum is capacious and has two obvious diverticulae projecting from it. The GI tract of the manatee, with its contents, can account for more than 20% of an individual’s weight. Urinary Tract The kidneys (C,D-KID) typically lie against the musculature of the back (B-HPX, hypaxial muscles), at or near the dorsal midline attachment of the diaphragm (crus, D-CRZ). In the manatee, the unusual placement of the diaphragm means that the kidneys lie against the diaphragm, not against hypaxial muscles. In many marine mammals, the kidneys are specialized as reniculate (multilobed) kidneys, where each lobe (renule) has all the components of a metanephric kidney. The reason that marine mammals possess reniculate kidneys is uncertain, but the fact that some large terrestrial mammals also possess reniculate kidneys has led to speculation that they are an adaptation associated simply with large body size (Vardy and Bryden, 1981), rather than for a marine lifestyle. Large body size may be important as the proximal convoluted tubules cannot be overlengthy and still conduct urine (Maluf and Gassmann, 1998). The kidneys are drained by separate ureters (D-URE), which carry urine to a medially and relatively ventrally positioned urinary bladder (C,D-BLD). The urinary bladder lies on the floor of the caudal abdominal cavity and, when distended, may extend as far forward as the umbilicus (A,B,C,D-UMB) in some species. The pelvic landmarks are less prominent in the fully aquatic mammals. In the manatee the bladder can be obscured by abdominal fat. Note that the renal arteries (D-REN) of cetaceans enter the cranial pole of the organ, and the ureters exit near the caudal pole, whereas in other marine mammals they enter and exit the hilus (typical of most mammals). Additionally, in manatees, there are accessory arteries on the surface of the kidney (Maluf, 1989). Genital Tract Pabst et al. (1999) noted that the reproductive organs tend to reflect phylogeny more than adaptations to a particular niche. If one were to examine the ventral aspect prior to removal of the skin and other layers, one would discover that, especially in the sirenians and some cetaceans, positions of male and female genital openings are obviously different, permitting easy determination of sex without dissection. In all cases, the female urogenital opening (AU/G) is relatively caudal, compared with the opening for the penis in males. One way to approach dissection of the reproductive tracts is to follow structures into the abdomen from the external openings. The position and general form of the female reproductive tracts are similar to those of terrestrial mammals (Boyd et al., 1999). The vagina (C,D-VAG) opens cranial to the anus (A,B,C,D-ANS) and leads to the uterus (C,D-UTR), which is bicornuate in marine mammal species. The body of the uterus is found on the midline and is located dorsal to the urinary bladder (the ventral aspect of the uterus rests against the bladder). The uterine horns (cornua) extend from the uterine body toward the lateral aspects of the abdominal cavity. Implantation of the fertilized egg and subsequent placental development take place in the walls of the uterine horns, usually in the ipsilateral horn to ovulation (see Chapter 11, Reproduction). Dimensions of uterine horns vary with reproductive history and age. Often the fetus may expand the pregnant horn to occupy a substantial portion of the abdominal cavity. The horns terminate 0839_frame_C09 Page 148 Tuesday, May 22, 2001 10:43 AM 148 CRC Handbook of Marine Mammal Medicine distally in an abrupt reduction in diameter and extend as uterine tubes (fallopian tubes) to paired ovaries (C,D-OVR). The uterus and ovaries are suspended from the dorsal abdominal wall by the broad ligaments. Uterine scars and ovarian structures may provide information about the reproductive history of the individual (Boyd et al., 1999; see Chapter 11, Reproduction). The ovaries of mature females may have one or more white or yellow-brown scars, called corpora albicantia and corpora lutea, respectively (see Chapter 11, Reproduction). Although ovaries are usually small solid organs, in sirenians they are relatively diffuse, with many follicles and more than one corpus albicans. The male reproductive tracts of marine mammals have the same fundamental components as those of “typical” mammals, but positional relationships may be significantly different. These differences are due to the testicond (ascrotal) position of the testes in many species (sea lion testes become scrotal when temperatures are elevated). The testes of some marine mammals are intra-abdominal* (DeSmet, 1977), whereas in phocids they are in the inguinal canal, covered by the oblique muscles and blubber (see Figure 2-20 in Pabst et al., 1999). The position of marine mammal testes creates certain thermal problems because spermatozoa do not survive well at body (core) temperatures; in some species, these problems are solved by circulatory adaptations mentioned below. The penis of marine mammals is retractable, and it normally lies within the body wall. General structure of the penis relates to phylogeny (Pabst et al., 1999). In cetaceans, it is fibroelastic type with a sigmoid flexure that is lost during erection, as seen in ruminants. Pinnipeds, sea otters and polar bears have a baculum within the penis, as do domestic dogs; in manatees it is muscular (see Chapter 11, Reproduction, and see Sexual Dimorphisms, below). Adrenal Glands In marine mammals, adrenal glands (D-ADR) lie cranial to the kidneys and caudal to the diaphragm, as in terrestrial mammals. Adrenal glands can be confused with lymph nodes, but if one slices the organ in half, an adrenal gland is easy to distinguish grossly by its distinct cortex and medulla. In contrast, lymph nodes are more uniform in appearance. Microscopic Anatomy of Structures Caudal to the Diaphragm Liver The histology of the liver of pinnipeds is quite similar to that of terrestrial mammals. In cetaceans, however, portal triads may have very thick-walled vessels (Hilton and Gaskin, 1978). Smooth muscle may also be found around some central veins (throttling veins) (Arey, 1941). Stainable iron (hemosiderosis) is common in neonatal harbor and northern elephant seals and in older otariids in captivity. Ito cells may be quite prominent in marine mammals, compatible with the presence of high vitamin A levels found in these livers (Rhodahl and Moore, 1943). Digestive System The oropharynx of pinnipeds and odontocetes, and the caudal part of the odontocete tongue, are richly endowed with minor mucous glands, which enter out onto the mucosal surface via ducts that are visible grossly as small pits. Microscopically, the nonglandular and glandular stomachs resemble the analogous structures in terrestrial mammals. Parietal cells are exception*The position of the testes in sea otters is scrotal, and the testes of polar bears are seasonally scrotal (Reynolds et al., in press). 0839_frame_C09 Page 149 Tuesday, May 22, 2001 10:43 AM Gross and Microscopic Anatomy 149 ally prominent in odontocetes. In sirenians, the cardiac gland is a submucosal mass that protrudes cranially from the greater curvature of the stomach; it has a complicated folded lumen lined by mucous surface cells overlying long gastric glands lined with mucous and parietal cells. The glands of the main sac are lined by mucous cells and a lesser number of parietal cells (Marsh et al., 1977; Reynolds and Rommel, 1996). Histologically, the intestines of marine mammals are also similar to those of domestic mammals with the following exceptions (Schumacher et al., 1995). The villi are said to be absent in the proximal duodenum in some cetaceans, and Brunner’s glands are variably present. Plicae rather than villi are often present, creating chevron shapes on cross sections of cetacean intestine. The light and electron microscopic appearance of the small intestine of small odontocetes has been described in detail (Harrison et al., 1977). Gut-associated lymphoid aggregates are present throughout the intestines and may be diffuse or nodular. They are especially numerous in the distal colon of odontocetes and baleen whales, where they form the anal tonsil (Cowan and Brownell, 1974; Romano et al., 1993). Urinary Tract Each reniculus has a histologically distinct cortex and medulla. Since cortex completely surrounds the medulla in the reniculi, ascending inflammation in one reniculus may spill over into the interstitium of an adjacent reniculus, giving the pattern of interstitial (hematogenous) nephritis. Thus, it is important to sample several reniculi from each kidney to assess pathological processes. In cetaceans there is normally a fibromuscular band at the corticomedullary junctions surrounding the medullary pyramid. Glomeruli of all species examined are of remarkably similar size (about one half the width of a 40× high dry field). Genital Tract The morphology of the reproductive tract of the female varies with the stages of estrus and gestation (see Chapter 11, Reproduction). A description of cyclic changes in some of the cetaceans is given in Harrison (1969a) and in some sirenians in Boyd et al. (1999). Morphological changes of the genital mucosa associated with the estrous cycle have not been studied in detail in marine mammals, other than the harbor seal (Bigg and Fisher, 1974). In this species (described here to illustrate the variation in appearance through the estrous cycle), during follicular development then regression, the uterine mucosa increases in height and pseudostratification and then decreases to simple cuboidal. Uterine gland epithelium increases in height and secretory activity, and glands become increasingly coiled. Vaginal epithelium “destratifies” to become a “transitional-type” epithelium only a few cells thick, with vaginal pits (glands) lined by columnar epithelium with apical secretory product (goblet cell-like). The endometrial luminal and glandular epithelium of the nongravid horn is secretory and declines to cuboidal by parturition. During this luteal phase, there are subnuclear lipid vacuoles in the glandular epithelium. The vaginal epithelium is transitional during early placentation, but increases in secretory activity to become lined with tall columnar mucous cells with fingerlike projections of the lamina propria replacing the mucosal pits. During lactation, the morphology of both uterine and vaginal epithelium changes again. In the first part of lactation, the surface and glandular uterine epithelium is cuboidal, then undergoes hypertrophy and hyperplasia during the latter half of lactation. Luminal epithelium is occasionally pseudostratified, and the uterine stroma of both horns is edematous. The patchy hyperplasia and pseudostratification might be mistaken for dysplasia. Vaginal epithelium is almost transitional during the first part of lactation but proliferates to stratified squamous nonkeratinizing cells covered by sloughing mucous cells by the end of lactation. The endometrium of the gray seal prior to implanation is described by Boshier (1979; 1981). 0839_frame_C09 Page 150 Tuesday, May 22, 2001 10:43 AM 150 CRC Handbook of Marine Mammal Medicine The placenta of pinnipeds is zonary, endotheliochorial, similar to that of domestic carnivores. In late gestation, it is often deep orange because of the marginal hematoma from which the fetus gains its iron stores in utero. After parturition and involution, old implantation sites may be visible grossly as dark areas in the mucosa, which are represented histologically by stromal hemosiderosis and arterial hyalinization. The placenta of cetaceans is diffuse epitheliochorial. The structure of the phocid corpus luteum is described by Sinha et al. (1972; 1977a). The prostate is the only accessory sex gland in pinnipeds and cetaceans (Harrison, 1969a). It is tubuloalveolar and has cuboidal to low-columnar to pseudostratified lining cells with basilar nuclei and pale apical cytoplasm. The fine structure of phocid testes and seminiferous tubules are described by Leatherland and Ronald (1979) and Sinha et al. (1977b), respectively. Adrenals Pinniped adrenals may have an undulating or pseudolobulated cortex. In cetaceans, however, pseudolobulation is extensive and is created by connective tissue septae extending from the capsule. Large nerves, ganglia, and many blood vessels are associated with the hilus and capsular surface of pinniped adrenals. Lymphoid and Hematopoietic Systems The capsules and trabeculae of pinniped lymph nodes are quite thick, and there is often abundant hilar and medullary connective tissue as well (Welsch, 1997). The degree of fibrosis seems to increase with age, and may be a function of chronic drainage reactions. Pinniped lymph nodes are organized like those of canids, having a peripheral subcapsular sinus, cortical follicular and interfollicular (paracortical) regions, and medullary cords and sinuses. Although some authors report that marine mammal lymphoid tissue is usually quiescent and lacks follicular development, secondary follicles are common in both peripheral and visceral lymph nodes of stranded pinnipeds, probably due to the common presence of skin wounds and visceral parasitism. In many stranded pinnipeds, the lymph nodes are sparsely but diffusely populated by lymphocytes, and the ghosts of germinal centers can be seen. Since this morphology is most common when the interval from death to post-mortem is prolonged, it has been interpreted to be a “washing out” of lymphocytes due to autolysis. The lymph nodes of some cetaceans are often deeply infolded or fused so that they appear to be organized similarly to the nodes of suids, whose follicular cortex is buried deep within the node and sinusoids and cords are located more toward the periphery. The correlation of anatomical location with nodal morphology has not been made for all species. The visceral nodes of the bottlenose dolphin have extensive smooth muscle in the capsule and trabeculae and have incomplete marginal sinuses (Cowan and Smith, 1999). The lymph nodes of the beluga are described by Romano et al. (1993). The elongated spleen of pinnipeds has a thick fibromuscular capsule and trabeculae with a sinusoidal pattern similar to that of canids. Periarteriolar reticular sheaths are more prominent in phocids than in otariids. The spherical spleen of cetaceans also has a thick capsule, which is fibrous externally and muscular internally, with the muscle cells extending into the thick trabeculae (Cowan and Smith, 1999). Extramedullar hematopoiesis is common in the spleens of pinniped and sea otter pups, but it seems to be uncommon in cetaceans. Nervous System A detailed description of marine mammal neuroanatomy is beyond the scope of this chapter; for a comparison of some marine mammal brains (D-BRN), see Pabst et al. (1999). Suffice it 0839_frame_C09 Page 151 Tuesday, May 22, 2001 10:43 AM Gross and Microscopic Anatomy 151 to say that the brains of cetaceans and pinnipeds are large and well developed and have complex gyri in the cerebral and cerebellar cortices that are relatively larger than similarly sized brains of terrestrial mammals (Flanigan, 1972). The cetacean cerebrum is globoid and the rostral lobes extend ventrally. Like higher primates, cetaceans have well-developed temporal lobes (ventrolateral aspects of the cortices) that make brain removal a challenge. The pinniped brain is similar in orientation to the canine brain except for the larger cerebellum. In pinnipeds, the pineal gland is very large (up to 1.5 cm in diameter), especially in neonates (Bryden et al., 1986) and the size varies seasonally (see Chapter 10, Endocrinology). The pineal gland is located on the dorsal aspect of the diencephalon between the thalami and may be attached to the falx cerebri when the calvarium is removed at necropsy. There are no published descriptions of the pineal in cetaceans, and whether or not it exists is unclear. The pituitary gland is relatively large in both cetaceans and pinnipeds (Harrison, 1969b; Leatherland and Roland, 1976; 1978; Griffiths and Bryden, 1986). It is located within a shallow sella tunica in cetaceans and is surrounded by reams of blood vessels making it difficult to remove on necropsy. In pinnipeds, it is often sheared off during removal of the brain, so care should be taken to cut the lip of bone partially covering it to remove it intact. The spinal cord of phocids is relatively shorter than that of otariids; only the cauda equina occupies the lumbar and sacral canal. The cauda equina of the harbor seal pup is similar to that of the dog, but as they grow older, the cord changes significantly. The cauda equina starts in the lumbocaudal region in manatees. The region surrounding the cord—the vertebral canal—is significantly enlarged in seals, cetaceans, and sirenians. The neural canal is filled mostly with vascular tissue in seals and cetaceans and mostly with venous and fatty tissue in manatees. Manatee brains have pronounced lissencephaly and large lateral ventricles (Reep et al., 1989). Circulatory Structures In general, blood vessels are named for the regions they feed or drain. Thus, the fully aquatic marine mammals (cetaceans and sirenians) lack femoral arteries, which supply the pelvic appendage. However, most organs in marine mammals are similar to those of terrestrial mammals, so their central blood supplies are also similar. The aorta (D-AOR) leaves the heart (D-HAR) as the ascending aorta, then forms the aortic arch (D-AAR) and roughly follows the vertebral column dorsal to the diaphragm as the thoracic aorta, which gives off segmental intercostal arteries and, in the case of cetaceans and manatees, feeds to the thoracic retia. Some of the segmental arteries of the dolphin anastomose at the base of the dorsal fin to form the single arteries that are arranged along the centerline of the dorsal fin (D-DFNaa). The aorta continues into the abdomen as the abdominal aorta, which gives off several paired (e.g., renal, gonadal) and unpaired (e.g., celiac, mesenteric) arteries. The caudal aorta follows the ventral aspect of the vertebrae in the tail; in the permanently aquatic marine mammals the caudal vessels are large when compared with the vessels in species with small tails. In the dolphin, the caudal arteries branch into dorsal and ventral superficial arrays of arteries (D-FLKaa; Elsner et al., 1974). In the permanently aquatic marine mammals, there are robust ventral chevron bones that form a canal in which the caudal aorta, its branches, and some veins (the caudal vascular bundle, D-CVB) are protected. This site is convenient in some species for venipuncture; however, note that it is an arteriovenous plexus, so samples collected may be mixed arterial and venous blood. Some of the diving mammals (e.g., seals, cetaceans, and sirenians) have few or no valves in their veins (Rommel et al., 1995); this adaptation simplifies blood collection because the blood can drain toward the site from both directions, although blood collection is complicated by the arteriovenous plexuses described above. Other exceptions to the general pattern of mammalian 0839_frame_C09 Page 152 Tuesday, May 22, 2001 10:43 AM 152 CRC Handbook of Marine Mammal Medicine circulation are associated with thermoregulation and diving. Countercurrent heat exchangers abound, and extensive arteriovenous anastomoses exist to permit two general objectives to be fulfilled: (1) regulating loss of heat to the external environment while keeping core temperatures high, and (2) permitting cool blood to reach specific organs (e.g., testes and epididymides, ovaries and uteri) that cannot sustain exposure to high body temperatures (see reviews by Rommel et al., 1998; Pabst et al., 1999). Mammals have three options for blood supply to the brain: the internal carotid, the external carotid, and the vertebral arteries. Some species use only one and others two, but the manatees use all three pathways. Cetaceans have a unique blood supply to the brain (D-BRN); the blood to the brain first enters the thoracic retia, a plexus of convoluted arteries in the dorsal thorax. Blood leaves the thoracic retia and enters the spinal retia, where it surrounds the spinal cord and enters the foramen magnum (McFarland et al., 1979). There are two working hypotheses for this convoluted path to the brain: (1) the elasticity of the retial system allows mechanical damping of the blood pulse pressure wave (McFarland et al., 1979; Shadwick and Gosline, 1994), and (2) the juxtaposition of the thoracic retia to the dorsal aspect of the lungs may provide thermal control of blood entering the spinal retia (Rommel et al., 1993b). Combined with cooled blood in the epidural veins, the spinal retia may provide some temperature control of the central nervous system (Rommel et al., 1993b). Carotid bodies, important in regulation of blood flow, have been documented in the harbor seal (Clarke et al., 1986). The Potential for Thermal Insult to Reproductive Organs Mammals maintain high and, in most species, relatively uniform core temperatures. Because they live in water, which conducts heat 25 times faster than air at the same temperature, many marine mammals have elevated metabolic rates and/or adaptations to reduce heat loss to the environment (Kooyman et al., 1981; Costa and Williams, 1999). Aquatic mammals with low metabolic rates must live in warm water or possess even more elaborate heat-conserving structures. Most mammalian tissues tolerate limited fluctuations in temperature, and some tissues, such as muscle, perform better at somewhat higher temperatures. However, reproductive tissues are particularly susceptible to thermal insult, and various mechanisms have evolved to protect them (VanDemark and Free, 1970; Blumberg and Moltz, 1988). In terrestrial mammals, production and storage of viable sperm requires a relatively narrow range of temperatures. Temperatures between 35 and 38°C can effectively block spermatogenesis (Cowles, 1958; 1965). Abdominal temperatures can detrimentally affect long-term storage of spermatozoa in the epididymides in many species (Bedford, 1977). In many mammals, the scrotum provides a cooler environment by allowing the sperm-producing tissues to be positioned outside the abdominal cavity, away from relatively high core temperatures. Additionally, in scrotal mammals, the pampiniform plexus can, via countercurrent heat exchange, reduce the temperature of arterial blood from the core to the testes and help keep testicular temperature below that of the core (Evans, 1993). The skin of the scrotum is well vascularized, has an abundance of sweat glands, and is highly innervated with temperature receptors. Muscles in the scrotal wall involuntarily contract and relax in response to cold and hot temperatures, respectively. The exposed scrotum provides a thermal window through which heat may be transferred to the environment, thereby regulating the temperature of sperm-producing tissues. Interestingly, the morphological adaptations for streamlining observed in some marine mammals create potentially threatening thermal conditions for the reproductive systems of diving mammals. The primary locomotory muscles of terrestrial mammals are appendicular, 0839_frame_C09 Page 153 Tuesday, May 22, 2001 10:43 AM Gross and Microscopic Anatomy 153 so much of the locomotory heat energy of the muscle is transferred to the environment rather than directed into the body cavities; this is not the case for ascrotal marine mammals, whose primary locomotory muscles surround the abdominal and pelvic cavities. A factor that may increase core temperature of marine mammals is change in blood flow patterns during diving. Marine mammals can dramatically redistribute their cardiac output during dives, resulting in severely reduced blood flow to some body tissues, such as muscles and viscera (Elsner and Gooden, 1983; Kooyman, 1985). In terrestrial mammals, redistributions of cardiac output in response to physiological conditions such as exercise, feeding, thermoregulation, and pregnancy are relatively well known (Elsner, 1969; Baker and Chapman, 1977; Baker, 1982; Blumberg and Moltz, 1988). For example, in humans, large increases in muscle temperature (as high as 1°C/min) have been measured during the ischemia at the onset of exercise (Saltin et al., 1968). Surprisingly, the magnitude of routine cardiovascular adjustments undergone by marine mammals during prolonged dives (Elsner, 1999) is approached in terrestrial mammals only during pathological conditions such as hyperthermia and hypovolemic shock. The axial locomotion of pinnipeds, cetaceans, and manatees requires a relatively large thermogenic muscle mass around the vertebral column and abdominal organs. Blubber insulates these thermogenic muscles, suggesting the potential for elevated temperatures at the reproductive systems, particularly during the ischemia of prolonged dives. The temporary absence of cooling blood through locomotory muscles increases the probability of severe thermal consequences for the diving mammal. Abdominal, or partly descended, testes (cryptorchidism) result in sterility in many domestic mammals and humans. Ascrotal testes are typical for many marine mammals, such as phocid seals, dolphins, and manatees. There are vascular adaptations that prevent deep-body hyperthermic insult in cetaceans and phocids (Rommel et al., 1998). In dolphins, cooled venous blood is delivered to an inguinal countercurrent heat exchanger to cool the testes and epididymides indirectly, whereas, in phocid seals, cooled venous blood is delivered to an inguinal venous plexus to cool the testes and epididymides directly. Similar structures prevent reproductive hyperthermic insult in females (Rommel et al., 1995). One additional vascular adaptation that may have significant influence on diving is the presence of cooled blood in the large vascular structures within the vertebral canal, adjacent to the spinal cord. The large epidural veins (dolphins, seals, and manatees) and spinal retia (dolphins) may influence spinal cord temperature and, thus, influence dive capabilities, by modifying regional metabolic rates (Rommel et al., 1993b). The central nervous system is temperature sensitive, and lowering cord temperature influences global metabolic responses. Skeleton Knowledge of the skeleton offers landmarks for soft tissue collection and provides an estimate of body size from partial remains (Rommel and Reynolds, in press). Traditionally, the postcranial skeleton is subdivided into axial components (the vertebral column, ribs, and sternabrae, which are “on” the midline) and appendicular components (the forelimbs, hind limbs, and pelvic girdle, which are “off ” the midline). The scapulae and humeri of the forelimbs are indirectly attached to the body, essentially by tensile elements (muscles and tendons); in contrast, the hind limbs are attached via a pelvis directly to the vertebral column and thus are able to transmit both tension and compression to the body. The skeleton supports and protects soft tissues, controls modes of locomotion, and determines overall body size and shape; the marrow of some bones may generate the precursors of certain blood cells. While the animal is alive, bones are continuously remodeled in response to biochemical and biomechanical demands and, thus, offer information that can help 0839_frame_C09 Page 154 Tuesday, May 22, 2001 10:43 AM 154 CRC Handbook of Marine Mammal Medicine biologists interpret events in the life history of the animal after its death. Skeletal elements contribute to fat (particularly in the cetaceans) and calcium (particularly in the sirenians) storage and thus influence buoyancy. The sea lion propels itself through the water by its forelimbs, and its skeletal components are relatively massive in that region. On land, its forelimbs can act as fulcra for shifting the center of mass by changing the shape of its neck and the trunk (for more, see English, 1976a,b; 1977). The permanently aquatic species locomote with a dorsoventral motion of the trunk and elongated tail. This dorsoventral motion of the axial skeleton is characteristic of almost all mammalian locomotion. In contrast, the seal uses lateral undulations of its trunk and hind flippers when swimming (like a fish), yet it may locomote on land with dorsoventral undulations, like its terrestrial ancestors. Relative motion between vertebrae is controlled, in part, by the size and shape of the intervertebral disks. The intervertebral disks resist the compression that skeletal muscles exert and tend to force vertebrae together. Intervertebral disks are composite structures, with a fibrous outer ring, the annulus fibrosus, and a semiliquid inner mass, the nucleus pulposus. The outermost fibers of the annulus are continuous with the fibers of the periosteum. The flexibility of the vertebral column depends, in part, on the thickness of the disks. Intervertebral disks are a substantial proportion (10 to 30%) of the length of the postcranial vertebral column. The intervertebral disks provide flexibility but are not “responsible” for the general curvature of the spine—the nonparallel vertebral body faces provide the spinal curvature. For convenience, the vertebral column is separated into five regions, each of which is defined by what is or is not attached to the vertebrae. These regions are cervical, thoracic, lumbar, sacral, and caudal. In some species, the distinctions between vertebrae from each region are unambiguous. However, in some other species the distinctions between adjacent regions are less obvious. This is particularly true in the permanently aquatic species, where there is little or no direct connection between the pelvic vestiges and the vertebral column. The vertebral formula varies within, as well as among, species. The number of vertebrae, excluding the caudal vertebrae, is surprisingly close to 30 in most mammals (Flower, 1885). Most mammals have seven cervical, or neck, vertebrae (sirenians and two-toed sloth have six and the three-toed sloth has nine), whereas the number of thoracic and lumbar vertebrae varies between species. The number of sacral vertebrae is commonly two to five, but there are exceptions. The number of caudal vertebrae varies widely—long tails usually have numerous caudal vertebrae. The cervical vertebrae are located cranial to the rib-bearing vertebrae of the thorax. Some cervical vertebrae have movable lateral processes known as cervical ribs, none of which makes contact with the sternum. Typically, the permanently aquatic marine mammals have short necks, even if they have seven cervical vertebrae. However, the external appearance of a short neck in seals is misleading. Close comparison of the seal and sea lion skeletons reveals that they have quite similar neck lengths, although the distribution of body mass is different. Seals often hold their heads close to the thorax, which causes a deep “S” curve in the neck. This provides the seals with a “slingshot potential” for grasping prey (or careless handlers). The shapes of the seal neck vertebrae are complex to allow this curve. Serial fusion (ankylosis) of two or more cervical vertebrae is common in the cetaceans, although in some cetaceans (e.g., the narwhal, beluga, and river dolphins), all the cervical vertebrae are unfused and provide considerable neck mobility. The rib-bearing vertebrae are the thoracic vertebrae, and the thoracic region is defined by the presence of movable ribs. The authors distinguish between vertebral ribs (E-VBR), which are associated with the vertebrae, and “sternal ribs” (E-SBR), which are associated with the sternum. This distinction is made because some odontocetes, unlike most other mammals, 0839_frame_C09 Page 155 Tuesday, May 22, 2001 10:43 AM Gross and Microscopic Anatomy 155 have bony rather than cartilaginous sternal ribs (bony “sternal ribs” are also found in the armadillo). “Costal cartilages” is an acceptable alternative term for sternal ribs if the sternal ribs are never ossified (calcification with old age does not count). Some thoracic vertebrae have ventral vertebral projections called hypapophyses (see the manatee, E-HYP)—not to be confused with chevron bones, which are intervertebral and not part of the caudal vertebrae. In the manatee, the diaphragm is firmly attached along the midline of the central tendon to hypapophyses. Hypapophyses also occur in some cetaceans (e.g., the pygmy and dwarf sperm whales, Kogia) in the caudal thorax and cranial lumbar regions. It is assumed that these hypapophyses increase the mechanical advantage of the hypaxial muscles much as do the chevrons (Rommel, 1990). The neural spines (E-NSP) of thoracic vertebrae of many mammals are often longer than those in any other region of the body. Long neural spines provide mechanical advantage to neck muscles that support a head cantilevered in front of the body. Terrestrial species with large heads tend to have long neural spines, but in aquatic mammals the buoyancy of water negates this reason for long neural spines. Ribs Embryologically, ribs and transverse processes develop from the same precursors. Thus, some aspects of ribs are similar to those of transverse processes (E-TPR). It is the formation of a movable joint that distinguishes a rib from a transverse process. An unfinished joint may be indicative of developmental age. In some species (i.e., the manatee) there may be a movable “rib” (pleurapophysis) on one side and an attached “transverse process” on the other side of the same (typically the last thoracic) vertebra (Rommel and Reynolds, 2000). Ribs may attach to their respective vertebrae at one or more locations (e.g., centrum, transverse process). Typically, the cranialmost ribs have two distinct regions of articulation (capitulum and tuberculum) with juxtaposed vertebrae and are referred to as double headed. The caudalmost ribs have single attachments and are referred to as single headed. In most mammals, the single-headed ribs have lost their tubercula and are attached to their vertebrae at the capitulum on the centrum. In contrast, the single-headed ribs of cetaceans lose their capitula and are attached to their respective vertebrae by their tubercula on the transverse processes (Rommel, 1990). The last ribs (E-LRB) often “float” free from attachment at one or both ends; these ribs tend to be significantly smaller than the ones cranial to them, and they are often lost in preparation of the skeleton. The ribs of some marine mammals are more flexible than those of their terrestrial counterparts; this flexibility is an adaptation to facilitate diving. Ribs are illustrated in layer E in the correct posture for a healthy animal. Note that all illustrated species but the manatees have oblique angles between the rib shaft and the long axis of the body. As the hydraulic pressures increase with depth, the ribs rotate to avoid bending with changes in thoracic cavity volume. Sternum The sternum (D,E-STR) is formed from bilaterally paired, serial elements called sternabrae. The paired elements fuse on the midline, occasionally imperfectly, leaving foramina in the sternum. The cranialmost sternal ribs (E-SRB, also called costal cartilages) extend from the vertebral ribs to articulate firmly with the sternum at the junctions between sternebrae. The first sternal rib articulates with the manubrium (C,D-MAN) cranial to the first intersternabral joint. The manubrium may have an elongate cartilaginous extension (e.g., in seals), and the first sternal rib is often different from the more caudal sternal ribs (typically larger and more robust). In some mysticetes, only the manubrium is formed, and only the first rib has a bony attachment to it. The subsequent ribs articulate with a massive cartilaginous structure that extends from the caudal 0839_frame_C09 Page 156 Tuesday, May 22, 2001 10:43 AM 156 CRC Handbook of Marine Mammal Medicine aspect of the manubrium (which may be referred to as a pseudosternum). The xiphoid process (E-XIP, last sternabra) is also different; it too may articulate with more than one (often many) sternal rib(s) and have a large cartilaginous extension. Postthoracic Vertebrae Some authors avoid the difficulties of defining the lumbar, sacral, and caudal regions in the permanently aquatic species by lumping them into one category—the postthoracic vertebrae; by “lumping,” these authors avoid some interesting comparisons. Generally, the lumbar vertebrae are trunk vertebrae that do not bear ribs, and the number of lumbar vertebrae is closely linked to the number of thoracic vertebrae, but not always. Note that the caudal vertebrae of cetaceans start with the start of the chevron bones, and extend to the tip of the tail (fluke notch, A-NOC), whereas manatee vertebrae stop 3 to 9% of the total body length (as much as 17 cm in a large specimen) from the fluke tip (E-LVR). Sacral Vertebrae There are at least two commonly accepted definitions for sacral vertebrae: (1) serial fusion of postlumbar vertebrae, only some of which may attach to the pelvis (the human os sacrum), and (2) only those that attach to the ilium, whether or not they are serially fused. Both definitions have merit. Within species, the number of serially ankylosed vertebrae may vary, particularly with age. Additional landmarks are the exit of spinal nerves from the neural canal and the foramina for segmental blood vessels. In species with a bony attachment between the vertebral column and the pelvis, the definition of sacral is easy. However, in the cetaceans and some sirenians (dugongs have a ligamentous attachment between the vertebral column and the pelvic vestiges), there are no sacral vertebrae by definition. Chevron Bones The chevron bones are ventral intervertebral ossifications in the caudal region. By definition, each is associated with the vertebra cranial to it (note that there is some controversy over which is the first caudal vertebra; see Rommel, 1990). Chevron bone pairs are juxtaposed (in manatees) or fused (in dolphins, but not always) at their ventral apexes and articulate dorsally with the vertebral column to form a triangular channel. Within the channel (hemal canal) are found the blood vessels to and from the tail. In some species, the ventral aspects of each chevron bone fuse and may continue as a robust ventral protection that can function to increase the mechanical advantage of the hypaxial muscles to ventroflex the tail. In some individuals, the first two or three chevrons may remain open ventrally but fuse serially on either side. Pectoral Limb Complex The forelimb skeleton includes the scapula, humerus, radius and ulna, and manus. The scapula is attached to the axial skeleton only by muscles. There is no functional clavicle in marine mammals (Strickler, 1978; Klima et al., 1980). The scapula consists of an essentially flat (slightly concave medially) blade with an elongate scapular spine extending laterally from it. The distal tip of the spine, if present, is the acromion. The scapular spine is roughly in the center of the scapular blade in most mammals. However, in cetaceans, the scapular spine is close to the cranial margin of the scapular blade, and both the acromion and coracoid extend beyond the leading edge of the blade. The humerus (E-HUM) has a ball-and-socket articulation in the glenoid fossa of the scapula— this is a very flexible joint. The humerus articulates distally with the radius (E-RAD) and ulna (E-ULN); this is also a flexible joint in most other mammals, but it is constrained in cetaceans. The olecranon is a proximal extension of the ulna that increases the mechanical advantage of the 0839_frame_C09 Page 157 Tuesday, May 22, 2001 10:43 AM Gross and Microscopic Anatomy 157 triceps muscles that extend the forelimb. In species like the sea lion, the olecranon is robust; however, in the cetacea, it is relatively small. The radius and ulna of manatees fuse at both ends as the animal ages. This fusion prevents axial twists that pronate and supinate the manus. The radius and ulna of cetaceans are also constrained but not typically fused. The distal radius and ulna articulate with the proximal aspect of the manus. The manus includes the carpals, metacarpals, and phalanges (English, 1976). There are five “columns” of phalanges, each of which is called a digit. The digits are numbered starting from the cranial aspect (the thumb, which is digit one, associated with the radius). In many of the marine mammals, the “long” bones of the pectoral limb (humerus, radius, and ulna) are relatively short, and the phalanges are elongated. Cetaceans are unique among mammals in that they have more than the maximum number of phalanges found in all other mammals; this condition is known as hyperphalangy (Howell, 1930). The number varies within each species—the bottlenose dolphin has a maximum number of nine digits. Pelvic Limb Complex The typical mammalian pelvis is made of bilaterally paired bones: ilium, ischium, pubis, and acetabular bone (the paired ossa coxarum), one to three caudal vertebrae, and the sacrum. Each of the halves of the pelvis attaches (via the ilium) to one or more sacral vertebrae. The crest of the ilium (C,E-ILC) is a prominent landmark that flares forward and outward beyond the region of attachment between the sacrum and the ilium. The ossa coxarum join ventrally along the midline at the pelvic symphysis, which incorporates the pubic bone cranially and the ischiatic bone caudally. In the permanently aquatic marine mammals, there is but a vestige of a pelvis (E-PEL) to which portions of the rectus abdominis muscles (B-REC) may attach. Additionally, the crura of the penis may be supported by these vestiges (Fagone et al., 2000). In some of the large whales, there is occasionally a vestige of a hind limb articulating with the pelvic vestige. The hind limb, if present, articulates with the vertebral column via a ball-and-socket joint at the hip. The proximal limb bone is the femur (C,E-FEM). The socket of the pelvis, the acetabulum, receives the head of the femur. Distally, the femur articulates with the tibia and the fibula (as the stifle joint). The tibia and fibula distally articulate with the pes, or foot. The pes is composed of the tarsals proximally, the metatarsals, and the phalanges distally. Note that the digits of the sea lion terminate a significant distance from the tips of the flipper. Sexual Dimorphisms In many mammals, the adult males are larger than the adult females. In marine mammals, this size difference is at its extreme in otariids, elephant seals, and the sperm whales. In contrast, the adult females of the baleen whales and some other species are larger than the adult males. In the permanently aquatic marine mammals, there may be sexual dimorphisms in the pelvic vestiges (Fagone et al., 2000). The penises of mammals are supported by crura consisting of a tough outer component (tunica albuginea) and the cavernous erectile central component (corpus cavernosum), which attach to the ischiatic bones of the pelvis. The muscles that engorge the penis with blood are also attached to the pelvis. Presumably, the mechanical forces associated with these muscles influence pelvic vestige size and shape, particularly in manatees. Males in some groups of mammals, particularly the carnivores, have a bone within the penis (the baculum) that helps support the penis. Growth rate of the os penis differs from that of the appendicular skeleton in some species (Miller et al., 1998). 0839_frame_C09 Page 158 Tuesday, May 22, 2001 10:43 AM 158 CRC Handbook of Marine Mammal Medicine Bone Marrow Bone marrow of cetceans is vertebral as well as costal. Because the marrow cavity of the bones of marine mammals generally retains abundant trabecular bone throughout life, it is best to examine the marrow histologically via impression smears of cut surface or in decalcified sections. Most manatee bones are amedullary (Fawcett, 1942), so usable marrow impression smears are restricted to vertebrae. Acknowledgments The authors thank Meghan Bolen, Judy Leiby, James Quinn, John Reynolds, Lisa Johnson, and Terry Spraker for reviewing the manuscript, Dan Cowan for information on parathyroids, and Frances Gulland and Rebecca Duerr at The Marine Mammal Center for helpful discussions. Anatomical illustrations were created with FastCAD (Evolution Computing, Tempe, AZ). References Arey, L.B., 1941, Throttling veins in the livers of certain mammals, Anat. Rec., 81: 21–33. Baker, J.R., 1989, Pollution-associated uterine lesions in grey seals from the Liverpool Bay area of the Irish Sea, Vet. Rec., 125: 303. Baker, M.A., 1982, Brain cooling in endotherms in heat and exercise, Annu. Rev. Physiol., 44: 85–96. Baker, M.A., and Chapman, L.W., 1977, Rapid cooling in exercising dogs, Science, 195: 781–783. 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Physiol., 47: 986–973. 0839_frame_C10 Page 165 Tuesday, May 22, 2001 11:08 AM 10 Endocrinology David J. St. Aubin Introduction The biochemicals classified as hormones are exceedingly potent agents, capable of profoundly influencing cellular functions to establish the optimum internal environment for a particular set of environmental challenges or survival needs. By definition, these chemicals are produced within well-defined glands or organs, secreted into blood or other extracellular media, and transported at least some distance to exert their effects on unrelated tissues. Endocrine systems are typically regulated through stimulatory and negative feedback mechanisms, often involving separate endocrine glands in a cascading sequence of hormone release originating from central neurological structures. Other biochemical stimuli, such as rising blood glucose or changes in the ratio of sodium to potassium (Na:K) in plasma, are equally capable of eliciting endocrine responses from the structures that are responsible for maintaining those constituents within appropriate physiological limits. The basic principles of vertebrate endocrinology, as presented in recent reference publications (Wilson et al., 1998), appear to hold for marine mammals. There are, nevertheless, some interesting adaptations, driven by the peculiar life histories of these animals, that represent important deviations from the norm for terrestrial mammals and need to be taken into account by both the researcher and the clinician. Some of these endocrine systems have received considerable attention in the literature, as extensively reviewed by Kirby (1990); for others, the available information is scant and deserves the attention of marine mammal physiologists and endocrinologists. The considerable and growing body of data on reproductive endocrinology will be examined in a separate chapter (see Chapter 11, Reproduction) focused on that specific aspect of marine mammal biology. Information on the status and role of various endocrine systems is invaluable to those seeking to understand better how marine mammals are able to survive the rigors of a most challenging environment. Prolonged fasts, deep dives, seasonally synchronized molting and breeding cycles, and an osmotically hostile medium, all require a metabolism finely tuned by endocrine controls. Breakdowns in these systems can significantly compromise the health and survival of the organism. The activity of specific endocrine organs, as measured by hormone levels in body fluids and excretions, can provide important information about the internal environment of the subject, and guide corrective therapy. Although large, the body of information on marine mammal endocrinology holds little regarding primary endocrinopathies, when compared with terrestrial mammals. More often, endocrine imbalances in marine mammals reflect perturbations in other systems, and the challenge is not only to establish what the primary cause might be, but also to recognize what physiological changes might be attributable to the secondary endocrine dysfunction. 0-8493-0839-9/01/$0.00+$1.50 © 2001 by CRC Press LLC 165 0839_frame_C10 Page 166 Tuesday, May 22, 2001 11:08 AM 166 CRC Handbook of Marine Mammal Medicine Sample Collection and Handling Blood The most commonly collected specimen for hormonal analysis is blood. Serum is preferred for most analyses, particularly for those in which anticoagulants have been identified as interfering with results. According to one manufacturer of radioimmunoassays (RIA) (Diagnostic Products Corporation, Los Angeles, CA), heparinized plasma yields satisfactory results, except for the measurement of free triiodothyronine (f T3), whereas EDTA-treated blood is generally unsuitable. Fasting is not usually a prerequisite for obtaining a sample for thyroid and adrenal hormone analysis, but highly lipemic samples collected during the absorptive phase after eating are unsuitable for thyroid hormone (TH) testing. For hormones such as cortisol, known to exhibit diurnal variation, it is important to standardize, or at least note, the time of day at which the specimen is collected to interpret the results properly. Most hormones, particularly steroids and TH, are quite stable in serum samples refrigerated for 2 to 3 days or stored frozen at −70°C for months. Thawed samples should not be refrozen. Saliva The measurement of hormones in saliva represents an attractive alternative as a noninvasive technique (Theodorou and Atkinson, 1998). Nevertheless, its collection requires either wellestablished behavioral control or full restraint, either of which can be used for the collection of blood samples. Laboratories are becoming better equipped to test saliva, and this is likely to result in more extensive reference data and established correlations with circulating levels of the hormone in question. One manufacturer of testing kits (Salimetrics LLC, State College, PA) recommends the use of plain, non-citric acid–treated, cotton Salivettes® (Sarstedt, Leicester, UK). Saliva samples should be frozen prior to assay to precipitate mucins. The approach has been investigated for monitoring reproductive hormones in marine mammals (Theodorou and Atkinson, 1998) (see Chapter 11, Reproduction), but there are insufficient data on other endocrine systems to establish its utility at this time. Feces Fecal analysis of corticosteroids and reproductive hormones has proved useful in monitoring the endocrine status of terrestrial mammals (Brown et al., 1994), and has been attempted in at least one study on cortisol in harbor seals (Phoca vitulina) (Gulland et al., 1999). Samples may be frozen for months prior to analysis. Cortisol was extracted in a solution of buffered saline and 50% ethanol containing 0.1% bovine serum albumin and 5% Tween 20 (Zymed Laboratories, Inc., San Francisco, CA), and then assayed using conventional radioimmunoassay techniques. Cortisol concentrations up to 1100 µg/kg were reported, but could not be correlated with plasma values obtained either at the approximate time of fecal collection or at the peak of adrenocortical stimulation on the previous day. Further studies are needed to allow the use and interpretation of fecal hormone data for marine mammals. Urine Hormones responsible for fluid and electrolyte balance, such as aldosterone and vasopressin, have been analyzed in urine samples of phocid seals (Hong et al., 1982). A 24-hour sample is optimal to integrate the daily fluctuations associated with consumption of food and water, which presents some impediment to investigations in marine mammals that cannot be confined or held out of water for the duration. Behavioral collection of urine has been established in 0839_frame_C10 Page 167 Tuesday, May 22, 2001 11:08 AM Endocrinology 167 cetaceans, but still cannot ensure that some of the daily urine production has not been lost into the environment. Samples for aldosterone determination should be refrigerated during or immediately after collection, and are stabilized with 1 g of boric acid/100 ml; they may be refrigerated for up to a week or stored frozen at −20°C for a month. No preservative is required for cortisol. Tissues Palmer and Atkinson (1998) established a methodology for analyzing the corticosteroid content of blubber biopsies, specimens that are routinely collected for genetic studies on free-ranging cetaceans, particularly large whales from which blood, saliva, and urine are virtually impossible to acquire. Once validated, relative to more established measures of circulating hormone concentrations, the approach could prove useful in field studies on mysticetes, among others. Pineal Gland Marine mammals exhibit strong seasonality in activities such as reproduction and molt. Synchronization of such events with appropriate environmental conditions is critical to optimizing survival, and likely requires the ability to sense cues that signal important seasonal events. Changes in air and water temperatures and daylength, particularly at midtemperate to high latitudes, can be pronounced enough to trigger significant annual events, such as migration in humpback whales (Megaptera novaeangliae) (Dawbin, 1966) (see Chapter 1, Sentinels). The hormone melatonin is considered to play a critical role in the integration of endocrine physiological systems with photoperiod in mammals (Goldman, 1983; Vivien-Roels and Pévet, 1983). Although at present of minimal clinical significance in marine mammals, the sporadic research that has been undertaken, particularly in pinnipeds, has identified the critical role of melatonin in early metabolism and subsequent seasonal activities. The principal source of melatonin is the pineal gland (epiphysis) typically located above the third ventricle of the brain. Other tissues, such as the retina, intestines, red blood cells, and salivary glands, contribute to circulating levels, and may represent significant sources in cetaceans, for which the very existence of a discrete pineal has been controversial (Flanigan, 1972). Nevertheless, Arvy (1970) and Behrmann (1990) have described the organ in several species of small odontocetes. This contrasts to the prominence of the gland in some pinnipeds, notably the Weddell seal (Leptonychotes weddellii) (Cuelo and Tramezzani, 1969; Bryden et al., 1986), northern fur seal (Callorhinus ursinus) (Elden et al., 1971), and northern (Mirounga angustirostris) (Bryden et al., 1994) and southern elephant seals (M. leonina) (Bryden et al., 1986; Little and Bryden, 1990). Earlier work on northern fur seals had recognized the pineal’s impressive dimensions and activity relative to those in humans, and suggested that further investigation might provide useful insights into the physiological role of melatonin in mammals (Elden et al., 1971). Weighing as much as 9 g in the newborn southern elephant seal (Little and Bryden, 1990), the gland can be roughly the size of the entire brain of a hamster, the species that has contributed most substantially to the understanding of melatonin physiology (Goldman, 1983). Elephant seals continue to show substantial changes in the size of their pineal throughout life. The gland is largest in the dark of winter, weighing up to 2 g/1000 kg of body weight, and regresses to less than half of that in nearly constant daylight in the summer (Griffiths et al., 1979; Griffiths and Bryden, 1981; Griffiths, 1985). No less remarkable are the fluctuations in circulating concentrations of melatonin that are most evident soon after birth in southern elephant seals (Table 1). Levels approaching 69,000 pg/ml have been recorded in neonates (Little and Bryden, 1990), with concentrations diminishing to 0839_frame_C10 Page 168 Tuesday, May 22, 2001 11:08 AM 168 CRC Handbook of Marine Mammal Medicine TABLE 1 Reported Concentrations (pg/ml) of Melatonin in Pinnipeds Species Cystophora cristata (hooded seal) Halichoerus grypus (gray seal) Leptonychotes weddellii (Weddell seal) Mirounga angustirostris (northern elephant seal) Mirounga leonina (southern elephant seal) Pagophilus groenlandicus (harp seal) Specimens Neonate, 24-h sample Neonate, 24-h sample Pup (4 d), 24-h sample Pup (10 d), 24-h sample Pup (0–10 d) day Pup (12–35 d) day Juvenile (60 d) Adult Pup (0–5 d) day Pup (0–5 d) night Pup (6–25 d) day Pup (4 wk) night Pup (4 wk) day Juvenile (10 wk) Adult Neonate 0–24 h Pup (0–5 d) Pup (6–20 d) Juvenile Postpubertal Adult Neonate (1–2 d) Pup (2 wk) Melatonin (pg/ml) 0–6000 100–7000 0–3000 0–450 50–>1000 50–220 53 5–12 695–1159 1200–>2318 <93 23–93 14–23 23–93 23 29–68,904 1275–4172 239–927 10–110 12–60 26 0–9000 0–160 Reference Stokkan et al., 1995 Stokkan et al., 1995 Stokkan et al., 1995 Stokkan et al., 1995 Bryden et al., 1986 Bryden et al., 1986 Barrell and Montgomery, 1989 Barrell and Montgomery, 1989 Bryden, 1994; Bryden et al., 1994 Bryden, 1994; Bryden et al., 1994 Bryden, 1994; Bryden et al., 1994 Bryden et al., 1994 Bryden et al., 1994 Bryden et al., 1994 Bryden et al., 1994 Little and Bryden, 1990 Bryden, 1994 Bryden, 1994 Griffiths et al., 1979 Griffiths and Bryden, 1981; Griffiths, 1985 Bryden et al., 1986 Stokkan et al., 1995 Stokkan et al., 1995 Note: Values given as a range of means from multiple publications, or either the mean or range (when available) from a single source. Some of the data were estimated from figures. pg/ml × 4.314 = pmol/l. less than 1000 pg/ml over the ensuing month (Bryden et al., 1986). Harp (Pagophilus groenlandicus), hooded (Cystophora cristata), and gray (Halichoerus grypus) seals show a similar pattern, with peak values of roughly 6000 to 9000 pg/ml. Since all these species give birth under relatively harsh environmental conditions, at least in the areas where they were studied, it has been suggested that, as in some other mammals (Heldmaier et al., 1981; Puig-Domingo et al., 1988), the hormone acts to enhance the production of T3 to stimulate nonshivering thermogenesis (NST) (Little and Bryden, 1990; Bryden, 1994). However, since the commonly recognized mechanism for NST involves brown adipose tissue, which has yet to be demonstrated in species such as Weddell and hooded seals, Stokkan et al. (1995) have suggested an alternative, but as yet untested, explanation to account for the extraordinarily high circulating levels of melatonin in these animals. The potent antioxidant properties of the hormone might protect the fetus from the detrimental effects of hypoxia experienced in utero during diving. Melatonin levels in plasma vary seasonally in southern elephant seals, with high concentrations inhibiting gonadotropic hormones in winter; lower activity in summer allows gonadal recrudescence to occur (Griffiths et al., 1979; Griffiths and Bryden, 1981; Griffiths, 1985). Circadian rhythms typical of other mammals are evident in several species (Griffiths et al., 1979; Bryden et al., 1994; Stokkan et al., 1995), and are abolished as expected under conditions of continuous daylight in southern elephant seals and Weddell seals (Griffiths et al., 1979; Barrell and Montgomery, 1989). 0839_frame_C10 Page 169 Tuesday, May 22, 2001 11:08 AM Endocrinology 169 Hypothalamus–Pituitary The endocrine connections linking higher centers of the central nervous system through to the pituitary gland have received little detailed study in marine mammals, and are presumed to function in a fashion similar to those in most other mammals. The organization of the pituitary itself is unremarkable, with distinguishable regions comparable to the pars distalis (adenohypophysis, anterior pituitary), pars nervosa (neurohypophysis, posterior pituitary), and pars intermedia (Harrison, 1969). The gland appears relatively immature in newborn elephant and harp seals (Leatherland, 1976; Leatherland and Ronald, 1978; Bryden, 1994), but well developed in the more precocious harbor seal (Amoroso et al., 1965). Immunohistochemical techniques have been used to identify the primary cell types typical of those for other mammals (Leatherland and Ronald, 1983; Bryden, 1994). Material derived from commercial whaling operations afforded the opportunity to isolate and characterize the adenohypophyseal hormones—adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), growth hormone (GH, somatotropin), lutenizing hormone (LH), and prolactin (PRL)—in a variety of mysticetes and also sperm whales (Physeter macrocephalus) (Kawauchi et al., 1978; Kawauchi and Tubokawa, 1979; Kawauchi, 1980). Considerable homology exists between the cetacean forms and those in other mammals. In fact, the amino acid sequence for ACTH from fin whales (Balaenoptera physalus) was found to be identical to that of humans (Kawauchi et al., 1978). Measurement of circulating levels of anterior pituitary hormones has seldom been reported. Most of the information available is for the gonadotropic hormones, considered elsewhere in this book (see Chapter 11, Reproduction). In view of the demonstrated homology between human and mysticete ACTH, analysis using conventional RIA systems would be expected to yield satisfactory results. Nevertheless, there are no published values for this hormone for any marine mammal. Commercially available reagents for measuring human TSH appear to be ineffective in detecting the hormone in belugas (Delphinapterus leucas) and bottlenose dolphins (Tursiops truncatus) (St. Aubin and Geraci, unpubl. data). John et al. (1980) used an RIA specific for ovine GH to monitor relative changes in GH-like protein in young harp seals. Since purified seal GH was unavailable to validate and calibrate the assay, no actual concentrations could be reported. The development and application of methodologies specific for the hormones in marine mammals would lead to greater insights into the regulation of these important endocrine pathways. Neurohypophyseal hormones principally include oxytocin (OT) and vasopressin. The latter will be reviewed in a subsequent section for its role in water balance. OT enhances smooth muscle contraction and plays a key role in parturition and milk flow during nursing. Injections of 15 to 50 IU of commercially available, synthetic hormone have been used to facilitate the collection of samples for studies on the energetic value and proximate content of milk from pinnipeds (Iverson et al., 1993; Lydersen et al., 1995; 1997). The effectiveness of the homologue suggests at least crude similarities in the role played by this hormone in pinnipeds and other mammals. Thyroid Gland In contrast to the patchy information on most endocrine systems in marine mammals, reports on TH abound for these species. The integral role of TH in regulating metabolism has perhaps fueled a more extensive inquiry, given the long-standing, but more recently tempered, views suggesting extraordinarily elevated metabolism in marine mammals (Lavigne et al., 1986). THs are among the more broadly conserved and uniformly evident hormones in vertebrates. Thus, assays utilizing RIA or enzyme-linked immunosorbent assay (ELISA) developed for humans 0839_frame_C10 Page 170 Tuesday, May 22, 2001 11:08 AM 170 CRC Handbook of Marine Mammal Medicine and other species have been extensively applied in the measurement of total thyroxine (tT4) and tT3, with apparently satisfactory results (Greenwood and Barlow, 1979). T4 is typically the form detected in highest concentration, but its ability to elicit cellular responses is generally less than for its principal metabolic derivative, T3. Activation of T4 to T3 is mediated by a suite of deiodinating enzymes occurring in, and sometimes specific to, various peripheral tissues. The enzymes vary in their kinetic properties and sensitivities to inhibitors such as propylthiouracil (PTU). The thyroid gland is the site of hormone synthesis and storage of both T4 and T3. It is the only endocrine gland that establishes a significant reserve of hormone that can be later metered into circulation to meet metabolic needs. The hormones are stored as part of a colloid matrix composed of thyroglobulin, which carries coupled iodinated tyrosine residues. The colloid is deposited extracellularly and contained within a follicle lined by thyrocytes, which can pinocytose the matrix and release the hormones as needed. Histological examination of marine mammal thyroid has revealed no important differences from this typical arrangement, but has shown marked variation in the apparent levels of activity of thyrocytes at various times during the development and life history of phocids (Harrison et al., 1962; Amoroso et al., 1965; Little, 1991) and cetaceans (Harrison, 1969; St. Aubin and Geraci, 1989). Early investigators were impressed by the size of the cetacean thyroid, particularly in its proportion to the weight of the animal. Belugas have three times more thyroid per unit body weight than a thoroughbred horse, and bottlenose dolphins have nearly twice as much as do humans (400 vs. 250 mg/kg) (Ridgway and Patton, 1971). This observation correlated well with assumptions that cetacean metabolic rate exceeded that predicted by Kleiber’s formula, presuming that the size of the gland reflected the amount of hormone released into circulation (Harrison and Young, 1970). Extensive measurements of both THs and reevaluation of assumptions about metabolic rate (see Chapter 36, Nutrition) have failed to support such a correlation. Nevertheless, the large reserve of hormone present in the beluga thyroid can sustain the marked elevation in circulating levels of TH that occurs during a brief period of thyroid hyperactivity in the summer period of estuarine occupation (St. Aubin and Geraci, 1989; St. Aubin et al., in press). An important consideration in the evaluation of thyroid status is the degree to which the hormones are bound by circulating proteins, principally thyroid binding globulin (TBG). Binding also occurs, but with lower affinity, to pre-albumin and albumin; efforts to demonstrate a pre-albumin binding protein in belugas and bottlenose dolphins have proved unsuccessful using methodologies established for other mammals (St. Aubin and Geraci, unpubl. data). It is presumed that the free, or unbound, hormone is responsible for regulating cellular processes, and that protein binding in circulation serves to deliver the hormone, maintain an available pool, and modulate the activity of metabolically potent substances such as TH (Ekins, 1986). The impact of TH can thus be regulated at a variety of levels, including rate of secretion from the thyroid gland, plasma binding capacity, rate of conversion to T3, and density of cellular receptors for the hormone. Although analysis of circulating levels represents the most readily obtained measure of thyroid status, it may yield misleading or confusing results if other elements are not taken into consideration. Blood concentrations of TH, both total and free hormone, have been reported for a number of marine mammal species (Tables 2 to 4). Variation in methodology, particularly with respect to the earlier literature, makes direct comparisons among species difficult. Nevertheless, certain patterns emerge. Levels of total T4 in cetaceans tend to be higher than for most other species, although concentrations in dolphins are roughly comparable to those in humans. Pinnipeds and polar bears (Ursus maritimus) show concentrations similar to those in most terrestrial mammals, but surprisingly are lower than in manatees (Trichechus manatus) which seems incongruous in light of the notoriously low metabolic rate of the latter (Gallivan and Best, 1980) 0839_frame_C10 Page 171 Tuesday, May 22, 2001 11:08 AM 171 Endocrinology TABLE 2 Reported Circulating Concentrations (µg/dl) of Thyroxine in Marine Mammals Species Specimens Thyroxine (µg/dl) Reference Cetaceans Balaenoptera physalus (fin whale) Delphinapterus leucas (beluga) Globicephala macrorhyncus (short-finned pilot whale) Inia geoffrensis (Amazon River dolphin) Lagenorhynchus obliquidens (Pacific whitesided dolphin) Orcinus orca (killer whale) Phocoena phocoena (harbor porpoise) Tursiops truncatus (bottlenose dolphin) Not specified 5.4 Kjeld and Olafsson, 1987 Various ages, both sexes Two males, age unspecified 8.0–19.2 4.3 St. Aubin and Geraci, 1988; 1989; 1992; St. Aubin et al., in press Ridgway et al., 1970 Various ages, both sexes 1.5 Ridgway et al., 1970 Five females, age unspecified 2.6–3.7 Ridgway et al., 1970 Two males, age unspecified Various ages, both sexes Various ages, both sexes 6.1 Ridgway et al., 1970 11.2 Koopman et al., 1995 7.4–13.6 Ridgway et al., 1970; Greenwood and Barlow, 1979; Orlov et al., 1988; St. Aubin et al., 1996 Pinnipeds Callorhinus ursinus (northern fur seal) Cystophora cristata (hooded seal) Halichoerus grypus (gray seal) Leptonychotes weddellii (Weddell seal) Mirounga angustirostris (northern elephant seal) Various ages, both sexes Neonates Neonates 2.8 St. Aubin, unpubl. data 4–9 Stokkan et al., 1995 3–10 Stokkan et al., 1995; Woldstad et al., 1999 Pups (4 d) Pups (10–14 d) 1–6 1.5–7.1 Juveniles (>2 wk) Adults 2.1–2.3 1.3–2.7 Adults and juveniles, molting Juveniles, both sexes 4.0 Stokkan et al., 1995; Woldstad et al., 1999 Engelhardt and Ferguson, 1980; Stokkan et al., 1995; Hall et al., 1998; Woldstad et al., 1999 Boily, 1996; Hall et al., 1998 Engelhardt and Ferguson, 1980; Boily, 1996; Hall et al., 1998 Boily, 1996 0.7 Schumacher et al., 1992 Pups (1–3 wk) Pups (4–10 wk) 3.3 3.5–4.3 Kirby, 1990 Kirby, 1990 Lactating Molting females 3.5 4.3 Kirby, 1990 Kirby, 1990 (Continued) 0839_frame_C10 Page 172 Tuesday, May 22, 2001 11:08 AM 172 CRC Handbook of Marine Mammal Medicine TABLE 2 Reported Circulating Concentrations (µg/dl) of Thyroxine in Marine Mammals (continued) Species Mirounga leonina (southern elephant seal) Pagophilus groenlandicus (harp seal) Specimens Phoca vitulina (harbor seal) Reference Neonate 2.9 Little, 1991 Weaned 1.3 Little, 1991 Neonates 1.3–19.1 Pups (<10 d) 1.4–20 Pups (2–3 wk) Lactating 4.6–6.5 0.4–6.1 Leatherland and Ronald, 1979; Engelhardt and Ferguson, 1980; Stokkan et al., 1995 Leatherland and Ronald, 1979; Engelhardt and Ferguson, 1980; Stokkan et al., 1995 Engelhardt and Ferguson, 1980 Leatherland and Ronald, 1979; Engelhardt and Ferguson, 1980 John et al., 1987 Adults, molting and 1–4 wk postmolt Adults Phoca largha (spotted seal) Thyroxine (µg/dl) 5.9, 4.6, 5.9 0.6–3 Juveniles Adults Juveniles, molting Adults, molting Neonates Pups (2–4 wk) Juveniles 0.2–3 1.2–3 0.5–4 0.5–5 8.2 4.1–4.8 0.6–4 Adults 0.5–3 Lactating Juveniles, molting 1.9–3.1 1.8–5 Adults, molting 0.5–1 Leatherland and Ronald, 1979; Engelhardt and Ferguson, 1980; John et al., 1987 Ashwell-Erickson et al., 1986 Ashwell-Erickson et al., 1986 Ashwell-Erickson et al., 1986 Ashwell-Erickson et al., 1986 Haulena et al., 1998 Haulena et al., 1998 Riviere et al., 1977; Ashwell-Erickson et al., 1986 Ronald and Thomson, 1981; Ashwell-Erickson et al., 1986; Brouwer et al., 1989; Renouf and Brotea, 1991; Renouf and Noseworthy, 1991 Haulena et al., 1998 Riviere et al., 1977; Ashwell-Erickson et al., 1986 Ashwell-Erickson et al., 1986 Sirenians Trichechus manatus (West Indian and Florida manatees) Captive Free-ranging 1.9–4.5 4.5–8.3 Ortiz et al., 2000 Ortiz et al., 2000 Sea Otter Enhydra lutris Pups Juveniles Adults 3.75 2.7 2.45 Williams et al., 1992 Williams et al., 1992 Williams et al., 1992 Polar Bear Ursus maritimus Adults 0.6–5.2 Leatherland and Ronald, 1981; Cattet, 2000 Note: Values given as a range of means from multiple publications, or either the mean or range (when available) from a single source. Some of the data were estimated from figures. µg/dl × 12.87 = nmol/l. 0839_frame_C10 Page 173 Tuesday, May 22, 2001 11:08 AM 173 Endocrinology TABLE 3 Reported Concentrations of Triiodothyronine (ng/dl) in Marine Mammals Species Triiodothyronine (ng/dl) Specimens Reference Cetaceans Delphinapterus leucas (beluga) Tursiops truncatus (bottlenose dolphin) Various ages, both sexes Adults, both sexes 59–177 83–165 St. Aubin and Geraci, 1988; 1989; St. Aubin et al., in press Greenwood and Barlow, 1979; Orlov et al., 1988; St. Aubin et al., 1996 Pinnipeds Callorhinus ursinus (northern fur seal) Cystophora cristata (hooded seal) Halichoerus grypus (gray seal) Leptonychotes weddellii (Weddell seal) Mirounga leonina (southern elephant seal) Pagophilus groenlandicus (harp seal) Various ages, both sexes Pups (1 d) 63 100–225 Neonates (1 d) Pups (4 d) 60–225 60–250 Pups (1–2 wk) 47–280 Post-weaned pups and juveniles Juveniles, molting Adults 44–130 Adults, molting Various ages, both sexes Neonates (6 h) Pups (14–20 d) 42 100 195 85 Little, 1991 Little, 1991 152 36–111 83–137 Pups (1–5 d) 60–360 Pups (7–10 d) Pups (2 wk) 130–226 60–170 Pups (3 wk) Adults 207–330 45–220 Adults, molting Stokkan et al., 1995 Stokkan et al., 1995 Stokkan et al., 1995; Woldstad et al., 1999 Engelhardt and Ferguson, 1980; Stokkan et al., 1995; Hall et al., 1998; Woldstad et al., 1999 Boily, 1996; Hall et al., 1998; Woldstad et al., 1999 Boily, 1996 Engelhardt and Ferguson, 1980; Boily, 1996; Hall et al., 1998 Boily, 1996 Schumacher et al., 1992 Neonates (9 h) Lactating St. Aubin, unpubl. data 45–120 227 Leatherland and Ronald, 1979; Engelhardt and Ferguson, 1980; John et al., 1987; Stokkan et al., 1995 Leatherland and Ronald, 1979; Engelhardt and Ferguson, 1980; Stokkan et al., 1995 Leatherland and Ronald, 1979 Engelhardt and Ferguson, 1980; Stokkan et al., 1995 Engelhardt and Ferguson, 1980 Leatherland and Ronald, 1979; Engelhardt and Ferguson, 1980; John et al., 1987 Leatherland and Ronald, 1979; Engelhardt and Ferguson, 1980 John et al., 1987 (Continued) 0839_frame_C10 Page 174 Tuesday, May 22, 2001 11:08 AM 174 CRC Handbook of Marine Mammal Medicine TABLE 3 Reported Concentrations of Triiodothyronine (ng/dl) in Marine Mammals (continued) Species Phoca largha (spotted seal) Phoca vitulina (harbor seal) Specimens Juveniles Adults Juveniles, molting Adults, molting Neonates (1 d) Pups (3–7 d) Pups (10–14 d) Pups (28 d) Juveniles Adults Pregnant Postpartum Juveniles, molting Adults, molting Triiodothyronine (ng/dl) 10–160 10–30 20–130 10–80 130 210 163 98 39–42 10–78 29 124 30–104 20–47 Reference Ashwell-Erickson et al., 1986 Ashwell-Erickson et al., 1986 Ashwell-Erickson et al., 1986 Ashwell-Erickson et al., 1986 Haulena et al., 1998 Haulena et al., 1998 Haulena et al., 1998 Haulena et al., 1998 Ashwell-Erickson et al., 1986; Renouf and Brotea, 1991 Ashwell-Erickson et al., 1986; Renouf and Brotea, 1991; Renouf and Noseworthy, 1991; Haulena et al., 1998 Brouwer et al., 1989 Ronald and Thomson, 1981 Ashwell-Erickson et al., 1986; Renouf and Brotea, 1991 Ashwell-Erickson et al., 1986; Renouf and Brotea, 1991 Sirenians Trichechus manatus (West Indian and Florida manatees) 140–160 Ortiz et al., 2000 Polar Bear Ursus maritimus 16–150 Leatherland and Ronald, 1983; Cattet, 2000 Note: Values given as a range of means from multiple publications, or either the mean or range (when available) from a single source. Some of the data were estimated from figures. ng/dl × 0.01536 = nmol/l. (see Chapter 36, Nutrition; Chapter 43, Manatees). Levels of T4 in sea otters (Enhydra lutris) reveal little of the very active metabolism of these animals (Williams et al., 1992). THs, particularly T4, appear to be cleared from circulation very rapidly in bottlenose dolphins, 15 times faster on average than in humans; a single study in a Pacific white-sided dolphin (Lagenorhynchus obliquidens) yielded results comparable to those in humans (Sterling et al., 1975). The authors suggested that low protein binding in bottlenose dolphins might account for the rapid loss from the circulation. However, free THs, expressed as a percentage of the total hormone concentration, are in fact lower in bottlenose dolphins than in humans and other mammals (St. Aubin et al., 1996). Removal of T4 through other metabolic pathways could play an important role in the dynamics of circulating TH in these species. A significant, but poorly understood, difference in THs in some marine mammals is their relatively high circulating levels of reverse T3 (rT3). The product of inner ring deiodination of T4 (outer ring deiodination of T4 yields T3), rT3 is considered to be an inactive metabolite found in blood in concentrations that are generally one third to one half those of T3. In cetaceans and harbor seals, however, rT3 concentrations are equivalent to or up to three times greater 0839_frame_C10 Page 175 Tuesday, May 22, 2001 11:08 AM 175 Endocrinology TABLE 4 Reported Concentrations of f T4 (ng/dl), f T3 (pg/ml), and rT3 (ng/ml) in Marine Mammals Species Specimens Concentration Reference f T4 Delphinapterus leucas (beluga) Globicephala macrorhynchus (short-finned pilot whale) Lagenorhyncus obliquidens (Pacific white-sided dolphin) Orcinus orca (killer whale) Tursiops truncatus (bottlenose dolphin) Callorhinus ursinus (northern fur seal) Halichoerus grypus (gray seal) Phoca largha (spotted seal) Phoca vitulina (harbor seal) Trichechus manatus (West Indian and Florida manatees) Various ages, both sexes Males, age unspecified 1.52 St. Aubin et al., in press 3.99 Ridgway et al., 1970 Both sexes, age unspecified 1.7–2.3 Ridgway et al., 1970 Male, age unspecified Various ages, both sexes Various ages, both sexes Pups (<10 d) Pups (>2 wk) Juveniles Adults Juveniles, molting Adults, molting Juveniles 2.78 Ridgway et al., 1970 1.36–3.58 0.25 Ridgway et al., 1970; St. Aubin et al., 1996 St. Aubin, unpubl. data 2–2.57 2–2.23 1.07 1.1–1.44 2.35 1.22 1–4 Hall et al., 1998; Woldstad et al., 1999 Hall et al., 1998; Woldstad et al., 1999 Boily, 1996 Boily, 1996; Hall et al., 1998 Boily, 1996 Boily, 1996 Ashwell-Erickson et al., 1986 Adults Adults, molting Neonates Pups (1–4 wk) Juvenile 1.5–3 1.2–5.5 2.18 1.0 1.5–2 Adult 1.50–1.93 Pregnant Postpartum Free-ranging Captive 1.0 1.4 1.33–1.59 0.5–1.13 Ashwell-Erickson et al., 1986 Ashwell-Erickson et al., 1986 Haulena et al., 1998 Haulena et al., 1998 Ashwell-Erickson et al., 1986; Renouf and Brotea, 1991 Renouf and Brotea, 1991; Renouf and Noseworthy, 1991 Brouwer et al., 1989 Haulena et al., 1998 Ortiz et al., 2000 Ortiz et al., 2000 f T3 Delphinapterus leucas (beluga) Tursiops truncatus (bottlenose dolphin) Halichoerus grypus (gray seal) Various ages, both sexes Adults, both sexes 1.68 St. Aubin et al., in press 1.29 St. Aubin et al., 1996 Preweaned pups Postweaned pups Adult female 0.87 0.84 0.90 Hall et al., 1998 Hall et al., 1998 Hall et al., 1998 (Continued) 0839_frame_C10 Page 176 Tuesday, May 22, 2001 11:08 AM 176 CRC Handbook of Marine Mammal Medicine TABLE 4 Reported Concentrations of fT4 (ng/dl), fT3 (pg/ml), and rT3 (ng/ml) in Marine Mammals (continued) Species Phoca vitulina (harbor seal) Specimens Neonates Pups (5–15 d) Pups (19–26 d) Postpartum Concentration 1.79 1.63–2.28 1.11–1.63 0.2–0.39 Reference Haulena et al., 1998 Haulena et al., 1998 Haulena et al., 1998 Haulena et al., 1998 rT3 Delphinapterus leucas (beluga) Tursiops truncatus (bottlenose dolphin) Callorhinus ursinus (northern fur seal) Phoca vitulina (harbor seal) Various ages, both sexes Adults, both sexes Various ages, both sexes Neonates Pups (5–25 d) Postpartum 4.0 St. Aubin et al., in press 1.81 St. Aubin et al., 1996 1.6 St. Aubin, unpubl. data 9.0 0.65–1.95 0.65–1.95 Haulena et al., 1998 Haulena et al., 1998 Haulena et al., 1998 Note: Values given as a range of means from multiple publications, or either the mean or range (when available) from a single source. Some of the data were estimated from figures. ng/dl × 12.87 = pmol/l for f T4; pg/ml × 1.536 = pmol/l for f T3; ng/ml × 1.536 = nmol/l for rT3. than T3 (St. Aubin et al. 1996; in press; Haulena et al., 1998). During the summer period of estuarine occupation in belugas, rT3 levels can reach 4.4 ng/ml, the highest reported for any adult mammal. The benefits of inactivating such a large proportion of T4 are unclear, but at the very least represent another option for managing the effects of circulating T4. Interpretation of TH levels in marine mammals, and particularly in pinnipeds, must take into account dynamic changes that occur in association with significant life-history events. Neonatal phocid seals typically show levels that are elevated above reference ranges for adults (Engelhardt and Ferguson, 1980; Stokkan et al., 1995; Haulena et al., 1998; Woldstad and Jenssen, 1999), a pattern similar to that in humans and domestic mammals. The elevations are consistent with histological evidence of hyperactivity in thyroid follicular cells, at least in harbor seals (Harrison et al., 1962; Amoroso et al., 1965) and elephant seals (Little, 1991); the harp seal shows no such correlation (Leatherland, 1976; Leatherland and Ronald, 1979). For many phocids, metabolically derived heat may be critical for survival until an insulative blubber layer is established, and the calorigenic effects of TH could readily explain the need for elevated levels at this time (Stokkan et al., 1995; Haulena et al., 1998). The levels decline during the first few weeks of life in most species, although a trend for T3 may not always be apparent. In southern elephant seals, high concentrations of melatonin are postulated to enhance the conversion of T4 to T3, thereby providing an additional stimulus to metabolism. Circulating levels of TH in marine mammals are also subject to considerable fluctuations throughout the year, particularly in phocid seals. Chief among the events associated with altered TH status is the molt (Riviere et al., 1977; Ashwell-Erickson et al., 1986; John et al., 1987; Boily, 1996). Among their many metabolic effects, THs are known to stimulate hair growth in terrestrial mammals, and presumably have the same effect in phocids. Elevated levels in TH are typically observed during the less obvious phase of follicular stimulation prior to the time of most extensive shedding, which may account for Renouf and Brotea’s (1991) inability to establish direct correlations with overt signs of molting. The profound changes in TH that have been documented in most phocids are indicative of the broad metabolic adjustments that occur 0839_frame_C10 Page 177 Tuesday, May 22, 2001 11:08 AM Endocrinology 177 during the molt (Ashwell-Erickson et al., 1986; Boily, 1996), and often correlate with other diagnostic signs of less than optimal health (Riviere, 1978). Seasonal variation in TH in harbor seals was associated with changes in appetite, fat accumulation, and metabolism (Renouf and Noseworthy, 1991). The only cetacean yet to be shown to have a comparable cycle in TH is the beluga (St. Aubin and Geraci, 1989), in which a concurrent stimulation of epidermal cell growth takes on virtually all manifestations of a molt (St. Aubin et al., 1990). Captive belugas, held under relatively constant environmental conditions, show neither the marked seasonal variations in TH nor the intense variation in epidermal cell turnover, although episodic sloughing does occur. Altered TH status has been associated with capture and handling in belugas (St. Aubin and Geraci, 1988; 1992), initially as part of stress-mediated changes in hormone secretion and metabolism. In other mammals, cortisol inhibits TSH secretion and also the monoiodinase responsible for producing much of the T3 in circulation, and these pathways appear to be similarly affected in belugas. Acclimation to captivity results in somewhat lower TH levels than observed even in the less active spring and fall seasons in the wild (St. Aubin et al., in press; St. Aubin and Ridgway, unpubl. data). Free-ranging female bottlenose dolphins have higher levels of tT4, f T4, and fT3 than their counterparts in captivity, possibly reflecting differences in their reproductive status (St. Aubin et al., 1996). No diurnal cycle was noted in T4 or T3 in neonatal harp and gray seals (Stokkan et al., 1995). However, belugas sampled at various times of day over a 4-year period showed a nadir in T4 concentration at 2200 hours, and a peak in T3 at 1400 hours (St. Aubin and Ridgway, unpubl. data). Thyroid stimulation tests have been performed in belugas (St. Aubin, 1987; St. Aubin and Geraci, 1992). Marked differences in response to 10 IU of bovine TSH were observed as a function of the time after capture the hormone was administered, with apparently diminished sensitivity over time. In three individuals, three doses given over a 58-hour period had no apparent adverse effect, and resulted in substantial elevation of both T4 and T3; no attempt was made to establish the optimum dosage through the use of graded doses of TSH. Pathological changes in thyroid have been described, though principally in association with other clinical problems (Greenwood and Barlow, 1979). There is some evidence that environmental contaminants acting as endocrine disrupters can upset TH balance (Brouwer et al., 1989; Hall et al., 1998) and produce histologically detectable abnormalities (Schumacher et al., 1993). Belugas from the St. Lawrence estuary in Canada, which are known to accumulate substantial burdens of organochlorine contaminants, among others (see Chapter 22, Toxicology), also show evidence of thyroid pathology (De Guise et al., 1994), although the association with contaminants is likely to remain circumstantial in the absence of experimental data in these species. Adrenal Gland The adrenal gland of marine mammals conforms to the same general architecture noted in terrestrial mammals, with a catecholamine-secreting medulla surrounded by a steroid-producing cortex. A prominent difference is the pseudolobulation of the cortex produced by septae of fibrous tissue arising from the capsule; these lobules are most extensively developed in cetaceans. The cortex is particularly well developed in fetal harbor seals, as a possible adaptation to precocious behavior and physiological accommodation in the neonate (Amoroso et al., 1965; Sucheston and Cannon, 1980). Within the cortex, the outermost layer, or zona glomerulosa, is most expansive, suggesting that the need to produce aldosterone for electrolyte homeostasis is critical at that time. 0839_frame_C10 Page 178 Tuesday, May 22, 2001 11:08 AM 178 CRC Handbook of Marine Mammal Medicine Few studies have examined catecholamine function and physiology in marine mammals. Attention has focused principally on the role of epinephrine and norepinephrine in the dive response (Hance et al., 1982; Hochachka et al., 1995; Lohman et al., 1998). Catecholamineinduced splenic contraction can contribute substantially to the circulating pool of erythrocytes, extending the aerobic dive limits for these animals. The hormones increase during dives of more than a few minutes in Weddell seals, and rapidly return to resting levels following the dive. Efforts to extract and identify steroids from adrenal tissues have yielded conflicting, sometimes puzzling, results regarding the types of steroids utilized by cetaceans and pinnipeds (DeRoos and Bern, 1961; Borruel et al., 1974; Carballeira et al., 1987). Nevertheless, virtually all studies on circulating corticosteroids have established the prominence of cortisol over corticosterone as the principal glucocorticoid (Sangalang and Freeman, 1976; Thomson and Geraci, 1986; Ortiz and Worthy, 2000), and the presence of aldosterone as the mineralocorticoid hormone. Establishing baseline values for constituents known to be influenced by stressors such as chase, capture, and restraint is challenging for the researcher, and clinicians are tasked with interpreting whether the efforts to obtain a blood sample for diagnostic purposes have produced misleading information. Captive bottlenose dolphins conditioned to allow unrestrained blood collection and those calmly approached and sampled within minutes have yielded specimens as close to baseline as can reasonably be expected (Thomson and Geraci, 1986; St. Aubin et al., 1996). Pinnipeds resting on ice floes or shorelines, or held in exhibits or research facilities, can sometimes be captured and sampled before circulating hormones change appreciably. Studies on the dynamics of corticosteroid release following stimulation by exogenous ACTH suggest that cortisol levels are elevated by 30 min (St. Aubin and Geraci, 1986; 1990; Thomson and Geraci, 1986). Even taking into account the possible artifact of capture-related elevations, the Weddell seal is distinguished among marine mammals, and indeed among most vertebrate species, by its extraordinarily high circulating concentrations of cortisol (Table 5) (Liggins et al., 1979; Barrell and Montgomery, 1989; Bartsh et al., 1992). No clear explanation has emerged to account for this conspicuous difference; it is not an adaptation necessary to diving, given the much lower levels in other pinnipeds, including the deep-diving elephant seal. Cortisol secretion tends to show a circadian cycle in mammals, with increasing levels during the morning hours in diurnal species, but there is little information on this point for marine mammals. Harbor seals show the highest concentrations at night, and the lowest in the early afternoon (Gardiner and Hall, 1997). No periodicity was evident in samples collected from Weddell seals exposed to continuous daylight; however, the study used pooled data from different seals sampled at different times and did not strictly follow changes in individual animals (Barrell and Montgomery, 1989). Belugas showed lower levels of cortisol between noon and midnight than during the rest of the day (St. Aubin and Ridgway, unpubl. data); a similar pattern was evident in a captive killer whale (Orcinus orca) (Suzuki et al., 1998). Other factors contributing to alterations in circulating cortisol levels include reproduction and molt. High levels of cortisol have been noted in molting seals, generally in an inverse relationship with thyroid hormones (Riviere et al., 1977; Ashwell-Erickson et al., 1986), although Boily (1996) found lower levels in molting gray seals. Cortisol concentrations are elevated in neonatal harp seals, decline within 3 days, and then return to the higher range by 3 weeks, at the time of lanugo shedding (Engelhardt and Ferguson, 1980). Cortisol is known to promote hair loss in terrestrial mammals. During late pregnancy and the early postpartum period in harbor seals, total corticosteroids were high and ranged widely, up to nearly 40 µg/dl (Raeside and Ronald, 1981), although Gardiner and Hall (1997) found no significant difference in cortisol levels between a pregnant and a nonpregnant captive harbor seal. Harp seals have higher levels while lactating than during the postlactation period (Engelhardt and Ferguson, 1980). 0839_frame_C10 Page 179 Tuesday, May 22, 2001 11:08 AM 179 Endocrinology TABLE 5 Reported Concentrations (µg/dl) of Cortisol in Marine Mammals Species Specimens Cortisol (µg/dl) Reference Cetaceans Balaenoptera acutorostrata (minke whale) Balaenoptera physalus (fin whale) Cephalorhynchus commersonii (Commerson’s dolphin) Delphinapterus leucas (beluga) Globicephala macrorhynchus (short-finned pilot whale) Grampus griseus (Risso’s dolphin) Inia geoffrensis (Amazon River dolphin) Kogia breviceps (pygmy sperm whale) Lagenorhynchus obliquidens (Pacific white-sided dolphin) Orcinus orca (killer whale) Phocoena phocoena (harbor porpoise) Phocoenoides dalli (Dall’s porpoise) Pseudorca crassidens (false killer whale) Stenella coeruleoalba (blue-white dolphin) Tursiops truncatus (bottlenose dolphin) Various ages, both sexes Not specified 0.33 Suzuki et al., 1998 1.0–1.2 Not specified 0.5 Kjeld and Olafsson, 1987; Kjeld and Theodórsdóttir, 1991 Suzuki et al., 1998 Various ages, both sexes Not specified 0.7–3.2 0.4–0.7 Suzuki et al., 1998; St. Aubin et al., in press Suzuki et al., 1998 Not specified 0.9 Suzuki et al., 1998 Not specified 0.8 Suzuki et al., 1998 Not specified 0.2 Suzuki et al., 1998 Not specified 0.8 Suzuki et al., 1998 Not specified 0.4 Suzuki et al., 1998 Not specified Both sexes, age unspecified Not specified 0.4 8.8 Suzuki et al., 1998 Koopman et al., 1995 0.7 Suzuki et al., 1998 Not specified 0.7 Suzuki et al., 1998 Not specified 0.5 Suzuki et al., 1998 Various ages, both sexes 0.6–3.6 Thompson and Geraci, 1986; Orlov et al., 1988; St. Aubin et al., 1996; Suzuki et al., 1998; Ortiz and Worthy, 2000 Pinnipeds Halichoerus grypus (gray seal) Leptonychotes weddellii (Weddell seal) Pups (1–2 wk) Juveniles, molting and non-molting Juveniles, molting Adult Adult male, breeding Adult 4.3 6.3–9.1 Engelhardt and Ferguson, 1980 Boily, 1996; Lohmann et al., 1998 4.5 3.6–5.9 Boily, 1996 Sangalang and Freeman, 1976; Engelhardt and Ferguson, 1980; Boily, 1996 Sangalang and Freeman, 1976; Engelhardt and Ferguson, 1980 Liggins et al., 1979; Barrell and Montgomery, 1989; Bartsh et al., 1992 21.2–35.4 69–153.9 (Continued) 0839_frame_C10 Page 180 Tuesday, May 22, 2001 11:08 AM 180 CRC Handbook of Marine Mammal Medicine TABLE 5 Reported Concentrations (µg/dl) of Cortisol in Marine Mammals (continued) Species Pagophilus groenlandicus (harp seal) Phoca hispida (ringed seal) Phoca largha (spotted seal) Phoca vitulina (harbor seal) Specimens Neonate Pups (<1 wk) Pups (3 wk) Juvenile Adult female Lactating Adult male Juvenile Juveniles Adults Cortisol (µg/dl) 5.3 1.8–2.5 5.5 11–15 3.6 8.2 11 12–20 4–16 7–24 Juveniles 3–8.6 Juveniles, molting 9–12 Adult female Prepartum (1–80 d) Postpartum (5 d) 8–16 6–16.4 39.2 Reference Engelhardt and Ferguson, 1980 Engelhardt and Ferguson, 1980 Engelhardt and Ferguson, 1980 St. Aubin and Geraci, 1986 Engelhardt and Ferguson, 1980 Engelhardt and Ferguson, 1980 Engelhardt and Ferguson, 1980 St. Aubin and Geraci, 1986 Ashwell-Erickson et al., 1986 Ashwell-Erickson et al., 1986 Riviere et al., 1977; Ashwell-Erickson et al., 1986; Gulland et al., 1999 Riviere et al., 1997; Ashwell-Erickson et al., 1986 Ashwell-Erickson et al., 1986 Raeside and Ronald, 1981 Raeside and Ronald, 1981 Sirenians Trichechus manatus (West Indian and Florida manatees) Age unspecified, both sexes Enhydra lutris Various ages, both sexes Ursus maritimus Adults, both sexes 0.15 Ortiz et al., 1998 Sea Otter 3.2–3.9 Williams et al., 1992 Polar Bear 6.9–54 Cattet, 2000 Note: Values given as a range of means from multiple publications, or either the mean or range (when available) from a single source. Some of the data were estimated from figures. µg/dl × 27.59 = nmol/l. Wild harbor seals show significant seasonal variation in cortisol levels, correlating with both breeding and molting; levels were lower during the breeding/molt season than at other times of the year (Gardiner and Hall, 1997). As for thyroid hormones, temporal associations between cortisol changes and overt signs of molt may be misleading, and asynchrony may simply reflect the slow development of follicular changes, either for hair loss or regrowth. The seasonal differences in cortisol were not evident in captive seals. Glucocorticoids have received the greatest attention in the literature for their role in the stress response; this subject is reviewed in more detail elsewhere in this volume (see Chapter 13, Stress). The basic physiology of glucocorticoid secretion has been investigated through the use of exogenous ACTH in cetaceans (Thomson and Geraci, 1986; St. Aubin and Geraci, 1990), phocids (St. Aubin and Geraci, 1986), and otariids (St. Aubin et al., unpubl. data). Gulland et al. (1999) used ACTH stimulation tests to assess adrenal function in harbor seals infected with an adrenotropic herpes virus. Dosages have ranged from 0.2 IU/kg in belugas, to 0.25 to 0839_frame_C10 Page 181 Tuesday, May 22, 2001 11:08 AM Endocrinology 181 0.4 IU/kg in bottlenose dolphins, to 1 IU/kg in harbor seal pups. Various synthetic (Cortrosyn®, Organon Canada, Ltd., Toronto, Ontario, and Repository Corticotropin, Austin, Jolliette, Canada) and natural porcine (ACTHar®, Harris Laboratories, Toronto, Canada) preparations have been effective in elevating serum cortisol levels within 30 to 60 min of administration. Systemic consequences of rising cortisol concentrations, including stress leukograms (leukocytosis, lymphopenia, and eosinopenia) and hyperglycemia, indicate that the pituitary– adrenal axis functions for the most part in accordance with the relationships established for other mammals. A significant difference, however, lies in the relatively low circulating levels of cortisol in cetaceans, and the modest increases observed following stimulation, a condition that confounds the use of cortisol as a diagnostic indicator of stress in these animals. Yet, the characteristic changes in other circulating constituents normally sensitive to elevations in cortisol suggest that even small changes may be clinically important. A possible explanation for the difference between pinnipeds and cetaceans in this respect is the hormone-binding capacity of the plasma. Whereas more than 90% of cortisol is bound in the Weddell seal (Liggins et al., 1979), studies on belugas and bottlenose dolphins indicate that the bound fraction represents 50% or less of the total hormone (St. Aubin and Geraci, unpubl. data). Small increments might therefore translate into relatively more free hormone and greater availability to exert effects on the organism. Although the importance of aldosterone is suggested by the prominence of the zona glomerulosa cells that produce it, particularly in young seals, its value in marine mammals is enigmatic. There would appear to be little advantage to conserving sodium in an environment in which the greater need would be to excrete it. Nevertheless, aldosterone is detectable in most samples drawn from marine mammals (Table 6). Levels tend to be higher in young phocids (Engelhardt and Ferguson, 1980), as might be expected from the histological appearance of the gland (Amoroso et al., 1965). Manatees, whether in the wild or in captivity, have higher aldosterone concentrations when in fresh than in salt water (Ortiz et al., 1998). By contrast, belugas sampled in fresh and marine waters showed wide ranges for plasma sodium and aldosterone concentrations (St. Aubin et al., in press), with no significant correlation between the two constituents and the environment in which they were sampled. This apparently casual approach to electrolyte regulation in belugas contrasts with significant clinical problems associated with both hyper- and hyponatremia in other species, particularly some phocid seals. Chronic salt deprivation produced widely fluctuating, but generally elevated, plasma levels of aldosterone in a ringed seal (Phoca hispida) that was able to maintain normonatremia (St. Aubin and Geraci, 1986). Salt deprivation in a second ringed seal resulted in mild hyponatremia (Na: 142 to 145 mEq/l), and slightly reduced but variable plasma aldosterone levels; a spontaneously hyponatremic harp seal with sodium concentrations of 115 to 130 mEq/l had low but still detectable aldosterone. Thus, while hyponatremia can occur in the presence of seemingly adequate levels of the hormone, it appears that sodium conservation in these phocids is achieved by increasing aldosterone. Elevated aldosterone during the postweaning fast in northern elephant seals appears to be a strategy to help conserve water, which is resorbed along with sodium (Ortiz et al., 2000). Electrolyte imbalance is not the sole stimulus for aldosterone secretion; angiotensin II (AII), which will be addressed later, and ACTH both play a role. It is the particular sensitivity of the zona glomerulosa to the latter that distinguishes marine from terrestrial mammals. In contrast to the modest increases found in humans and other mammals, elevations in aldosterone as high as sevenfold have been noted in ringed (St. Aubin and Geraci, 1986) and harbor (Gulland et al., 1999) seals, northern fur seals (St. Aubin et al., unpubl. data), bottlenose dolphins (Thomson and Geraci, 1986), and belugas (St. Aubin and Geraci, 1990). Sodium conservation during times of stress apparently is an important requirement shared by a variety of species adapted to the 0839_frame_C10 Page 182 Wednesday, May 23, 2001 10:43 AM 182 CRC Handbook of Marine Mammal Medicine TABLE 6 Reported Concentrations of Aldosterone (pg/ml) in Marine Mammals Species Aldosterone (pg/ml) Specimens Reference Cetaceans Balaenoptera physalus (fin whale) Delphinapterus leucas (beluga) Tursiops truncatus (bottlenose dolphin) Not specified 17–168 Various ages, both sexes Adults, both sexes 203–450 3–677 Kjeld and Olafsson, 1987; Kjeld and Theodórsdóttir, 1991 St. Aubin and Geraci, 1989; St. Aubin et al., 2001 Malvin et al., 1978; Thomson and Geraci, 1986; St. Aubin et al., 1996 Pinnipeds Mirounga angustirostris (northern elephant seal) Pagophilus groenlandicus (harp seal) Phoca hispida (ringed seal) Phoca vitulina (harbor seal) Halichoerus grypus (gray seal) Zalophus californianus (California sea lion) Pups, at weaning Pups, fasting 5–7 wk Neonates Pups (<2 wk) Juveniles Adults Adults Juveniles 220 1000 2250 600–1180 300 400–1200 140–1040 750–1110 Adults 1400–3200 Juveniles 140–310 Ortiz et al., 2000 Ortiz et al., 2000 Engelhardt and Ferguson, 1980 Engelhardt and Ferguson, 1980 Engelhardt and Ferguson, 1980 Engelhardt and Ferguson, 1980 St. Aubin and Geraci, 1986 Gulland et al., 1999 Sangalang and Freeman, 1976 Malvin et al., 1978 Sirenians Trichechus manatus (West Indian and Florida manatees) Both sexes (fresh water) Both sexes (brackish and salt water) 660 37–95 Ortiz et al., 1998 Ortiz et al., 1998 Note: Values given as a range of means from multiple publications, or either the mean or range (when available) from a single source. Some of the data were estimated from figures. pg/ml × 2.775 = pmol/l. marine environment. In ringed seals stressed by salt restriction, the aldosterone response to ACTH stimulation is exaggerated until the zona glomerulosa is exhausted (Figure 1). Pinniped hyponatremia, which can occur under conditions other than Na deprivation, may thus be a consequence of adrenal failure precipitated by chronic stress (Geraci, 1972; St. Aubin and Geraci, 1986). Osmoregulatory Hormones The classification of a subset of mammals as “marine” might suggest the presence of hormonemediated physiological adaptations to cope with a substantially hypertonic environment. In fact, with the exception of the large size of the kidney in the sea otter, renal tubular morphology and function in marine mammals are unremarkable, and render unnecessary the requirement for unusual endocrine pathways to manage water and electrolytes. Nevertheless, other aspects of marine mammal life histories, such as prolonged fasting in pinnipeds, place particular demands on these systems and have been the subject of numerous investigations. It is these adaptations that represent the more important concerns for the clinician. 0839_frame_C10 Page 183 Tuesday, May 22, 2001 11:08 AM Endocrinology 183 FIGURE 1 Plasma aldosterone concentration in five seals following intramuscular injection of ACTH. Two saltsupplemented ringed seals maintained in salt water served as normal controls ( ------ and ■ ------■). One ringed seal remained normonatremic when salt deprived (———), while another became hyponatremic (------). A harp seal spontaneously developed hyponatremia while being held in salt water and receiving dietary salt supplements (------). (Redrawn from St. Aubin and Geraci, 1986.) Vasopressin An antidiuretic hormone (ADH) was demonstrated in pituitaries from commercially harvested whales by researchers in the 1930s. Specific RIAs on seal pituitary extracts (Dogterom et al., 1980) and plasma from various pinnipeds, cetaceans, and sirenians (Table 7) suggest that the form elaborated by marine mammals is arginine vasopressin (AVP). In general, the range of reported AVP concentrations noted in pinnipeds and cetaceans is higher than that in manatees. None of the reported values is unusual relative to most other mammals. The dynamics of AVP during various physiological stresses challenge conventional expectations based on the role of this hormone in other mammals. During their prolonged postweaning fast, northern elephant seal pups showed declining levels of both AVP and urinary output (Ortiz et al., 1996); a subsequent study on the same species found no change in AVP during the fast (Ortiz et al., 2000). The hormone thus appears to be inconsequential in water conservation at this time. In fasted gray seals, AVP levels increased as much as threefold, an expected response that is likely tied to the concurrently increasing urinary osmolality (Skog and Folkow, 1994). However, water loading in gray seals failed to suppress AVP, and the excess fluid was cleared in a large volume of dilute urine despite the persistence of elevated AVP levels in circulation. A significant role for AVP could not be demonstrated in bottlenose dolphins in an early study monitoring urinary flow and osmolality (Malvin et al., 1971). Other physiological actions of AVP have been explored. Ortiz and Worthy (2000) considered the relationship between AVP and adrenal corticosteroids during capture stress in bottlenose dolphins; the lack of correlation suggested that AVP did not induce changes in ACTH, as it does in other mammals. Bradycardia during resting apnea in Weddell and northern elephant 0839_frame_C10 Page 184 Tuesday, May 22, 2001 11:08 AM 184 CRC Handbook of Marine Mammal Medicine TABLE 7 Reported Concentrations of Arginine Vasopressin (AVP) (pg/ml), Angiotensin II (AII) (pg/ml), and Atrial Natriuretic Peptide (ANP) (pg/ml) in Marine Mammals Species Tursiops truncatus (bottlenose dolphin) Eumetopias jubatus (Steller sea lion) Specimens AVP AII ANP Reference Both sexes, ages unspecified Pups 3.3 — — Ortiz and Worthy, 2000 7.2 46.9 Yearling, subadult Adults 6.2–6.5 20.5–24.6 Leptonychotes weddellii (Weddell seal) Mirounga angustirostris (northern elephant seal) Phoca hispida (ringed seal) Phoca vitulina (harbor seal) Various ages 3.2–7.2 12.2–39.6 12.5–30.6 Pups 1.5–28 16.5–33.2 20.9–26.3 Adults 9.3 14.0 Various ages 7.2–13.3 29.0 Trichechus manatus (West Indian and Florida manatees) Fresh water Salt water and brackish water (wild) Salt water (captive) 0.6–1.1 2.1–2.5 — — — — Zenteno-Savin and Castellini, 1998b Zenteno-Savin and Castellini, 1998b Zenteno-Savin and Castellini, 1998b Zenteno-Savin and Castellini, 1998b Zenteno-Savin and Castellini, 1998b Zenteno-Savin and Castellini, 1998a,b Zenteno-Savin and Castellini, 1998a,b; Ortiz et al., 1996; 2000 Zenteno-Savin and Castellini, 1998b Zenteno-Savin and Castellini, 1998b; Ellsworth et al., 1999 Ortiz et al., 1998 Ortiz et al., 1998 0.5 — — Ortiz et al., 1998 Zalophus californianus (California sea lion) 88.3 6.5–32 14.2 55.8 139.3 Pups 4.7 7.6 26.9 Adult 10.2 8.4 31.7 126.8 12.2–66.8 Note: Values given as a range of means from multiple publications, or either the mean or range (when available) from a single source. seals is associated with rapid decreases in AVP, a response that develops with age in the latter species (Zenteno-Savin and Castellini, 1998a) The closely related Baikal (Phoca sibirica) and ringed seals were studied for evidence of hormonal differences associated with specific needs for water conservation in their respective environments (Hong et al., 1982). Urinary ADH (AVP) concentration, expressed relative to that of creatinine, was similar in both species, and increased during water deprivation and fasting. After water loading, the hormone was undetectable. Thus, the Baikal seal exhibited no obvious adaptations in this mode of water management after an estimated half-million years of isolation in fresh water. Manatees naturally occur in habitats of varying salinity and, in the wild, show differences in blood AVP consistent with the expected need to conserve or eliminate water (see Table 7) (Ortiz et al., 1998). Paradoxically, captive manatees in salt water have lower AVP concentrations than those in fresh water, although the differences are small and insignificant. Perhaps the low salt content of the lettuce diet offered to the captive animals, compared with that in the natural marine vegetation, can account for the reduced need for water conservation in the former environment (see Chapter 36, Nutrition). Overall, plasma AVP and osmolality were significantly correlated. 0839_frame_C10 Page 185 Tuesday, May 22, 2001 11:08 AM Endocrinology 185 Renin–Angiotensin System The scant information on the renin–angiotensin system (RAS) in marine mammals is surprising in light of the profound changes in blood pressure and flow associated with at least some stages of the dive response. Secreted from juxtaglomerular cells of the kidney in response to hypotension in afferent arterioles, renin converts angiotensin I released from lung cells to AII, a potent vasoconstrictor. Renin activity is measured according to the rate of production of AII, while AII levels are determined directly by RIA. A recent survey of AII concentrations in pinnipeds found concentrations similar to those in most other mammals (see Table 7) (Zenteno-Savin and Castellini, 1998b). Early studies by Malvin and co-workers (Malvin and Vander, 1967; Malvin et al., 1978) focused on the RAS from the perspective of its role in osmoregulation in cetaceans and pinnipeds. Renin and aldosterone levels were significantly correlated in bottlenose dolphins, California sea lions (Zalophus californianus), and northern elephant seals, suggesting that this arm of aldosterone control is functional in at least some marine mammals (Malvin et al., 1978; Ortiz et al., 2000). The only insight into the dynamics of AII during diving comes from observations during apnea in Weddell and northern elephant seals (Zenteno-Savin and Castellini, 1998a). The observed decrease in AII levels appears to be inconsistent with a presumed rise in renin resulting from reduced blood flow to the kidney. The authors speculated that a response in AII might be delayed until circulation to the kidney is reestablished and renin is delivered systemically. Atrial Natriuretic Peptide First described in the literature as atrial natriuretic “factor” in the mid-1980s, this substance received growing attention in the human medical literature, including its characterization as a peptide and subsequent renaming as atrial natriuretic peptide (ANP). Consideration of its presence in marine mammals began with the demonstration of characteristic secretory granules in cardiomyocytes of ringed, harp, and northern elephant seals (Pfeiffer and Viers, 1995; Tagoe et al., 1998). The significance of the osmoregulatory function of this hormone was questionable in elephant seals, particularly, because of the sparseness of the structure. Investigation of the activity of the hormone in marine mammals is limited to a survey of circulating levels in some pinnipeds (Zenteno-Savin and Castellini, 1998b) and two functional studies. “Resting” levels are comparable to those measured in other mammals (see Table 7). Concentrations increase during apnea in Weddell seals, but not northern elephant seals (ZentenoSavin and Castellini, 1998a). The only experiment to examine the osmoregulatory action of this hormone yielded inconclusive results (Ellsworth et al., 1999). Named for its action, ANP responds more consistently to volumetric expansion, and the resultant atrial stretch, than to sodium loading, although levels do increase with sodium burden. The consequence of natriuresis serves to rectify hypervolemia rather than correct hypernatremia. Nevertheless, intravenous administration of up to 2 l of normal saline in a 1-hour period in adult harbor seals failed to consistently produce the expected increase in circulating ANP. It was postulated that the large, distensible vascular reservoirs in these animals dampened the intended stimulus, which had produced consistent changes in comparably sized humans. The functional significance of this hormone in phocids, at least, remains a question for further research. Endocrine Pancreas Insulin extracted from mysticete and sperm whale pancreas was found to have the same amino acid sequence as the porcine hormone, and to differ from the human form by only a single amino acid (Hama et al., 1964). Although the structure of glucagon has not been reported, it 0839_frame_C10 Page 186 Tuesday, May 22, 2001 11:08 AM 186 CRC Handbook of Marine Mammal Medicine TABLE 8 Reported Concentrations of Insulin (µU/ml) and Glucagon (pg/ml) in Marine Mammals Species Tursiops truncatus (bottlenose dolphin) Mirounga angustirostris (northern elephant seal) Phoca vitulina (harbor seal) Ursus maritimus (polar bear) Specimens Insulin Glucagon Reference 12–70 h fast Postprandial Not specified Pup (<3 wk) Weaned (2– 4 wk) molting, fasting Weaned (4–11 wk) fasting Lactating (1–4 wk) Adult, molting Pre-dive 11.2 12.3 10 9.9–11.3 9.2 94 117 — 195–844 153–346 Patton et al., 1977 Patton et al., 1977 Orlov et al., 1988 Kirby, 1990 Kirby, 1990 7.2–8.1 8.9–11.9 4.3 4–12 179–363 — 145–379 30–75 Kirby, 1990 Kirby, 1990 Kirby, 1990 Robin et al., 1981 3–96 18–637 Adults, feeding and fasting Cattet, 2000 Note: Values given as a range of means from multiple publications, or either the mean or range (when available) from a single source. is similar enough to that in other mammals to be measured using methods developed for other species. Both hormones are presumed to function in marine mammals as they do in other mammals. With a diet typically very low in carbohydrates, marine mammals sustain their glucose requirements principally through gluconeogenesis. As such, the hormones responsible for glucose homeostasis, insulin and glucagon, are balanced to deliver glucose into circulation rather than promote its uptake. The ratio of insulin to glucagon is consequently very low in virtually all groups studied (Table 8). The exception is the polar bear, in which insulin concentrations invariably exceed those of glucagon. Given that the polar bear’s diet for much of the year is also devoid of carbohydrate, the reversed relationship probably reflects a physiology more reminiscent of that of a terrestrial mammal. Insulin levels in harbor seals, elephant seals, and bottlenose dolphins were mostly unaffected during glucose tolerance tests, but were increased in the latter following protein meals and oral arginine (Ridgway et al., 1970; Patton, 1977; Patton et al., 1977; Kirby, 1990). The blunted response in these animals undermines the utility of conventional approaches to assess pancreatic function. Such information might be particularly useful in species such as harbor porpoises, which commonly show extensive pancreatic fibrosis as a result of trematode infections. The more important role for insulin and glucagon in marine mammals is maintaining circulating levels of glucose for delivery to the brain during dives. The ratio of insulin with respect to glucagon falls during voluntary dives in Weddell seals and contributes to hyperglycemia at the end of the dive (Hochachka et al., 1995). Future Studies Although the basic framework of marine mammal endocrinology has essentially been described, intriguing questions remain regarding the dynamics of some of these systems during physiologically challenging conditions such as diving and fasting. The measurement of circulating levels is only one index of hormone activity, and can sometimes be misleading. Binding proteins, metabolic clearance rate, and cell receptor density all play a role in modulating the actions of hormones, but the information on these points for marine mammals is sparse or nonexistent. The development of specific assays for peptide hormones will lead to a better 0839_frame_C10 Page 187 Tuesday, May 22, 2001 11:08 AM Endocrinology 187 understanding of factors, such as GH and tropic hormones, that may show important changes during the life history of these animals. Advances in the fundamental endocrinology of marine mammals will also improve our ability to recognize the effects of environmental contaminants that can disrupt endocrine systems. Acknowledgments The author is grateful to Pauline Schwalm for her assistance in tabulating the reported hormone data and to Shannon Atkinson and Ailsa Hall for their helpful reviews. Data on thyroid and adrenal hormones in northern fur seals at Mystic Aquarium were collected in collaboration with Thom Lembo and Larry Dunn, with support from the staff of the Departments of Husbandry and Research and Veterinary Services. These studies were funded by Mystic Aquarium and the Bernice Barbour Foundation. The Office of Naval Research and the Naval Ocean Systems Center (now NCCOSC RDTE), through Sam Ridgway, supported the research on hormone cycles in captive belugas. 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Endocrinol., 29: 419–422. 0839_frame_C10 Page 192 Tuesday, May 22, 2001 11:08 AM 192 CRC Handbook of Marine Mammal Medicine Schumacher, U., Raugh, G., Plötz, J., and Welsch, U., 1992, Basic biochemical data on blood from antarctic Weddell seals (Leptonychotes weddellii): Ions, lipids, enzymes, serum proteins and thyroid hormones, Comp. Biochem. Physiol. A, 102: 449–451. Schumacher, U., Zahler, S., Heidemann, G., Skirnisson, K., and Welsch, U., 1993, Histological investigations on the thyroid glands of marine mammals and the possible implications of marine pollution, J. Wildl. Dis., 29: 103–108. Skog, E.B., and Folkow, L.P., 1994, Nasal heat and water exchange is not an effector mechanism for water balance regulation in grey seals, Acta Physiol. Scand., 151: 233–240. Sterling, K., Milch, P.O., and Ridgway, S.H., 1975, The day of the dolphin: Thyroid hormone metabolism in marine mammals, in Thyroid Hormone Metabolism, Harland, W.A., and Orr, J.S. (Eds.), Academic Press, London, 241–248. 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Mammal Sci., 14: 304–310. Thomson, C.A., and Geraci, J.R., 1986, Cortisol, aldosterone, and leucocytes in the stress response of bottlenose dolphins, Tursiops truncatus, Can. J. Fish. Aquat. Sci., 43: 1010–1016. Vivien-Roels, B., and Pévet, P., 1983, The pineal gland and the synchronization of reproductive cycles with variations of the environmental climatic conditions, with special reference to temperature, Pineal Res. Rev., 1: 91–143. Williams, T.D., Rebar, A.H., Teclaw, R.F., and Yoos, P.E., 1992, Influence of age, sex, capture technique, and restraint on hematologic measurements and serum chemistries of wild California sea otters, Vet. Clin. Pathol., 21: 106–110. Wilson, J.D., Foster, D.W., Kronenberg, H.M., and Larsen, P.R. (Eds.), 1998, Williams Textbook of Endocrinology, W.B. Saunders, Philadelphia, 1819. Woldstad, S., and Jenssen, B.M., 1999, Thyroid hormones in grey seal pups (Halichoerus grypus), Comp. Biochem. Physiol. A, 122: 157–162. Young, B.A., and Harrison, R.J., 1970, Ultrastructure of the dolphin adenohypophysis, Z. Zellforsch., 103: 475–482. Zenteno-Savin, T., and Castellini, M.A., 1998a, Changes in the plasma levels of vasoactive hormones during apnea in seals, Comp. Biochem. Physiol. C, 119: 7–12. Zenteno-Savin, T., and Castellini, M.A., 1998b, Plasma angiotensin II, arginine vasopressin and atrial natriuretic peptide in free ranging and captive seals and sea lions, Comp. Biochem. Physiol. C, 119: 1–6. 0839_frame_C11 Page 193 Tuesday, May 22, 2001 11:09 AM 11 Reproduction Todd R. Robeck, Shannon K. C. Atkinson, and Fiona Brook Introduction The reproductive physiology of marine mammals is an extremely diverse topic; yet the small amount of information that has been collected has come from only a few species of cetaceans and pinnipeds. As a result, generalizations are made concerning the reproductive function of entire families based on information obtained from these few species. These generalizations must be interpreted with caution, as important differences exist among species within each family. In addition, this chapter focuses on reproductive aspects of species most likely to be encountered by veterinarians working with animals in captivity. This chapter assumes the reader has a basic knowledge of the physiology of mammalian reproduction. Reviews by Harrison and Ridgway (1971), Richkind and Ridgway (1975), Hill and Gilmartin (1977), Kirby (1982), Sawyer-Steffan et al. (1983), Kirby and Ridgway (1984), Schroeder and Keller (1989; 1990), and Schroeder (1990a,b), documented work with bottlenose dolphins (Tursiops truncatus). Perrin et al. (1984) reviewed cetacean reproduction. For pinnipeds, Riedman (1990) provided useful tables on reproductive timing and maternal care, and a review of reproduction by Atkinson (1997) focused primarily on phocids. Most recently, Boyd et al. (1999) reviewed reproductive physiology, timing of reproduction, and different lifehistory strategies for pinnipeds, sirenians, and cetaceans. Physiology of Reproduction Although the reproductive function of mammals varies among species, the hormones involved and their general functions tend to be conserved across the mammalian class. A general review of the control of reproduction, with emphasis on the estrous cycle, will give the reader a foundation on which other reproductive processes can be discussed in both the male and female. If a more detailed understanding of the physiology of these processes is desired, there are a number of good reference books available (Knobil and Neill, 1988; Cupps, 1991; Youngquist, 1997). Mammalian reproduction is regulated by a series of neurological and hormonal feedback mechanisms involving the hypothalamus, pituitary, and gonads. These three organs are commonly referred to as the hypothalamic–pituitary–gonadal axis (see Chapter 10, Endocrinology). The effects that photoperiod and other environmental stimuli have on reproductive events 0-8493-0839-9/01/$0.00+$1.50 © 2001 by CRC Press LLC 193 0839_frame_C11 Page 194 Tuesday, May 22, 2001 11:09 AM 194 CRC Handbook of Marine Mammal Medicine provide evidence that neurological transduction of these stimuli in the brain leads to control of reproductive events. Most of this transduction appears to occur in the hypothalamus and associated nuclei where neurons originate that secrete hypophysiotropic hormones into the hypophyseal portal system. These hormones control the anterior pituitary gland. Gonadotropin-releasing hormone (GnRH) is one of these hormones and is of primary importance in regulating reproductive endocrinological events. GnRH receptor binding in the anterior pituitary causes luteinizing hormone (LH) and follicle-stimulating hormone (FSH) to be released into circulation. GnRH secretion is important for reproductive control and is pulsatile in nature. Secretion of GnRH is mediated by a pulse generator located in the mediobasal hypothalamus. The episodic generation of GnRH translates into a subsequent pulsatile release of LH and FSH from the anterior pituitary. The significance of the episodic secretion is apparent when comparing the effects of exogenous GnRH delivered as a constant infusion or as a pulse infusion (Ganong, 1991). GnRH receptors in the anterior pituitary rapidly downregulate in both numbers and sensitivity when exposed to continual GnRH input and upregulate when GnRH concentration is low. Thus, constant GnRH infusion first stimulates LH release; then, as receptor sensitivity decreases, GnRH will inhibit LH release (Nett et al., 1981; Conn et al., 1988; Blue et al., 1991). This response is the basis for the use of GnRH agonist as contraception agents and will be discussed further below. Control of GnRH release is mediated by neurological input and feedback from gonadal hormones. Feedback appears to have direct effects on the pulse generator by causing changes in the amplitude and frequency of GnRH release. The basic model for this control is based on primate research, but the control appears to be similar in most mammalian species. During the early follicular phase of the estrous cycle, FSH production is slightly elevated. This increase in FSH production results in follicular recruitment and growth and causes an increase in LH receptor concentrations in the follicle(s) (Brown et al., 1986). Estrogen has also been positively correlated with numbers of LH receptors in the preovulatory follicle. As the follicles continue to expand or grow, estrogen is produced through paracrine interactions between thecal and granulosa cells that line the follicle. Increased estrogen production initially inhibits both FSH and LH secretion from the pituitary. As the follicle(s) approaches preovulatory stage, estrogens reaching maximal production (the preovulatory estrogen surge) exert a positive effect on frequency and amplitude of GnRH secretion resulting in the preovulatory LH surge. LH causes the follicle to produce a small two-subunit glycoprotein, called inhibin. Inhibin not only suppresses FSH production, but increases thecal cell sensitivity to LH in the preovulatory follicle (Baird and Smith, 1993). This combination of increased LH receptors and increased sensitivity to LH ensures an adequate response to the LH surge and ovulation. Once ovulation occurs, granulosa and thecal cells are converted to progesterone-secreting large and small luteal cells, respectively (Hendricks, 1991). These morphologically different luteal cells appear to have different functions in the corpus luteum (CL) and have been shown to have different secretory capacities. The luteal cells of the recently ruptured follicle rapidly organize into the CL. Progesterone, and to a smaller extent estrogen, produced by the CL inhibit LH and FSH secretion by decreasing the frequency of GnRH release from the hypothalamus. If the cycle is nonfertile, the uterus releases a series of five to eight pulses of prostaglandin F2α , which, in turn, result in luteal regression. The release of prostaglandin, at least in ruminants, appears to be initiated by pulsatile oxytocin release from the neurohypophysis, encouraged by release of oxytocin from the CL, and a concomitant decrease in circulating progesterone and estrogen (Silvia et al., 1991). The decrease in progesterone and estrogen allows the GnRH pulse generator once again to increase in frequency and amplitude, resulting in FSH and LH secretion and initiating folliculogenesis of the next cycle. 0839_frame_C11 Page 195 Tuesday, May 22, 2001 11:09 AM Reproduction 195 The pineal gland influences reproductive function by transducing photoperiodic messages to chemical messages through innervation in the superior ganglia (Lindsay, 1991). In response to changes in photoperiod, the pineal gland releases melatonin. The increase in melatonin appears to inhibit reproduction by affecting the pulsatile release of LH. Melatonin is synthesized only during dark hours, and its production can be inhibited by nocturnal exposure to artificial light. Prolactin may play an important role in regulating seasonality, but this remains to be determined. Pinniped Reproduction Pinniped reproduction has recently been reviewed by Atkinson (1997) and Boyd et al. (1999). This chapter summarizes basic reproductive physiology and focuses on clinically significant parameters. The tremendous variability that exists among the three pinniped families (phocidae, otariidae, and odobenidae) and the lack of information on their reproductive physiology preclude any detailed discussion concerning any one species. Instead, gross generalizations have often been made out of necessity. Research with harbor seals (Phoca vitulina) will often be used as an example of normal phocid reproductive parameters. As this approach may be misleading, any reader who truly wants a deeper appreciation of a particular species is advised to use this chapter as a beginning, or foundation, for further inquiry. Female Pinniped Reproduction Reproductive Cycle For this chapter, the reproductive cycle is defined as the period during which all major components of reproduction are experienced. These components arbitrarily begin with a fertile estrous period (which includes estrus, ovulation, and conception), followed by gestation, lactation, anestrus, and back to a fertile estrous cycle. As most marine mammals have some seasonal component to their reproductive events, and as seasonality has a direct impact on when a fertile estrus can occur, seasonality of reproductive events is included in this discussion. The reproductive cycle of pinnipeds is dominated by three basic phases: estrus, embryonic diapause, and fetal growth and development (Boyd et al., 1999). Embryonic diapause, or delayed implantation, was recognized in pinnipeds as early as 1940 (Harrison, 1968). Pinnipeds are classified as having obligate embryonic diapause (Renfree and Calaby, 1981). The time when the embryo resumes cellular divisions is a critical point during embryonic development of the fetus and, in nonpregnant females, is a period of reactivation of sexual activity. Understanding this phenomenon is important when attempting to diagnose pregnancy in these species. It appears that most if not all pinnipeds have either postpartum or postlactational estrus periods. Otariids generally have a postpartum estrus 6 to 12 days after birth. California sea lions (Zalophus californianus), however, appear to be exceptions among otariids in that their estrous period is approximately 1 month after birth (Heath, 1985). In phocids, estrus begins toward the end of lactation, or after weaning (Riedman, 1990; Atkinson, 1997). Harbor seal lactation can last 21 to 42 days, with estrus occurring after that time (Bigg, 1969; 1973). Estrus can last from 1 to 9 weeks, with some animals being induced ovulators. In walrus (Odobenus rosmarus), an approximate 4-month postpartum estrus occurs in late summer; however, conception cannot occur because males are infertile at this time. The females have a second midlactational estrus approximately 6 months later, around February, during peak male fertility (Fay et al., 1981; Riedman, 1990). Thus, walrus are polyestrous, but functionally monoestrous. The potential fertility of the late-summer postpartum estrus is unknown. For a summary of reproductive events in pinnipeds, see Table 1. 0839_frame_C11 Page 196 Tuesday, May 22, 2001 11:09 AM 196 CRC Handbook of Marine Mammal Medicine TABLE 1 Reproductive Characteristics of Three Species of Pinnipeds Species Reproductive Characteristics Harbor Seal a (Phoca vitulina) d California Sea Lion b (Zalophus californianus) Walrus c (Odobenus rosmarus) Mid-April to mid-June, peak May Midlactation Pupping period Early May Late May, early June Timing of ovulation End of lactation Conception Duration of lactation Delayed implantation period Delayed implantation Postimplantation gestation Total gestation interval June 21–42 days July–Aug. Approx. 28 days postpartum Late June, early July 6–12 months July–Sept. 1.5–3 months Sept.–May 3 months Oct.–May 4–5 months Aug.–May 11 months 11 months 15 months Jan.–March (peak Feb.) 24+ months March–July a Sources: Bigg, 1969; Bigg, 1973; Gardiner et al., 1999; Odell, pers. comm. Sources: Odell, 1981; Odell, pers. comm. c Sources: Fay et al., 1981; Fay, 1981. d These pupping data are based on U.S. captive animal observations. Time intervals are consistent, but timing of the reproductive cycle varies with latitude. For example, postimplantation gestation lasts 8.5 months for all harbor seals, but on the West Coast of North America, pups are born in February in Mexico and in July in Alaska. b Estrous Cycle The onset of the estrous cycle of pinnipeds is closely tied to the annual reproductive cycle (or biennual in the case of the walrus). Available data suggest that otariids and phocids are monestrous, spontaneous ovulators, and if pregnancy does not occur, they do not have a second estrous period until the following year. The known exception to this generality is the Hawaiian monk seal (Monachus schauinslandi), which has been shown to exhibit polyestrous activity (Iwasa et al., 1997; Iwasa and Atkinson, 1997). This may be a result of the animal’s subtropical environment and lesser dependence on a well-defined annual reproductive cycle than other species, or a reflection of true reproductive potential of the phocids (Atkinson and Gilmartin, 1992; Pietraszek and Atkinson, 1994). It can be assumed that the onset of parturition and the subsequent diminishing levels of circulating progesterone are the triggers that cause the pinniped hypothalamus to begin increased GnRH secretion, LH and FSH release, and to initiate follicular recruitment and development. In northern fur seals (Callorhinus ursinus), follicular recruitment begins in February in the nongravid ovary, before parturition and ovulation in July (Craig, 1964). In phocids, follicular recruitment starts in close proximity to parturition, resulting in mid- to late-lactational ovulation. This difference may reflect differences between phocids and otariids in the functional life span of the CL. In otariids, the CL begins regressing, and blood progesterone levels begin to fall in February, coincident with follicular recrudescence in the opposite ovary (Kiyota et al., 1999). In seals, the CL regresses and circulating progesterone declines rapidly after parturition (Boyd, 1983; Iwasa et al., 1997). In most phocids and otariids, follicular growth and ovulation occur on alternating ovaries during subsequent pregnancies. It appears that the presence of a CL inhibits follicular activity on the ipsilateral ovary to a greater degree than the contralateral ovary due to some local or paracrine effect (Craig, 1964; Amoroso et al., 1965; Boyd, 1983). 0839_frame_C11 Page 197 Tuesday, May 22, 2001 11:09 AM Reproduction 197 Follicular maturation continues either during late pregnancy or lactation, and results in a rapid rise in estrogen production, presumptive LH surge, and ovulation. This rise in estrogen that has been observed in northern fur seals lasts less than 5 days and reaches circulating concentrations greater than 30 pg/ml (Kiyota et al., 1999). Estrus in Hawaiian monk seals lasts 2 to 6 days (Atkinson et al., 1994; Pietraszek and Atkinson, 1994). In the northern fur seal, multiple follicular recruitment results in approximately four Graffian follicles greater than 10 mm in diameter around parturition. From this group of follicles, one is selected and ovulates 3 to 5 days after parturition (Craig, 1964). Pregnancy and Pseudopregnancy Pregnancy in pinnipeds can be divided into five distinctly important events: (1) conception, (2) embryonic diapause, (3) embryo reactivation and implantation, (4) fetal development, and (5) parturition. In otariids, it appears that an obligate pseudopregnancy ensues after ovulation, regardless of the presence of a normal blastocyst (Boyd, 1991; Atkinson, 1997). However, after the 4 months physiologically allotted for embryonic diapause, uterine development and placental formation can only occur if a functional blastocyst is present. The specific period during embryonic diapause or gestation when maternal recognition of pregnancy (MRP) occurs is unknown. After implantation, and during the latter part of gestation, it is believed that placental gonadotropin, acting via fetal gonads, results in placental production of estrogens and progesterone. This fetal–placental unit is then believed to be responsible for the maintenance of pregnancy, and for triggering parturition. Fetal production of adrenal or gonadal hormones results in hypertrophy of these organs, which are similar in size at birth to adult organs, but rapidly regress in size until puberty. Despite circumstantial evidence for the importance of placental steroid production, some conflicting data recently have been obtained. A complete monitoring of pinniped serum progesterone and estrogen was done by Kiyota et al. (1999) on four northern fur seals during 2 consecutive years. They observed an initial rise in progesterone to 20 to 30 ng/ml in July, indicating ovulation. Progesterone concentrations dropped to 5 to 10 ng/ml during embryonic diapause from August through October, and increased again in November to 25 to 35 ng/ml (similar to observations in wild fur seals; Daniel, 1974). This pattern was observed in seven cycles, but only two resulted in pregnancy. One of the cycles had an initial progesterone spike of around 8 ng/ml that rapidly dropped to slightly over 1 ng/ml. The five hormonal profiles in nonpregnant fur seals studied by Kiyota et al. (1999) that appeared similar to the two hormonal profiles of pregnant animals provide evidence that otariids exhibit an obligatory pseudopregnancy beyond the period of normal implantation. That is, maintenance of the CL is not dependent upon maternal recognition of pregnancy or an embryonic product. The presence of circulating progesterone also contradicts Laws’ (1955) assumption that the CL was nonfunctional in late gestation. In contrast, endocrine data from the harbor seal show evidence for pseudopregnancy that only lasts through diapause, with blood progesterone levels declining rapidly after the window of implantation has occurred in nonpregnant seals (Reijnders, 1991; Atkinson, 1997). High circulating levels of progesterone (greater than 3 ng/ml) have been observed for long periods of time in nonpregnant captive walrus. However, no serial sampling has been done to define the duration of this pseudopregnancy. Placental gonadotrophins isolated by Hobson and Boyd (1984) also appear to be required for CL function in some species. Since pinnipeds are an extremely diverse group of animals, it would be safe to assume that extreme species variations can occur, and fetal–placental or maternal control of luteal function should be addressed on a species-by-species basis. 0839_frame_C11 Page 198 Tuesday, May 22, 2001 11:09 AM 198 CRC Handbook of Marine Mammal Medicine Embryonic Diapause and Reactivation Embryonic diapause in pinnipeds appears to be regulated maternally. During early postconception, the embryo divides at a normal (compared with mammals without diapause) rate until the blastocyst stage around day 5 to 8. At this point, cellular divisions, as determined by a mitotic index, decline rapidly to a point where the embryo doubles in cell numbers every 50 to 60 days (Daniel, 1971). The embryo remains in this slow period of growth for 2 to 4 months (species dependent) until it is reactivated by maternal physiology. During this slow-growth period, the embryo remains in its zona pellucida and does not hatch until after reactivation (Harrison, 1968). Reactivation of the blastocyst appears to be controlled by photoperiod, with most animals implanting during a decreasing photoperiod. Water temperature and nutritional availability may also be important factors regulating pinniped reproductive cycles (Atkinson, 1997). Research into photoperiodic control of reproduction in other mammals has found that an animal does not have to be exposed to a continual light/dark cycle, but has windows of receptivity when exposure to light or dark can define the endocrine response. Thus, exposure to a 1-hour “pulse” of light during the receptive period approximately 9.5 hours after the onset of darkness can be enough to induce early reactivation of reproductive activity in the mare (Sharp et al., 1997). In the same manner, it has been postulated that pinniped parturition and blastocyst reactivation are controlled by the date they are exposed to a particular length of day, generally during a decrease in day length (Temte, 1991). This time appears to vary slightly with each species, but generally occurs around the autumn equinox, when the day length is 12 hours. Recent research in harbor seals demonstrated a significant decrease in pituitary sensitivity to LH during winter and spring (Gardiner et al., 1999). This decreased sensititivity to LH is consistent with animals whose reproduction is under photoperiodic control. During reactivation of the blastocyst, the quantity and molecular weight of uterine protein secretions increase. The increase in uterine protein secretion corresponds to an eight-to ninefold increase in blastocyst mitotic activity, with cell number doubling every 12 hours (Harrison, 1968). A protein, possibly related to blastokinin, is believed to be responsible for blastocyst reactivation. Concurrent with uterine protein secretion, and possibly regulated by photoperiod, progesterone and estrogen increase dramatically. The estrogen increase has been described as a “surge,” and may represent follicular activity on the ovaries prior to reactivation (Temte, 1985). These follicles quickly become atretic after implantation, but the estrogen surge may prime the pituitary to secrete more LH, causing the luteotrophic effects required for CL stimulation and the resulting increase in progesterone secretion. The estrogen increase may also be required to increase uterine progesterone receptors, thus increasing sensitivity to progesterone and ensuring the proper endometrial response to progesterone. Progesterone causes the uterus to prepare for approaching implantation. In harbor seals in the United Kingdom, implantation occurs in November and, by December, placentation has been established. The exact timing varies with latitude. Implantation Although the preimplantation estrogen surge was observed in harbor seals by earlier researchers, until recently, it had not been observed in other pinniped species. This lack of duplicative research left many questioning its existence. However, Kiyota et al. (1999) observed an estrogen “surge” in northern fur seals associated with implantation in November. Unfortunately, they did not clarify if the surge was documented in animals that were pregnant, or nonpregnant, or both. Since the surge was not observed in all of the animals sampled, they felt that their sampling frequency of every 5 days was insufficient to determine whether it was inconsistently observed, or whether they just missed it. 0839_frame_C11 Page 199 Tuesday, May 22, 2001 11:09 AM Reproduction 199 FIGURE 1 An approximately 8-month-old Odobenus rosmarus fetus. The dotted line represents a biparital measurement of 6.1 cm. Fetus is estimated between 90 and 120 days post-reactivation. (From T. Robeck, unpubl. data.) As fetal development proceeds, and in support of placental–fetal maintenance of pregnancy, progesterone secretion declines slightly until parturition occurs. Fetal gonads hypertrophy and are believed to be responsible for secretion of important steroid precursors that are converted to estrogens by the placenta. In addition, placental chorionic gonadotropin (CG) production is believed to be essential for CL maintenance. It was hypothesized that nonpregnant pinnipeds have an obligate pseudopregnancy interval equal to the period during gestation prior to placental gonadotropin production. CLs in pregnant animals will have a third surge (the second surge occurs at implantation) of luteal activity in response to placental CG production, resulting in continued production of progesterone until parturition. However, CG concentrations in the placenta are extremely low when compared with other species that rely on CG for luteal maintenance (Hobson and Boyd, 1984; Hobson and Wide, 1986). Recent research in northern fur seals demonstrates similar hormonal profiles in pregnant and pseudopregnant animals (Kiyota et al., 1999), while research on harbor seals continues to provide support for this theory of extra-hypophyseal or placental support of the CL (Hobson and Boyd, 1984; Reijnders, 1991; Gardiner et al., 1999). These differences demonstrate the lack of understanding of mechanisms involved in maintenance of pregnancy in pinnipeds and the differences between phocids and otariids. Pregnancy Diagnosis Ultrasound diagnosis of pregnancy has been used successfully in mid- to late-gestation in a variety of pinnipeds, but will not detect the fetus during embryonic diapause. Elevated progesterone levels are a useful indication of pregnancy, although values have only been published for a limited number of species. Levels also may be elevated during pseudopregnancy and in nonpregnant animals (levels greater than 3 ng/ml were observed in a nonpregnant captive walrus). The practitioner is advised to use a combination of high progesterone and ultrasound to detect pregnancy (i.e., after embryonic reactivation) (Figure 1). Induction of Parturition Cloprostenol, a synthetic prostaglandin F2α (Estrumate®, Mobay, Shawnee, KS), has been used on California sea lions with full-term fetuses to induce abortion (Gulland, 2000). Five animals that had been exposed to domoic acid (with dead fetuses) were given 500 mg cloprostenol intramuscularly (IM) and delivered fetuses 36 to 40 hours later. 0839_frame_C11 Page 200 Tuesday, May 22, 2001 11:09 AM 200 CRC Handbook of Marine Mammal Medicine Lactation As with most eutherian mammals, prolactin and oxytocin appear to be crucial hormones for regulating lactation. No single prolactin-releasing hormone has been identified, but a number of neuropeptides in the hypothalamus, including vasoactive intestinal polypeptide, thyrotropinreleasing hormone (TRH), and prolactin-releasing factor (perhaps identical to TRH), may all function in this capacity (Norman and Litwack, 1987; Ganong, 1991). Prolactin secretion is increased by neurogenic stimulation via suckling. Prolactin appears essential for mammary gland secretory cell development, and increases in otariids 1 to 2 days prior to parturition, peaking 0 to 3 days postpartum (Boyd, 1991). However, unlike in mink (Mustela vison), in which a preimplantation rise in prolactin is believed to be involved with reactivation, prolactin concentrations decreased to undetectable levels toward the end of lactation and embryonic diapause (Boyd, 1991). In carnivores, prolactin has been identified as playing a role in the development of the CL. In otariids, it appears that prolactin may play a role in both ovulation and CL formation; however, additional data are needed to determine this. Oxytocin (synergistically with prolactin or somatotropin and cortisol) is believed to be essential for the maintenance of lactation. This hormone is secreted via the neurohypophysis in response to suckling stimuli. Once released, oxytocin is important for milk letdown. During this process, myoepithelial cells surrounding the alveoli contract, forcing milk out of the glands. In addition, oxytocin causes relaxation of smooth muscles surrounding the ducts and teat cisterns, resulting in space for milk ejected from the alveoli. Thus, suckling animals only have to overcome the teat sphincter resistance to nurse effectively (Baldwin and Miller, 1991). Generally, continued suckling stimulation, and subsequent oxytocin release, is required to maintain milk production. Indeed, phocid females, whose lactations last from 4 to 60 days, will spend almost the entire time with the pup during this period, with short or no intervals for feeding. However, otariids whose lactation period lasts 4 to 12 months will often leave the pup for feeding from 1 to 8 days. Thus, continual suckling is not required for maintenance of lactation in otariids. During periods of low or no stimuli, milk production slows down or stops; however, under the influence of prolactin, mammary glands do not involute. Once suckling recurs, milk is let down, most likely via oxytocin secretion, and milk production increases or is reinitiated (Boyd, 1991). Milk Collection Oxytocin has been used on a variety of pinnipeds to enhance collection of milk samples for research purposes. Intramuscular injection of 20 Posterior Pituitary Units (USP) of oxytocin will facilitate collection of milk by stimulating milk let down from the teat. Unlike many species of cetacea, pinnipeds do not have a lactational or suckling suppression of estrus. In fact, all pinniped species undergo estrus toward the end of, or during, lactation. Male Pinniped Reproduction Anatomy The reproductive anatomy of male pinnipeds varies with the family. Phocids and odobenids have para-abdominal testes that lie below the blubber layer adjacent to the abdominal musculature, while the otariids have scrotal testes. Some otariids are seasonally scrotal; that is, their testes descend into the scrotum only during the breeding season. Testis size in all marine mammals is proportional to body mass and, in most cases, body length (Kenagy and Trombulak, 1986). Testis size is also related to the mating system and/or the length of the breeding season, with relatively large testes in species that have high rates of copulatory activity and associated high rates of spermatogenesis. 0839_frame_C11 Page 201 Tuesday, May 22, 2001 11:09 AM Reproduction 201 Animals with scrotal testes are able to lower and raise the testes using the cremaster and dartos muscles. This scrotal agility protects the sperm from cold shock of the surrounding aquatic environment as well as physically protecting the testes when the animal is moving on land. In ascrotal species, protection of the testes and developing spermatocytes from hyperthermia is accomplished through a direct vascular heat-exchange mechanism using arteriovenous anastomoses. The anatomy of the arteriovenous anastomoses allows cool blood from the skin and flippers to flow directly to the testicular artery, preventing hyperthermic insult to the developing sperm (Rommel et al., 1995) (see Chapter 9, Anatomy). All of the pinnipeds, polar bears (Ursus maritimus), and sea otters (Enhydra lutris) have bacula, or penis bones. The distal end of the baculum is morphologically variable and differs substantially among species (Morejohn, 1975). Most of the phocids are aquatic copulators with relatively large bacula, which may function either to prevent water damage to sperm cells after ejaculation or to increase sperm competition in species where the female mates with more than one male (Miller et al., 1999). Most otariids are of large body size and are terrestrial copulators; bacula in otariids are relatively small; bacular fractures have been reported in otariids. Most of the growth in bacular length is achieved by puberty; however, bacular mass and density continue to increase for another decade. Sexual Maturity Sexual maturity in male pinnipeds tends to occur at 2 to 7 years of age (Atkinson, 1997; Boyd et al., 1999). Diagnostic measures of puberty are the relative weight of the testes, an increase in the circulating concentrations of testosterone, and active spermatogenesis. Bacular mass and length also increase during puberty. Testosterone concentrations have been measured in many pinnipeds (Noonan et al., 1991; Atkinson and Gilmartin, 1992), and in all species the concentrations increase around the time of sexual maturity. Histological evidence of sexual maturity can be measured in the diameter of the seminiferous tubules, proportion of the tubules to interstitium, and the presence, abundance, and maturation of spermatocytes in the tubules. Although the age of puberty may occur early in life, many pinnipeds are not behaviorally capable of breeding until 8 to 10 years of age (Atkinson, 1997). In sexually dimorphic species, the secondary sexual characteristics generally become obvious during and after puberty. Examples in pinnipeds include increased body size, a developed sagittal crest, elongated proboscis or hood, calloused chest shield, development of a musky odor, and/or more or elongated guard hairs on the neck and shoulders. In some species, the secondary sexual characteristics are only fully developed in males that are both physiologically and behaviorally mature. Seasonality Pinnipeds are seasonally fertile, with the length of the fertile season greatest in tropical animals and shortest in temperate animals (Atkinson and Gilmartin, 1992). Seasonality is associated with increased size of the testes and accessory reproductive glands, increased testicular and circulating testosterone concentrations, and spermatogenesis during the breeding season (Griffiths, 1984a,b). Increased size and mass of the testes are due to increased diameter of the seminiferous tubules and the epididymis. Decreases in testicular tissue and associated glands during the nonbreeding season are thought to be due to the shrinkage of the anterior pituitary cells that produce gonadotropins. This theory has recently been supported by the significant seasonal decrease in pituitary release of LH in response to an exogenous GnRH challenge (Gardiner et al., 1999). The lack of gonadotrophic support via decreased LH and FSH release from the pituitary leads to seasonal atrophy of the testes, and testosterone concentrations decline to baseline during the nonbreeding season (Frick et al., 1977; Gardiner et al., 1999). 0839_frame_C11 Page 202 Tuesday, May 22, 2001 11:09 AM 202 CRC Handbook of Marine Mammal Medicine Mature sperm in both the seminiferous tubules and epididymis and elevated testosterone concentrations are apparent preceding the breeding period in several species of pinnipeds. Spermatogenesis usually lags behind testosterone production by 1 to 3 months, as production of testosterone by testicular Leydig cells is necessary for germ-cell differentiation in the seminiferous tubules. During seasonal quiescence, spermatogenesis ceases. In addition, at least in gray seals (Halichoerus grypus), the seminiferous tubules undergo involution, resulting in a decrease in both testicular dimension and mass (Griffiths, 1984a). Contraception and Control of Aggression A common concern in facilities housing marine mammals is the control of fertility in captive animals. Three particular species for which fertility control has become a concern are bottlenose dolphin, California sea lion, and harbor seal. All of these species can be prolific breeders in the captive setting. The most common methods of reducing fertility have been physical separation, castration of males, and contraception of female animals. Sexual behaviors are often associated with territorial or aggressive behaviors. The need to control behavior is obvious in the captive setting. It also is important in the management of declining species in which male aggression inhibits the recovery of the species. Sexual behaviors may be as obvious as approaching, chasing, and nudging of females, vocalizations, and agonistic threats to neighboring males. Many intraspecific acts of aggression indicate a form of dominance. In several pinniped species, territorial and/or aggressive behaviors occur when testosterone concentrations are increasing, suggesting a behavioral role for the elevated hormone concentrations (Atkinson and Gilmartin, 1992; Theodorou and Atkinson, 1998). Increased testosterone concentrations usually coincide with the seasonal approach of the breeding season. In many species, the ability of an adult male to maintain rank and access to estrus females correlates with age and territorial behavior. Females For female pinnipeds, the majority of research, sparse as it may be, has been conducted on phocids. Research has focused on the use of porcine zona pellucida vaccine (PCP). PCP vaccine uses sperm-binding sites on the porcine zona pellucida as a source of antigen. Thus, the vaccine causes an autoimmune antibody response directed against recently ovulated ova that blocks sperm binding. Without sperm binding, the degranulation reaction cannot occur and sperm are unable to penetrate the zona to fertilize the ova. This vaccine has been effectively applied to a number of captive and wild hoofstock (Kirkpatrick et al., 1982; 1990; 1996). Its practical application to wild pinniped populations was hindered because of the requirement for up to four booster vaccinations. Recent improvements to the delivery system, however, have resulted in effective contraception after single-dose administration in wild seals (Brown et al., 1997a,b). Although this vaccine may have its use in captive populations, and a large body of evidence suggests that in some species it may not be reversible, and it has been associated with negative ovarian and systemic inflammatory side effects in canids and felids (Mahi-Brown et al., 1988; Asa, 2000). Males Castration has been used routinely to prevent breeding of captive harbor seals and captive California sea lions. Recently, GnRH agonists have been applied to male marine mammals in an effort to reduce fertility and control aggression (Atkinson et al., 1993; 1998; Briggs, 2000). In the male, episodic pulses of GnRH occur at regular, species-specific frequencies (Sisk and Desjardins, 1986) concurrent with cyclic changes in GnRH secretion frequency and amplitude observed in females (Ganong, 1991; Mariana et al., 1991). The periodicity of the pulse rate of GnRH secretion is 0839_frame_C11 Page 203 Tuesday, May 22, 2001 11:09 AM Reproduction 203 important for normal reproductive function. This is evident when comparisons are made between steady infusions or pulsatile infusions of GnRH. Since GnRH regulates its own receptor production at the pituitary, receptor production is high when ligand is low and low when ligand is high, and receptor changes can occur rapidly. Constant infusions of GnRH result in constant downregulation of receptors (Conn et al., 1987). Thus, when GnRH is administered in a constant fashion, there is an initial dramatic increase in LH secretion from the pituitary, and subsequent LH secretion becomes refractory to GnRH as the pituitary receptors for GnRH are reduced (Sundaram et al., 1982; Mann et al., 1984; Schurmayer et al., 1984). In addition to the initial post-GnRH agonist administration surge of LH, a temporally associated testosterone increase is also observed (Belanger Anclair et al., 1980). After 3 to 4 days of constant infusion of GnRH agonist, basal levels of testosterone can double, declining to baseline, or less than baseline, as the pituitary becomes desensitized to GnRH around day 10 (Chrisp and Goa, 1990). Depression of testosterone synthesis and secretion beyond day 10 requires continued, steady administration of the agonist. When administered to Hawaiian monk seals, GnRH agonists (D-Trp-6-LHRH and D-Ala-6LHRH) have reduced circulating testosterone concentrations to castrate levels by approximately 2 weeks after injection, with results lasting approximately 2 months (Atkinson et al., 1993; 1998). As predicted, reduction in circulating testosterone concentrations was preceded by a dramatic elevation in testosterone concentrations; however, LH concentrations have never been measured to evaluate exactly when the pituitary becomes refractive (Atkinson, unpubl. data). Doses of 2.5 to 11.25 mg of the GnRH agonist incorporated into microlatex beads were administered to Hawaiian monk seals, with similar results after all doses. Harbor seals and northern elephant seals (Mirounga angustirostris) exhibited similar responses; however, the northern elephant seals required 40 mg to produce a discernible effect on testosterone concentrations (Atkinson, Yochem, and Stewart, unpubl. data). The effects of GnRH agonists on fertility have been demonstrated in two facilities that house harbor seals. After annual treatment of males, no offspring have occurred. Reproductive Abnormalities in Pinnipeds Very little information is available concerning pathological conditions of reproductive events. Reijnders (1986) showed reduced reproductive rates in harbor seals fed fish from polluted waters, and Gilmartin et al. (1976) demonstrated an association between maternal and fetal concentrations of pesticides and premature births in California sea lions (see Chapter 22, Toxicology). High tissue concentrations of polychlorinated biphenyls and reproductive tract abnormalities including uterine stenosis have been described in gray, harbor, and ringed (Phoca hispida) seals (see Chapter 22, Toxicology). The mechanisms for these changes are unknown, but pregnancy rates of seals in the Gulf of Bothnia decreased from a normal of 60 to 90%, to as low as 25% (Boyd et al., 1999). Rates of dystocia in captive-bred animals have not been determined; however, they appear to be low since no cases have been reported. Stillbirths occur infrequently, with no data available on causes or incidence of occurrence. In a few species of pinnipeds, mobbing behavior is observed, in which groups of males attempt a mass mating, typically with an adult female or juvenile seal of either sex. In Hawaiian monk seals, the behavior is primarily targeted at female seals that are periovulatory, and is concurrent with a seasonal rise in testosterone concentrations (Atkinson et al., 1994). In northern elephant seals, the females are thought to submit to the mobbing behavior as they leave the territory of the dominant male, returning to the sea. These behaviors have yet to be documented in captive animals; however, the species in which the behaviors have been demonstrated are not commonly maintained in captivity. 0839_frame_C11 Page 204 Tuesday, May 22, 2001 11:09 AM 204 CRC Handbook of Marine Mammal Medicine Cetacean Reproduction The majority of cetaceans housed in zoological settings can be divided into two different taxonomic families: Delphinidae and Monodontidae. The most commonly displayed Delphinidae include the bottlenose dolphin, the killer whale (Orcinus orca), the white-sided dolphins (Lagenorhynchus obliquidens and L. acutus), and the false killer whale (Pseudorca crassidens). The only Monodontidae displayed is the white whale, or beluga (Delphinapterus leucus). The diverse reproductive strategies and physiology among the Delphinidae alone demonstrate the importance of learning basic reproductive physiology for each species. Inefficiency and inaccuracy can occur when using one species as a model for reproductive patterns in another. As with pinnipeds, the amount of information available for each species varies tremendously, which reflects the lack of systematic research that has been conducted with most cetacean species. Before advances in manipulation and control of reproduction can occur, these systematic studies must be conducted. Female Cetacean Reproduction Reproductive Maturity Bottlenose Dolphin The age of sexual maturity of the Tursiops truncatus aduncus subspecies of bottlenose dolphins in the wild was estimated at over 10 years for females (Ross, 1977). Brook (1997) documented first ovulation in two captive T. t. aduncas at 6 to 7 years of age. The youngest captive bottlenose dolphin to give birth was 4 years of age; however, the majority first gave birth at 7 to 10 years (Duffield et al., 2000). In wild animals, the youngest female observed to calve was 6 years old, and the majority of females gave birth at 8 years of age (Wells, 2000). White-Sided Dolphin Sergeant et al. (1980) and Rogan et al. (1997) estimated sexual maturity for Atlantic whitesided dolphins (L. acutus) at around 218 cm in length and 6 to 8 years of age. The authors observed a captive dolphin conceive at 3 years of age and deliver a healthy calf 1 year later (Dalton and Robeck, unpubl. data). The lack of data from wild animals precludes one from determining whether reproductive capabilities of this animal were accelerated by an increased plane of nutrition or if normal reproductive potential is as early as 3 years. Killer Whale In the wild, sexual maturity was estimated at 8 to 10 years of age and greater than 3 m in length (Christensen, 1984). The average age at which captive killer whales first exhibited luteal activity was 9.06 ± 2.1 years (range 5.8 to 12 years, n = 9) and first conception was observed at 11.7 ± 2.9 years (range 6 to 14 years, n = 9). The average age of first calving in wild killer whales off the northwest coast of the United States was 14.9 years (Olesiuk et al., 1990). During analysis of urinary endocrine data in captive killer whales, Walker et al. (1988) and Robeck (1996) observed short transient elevations in estrogen conjugates (EC) without luteal phases, or with short luteal phases in young animals, which may have represented normal endocrine activity during reproductive maturation (Robeck, 1996). The short spikes of EC without subsequent immunoreactive pregnanediol-3-glucuronide (iPdG) appear similar to reproductive endocrine characteristics exhibited by primates during sexual maturation (Plant, 1988). Low progesterone levels and irregular short luteal phase lengths during sexual maturation also have been observed in the ovine and primate (Goodman, 1988; Plant, 1988). 0839_frame_C11 Page 205 Tuesday, May 22, 2001 11:09 AM Reproduction 205 False Killer Whale False killer whales were thought to attain sexual maturity at 3.7 to 4.3 m in length, and 8 to 14 years of age (Purves and Pilleri, 1978). In agreement with these data, Atkinson et al. (1999) did not observe any evidence of ovarian activity in a 6-year-old, 3.15-m female. However, another facility has recently had a 5-year-old, 347-kg, 3.4-m false killer whale conceive, although the outcome of this pregnancy is still pending (Walsh, pers. comm.). For captive false killer whales, body length at first conception is close to lengths observed in mature wild females. Beluga Sexual maturity in white whales has been estimated at 6 to 7 years in both captive and wild populations (Braham, 1984; Calle et al., 1996). Females in captivity have conceived up to 20 years of age. This correlates with the estimated age of senescence for wild populations of 21 years (Brodie, 1971a). Reproductive Cycle Most Monodontidae or Delphinidae exhibit seasonal reproductive activity or show seasonal trends that may reflect adaptations to food sources or climate. Photoperiod is thought to provide an environmental cue to seasonal breeders. For a species to be considered a seasonal breeder regulated by photoperiod, it must have repeatable patterns of reproductive quiescence that correlate with increasing or decreasing changes in light. In addition, physiological evidence of changes in pituitary sensitivity to gonadotropic hormones must exist. As shall be seen, two species pass the criteria for seasonal quiescence, the Pacific white-sided dolphin and the beluga; however, no data exist on seasonal pituitary down regulation. Bottlenose Dolphin The bottlenose dolphin can be defined loosely as seasonally polyestrous (Kirby and Ridgway, 1984; Robeck et al., 1994a; Robeck, 2000). Most estrous cycling activity occurs spring through fall, but births have occurred in every month of the year. When cycling, individual animals can cycle one or more times during the year. If animals are in a breeding colony, the majority will get pregnant on the first or second estrus. Gestation for bottlenose dolphins is estimated at 12 months, and lactation can last up to 2 years or more for wild animals. Lactational suppression of estrus does occur; however, there appears to be a threshold level. When daily suckling decreases below a certain time period, usually after 1 year, ovarian activity will resume (West et al., 2000). Thus, the entire reproductive cycle or calving interval may last 3 to 4 years. Wells (2000) describes a calving interval for wild populations that varies with age class and ranges from 3 to 6 years. Females in their twenties produce calves most frequently, while younger and older females have longer calving intervals. This age-related change in fecundity is also described for captive populations (Duffield et al., 2000). In wild animals, age-associated fecundity rates may be a reflection of social status in younger animals, and reduced fertility in older animals. These factors may also play a role with captive populations; however, controlled access to females of certain age classes by males often biases captive breeding results. Managers of breeding colonies should be aware of bottlenose dolphin reproductive potential, and should try to maintain colonies that mimic natural social groupings (Wells, 2000). These natural social groups contain three basic units: (1) female/nursery groups consisting of mothers with their most recent calves; (2) juveniles in mixed-gender groups forming temporary relationships; and (3) adult males, as individuals or in pairs with strong bonds (Wells et al., 1999; Wells, 2000). White-Sided Dolphin The Atlantic white-sided dolphin is believed to cycle in August and September and calf in June and July, suggesting an 11-month gestation period. The Pacific white-sided dolphin is seasonally polyestrous with estrous activity and births occurring from July through September in the 0839_frame_C11 Page 206 Tuesday, May 22, 2001 11:09 AM 206 CRC Handbook of Marine Mammal Medicine United States. No information is available describing physiological control of their seasonality. Captive Pacific white-sided dolphins have exhibited an approximately 12-month gestation period (Dalton and Robeck, unpubl. data). Killer Whale Killer whales are polyestrous. Estrus and conception occur throughout the year, with a slight, nonsignificant, seasonal increase in activity during the spring from March through August (Matsue et al., 1971; Robeck et al., 1993). Nonlactational periods of anestrus have ranged from 3 to 24 months in mature healthy females (Duffield et al., 1995; Robeck, 1996). Duffield et al. (1995) used biweekly progesterone data to describe a calving interval in captive killer whales of 32 to 58 months. Robeck (1996) found that the mean calving interval in females that were nonsuccessful at calf rearing (due to stillbirth or unsuccessful nursing) was 33 months, whereas in females that nursed successfully it was 50 months. The minimum calving interval observed for resident wild killer whales off the northern Pacific Coast of the Unites States was 36 months (Balcomb et al., 1982). Recent estimates from resident whales of this region place calving intervals from 24 months to 12 years (Olesiuk et al., 1990), with the average calving interval for wild populations estimated at 8.6 years (Balcomb et al., 1982) and 10.3 years (Bigg, 1982). The reduced calving interval of captive whales compared with wild whales is probably explained, to some extent, by nutritional and environmental differences (Matkin and Leatherwood, 1986). A decrease in reproductive productivity in response to adverse or seasonal nutritional and environmental conditions is well documented in other species (Bronson, 1988). Another possible explanation for the calving interval differences is that early postpartum or peripartum neonatal calf mortality might go unnoticed in wild killer whales. False Killer Whale The false killer whale is polyestrous with no strong evidence for seasonality (Robeck et al., 1994b; Atkinson et al., 1999). Information on wild animals suggests that they can become pregnant any time of the year and have an estimated gestation period of 12 to 15 months (Comrie and Adams, 1938; Purves and Pilleri, 1978). Robeck et al. (1994b) described a gestation period of 14 months in a captive animal that produced a normal calf. If gestation lasts 14 months and lactation 6 to 12 months (with lactational anestrus), one could estimate a calving interval of 2.5 to 3.5 years. Atkinson et al. (1999) noted possible pseudopregnancy and prolonged anestrus in captive false killer whales with no access to males. Beluga The beluga is seasonally polyestrous, breeding in the wild in April and May (Brodie, 1971b). Captive animals have conceived from February to June (Calle et al., 1996). This difference may be the result of latitudinal differences and associated photoperiod effects on breeding activity, although there is no evidence to confirm this. Calving in wild belugas has been observed from July to September, and in captive animals from May through September. Gestation lengths have been estimated at 14.5 months for wild populations and 15 to 17 months for captive ones (Brodie, 1971a; Calle et al., 1996). Animals have been observed to nurse for 2 years, and at least one animal conceived during the spring season after a previous summer birth; thus, lactational anestrus may not occur in this species. The calving interval for captive animals is as little as 3 years (Brodie, 1971a; Seargent, 1973; 1980; Braham, 1984). Estrous Cycle and Ovarian Physiology Bottlenose Dolphin Most of the published information on cetacean reproduction concerns the most common cetacean in captivity, the bottlenose dolphin (Robeck et al., 1994a). Existing endocrine data has come from weekly or biweekly blood sampling of trained captive animals. This type of 0839_frame_C11 Page 207 Tuesday, May 22, 2001 11:09 AM Reproduction 207 FIGURE 2 Mean (±S.D.)(n = 35 estrous cycles) pattern of development for the dominant follicle prior to ovulation in four T. t. aduncus. FD = Follicle diameter. (From F. Brook, Hong Kong Polytechnic University, Kowloon, Hong Kong, 1997, 339.) sampling frequency is sufficient to describe seasonality or estrous cycle patterns, but is not adequate to the pulsatile endocrine activity that occurs in proximity to ovulation, or other important ovarian events. At best, one could hope to catch an estrogen surge, but without serial sampling, few to no conclusions can be drawn. Urinary and fecal sampling or other noninvasive techniques that can be performed daily offer the best hope for describing and eventually predicting ovarian and endocrine relationships. Urinary endocrine monitoring offers great promise, but, until recently, the only species that had been reliably trained for this procedure was the killer whale, although many facilities have now reported success in training bottlenose dolphins. A wealth of information on bottlenose dolphin reproductive physiology and follicular dynamics has recently been collected through the use of sonographic ovarian analysis (Brook, 1997; 2000; Robeck et al., 1998; 2000). However, this technique, has yet to be combined in adequate endocrine monitoring to describe how hormonal events relate to ovulation. Harrison and Ridgway (1971) reported on the gonadal activity of 22 female bottlenose dolphins. In these animals, most of the follicles were 2 mm or less in diameter with no follicles greater than 5 mm, although there was an accessory CL formed from a luteinized follicle 10 mm in diameter. Brook (1997) used ultrasonography to follow follicular activity in bottlenose dolphins (T. t. aduncus) and provide the first real-time description of folliculogenesis in cetaceans. Multiple 2- to 3 mm-diameter follicles were often observed on the ovary, regardless of ovarian activity. Once a follicle was larger than 3 mm, it could be classified as developing. In 32% of observed cycles (n = 37), more than one follicle developed beyond 4 mm in diameter. The dominant or primary follicle appeared 1 to 2 days prior to ovulation, when it was distinguished from other follicles by its size. Only one follicle was seen to ovulate, subordinate follicles regressing either before or just after ovulation. Ovulation occurred at a mean of 8 days after the dominant follicle reached 10 mm in diameter (Figure 2). Preovulatory follicles ranged in size from 18 to 28 mm, with a mean of 20.9 mm (Figure 2). 0839_frame_C11 Page 208 Tuesday, May 22, 2001 11:09 AM 208 CRC Handbook of Marine Mammal Medicine There appeared to be a loose correlation between the size of the dolphin and the size of the preovulatory follicle in this population, although the number of females studied in detail was small and this remains to be confirmed. There is evidently significant individual variation in preovulatory follicle size and it is essential to assess each animal over time in order to use follicular size to predict ovulation. The maximum diameter of “normal” CLs (i.e., not associated with pregnancy or pseudopregnancy) observed ranged from 21 to 36 mm. Again, the largest CLs were seen in the larger females. Estrous cycle length in T. t. aduncas is about 30 days. For T. t. truncatus, cycle lengths of 21 to 42 days have been estimated from serum hormone levels (Sawyer-Steffan and Kirby, 1980; Kirby and Ridgway, 1984; Schroeder, 1990b). Periods of anestrus not associated with gestation or lactation occur in Tursiops (Brook, 1997). At these times, ovulation does not occur and the ovaries appear to “shut down.” Periods of anestrus of up to 27 months have been documented in T. t. aduncas, but the significance of this phenomenon is not understood. Killer Whale The only cetacean species in which detailed information on gonadotropic hormones has been collected is the killer whale. Walker et al. (1988) used urinary progesterone and estrogen metabolites, and bioactive FSH, to describe endocrine changes that occurred during two estrous cycles. Based on their results, they predicted a wave of follicular activity that begins before peak estrogen levels, but the temporal relationship between peak plasma estrogen and ovulation could not be determined. Urinary LH levels can be quantitatively detected in killer whales although twice-daily urine samples are necessary to describe the LH peak or surge consistently (Robeck et al., 1990; Robeck, 1996). Recent data suggest the LH surge occurs around 12 hours after the peak estrogen surge (Robeck et al., unpubl. data). The mean estrous cycle length based on the beginning of luteal phases was 41.2 days (range 19 to 49 days), the follicular phase lasts around 18 days, and the luteal phase lasts around 20 days (Robeck, 1996). Anestrus periods as long as 2 years, which are not associated with gestation or lactation, have been observed in killer whales (Robeck, unpubl. data). Copulatory activity of killer whales has been compared with qualitative estimates of vaginal mucus secretion and endocrine data (Robeck, 1996). A higher percentage of mucus secretions and copulations occurred around peak levels of EC rather than peak levels of LH, and heavy vaginal mucus secretion was often associated with estrus or receptivity. Although mild mucus secretion occurred during various phases of the estrous cycle, all of the heavy vaginal secretion occurred during periods of detectable EC. Thus, it appears that in the killer whale, as with other terrestrial species, estrogens, presumably produced from developing follicles, are responsible for stimulating sexual activity (probably by changing female receptivity) and producing secretory changes (i.e., vaginal and cervical mucus secretions) required for conception. Limited observations with killer whale ovaries suggest a different pattern for developing follicles from that in the bottlenose dolphin. Follicles destined to ovulate appear to develop over two cycles, with the size of the follicle ranging from 2.5 to 4.5 cm at the start of the follicular phase (Figure 3). As many as four preovulatory follicles have been observed on the ipsilateral ovary and at least two on the contralateral ovary (Robeck, unpubl. data). More observations are needed to understand better the range of patterns that naturally occur in this species. False Killer Whale Prolonged periods of elevated serum progesterone or pseudopregnancy may occur with regularity in false killer whales. Serum progesterone and hydrolyzed conjugated progesterone in daily urine samples from two female false killer whales indicated prolonged luteal or pseudopregnant periods of elevated progestin for 378, 202, 36, and 24 days (Robeck et al., 0839_frame_C11 Page 209 Tuesday, May 22, 2001 11:09 AM Reproduction 209 FIGURE 3 Killer whale with a large dominant follicle approximately 3.5 cm in diameter. This follicle was 12 days from an ovulation that resulted in an artificial insemination (with cooled transported semen) pregnancy. The dotted lines represent the ovarian length (9.9 cm). (From T. Robeck, unpubl. data.) 1994b). Atkinson et al. (1999) measured weekly serum progesterone concentrations and observed a prolonged period of ovarian activity from March to December. Periods of anestrus not associated with gestation or lactation of 3 to 10 months have been observed in false killer whales (Atkinson et al., 1999). Suckling (Lactational) Suppression of Estrus During a 10-year period of observations on one group of bottlenose dolphins (T. t. aduncus), ovulation during lactation was never observed (Brook, unpubl. data). On one occasion, a female was accompanied by a 1.5-year-old calf, but suckling was not observed for several weeks. This animal was seen to ovulate once, but then her calf slid over the enclosure wall and stranded on the poolside. Although physically unharmed, intensive suckling behavior resumed when the calf was returned to the mother, and continued for some time. The mother did not cycle again for several months until suckling stopped again. Robeck (1996) provides strong evidence of lactational, or suckling, suppression of estrous activity in killer whales. There were significant differences between postpartum return to estrus in lactating (mean 481.4 days; range 159 to 983 days) and nonlactating (mean 65.8 days; range 31 to 122 days) females. Lactation alone does not suppress estrus. This was demonstrated by West et al. (2000) when they collected milk samples from lactating dolphins with or without suckling calves for up to 402 days postpartum. Although these dolphins were lactating, cycling began after the calf had been weaned, or, if the calf was stillborn, within a relatively short period. When an animal is lactating, total suckling time can drop below the minimum threshold duration of stimuli required to suppress estrus, and the animal will return to estrus. This usually occurs in females with older calves that obtain most of their nutrition from fish, but will still occasionally nurse when presented with the opportunity. This threshold effect may be related either to decreased sucking stimuli or to a built-in time clock that reduces the hypothalamic inhibitory effects of suckling stimuli after a certain period postpartum, or a combination of the two. In general, lactational alteration of reproductive function is believed to be caused by suckling stimuli, which suppresses gonadotropin (particularly LH) secretion, preventing normal follicular maturation and ovulation (McNeilly, 1988). In dolphins and 0839_frame_C11 Page 210 Tuesday, May 22, 2001 11:09 AM 210 CRC Handbook of Marine Mammal Medicine killer whales, therefore, it appears that suckling (which also helps to maintain lactation) plays an important role in regulating the calving interval. Corpora Albicantia and Asymmetry of Ovulation Histological changes in ovarian structures in the bottlenose dolphin and other delphinids have been described in detail (Harrison, 1969; Benirschke et al., 1980; Perrin and Reilly, 1984). Corpora albicantia (CA) are believed to be retained indefinitely in pilot whales (Globicephala macrorhynchus), but are only retained when they have originated from corpora lutea of pregnancy in other species, such as bottlenose dolphins and Stenella spp. (Harrison, 1969; Marsh and Kasuya, 1984; Perrin and Reilly, 1984). This has recently been confirmed by analysis of ovaries from a bottlenose dolphin whose entire reproductive history, including ovulations and pregnancies, was documented by ultrasound (Brook, unpubl. data). Based on histological identification of CA, ovulation and pregnancy in the bottlenose dolphin occurred in the left ovary and left uterine horn more than 68% of the time (Ohsumi, 1964; Harrison and Ridgway, 1971). Brook (1997) found similar asymmetry with respect to ovulation in T. t. aduncus. Asymmetry exists in other cetaceans; yet the physiological mechanisms for this are unknown (Ohsumi, 1964; Perrin and Reilly, 1984; Bryden and Harrison, 1986). Pseudopregnancy Pseudopregnancy occurs in bottlenose dolphins, killer whales, and false killer whales. The cause of pseudopregnancy in delphinids is unknown and may be multifactorial. In terrestrial species (without obligate embryonic diapause), the most common cause is early embryonic loss after the embryo has released pregnancy-specific proteins that are involved with MRP. Thus, the maternal uterus “believes” it is pregnant, release of prostaglandin is inhibited, and the CL maintains secretion. For pseudopregnancy to continue for any significant duration, however, there must be a source of gonadotropins to maintain the CL. As discussed below, in killer whales, it appears that at least early pituitary LH is responsible for CL growth and development. If fetal death occurs after placental formation, it may be a local source (Hobson and Wide, 1986). Using ultrasound, Jensen (2000) described early fetal abortion in a bottlenose dolphin. Although data are inconclusive, it appears that the fetus died approximately 3 to 4 weeks before CL progesterone secretion stopped, and that the abortion of the dead fetus coincided with basal progesterone concentrations. Ultrasound evaluation of early embryonic loss and how, or if, the timing of such events affects the endocrine system may help determine whether it plays a role in pseudopregnancy. Pseudopregnancy occurs with some frequency in females without access to males. If pseudopregnancy only occurred in females without access to males, then it would be easy to blame the unnatural social groups found in managed environments as the cause for these conditions. Cowan (2000), however, reported a number of wild dolphins having luteal cysts that could result in pseudopregnancy. In killer whales, pseudopregnancy tends to occur in animals that have cycled multiple times (more than four cycles) without becoming pregnant. It is not dependent on age, but once an animal has experienced pseudopregnancy, it appears more likely to experience it a second time. Although killer whales will cycle multiple times during a season, this polyestrous activity occurs only in the absence of a fertile male, and as such, would probably not occur in wild populations. The rate of pseudopregnancy among wild animals is not known. 0839_frame_C11 Page 211 Tuesday, May 22, 2001 11:09 AM Reproduction 211 Management of pseudopregnancy via prostaglandin F2α administration is a viable option for returning females to the breeding pool and maximizing their reproductive potential (Robeck et al., 2000). Pregnancy Bottlenose Dolphin The use of ultrasound to monitor pregnancy in captive cetaceans provides valuable data on fetal morphology, development, and well-being and on maternal gestation length in bottlenose dolphins, although there remains a need for normal data (see Chapter 26, Ultrasonography) (Williamson et al., 1990; Taverne, 1991; Brook, 1994; Stone et al., 1999; Sweeney et al., 2000). Gestation periods in Delphinidae vary. The gestation period for bottlenose dolphins has been estimated at 12+ months. Recent data in T. t. aduncus with known conception dates places these values at 370 ± 11 days (Brook, 1997). Plasma progesterone levels recorded during pregnancy in bottlenose dolphins range from 2.0 to 56.0 ng/ml (Sawyer-Steffan and Kirby, 1980; Schroeder and Keller, 1989). Killer Whale Robeck (1996) used high-performance liquid chromatography (HPLC) to describe progesterone metabolite secretion during the luteal phase, and early, mid, and late pregnancy in killer whales. The presence of only one major immunoreactive metabolite during these periods provides evidence for the presence of a single source of progesterone. These data support the commonly proposed hypothesis that maintenance of pregnancy relies heavily on luteal production of progesterone. Recent data demonstrated an increase in the frequency of LH surges soon after conception but after initial luteal progesterone levels had begun to increase (Robeck, 1996). This increase in high-amplitude LH secretion was not observed during the luteal phases of nonconceptive cycles. A similar increase in LH secretion during the early luteal phase has been observed in Asian elephants (Elephas maximus) (Brown et al., 1991). However, unlike the killer whale, this increased LH secretion is not limited to conceptive cycles. The hypothesized significance of these early-luteal-phase LH surges in the elephant was to aid in the formation of a critical mass of luteal tissue necessary for the maintenance of pregnancy (Brown et al., 1991). Since these LH surges were observed after progesterone had begun to rise, they may be needed for stimulating maintenance of the existing CL or for formation of accessory luteal structures. Similarly, the killer whale may require additional LH release for correct formation of the CL of pregnancy (Robeck, 1996). This differential secretion of LH during the early progesterone secretion of a conceptive cycle rather than a nonconceptive cycle should result in the formation of two different types of CL structures. The CL that has been supported by additional LH secretion should theoretically be developed to a greater degree than the one without this additional stimulation. This theory is supported by the presence of two types of luteal scars or CA on the ovaries of odontocetes (Harrison, 1969; Fisher and Harrison, 1970; Harrison et al., 1972). Type 1 CAs are typically 5 to 10 mm in diameter and represent the remnants of a well-vascularized and organized CL. Type 2 CAs are usually smaller, 3 to 5 mm, and appear to represent a less well developed or organized CL. Although there is some debate over the significance of these histologically distinct CAs, type 1 CAs are believed to be associated with pregnancy and type 2 CAs are believed to represent anovulatory luteinized follicles or nonconceptive ovulations, (Harrison and Ridgway, 1971; Gaskin et al., 1984; Marsh and Kasuya, 1984). 0839_frame_C11 Page 212 Tuesday, May 22, 2001 11:09 AM 212 CRC Handbook of Marine Mammal Medicine Beluga Gestation length in the beluga has been estimated as 14.5 to 17 months. Little is known about the physiology of pregnancy in this species. Pregnancy appears to be maintained by the CL of pregnancy. Accessory corpora (luteinized follicles) have been found in 11 to 15% of pregnant belugas, but because of their low incidence, they are obviously not required to maintain pregnancy. They also indicate that follicular growth can occur in pregnant animals, possibly during the next breeding season, when they would normally come into estrus. Calle et al. (1996) pooled mean monthly gestational plasma hormone levels for captive animals; progesterone levels ranged from 0.97 ± 1.14 to 42.86 ± 12.00 ng/ml and estrogen ranged from 13.93 ± 11.62 to 30.62 ± 12.43 pg/ml. Pregnancy Diagnosis Pseudopregnancy occurs with such regularity in dolphins, killer whales, and false killer whales that an animal cannot be confirmed pregnant without the use of ultrasonography (see Chapter 26, Ultrasonography). Despite the regular occurrence of pseudopregnancy, it is still a relatively newly described phenomenon that undoubtedly has always occurred, and may have led to an overestimation of abortion rates. Because of its recent recognition, and the slow integration of ultrasound into clinical practice, data are insufficient to allow accurate descriptions of its frequency and to determine which class of animal is most susceptible. Parturition The mechanism of control of parturition in cetaceans is unknown; however, there appears to be an interaction between hormones produced by the fetal–placental uterine axis. Six major hormones, and probably others, appear to be intertwined during the induction of parturition. These hormones include estrogens, progesterone, adrenal steroids, oxytocin, relaxin, and prostaglandins. Stages of Parturition Early stages of pregnancy generally have similar behavioral components. The most common behavioral signs are listed in Table 2. The table was designed as a quick reference to some important periparturient events. Many of these events, such as first nursing, can have extreme variability in length, so it is important to remember these guidelines cannot replace careful clinical observation of each situation. For example, if when using the table to determine interval to first nursing, it may be comforting to know that to the authors’ knowledge bottlenose dolphin calves have lived even after failing to nurse for up to 48 hours. However, the level of comfort of the clinician attending a parturient cetacean should be dependent upon the behavior, condition, and activity of the cow and calf. A predictor of parturition not on the table is a decrease in rectal temperature 24 hours prior to stage-two labor. These data have recently been collected for both the bottlenose dolphin and the killer whale. It requires minimal training to condition the animals to obtain a daily body temperature and may provide an objective indicator for predicting parturition (Katsumata et al., 1999a; Terasawa et al., 1999). Recognition of the onset of parturition is an important management tool. Most reproductiverelated problems (dystocia, stillbirth, weak calf, poor maternal care) occur, and can be observed, in the first few hours after delivery. Induction of Parturition Although the hormonal control of parturition is not understood, administration of hormones in appropriate combinations can result in the induction of parturition, sometimes with less-than-satisfactory results (Catchpole, 1991). Induction of parturition should not be 115 min (n = 1) >5 h F (93%, n = 15) 8–12 Usually after stage 3, >12 h <48 h 188.8 (20–600) min F (98.1%) <12 Often after stage 3, <12 h Lactation (months) Birth to first fish (months) Calf behavior critical, <36 h 26.6 (18 to 36) 5.8 (2.5 to 27) 94.3 (45–240) min 15–24 3–6 228 min (n = 1) Length of stage-2 labor For animals with live calves For animals with dead calves Presentation Birth to stage 3 (h) Birth to nursing Normal Maximum Flukes appear, VD 8–12 2–3 48 h F (100%, n = 5) 6–20 <15 h See bottlenose dolphin Unknown 12 months See bottlenose dolphin 29–35 days 12 months MD, VD, DA, CT, DBT Estrous cycle length Gestation length Stage-1 labor signs (within 24 h of stage 2) Stage-2 labor signs Seasonal polyestrous July–Oct. Polyestrous All year, peak spring–fall 39–45 days 17 months See bottlenose dolphin See bottlenose dolphin 60–240 min White-Sided Dolphin (Lagenorhynchus c obliquidens) Polyestrous All year, peak spring–fall Killer Whale b (Orcinus orca) Reproductive pattern Period of activity Characteristic Bottlenose Dolphin a (Tursiops truncatus) TABLE 2 Reproductive Parameters of Cetaceans 18 Unknown F (n = 1) 5.8 7h ? 165 min (n = 1) Flukes appear Variable 14 months Arching Polyestrous All year False Killer Whale d (Pseudorca crassidens) f g (Continued) 24–36 10 (6–23) 33 h F (14% HF) 6.2–8.3 <18 h >2 days See bottlenose dolphin 392 (136–870) min Unknown 14.5 months Arching, VD Seasonal polyestrous Feb.–June Beluga (Delphinapterus e leucas) 0839_frame_C11 Page 213 Tuesday, May 22, 2001 11:09 AM Reproduction 213 2.5 yr 4 yr 5.8 yr 5.8–12 yr 10 yr 10–12 yr 2.9 yr 3.6 yr 4 yr 7–10 yr 8 yr 8–10 yr Killer Whale b (Orcinus orca) 6–8 yr Unknown 3 yr 3 yr 3–6 yr Unknown White-Sided Dolphin (Lagenorhynchus c obliquidens) 8–14 yr Unknown Unknown 5 yr 8–14 yr Unknown False Killer Whale d (Pseudorca crassidens) 8–9 yrs 3 yr 6 yr 6–7 yr Unknown Beluga (Delphinapterus e leucas) 214 Key: CI = calving interval; CT = contractions; DA = decreased appetite; DBT = decrease in basal temperature; F = flukes first; HF = head first; MD = milk discharge; VD = vaginal discharge. a From Andrews et al., 1997; Duffield et al., 2000; Joseph et al., 2000; Sweeney et al., 2000; Wells, 2000. b From Duffield et al., 1995; Robeck, 1996; McBain, Reidarson and Walsh, pers. comm. c From Sergeant et al., 1980, Dalton et al. 1995; Rogan et al., 1997. d From Comrie and Adams, 1938; Purves and Pilleri, 1978; Robeck et al., 1994b; Atkinson et al., 1999; Walsh, M. 2000; pers. comm. e From Brodie, 1971a; Braham, 1984; Dalton et al., 1994; 1996; Calle et al., 1996. f Both calves had to be manually extracted. g The authors have had one calf go 5 days without nursing; however, intensive management and intravenous IgG were required to keep the calf alive. CI, nonviable calf CI, viable calf Youngest mature female Sexual maturity: female Youngest mature male (sired a calf) Sexual maturity: male Characteristic Bottlenose Dolphin a (Tursiops truncatus) TABLE 2 Reproductive Parameters of Cetaceans (Continued) 0839_frame_C11 Page 214 Tuesday, May 22, 2001 11:09 AM CRC Handbook of Marine Mammal Medicine 0839_frame_C11 Page 215 Tuesday, May 22, 2001 11:09 AM Reproduction 215 attempted, therefore, unless the clinician feels it is the only recourse available. With a wide range in gestational lengths within species, and a usually speculative conception date, induction of “overdue” calves is never indicated. In the authors’ clinical experience, and in most cases, attempting to induce delivery of an apparently dead, in utero fetus is not indicated. If uterine infection is the cause of the dead fetus, the cow can be placed on antibiotics until she aborts the fetus. At that time, uterine, placental, and fetal cultures can be obtained to ensure effective treatment. In addition, the postpartum uterus is easily catheterized for local treatment. However, if the clinician feels induction is necessary, prostaglandin F2α has been used successfully to induce parturition in a beluga (Robeck, unpublished data). In this case, 40 mg PgF2α IM, BID, for 4 days caused progesterone to decrease to less than 1 ng/ml and stage-two labor to commence 7 days after the final injection. Another attempt at induction of parturition was of a midterm fetus in a bottlenose dolphin. The animal had a serious systemic infection, and based on a history of difficult pregnancies, it was believed that the fetus posed a risk to the cow’s health. Thus, multiple doses of prostaglandin F2α were administered until a response was observed. No response in circulating progesterone was observed until a single dose of 60 mg was used. The animal finally went into labor, but was unable to pass the calf and died during manual extraction. The reader must understand that the efficacy of these protocols is not well established, so sound clinical judgment should be employed. Early (60-day or less) unwanted pregnancies may be a situation where the chance of success at inducing abortion is greater than the risks. Because prostaglandin has been effective for CL lysis in pseudopregnant animals (Robeck et al., 2000), one can only speculate that application of these protocols in early gestation might be successful. Male Cetacean Reproduction Sexual Maturity Bottlenose Dolphin Postmortem assessment of sexual maturity in males is based on testis weight, diameter of the seminiferous tubules, presence of spermatozoa in the seminiferous tubules, and presence of seminal fluid in the epididymis (Perrin and Reilly, 1984). Observations of the gonads of bottlenose dolphins from Florida waters suggested that the age of sexual maturity for males was 10 to 13 years (Seargent et al., 1973; Perrin and Reilly, 1984; Cockcroft and Ross, 1990), but may begin as early as 9 years (Cockcroft and Ross, 1990). Males recovered on the east coast of South Africa were estimated to attain sexual maturity at 14.5 years of age (Cockcroft and Ross, 1990). Normal ejaculate was obtained from a 7-year-old captive T. t. aduncus (Brook, 1997). Captive animals are maintained under artificial social conditions that often allow younger animals opportunities to breed successfully. In the wild, the presence of a physically dominating male appears to exclude reproductively mature, but physically immature, males from successfully mating until they reach at least 20 years of age (Duffield and Wells, 1991). White-Sided Dolphin Sexual maturity occurs when males reach 2 to 4 m in length and 6 to 8 years of age (Sergeant et al., 1980; Rogan et al., 1997). Killer Whale Wild killer whales have been estimated to reach sexual maturity at 15 to 16 years of age and 6 to 7 m in length (Bigg, 1982; Christensen, 1984). By evaluating serum testosterone in biweekly samples, Robeck et al. (1995) concluded that male killer whales are fertile as early as 10 years of age. Younger animals, however, were not included in the study, so the earliest age when mature testosterone levels were produced could not be determined. Katsumata et al. (1999b) 0839_frame_C11 Page 216 Tuesday, May 22, 2001 11:09 AM 216 CRC Handbook of Marine Mammal Medicine used similar methods to estimate age of sexual maturity (based on testosterone) in one male at 12 years of age. Testosterone concentrations for this animal were below 1 ng/ml until reaching maturity at 12 years. Beluga Sexual maturity was estimated at 8 to 9 years of age in belugas (Brodie, 1971a). Seasonality Influences on male seasonality have yet to be investigated. Presence or absence of mature cycling females and other males, dominance hierarchies, size of the breeding population, and environmental or nutritional cues may all play some role in modification of seasonal levels of fertility. Bottlenose Dolphin Harrison and Ridgway (1971) found evidence for seasonal variation in testosterone levels of bottlenose dolphins, which were elevated to 14 to 24 ng/ml in September and October, as well as in April and May. Peak testosterone levels correlated well with peak breeding activity. Schroeder and Keller (1989) measured serum testosterone levels and sperm production in a 19-year-old bottlenose dolphin. Blood samples were collected twice monthly, and ejaculate was obtained twice weekly, over a 28-month period. Testosterone levels ranged from 1.1 to 54.1 ng/ml, with increasing levels from April to a peak in July in two consecutive seasons (Schroeder, 1990b). Peak sperm production and density, however, occurred during the breeding season, late August through October, when testosterone levels were lowest. Other seasonally reproductive species exhibit peak sperm production after serum testosterone peaks (Byers et al., 1983; Asher et al., 1987; Matsubayashi et al., 1991). This delay may represent the observed inhibitory effects that high testosterone can have on spermatogenesis (Matsumoto, 1990; Tom et al., 1991). Submaximal concentrations of testosterone may be required for optimum sperm recruitment. This is supported by the observation that normal spermatogenesis can occur in the presence of low intratesticular testosterone concentrations (Cunningham and Huckins, 1979). The delay may also represent the normal lag time from spermatocyte recruitment (which is maximally stimulated during peak testosterone) to sperm maturation in dolphins (Byers et al., 1983; Asher et al., 1987). Kirby (1990) summarized data of serum testosterone levels in bottlenose dolphins and reported that twice weekly samples from five male dolphins over periods of 6 to 24 months allowed classification of individuals as immature, pubescent, or sexually mature. Testosterone levels in mature animals (13 to 15 years of age) fluctuated between 2 and 5 ng/ml, rising above 10 ng/ml in the breeding season. Puberty in males has been estimated as the time when testosterone levels first rise from less than 1 ng/ml to 10 ng/ml. In contrast, Brook et al. (2000) and Brook (1997) determined that mature male T. t. aduncus can exhibit testosterone levels below 1.0 ng/ml, and found sonographic testicular echo texture a more reliable indicator of maturation. More significantly, they did not find changes in testicular echo pattern with season, and only a slightly seasonal pattern of testosterone production. Data from Brook et al. (1996; 2000) support the basic presumption that temperate animals would have less nutritional or environmental pressures for the development of seasonal breeding patterns. Thus, although numerous studies show increases in fecundity during predictable periods, dolphins remain fertile throughout the year, and can only be classified as facultatively seasonally polyestrous. Social patterns as opposed to environmental patterns (photoperiod, temperature) may have been the overwhelming pressure behind the development of the slightly seasonal trends. White-Sided Dolphin Research conducted in Japan indicated that at least one male Pacific white-sided dolphin had a well-defined breeding seasonal where sperm was only collected from May to September 0839_frame_C11 Page 217 Tuesday, May 22, 2001 11:09 AM Reproduction 217 (Yoshioka et al., 2000). The data included testosterone levels and sperm production and illus8 trated peak sperm concentrations in June of 19.3 × 10 /ml (mean from May to September = 8 3.8 × 10 /ml ± 0.65) to total azospermia from November through April. Although data are limited and different from bottlenose dolphins or seasonal breeders, peak testosterone occurred simultaneously with peak sperm production in May and June. From this information and assuming it holds true for the species as a whole, Pacific white-sided dolphins have a short seasonal reproductive period, synchronized with female cyclicity and regulated by an unknown physiological mechanisms. Killer Whale No significant seasonal changes in testosterone levels were observed in biweekly serum samples from five male killer whales 10 years old or older, although mean testosterone was significantly lower in October. Testosterone concentrations ranged from a low in October of 1.4 ng/ml to a high in April of 2.2 ng/ml, with peak levels occurring from March to July (Robeck et al., 1995). As would be expected, no significant seasonal patterns have been observed in sperm concentration voluntarily collected from a captive male killer whale (Robeck, unpubl. data). Thus, in agreement with observed calving periods and female cyclic activity, killer whale males appear to be fertile throughout the year, with possible peak fertility occurring in the spring and summer (Robeck et al., 1995; Katsumata et al., 1999b). False Killer Whale The only reproductive data from male false killer whales are testosterone levels that have no obvious seasonal trend (Robeck et al., 1994b). Beluga In 11 captive male belugas 3 to 21 years old, mean circulating testosterone concentrations were lowest in September (0.9 ng/ml) and highest 6 months later in March (4.95 ng/ml) (Dalton et al., 1994). Mean testosterone levels gradually rose throughout the fall and were elevated (>3.5 ng/ml) from January through April, then declined to the nadir in September (Calle et al., 2000). The relationship between circulating testosterone and spermatozoa production is unknown, although if belugas are physiologically similar to other seasonal mammals, sperm production should peak 1 to 2 months after peak testosterone. If this proves true, captive beluga males should have peak sperm production in May or June. Contraception and Control of Aggression Females To the best of the authors’ knowledge, the only method of contraception attempted in female Delphinidae involves the use of the oral progestin, altrenogest (Regu-Mate®, Hoechst Roussel Vet, Melbourne, Australia), which is a relatively safe contraceptive. Altrenogest has been used effectively in several different animals to regulate the estrous cycle without producing any detrimental side effects (e.g., reduced fertility or abnormal behavioral patterns). It was developed for use in the mare (Squires et al., 1979; 1983; Webel and Squires, 1982), but has since proved effective in the sow (Kraeling et al., 1981; Stevenson and Davis, 1982), the giraffe, and the okapi (Loskutoff, pers. comm.). Regu-Mate has been used long term without any clinical evidence of damaged fertility in a killer whale (Young and Huff, 1996), Pacific white-sided dolphin, and bottlenose dolphins (Asa, 2000; Dougherty et al., 2000). It must be administered daily (0.05 mg/kg) by mouth and should (although no data exist to confirm this) be effective after 2 days of administration. Progestins typically do not inhibit follicular growth; thus animals on Regu-Mate may still exhibit behavioral estrus. 0839_frame_C11 Page 218 Tuesday, May 22, 2001 11:09 AM 218 CRC Handbook of Marine Mammal Medicine Males Most efforts in marine mammal contraception have been primarily to control fertility and aggression in males. In male bottlenose dolphins, the GnRH agonist leuprolide acetate (Lupron®, Tap Pharmaceuticals, Inc., Deerfield, IL) has been successfully used to cause azospermia and is currently the only recommended form of contraception for male bottlenose dolphins (Briggs, 2000). Its mechanism of action has previously been discussed in the pinniped contraception section. If the primary objective for its administration is the reduction of circulating testosterone and related aggression, then the clinician should understand that initial serum testosterone concentration may double, and a measured increase in aggression may be observed. Serum testosterone should subside by day 10, and reach basal concentrations from day 14 to 20. The major disadvantages of its use include the need for monthly or bimonthly injections, and cost. A newer generation of GnRH agonist, Deslorelin (Peptech Ltd, North Ryde NSW, Australia) has shown good activity for as long as a year in carnivores (Jochle, pers. comm.). Its application for marine mammals is under investigation. Reproductive Abnormalities in Cetaceans Cystic follicles with varying degrees of luteinization were reported in the short-finned pilot whale (Marsh and Kasuya, 1984). Cystic follicles have been known to produce estrogens and progesterone depending on the degree of luteinization that occurs (Youngquist, 1986; Carriere et al., 1995). By using ultrasound, cystic follicles have since been visualized in bottlenose (Brook, unpubl. data; Jensen, unpubl. data; Robeck, unpubl. data) and Pacific white-sided dolphins (Robeck, unpubl. data). Luteinized cystic follicles may be partially responsible for pseudopregnancy that can occur in at least three delphinid species (Figure 4). Prolonged luteal phases in domestic animals have been associated with uterine infection or inflammation, early embryonic loss, and diestrus ovulations (Hinrichs, 1977). No clinical evidence exists to suggest an inflammatory process as causing prolonged or erratic luteal phases in the authors’ cases, although frequent and timely ultrasound examinations during ovarian activity may help explain these phenomena. FIGURE 4 Luteinized follicular cyst in a T. t. aduncus. (From F. Brook, unpubl. data.) 0839_frame_C11 Page 219 Tuesday, May 22, 2001 11:09 AM Reproduction 219 Dystocia with fetal death has occurred in cetaceans. In these situations, intervention was usually delayed until fetal death had occurred, so the only remaining concern was for the cow. Chapter 30, Intensive Care, reviews treatments that have been used for dystocia in various cetacean species. By far the most frequent pathology associated with reproduction is stillbirth. A recent survey revealed 8% abortion and 8.8% stillbirth rates in bottlenose dolphins from 1995 through 2000 (Joseph et al., 2000). Only a few females were responsible for a high percentage of stillbirths and neonatal deaths. These females should be identified, and environmental or physiological conditions that may be contributing to poor reproductive success should be changed. Furthermore, as Miller and Bossart (2000) point out in their review of reproductive-related pathology in bottlenose dolphins, the fetus and placenta should be submitted for culture and histology, in an effort to determine potential infectious causes for reproductive failures. Artificial Insemination Artificial insemination (AI) can be an important and powerful tool for genetic management of captive populations. However, it is usually most effective when applied to populations that are reproducing successfully. AI does not replace, but rather enhances, reproductive efficiency. Neither can it be viewed as the sole solution to infertility or other reproductive abnormalities (Lasley and Anderson, 1991; Wildt, 2000). AI has recently been successful in at least three individuals of two different species; killer whales and bottlenose dolphins (T. t. aduncus) (Robeck unpubl. data; Brook, unpubl. data). The development of AI in these two species is of no surprise as they are the two cetacean species in which most of the basic reproductive physiological research has occurred. Although these successes provide insight into what might be accomplished when these techniques become routine, many challenges remain before that vision can be realized. There are many techniques that must be improved or investigated (depending upon the species) before AI can be developed in other cetacean species. These techniques include semen collection, handling, and storage, ovulation detection, estrus synchronization, and insemination techniques. Perhaps the biggest obstacle to applying any successful AI techniques to cetaceans is the intense management that must occur. It is the job of investigators, not only to develop AI and related technologies, but also to use methodologies that can be applied to a wide range of husbandry situations with minimal additional equipment and training. Once this has been accomplished, assisted reproductive techniques will truly make an impact on captive cetacean management. Semen Collection and Storage Much has been written about early successes in freezing semen (Hill and Gilmartin, 1977; Fleming et al., 1981; Seager et al., 1981; Schroeder and Keller, 1989). This section reviews some of this work, but focuses on recent and current, often unpublished, work that has been performed since these earlier trials. The sensitivity of semen to cryopreservation and to various cryopreservation methods varies among species and individuals (Watson, 1979; Senger, 1986; Howard et al., 1991). Schroeder (1990b) found the post-thaw motility of semen frozen with lactose-based egg yolk extender to be greater than that of semen frozen in a fructose-based extender. His extender was composed of 11% lactose or fructose, 6% glycerol, and 20% egg yolk (1000 IU/ml Penicillin G and 1.25 mg/ml streptomycin sulfate were included in the extender). Few other studies with dolphin semen have attempted to evaluate and/or compare other major variables that can have important influences on the success of cryopreservation attempts. These variables include the effects of cryoprotectants and diluents on in vitro longevity at varying 0839_frame_C11 Page 220 Tuesday, May 22, 2001 11:09 AM 220 CRC Handbook of Marine Mammal Medicine TABLE 3 A Simple Method for Cryopreservation of Cetacean Semen 1. Warm Extender A (without glycerol) to 35°C in water bath in preparation for extension. 2. Once semen is in the laboratory, determine total motility (TM), percent progressive motility (PPM), and rate of forward motility (RFM). TM and PPM are determined by visual 6 estimation of extender-diluted semen (diluted to ∼25 × 10 ). RFM is judged on a scale of 0 to 5: 0 = no forward motility; 1 = little forward movement; 2 = movement and poor progression; 3 = slow forward progression; 4 = steady forward progression; 5 = rapid forward progression. 3. Slowly (over 5 min) dilute semen with equal volume of extender (1:1 dilution). Take care to mix semen gently while the extender is added. 4. Place diluted semen into a conical vial and store at 5°C for 2 hours. 5. Place a volume of Extender A that is equal in volume and initial temperature to the extended semen in the refrigerator at the same time. The temperature of these two vials (extender and extended semen) should remain the same. 6. Place a vial of Extender B (with 14% glycerol) into the refrigerator. The vial should contain enough glycerolated Extender B to extend the maximum amount of Extender A 1:1. 7. Determine the concentration of the raw semen. Based on this concentration, determine how much additional extender (Extender A) must be added to make the concentration 200 to 300 million sperm/ml. Slowly add the necessary amount of Extender fraction A to achieve the desired concentration. 8. Place extended semen and Extender B in an ice water bath for 30 min; then slowly add an equal volume of Extender B to Extender A (a ratio of 1:1). 9. Incubate the glycerolated semen at 3°C (ice-water bath) for 1 hour. 10. Fill straws with semen, minimizing exposure to the warm air, seal, and then place back into ice-water bath until all straws are filled. Float Styrofoam platform in liquid nitrogen. Dry and load straws on freezing rack and place on floating lid. Straws should be approximately 8 cm above liquid nitrogen. After 10 min, plunge into liquid nitrogen. temperatures, cooling rates, alternative freezing methods (straws vs. pellets), freezing curves, varying thaw temperatures, and effects of cryopreservation on acrosomal and/or plasma membrane integrity (Pursel and Park, 1985; Pontbriand et al., 1989; Bwanga, 1991; Pickett et al., 1992; Curry, 2000; Holt, 2000). Recently, Yoshioka et al. (2000) evaluated the effects of extender composition on post-thaw motility when Pacific white-sided and bottlenose dolphin sperm were frozen using the pelleting method described by Schroeder and Keller (1990). They found that the non-sugar-based extenders (egg yolk citrate) resulted in significant increases in post-thaw motility in both species. Durrant et al. (2000) provided the first descriptions of the effects of different freezing rates, incubation times with glycerol either prior to or after cooling, and freezing with or without cooling below room temperature. Their most effective freezing protocol was a medium freezing rate (12.8°C/min) with glycerol (4%) added prior to freezing after a 30 min cool to 4°C. They also illustrate the importance of comparing postfreezing motility score values to prefreezing values as a percentage. This eliminates the effect that differences in ejaculate quality between and within animals can have on post-thaw motility. Ongoing work with killer whale and Pacific white-sided dolphin semen indicates that they can be frozen successfully using straws. A simple method being developed for killer whales that has also been successful in Pacific white-sided dolphins is outlined in Table 3. It must be remembered when applying this method to a novel species that freezing curves and the most effective extender will vary with each species. Table 3 provides only a beginning. Postthaw motility as high as 70% in both species has been recorded (Robeck, unpubl. data). Ongoing research in Japan has shown high post-thaw motility when pelleting semen from 0839_frame_C11 Page 221 Tuesday, May 22, 2001 11:09 AM Reproduction 221 Pacific white-sided dolphins (Yoshioka et al., 2000). Pelleting dolphin semen has a high rate of success, and methods have been discussed above, with details provided in the previous references. No methods have been published, however, that detail freezing cetacean semen in straws. Epididymal spermatozoa remain structurally intact, retaining motility in the tail of the epididymis for hours after death (Hopkins et al., 1988; Marmar, 1998). Successful collection and storage of post-mortem epididymal spermatozoa has been accomplished in a few species (Howard et al., 1986; Hopkins et al., 1988; Goodrowe and Hay, 1993). The concentration and motility of the spermatozoa vary with species, health of the animal before its death (traumatic event vs. chronic debilitation), length of time after death it is collected, environmental conditions at death, and handling of gonads once collected. Cornell and Leibo (pers. comm.) were able to collect and cryopreserve epididymal spermatozoa with 10% motility 24 hours post-mortem from a male bottlenose dolphin. After 72 hours at 4°C in Test-Y (Graham et al., 1972) extender they cryopreserved (at −10°C/min) four straws of semen in Test-Y and 10% glycerol. After 10 min at −196°C, they thawed (250°C/min) and evaluated the semen. The thawed semen had a 3 to 5% post-thaw motility. The ability to collect spermatozoa from wild or captive animals that die incidentally could provide managers another method to store and judiciously to infuse genetic material into captive populations (Wildt, 1989; Wildt et al., 1997; Kraemer, 2000). Manipulation and Control of Ovulation Populations of bottlenose dolphins tend to exhibit seasonally bimodal peaks of reproductive activity or calf production. However, individual animals within these populations can be polyestrus throughout the year, anestrous, or pseudopregnant. Attempts to maximize the reproductive potential of these populations are difficult when potential breeding females are experiencing anestrus or pseudopregnancy. In addition, unpredictable estrous patterns reduce reproductive managers’ control of potential breeding events. Two basic methods of controlling ovulation in any mammalian species include induction of ovulation and estrus synchronization. Induction of Ovulation Multiple attempts to induce ovulation in dolphins with exogenous gonadotropins have been performed with wide variations in response (Sawyer-Steffan et al., 1983; Schroeder and Keller, 1990). Because success was defined as elevated serum progesterone concentrations posttreatment, the authors were unable to determine if elevated progesterone reflected normal postovulatory luteinization. Similar doses of exogenous gonadotropins in other species commonly result in multiple ovulation, follicular luteinization, or other ovarian abnormalities (Hansel, 1985; Sreenan, 1988). In an effort to determine whether induced ovulation was normal, Schroeder and Keller (1990) allowed a reproductively successful male dolphin access to five exogenously induced females. Although breeding activity was observed, none of the females became pregnant. Two animals in this group were diagnosed as having persistent CLs. The lack of postinduction pregnancy after natural insemination and ovarian abnormalities (persistent CLs) provides strong evidence that these protocols were not effective. Robeck et al. (1998) used transabdominal ultrasonography to evaluate the response of bottlenose dolphins to ovulation-induction protocols. The results indicated that (1) bottlenose dolphins can be sensitive to exogenous gonadotropins, as multiple follicular recruitment of follicles occurred; (2) no physical evidence of ovulation was detected, but if ovulation were to occur, there was a good potential for multiple ovulations; and (3) until further ultrasonographic studies can be conducted to evaluate the effects of titrated doses of exogenous gonadotropins, induction protocols should be considered unsuitable for AI procedures in bottlenose dolphins. 0839_frame_C11 Page 222 Tuesday, May 22, 2001 11:09 AM 222 CRC Handbook of Marine Mammal Medicine Robeck et al. (2000) attempted ovulation induction with three additional animals. Prior to exogenous gonadotropin administration, however, ultrasound was used to classify the dolphins as either anestrus or cycling. Cycling animals had follicles >5 mm (Brook, 1997). All three animals were placed on altrenogest at 1.5 ml/50 kg (110 lb), PO (oral), SID, for 16 days. After a single dose of 1500 IU of PG600® (Intervet America, Inc., Millsboro, DE) IM and 17.6 mg FSH intramuscularly (IM) on day 14, animal 2 and 3 responded with increased cortical activity, or antral follicle activity. After a second dose of 1800 IU PG600 was administered on day 22, animal 2 exhibited no ovarian change, and animal 3 had grown three follicles >25 mm. Animal 3 was administered two doses of 100 µg of GnRH (Cystorelin®, Merial Ltd, Harlow, Essex, U.K.) 10 days apart. GnRH administration did not stimulate ovulation despite the presence of follicles similar in size to preovulatory follicles previously characterized for T. t. aduncus (Brook, 1997). This may indicate that either the follicle was not preovulatory and/or that the dose of GnRH was ineffective. Administration of GnRH to animals that have nonpreovulatory follicles usually results in luteinization (Hennington et al., 1982; Valle et al., 1986). However, with this animal neither luteinization nor ovulation occurred, which probably indicated an insufficient dose of GnRH. These attempts at inducing ovulation in dolphins indicate that further investigations are needed to evaluate the differential sensitivity of the dolphin hypothalamic–pituitary–ovarian (HPO) axis to exogenous gonadotropins during anestrus or estrus, and at different stages of follicular growth. Synchronization of Ovulation Attempts at synchronizing ovulation are most effective when used with normal, cycling animals. The ovarian response to exogenous hormones is variable both among and within species. In domestic species, the most effective methodologies use progestagens (Davis et al., 1979; Squires et al., 1979; Wright and Malmo, 1992) with or without estrogens (estradiol valerate) (Heersche et al., 1979; Odde, 1990) and/or prostaglandin F2α (Bunch et al., 1977; King et al., 1982; Odde, 1990). In some domestic species synthetic or natural prostaglandin F2α are commonly used in ovulatory synchronization protocols because of their luteolytic effect on receptive CL (between day 5 and 15 of the estrous cycle in cattle) (King et al., 1982). The many methodologies employed for estrus synchronization in various species are beyond the scope of this chapter (for review, see Wright, 1981; Odde, 1990; Wright and Malmo, 1992). Recently, the oral progestin altrenogest (Regu-Mate) has been evaluated as an estrus (ovulatory) synchronization tool in killer whales and bottlenose dolphins (Robeck, 2000; Robeck et al., 2000). In these studies, three dolphins and two killer whales were placed on Regu-Mate for as long as 31 days. Both of the killer whales and one of the dolphins were cycling prior to administration of the hormone. The time from progesterone withdrawal to estrus in the dolphins and killer whales was a mean 17.6 and 21.3 days, respectively. In both dolphins and killer whales, Regu-Mate appeared to cause a delay or suppression of ovarian activity after the hormone was withdrawn. The mean length of this suppression appeared to be similar to the length of the animals’ normal luteal phase. After this interval was reached, folliculogenesis and ovulation often occurred. All three dolphins placed on Regu-Mate returned to estrus within 1 week of each other. Although this interval is prolonged and too variable compared with traditional estrus-synchronization methods, it was effective for coordinating ovulation in a group of females during intensive AI trials. Receptivity of the cetacean CL to luteolytic doses of prostaglandin F2α is currently under investigation. Thus far, limited data indicate that PGF2α can be effective at disrupting normal CL function (Robeck et al., 2000). Three sonographically diagnosed nonpregnant animals with persistently elevated progesterone were administered an initial dose of 25 mg Lutalyse® (Pharmacia 0839_frame_C11 Page 223 Tuesday, May 22, 2001 11:09 AM Reproduction 223 & Upjohn Co., Peapack, NJ) SID or BID (twice a day). Serum progesterone was determined 1 week after the initial dose. Two animals responded after the initial dose; the other one had to be given two additional doses of 25 mg Lutalyse 6 hours apart. Two of the animals went on to cycle normally, and have since become pregnant. Side effects of PgF2α administration generally consisted of apparent abdominal discomfort, nausea, and, on two occasions, inappetence for the remainder of the day. All obvious abdominal discomfort was gone within 1 hour, and all animals returned to normal behavior by the following day. The data suggest that nonpregnant animals with a history of elevated progesterone (>3 ng/ml) should be considered candidates for prostaglandin treatment. The results also demonstrate that these hormones can be administered safely. Further research is required to determine when and if a CL of diestrus is sensitive to exogenous prostaglandin F2α and what effect it will have on subsequent cycles. Insemination Techniques Schroeder and Keller (1990) attempted to artificially inseminate five bottlenose dolphins in conjunction with the ovulation-induction protocol described above. For the procedure, freshly collected semen was placed external to the cervix in the spermathecal recess of the female using a flexible fiber optic laryngoscope (Schroeder, 1985; 1990; Schroeder and Keller, 1990). Based on serum progesterone levels, two of the five artificially inseminated animals were diagnosed as pregnant. Both pregnancies were believed to have spontaneously terminated in the first trimester. As was mentioned above, recent evidence using ultrasound indicates that when dolphins respond to exogenous gonadotropins, they do so with multiple follicular development. This increases the likelihood of multiple ovulation. Obviously, multiple ovulation, and potentially multiple embryos, would not be advantageous. Thus, without further research, ovulationinduction trials should be considered inappropriate for artificial or natural breeding. Recently, AI using cooled, transported semen and fresh, extended semen has been successful in the killer whale and the bottlenose dolphin, respectively (Robeck, unpubl. data; Brook, unpubl. data) (Figure 5). Each method used different indicators for determining when insemination should occur in relationship to ovulation. With the killer whale, urinary endocrine data were used to determine when the preovulatory estrogen surge had occurred. How this hormonal event relates to ovulation has yet to be determined, but ongoing ultrasonographic examinations should help determine this association. With bottlenose dolphins, ultrasonographic follicular evaluation was used to estimate the time of ovulation (Figure 6). Both FIGURE 5 A 72-day-old T. t. aduncus fetus that was conceived through artificial insemination using fresh extended semen from a male located on site. The white arrows represent the CL of pregnancy. ac = amniotic cavity. (From F. Brook et al., unpubl. data.) 0839_frame_C11 Page 224 Tuesday, May 22, 2001 11:09 AM 224 CRC Handbook of Marine Mammal Medicine FIGURE 6 Pre- and postovulation in a female T. t. aduncus. The sonogram on the left shows a 2-cm preovulatory follicle. The sonogram on the right was taken 12 hours after the one on the left. Fluid can still be seen in the recently ovulated follicle (black arrow). White arrows indicate ovarian dimensions. (From F. Brook, unpubl. data.) techniques require intensive monitoring and have their limitations. Endocrine data require that (1) the animal be trained for urine collection; (2) an assay system be validated for the species in question; (3) the assay be rapid and provide results twice daily; and (4) the animal should have extensive hormonal profiling prior to inseminations. This profiling will help the manager predict when the animal will return to estrus and how long, generally, the animal will be in estrus before the estrogen surge occurs. Similar intensive animal monitoring is required when relying on ultrasonography. For this procedure to be effective (1) animals must be trained for regular voluntary sonogram exams or be restrained for the procedure; (2) there must be an ultrasound unit of minimal quality on site; and (3) the normal range of preovulatory follicular size for each animal should be determined. Future Applications Kraemer (2000) and Wildt (2000) give good descriptions of current reproductive biotechnology and its realistic applications in exotic species. The short-term applications of biotechnology revolve around AI. The use of these technologies takes on more significance for long-term genetic management as the procedures become more refined, and can be applied in many different situations. Refinements of AI sophistication include successful insemination with fresh extended, cooled, transported, frozen, post-mortem epididymal, and sexed semen. The only successful method of AI in bottlenose dolphins relied on the most basic form. This involves collecting semen from the male on site (the male is usually in a different holding pool than the female), extending the semen to help protect and provide nutrients to maintain viability, and inseminating within a few hours of collection. Although this method has limited application for marine mammals as a whole, it enables park managers to house mature males and females separately. This type of social arrangement is most often observed in wild populations, and may have other benefits for population management (Wells, 2000). The second level of improvement for AI technologies is the use of cooled transported semen. This method, which was recently validated in killer whales, opens the door for 0839_frame_C11 Page 225 Tuesday, May 22, 2001 11:09 AM Reproduction 225 meaningful application of AI to the marine mammal community (Robeck, unpubl. data). This method is so effective that the equine AI industry has been built around its use (Samper, 1997). This method involves collection, extension, and shipping of semen to an off-site location. Most shipping systems are designed to cool the semen in transit to provide longer periods of viability. Once on location, the cooled semen is deposited into the female that is approaching ovulation. For this system to work, managers must be able to predict when the female will ovulate within a couple of days, be able to collect semen routinely from the donor male, and develop extenders that will allow semen to remain viable during, and for at least 3 days after, the cooling process. The next area of progress in AI technology is in the successful use of cryopreserved semen. Developing this methodology requires tremendous effort to develop a system that will allow managers to cryopreserve semen with minimal loss in its fertility. Although most research with cryopreserved semen uses post-thaw motility and membrane integrity to evaluate success of the procedure, the ultimate and often only meaningful test is to determine its fertility. Once it can be demonstrated that frozen semen can be used to inseminate a marine mammal successfully, the door will be opened for long-term genetic management. This technique will allow shipment of semen across international borders, thus effectively opening up the captive gene pool to worldwide contributions. It will also allow long-term storage of valuable genetic material that can be selectively reintroduced into the gene pool generations after the donor is deceased. It may also allow the use of cryopreserved semen collected postmortem from the epididymis of stranded delphinids. Harvesting of genetic material from animals that would otherwise be lost to wild populations would greatly increase the genetic diversity of captive populations without the need for additional wild live captures. This technique has already been used successfully in domestic and exotic terrestrial animals and has been applied for in vitro fertilization techniques in minke whales (Balaenoptera acutorostrata) (Fukui et al., 1997a,b). Finally, use of sex-determined semen (sorted by chromosomal content) for successful AI in marine mammals would revolutionize animal management procedures. The ability to control sex ratios would allow optimum utilization of the limited resources available to managed species. Although in its infancy, this technique uses flow cytometry to sort semen based on nuclear content or X- vs. Y-bearing spermatozoa. The current sorting rates are around 900 live sperm/second; thus, the technique produces far fewer sperm than are required for transcervical inseminations. However, the technique has been used together with laparoscopic insemination in cattle to produce conception rates of 30% with liquid semen and 51% with frozen semen, and producing 97% females (Seidel et al., 1999). This technique appears to be a long way from being implemented in marine mammals; however, the recent abilities to perform laproscopic procedures (see Chapter 27, Endoscopy) and to monitor ovarian activity place this procedure within reach, although many challenges still remain before the sorting system is validated for delphinid semen. Acknowledgments The authors thank Denise Greig for editorial assistance in preparing this chapter for publication. In addition, the authors acknowledge all those, far too numerous to mention individually, whose work has contributed to their increasing knowledge of reproduction in marine mammals and, in particular, those colleagues who have directly supported the authors’ efforts over the years. 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Squires, E.L., Heesemann, S.K., Webel, S.K., Shideler, K., and Voss, J.L., 1983, Relationship of altrenogist to ovarian activity, hormone concentrations and fertility of mares, J. Anim. Sci., 56: 901–909. Sreenan, J.M., 1988, Embryo transfer: Its uses and recent developments, Vet. Rec., 122: 624–629. Stevenson, J.S., and Davis, D.L., 1982, Estrus synchronization and fertility in gilts after 14- or 18-day feeding of altrenogest beginning at estrus or diestrus, J. Anim. Sci., 55: 119–123. Stone, L.R., Johnson, R.L., Sweeney, J.C., and Lewis, M.L., 1999, Fetal ultrasonography in dolphins with emphasis on gestational aging, in Zoo and Wild Animal Medicine: Current Therapy 4, Fowler, M.E., and Miller, E.R. (Eds.), W.B. Saunders, Philadelphia, 501–506. Sundaram, K., Connell, K.G., Bardin, C.W., Samojlik, E., and Schally, A.V., 1982, Inhibition of pituitarytesticular function with [D-Trp] luteinizing hormone-releasing hormone in rhesus monkeys, Endocrinology, 110: 1308–1314. Sweeney, J.C., Krames, B., Krames, J., and Stone, R., 2000, Stages of parturition, normal early calf development, and food energy requirements of the cow, in Report from the Bottlenose Dolphin Breeding Workshop, Duffield, D.A., and Robeck, T.R. (Eds.), American Zoological Association Marine Mammal Taxon Advisory Group, Silver Spring, MD, 289–296. Taverne, M.A.M., 1991, Applications of two-dimensional ultrasound in animal reproduction, Wien. Tierärztl. Monatsschr., 78: 341–345. Temte, J.L., 1985, Photoperiod and delayed implantation in the northern fur seal (Callorhinus ursinus), J. Reprod. Fertil., 73: 127–131. Temte, J.L., 1991, Precise birth timing in captive harbor seals (Phoca vitulina) and California sea lions (Zalophus californianus), Mar. Mammal Sci., 7: 145–156. Terasawa, F., Yokoyama, Y., and Kitamure, M., 1999, Rectal temperature before and after parturition in bottlenose dolphins, Zoo Biol., 18: 153–156. Theodorou, J., and Atkinson, S., 1998, Monitoring total androgen concentrations in saliva from captive Hawaiian monk seals (Monachus schauinslandi), Mar. Mammal Sci., 14: 304–310. Tom, L., Bhasin, S., Salameh, W., Peterson, M., Steiner, B., and Swerdloff, R.S., 1991, Male contraception: Combined GnRH antagonist and testosterone enanthate, Clin. Res., 39: 91A. Valle, E.R., Cruz, L.C., Cmarik, G.F., Ott, R.S., Peterson, L.A., and Kesler, D.J., 1986, The effect of GnRH and its method of administration on ovulation response, corpus luteum function and fertility of beef heifers synchronized with norgestomet and PGF 2α, J. Anim. Sci., 63: 132. Walker, L.A., Cornell, L., Dahl, K.D., Czekala, N.M., Dargen, C.M., Joseph, B.E., Hsueh, A.J.W., and Lasley, B.L., 1988, Urinary concentrations of ovarian steroid hormone metabolites and bioactive follicle-stimulating hormone in killer whales (Orcinus orca) during ovarian cycles and pregnancy, Biol. Reprod., 39: 1013–1020. 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Youngquist, R.S., 1986, Cystic follicular degeneration in the cow, in Current Therapy in Theriogenology, Morrow, D.A. (Ed.), W.B. Saunders, Philadelphia, 243–246. Youngquist, R.S., 1997, Current Therapy in Large Animal Theriogenology, W.B. Saunders, Philadelphia. 0839_frame_C12.fm Page 237 Tuesday, April 9, 2002 1:36 PM 12 Immunology Donald P. King, Brian M. Aldridge, Suzanne Kennedy-Stoskopf, and Jeffrey L. Stott Introduction The immune system is primarily a series of defense mechanisms that function to protect the body against the potential harmful effects of foreign microorganisms. In recent years, there have been rapid advances in the field of immunology. With these advances have come new methods for preventing and treating infectious disease. Although marine mammal immunology is a relatively recent field of scientific endeavor, it is already possible to perform reliable and pertinent studies to address specific aspects of health and disease in these species. Immune system monitoring and serological diagnostic assays have clear roles in the management of disease in individual marine mammals. In addition to clinical assessment, there are a number of other reasons to consider immunological parameters in marine mammals. The concept that the status and well-being of the aquatic environment are reflected in the immune systems of marine mammals has gained considerable acceptance within the last decade. Furthermore, there has also been a strong interest in genetic markers of immunological diversity, since many believe that the successful management of endangered populations may require assessment of genetic diversity. This chapter reviews the most recent advances in marine mammal immunology and immunodiagnostics. It concludes with what might be considered a typical approach to defining immunological dysfunction in a marine mammal. To date, clinical and experimental evidence support the notion that the immune systems of marine mammals share all the major identifiable components that have been described in detail for key terrestrial species, such as humans and rodents. However, it is likely that marine mammals possess some unique immunological features that reflect the adaptations required for survival and function in the aquatic environment. These adaptions may, in turn, reflect the spectrum of microbial pathogens that inhabit marine ecosystems, or may comprise homeostatic mechanisms that maintain immune function despite physiological extremes, such as hypoxia, hyperbaric pressures, or cold temperatures, that have been shown to be immunosuppressive in other species (Shinomiya, 1994; Knowles et al., 1996; Shepard and Shek, 1998; Brenner et al., 1999). Until more-detailed studies are performed, and immunological adaptations to the marine environment are documented, it is useful to refer to a generalized model of a mammalian immune system to understand how marine mammals mount a protective response to invading pathogens. 0-8493-0839-9/01/$0.00+$1.50 © 2001 by CRC Press LLC 237 0839_frame_C12.fm Page 238 Tuesday, May 22, 2001 11:09 AM 238 CRC Handbook of Marine Mammal Medicine Overview of the Immune System The immune system has been classically divided into the innate and adaptive (or acquired) immune responses. Whereas the innate immune system is static relative to the quantity and quality of a response, the adaptive response gains quantity and quality (immunological memory) upon repeated exposure to the pathogen. Although these divisions are descriptively useful, it is important to realize that successful host defense responses rely on close orchestration between these two arms. To help the reader fully appreciate the progression of the immunological processes involved in pathogen clearance and host protection, a general inflammatory response will be described first, followed by the sequence of events that would occur following exposure to a pathogen. Innate Immunity and the Inflammatory Response The normal, healthy, mammalian host is exposed to a vast number of potentially pathogenic microorganisms each day. Since clinical infectious disease is relatively uncommon in normal individuals, defense against these organisms must be a constant process. The majority of microorganisms are repelled by innate host defenses that include nonimmunological anatomical and physiological barriers (e.g., mucociliary blanket), antimicrobial factors (e.g., complement, lysozyme, lactoferrin, defensins, and reactive oxygen and nitrogen intermediates), and immunological effector cells (e.g., neutrophils, eosinophils, macrophages, and natural killer, or NK, cells). Many of these immune mechanisms act immediately following microbial invasion, particularly against those pathogens possessing identifiable structures such as lipopolysaccharide (LPS) present on Gram-negative bacteria, or double-stranded viral RNA. To be effective, the mammalian immune system possesses molecules capable of recognizing and neutralizing an enormous repertoire of infectious agents. Recognition is one of the key steps in the stimulation of early-induced immune responses that function to keep the infection under control, while the antigen-specific cells of the adaptive immune response are recruited and activated. At many portals for potential infection (e.g., mucosal surfaces), there are a number of locally produced antimicrobial peptides and cells that are sufficient to repel or eliminate a small pathogen load. However, in the event of an infection that can overwhelm these in situ defense mechanisms, an inflammatory process is initiated, aimed at destroying and eliminating the offending pathogen and at healing damaged tissues. Occasionally, the nature or extent of the localized inflammation may be severe enough to evoke a number of systemic inflammatory processes termed the acute-phase response, which serves to produce inflammatory mediators and recruit more inflammatory cells to the site of infection. The most important effector cells in these early phases of the immune response are phagocytes (tissue macrophages and migrating neutrophils). These not only trap, engulf, and destroy microbes, but also secrete cytokines that initiate the systemic acute-phase response and recruit additional leukocytes to magnify the local inflammatory response. The recruitment of cells involves chemotaxis and an increase in vascular endothelial cell and immune cell-adhesion molecule expression. These factors, in conjunction with an increased local blood flow and increased vascular permeability, lead to an accumulation of leukocytes, immunoglobulins, and other blood proteins in the infected tissue. Adaptive Immune Response If a pathogen evades or overwhelms the innate defense mechanisms of the host, causing the foreign antigen to persist beyond the first several days of infection, an adaptive immune response is initiated. In contrast to the innate immune responses, the adaptive response produces effector cells (B- and T-lymphocytes) and molecules (immunoglobulins), which are highly specific to the 0839_frame_C12.fm Page 239 Tuesday, May 22, 2001 11:09 AM Immunology 239 antigens of the invading microbe. In addition, the antigen-specific lymphocytes of the adaptive immune response are capable of swift clonal expansion and of a more rapid and effective immune response on subsequent exposures to the pathogen (immunological memory). The trigger for the adaptive immune response, the activation and proliferation of lymphocytes, takes place in organized lymphoid tissues. There are three major portals by which an invading pathogen can enter the host, namely, via a mucosal surface (respiratory tract, gastrointestinal tract), through the skin, or by direct inoculation into the bloodstream. At each of these portals are organized lymphoid tissues (mucosal-associated lymphoid tissue, regional lymph nodes, and spleen, respectively), which provide the organized microenvironment in which the intricate events of the adaptive immune response are closely coordinated. Microscopic investigations of the marine mammal immune system reveal that the morphology of the lymphoid organs is similar to terrestrial mammals, but with a few unique attributes (Romano et al., 1993; 1994; Cowan and Smith, 1995; 1999; Cowan, 1999; Smith et al., 1999). At these lymphoid sites, pathogens are trapped and engulfed by phagocytic cells. Some of the lymphoid cells are specialized for processing microbial antigens into small peptides, and presenting these peptides in association with highly polymorphic glycoproteins, called major histocompatibility (MHC) proteins, on their cell surface. The ability of the immune system to recognize and respond to such a vast array of foreign proteins is determined to a large degree by the number and structural diversity of the MHC molecules present in an individual. The polymorphic nature of these MHC proteins ensures maintenance of the host’s immunological vigor by minimizing the ability of a pathogen to avoid presentation by selective mutation. It is speculated that genetically restricted species, such as those that have been subjected to a “population bottleneck,” will lack MHC diversity. This is an area of increasing interest among marine mammal researchers (Gyllensten et al., 1990; Slade, 1992; Murray and White, 1998; Hoelzel et al., 1999; Zhong et al., 1999). The immunogenic peptides of the invading pathogens bound to the cell-surface MHC molecules are recognized by the highly specific receptors of T-helper lymphocytes, which by specific patterns of cytokine secretion stimulate either B lymphocyte expansion and antibody production (humoral immunity) or activation of macrophages (delayed-type hypersensitivity), and expansion and activation of cytotoxic T lymphocytes. The subsets of lymphocytes with these polarized patterns of cytokine production are T-helper1 and T-helper2 cells, respectively. Cytokines The initiation, maintenance, and amplification of the immune response are regulated by soluble mediators called cytokines. Cytokines are the soluble messengers of the immune system and have the capacity to regulate many different cells in an autocrine, paracrine, and endocrine fashion. The predominant proinflammatory cytokines are interleukin-6 (IL-6), IL-1, and tumor necrosis factor alpha (TNF-α). These cytokines have a number of systemic effects, including body temperature elevation (fever), neutrophil mobilization, and stimulation of acute-phase protein production in the liver. Cytokines can also be immune effectors. Interferon-α (IFN-α) and INF-β are produced by a number of different cell-types following viral infection. They interfere with viral replication and can therefore limit the spread of viruses to uninfected cells. Additional cytokines such as IL-2, IL-4, IL-5, IL-10, IL-12, IL-15, and IFN-γ are pivotal in directing the development of both humoral and cellular immune responses. By using existing biological assays, it is possible to assay cytokine-like activity in mitogen-stimulated cultures (King et al., 1993b; 1995). Furthermore, cytokine transcripts from a number of marine mammals have been recently cloned and the DNA sequence determined (Table 1). The identification of these sequences will facilitate the development of molecular techniques for examining 0839_frame_C12.fm Page 240 Tuesday, May 22, 2001 11:09 AM 240 CRC Handbook of Marine Mammal Medicine TABLE 1 Published Marine Mammal Cytokines Cytokine Species a cDNA Clone (base pairs) GSDB Accession Number Reference IL-1α Bottlenose dolphin (Tursiops truncatus) 906 AB028215 Inoue et al., 1999c IL-1β Bottlenose dolphin (T. truncatus) 818 AB028216 Inoue et al., 1999c IL-2 Killer whale (Orcinus orca) Beluga (Delphinapterus leucas) Northern elephant seal (Mirounga angustirostris) Gray seal (Halichoerus grypus) Manatee (Trichechus manatus latirostris) 455 AF009570 Ness et al., 1998 465 AF072870 658 U79187 St-Laurent et al., 1999 Shoda et al., 1998 468 AF072871 450 U09420 St-Laurent et al., 1999 Cashman et al., 1996 IL-4 Bottlenose dolphin (T. truncatus) 528 AB020732 IL-6 Killer whale (O. orca) Beluga (D. leucas) Harbor seal (Phoca vitulina) Southern sea otter (Enhydra lutris nereis) 670 L46803 King et al., 1996 627 AF076643 682 L46802 St-Laurent et al., 1999 King et al., 1996 676 L46804 King et al., 1996 Herman, unpubl. data IL-10 Killer whale (O. orca) 548 U93260 IFN-γ Killer whale (O. orca) Bottlenose dolphin (T. truncatus) 144 — 548 AB022044 a Inoue et al., 1999b King, unpubl. data Inoue et al., 1999a Genome Sequence Database. cytokine gene expression during infectious disease. These techniques have great potential for improving the ability to measure immune cell activity in marine mammals. Immunodiagnostics Inflammation Monitoring the changes associated with inflammation is a key component of diagnostic tests that establish the overall health of an animal. Unfortunately, in part because of the presence of blubber, the cardinal signs classically used to define inflammation in humans and domestic species can be difficult to recognize in some marine mammals. However, experimental and clinical data from human and veterinary medicine demonstrate that changes in the concentrations of specific proteins (collectively referred to as acute-phase proteins) can aid the detection and quantification of inflammation. In human medicine, the acute-phase response can be assessed by measuring the erythrocyte sedimentation rate (ESR) of blood collected into anticoagulant. This method is, in part, an 0839_frame_C12.fm Page 241 Tuesday, May 22, 2001 11:09 AM Immunology 241 indirect measurement of fibrinogen and has been successfully adapted for use in a variety of cetacean species. Unfortunately, as a result of variable serum lipid content, this method is unreliable for detecting inflammation in pinnipeds. Determination of serum iron concentration can also be used as an indirect measure of inflammation in cetaceans, but has not been evaluated in pinnipeds (see Chapter 19, Clinical Pathology). Although these methods are widely used in marine mammal medicine, current efforts are directed at identifying inflammation at earlier stages. The characteristics of ideal markers are that they exhibit dramatic changes in serum concentrations early in a systemic inflammatory response and that they are not influenced by other physiological changes, such as malnutrition or handling stresses. Recent approaches in this field employ the measurement of the specific protein mediators of the acute-phase response such as IL-6. This cytokine is produced by macrophages at the site of tissue damage and injury. Unlike most other cytokines that possess only local activity, IL-6 enters the systemic circulation and is a key player in the induction of acute-phase protein synthesis in the liver. Recent studies using a murine bioassay system have suggested that IL-6 may prove to be a valuable indicator of inflammation in a number of marine mammal species (King et al., 1993b). Of the many acute-phase proteins, C-reactive protein (CRP) and serum amyloid A have been targeted for use in clinical medicine. The potential utility of CRP has been highlighted in a recent study in harbor seals (Phoca vitulina) that measured increases of CRP in excess of 100-fold associated with clinical signs of inflammatory disease, compared with apparently healthy animals (Funke et al., 1997). Cellular Immunity Classical differential white blood cell counts can morphologically distinguish and enumerate major leukocyte subpopulations into lymphocytes, monocytes, eosinophils, and neutrophils (see Chapter 19, Clinical Pathology). These cells, although ultimately derived from the same progenitor bone marrow stem cell population, make different functional contributions to the immune system. There is a wide range of immunological techniques that can be used to evaluate the cellular immune system. Broadly speaking, these assays can be divided into those that measure the phenotypic qualities of leukocytes (lymphocyte subpopulations and the cellsurface density of adhesion proteins) and those that assess functional aspects of the cells. Recently, a major use of these assays in marine mammals has been to examine immunological dysfunction arising from the presence of environmental pollutants (de Swart et al., 1995; 1996; Lahvis et al., 1995; Ross et al., 1995a,b). Furthermore, since the immune system is acutely sensitive, these methods have the potential to measure the influence of many internal and external stresses that affect marine mammals (see Chapter 13, Stress). The peripheral blood represents the most convenient sampling window for the assessment of the cellular immune system. In many circumstances, cells can also be isolated from tissues, such as spleen and lymph nodes collected during post-mortem examination, and can be subsequently used in phenotypic assays and/or in vitro functional testing. Tissues and blood collected into anticoagulant should be transported to the laboratory and used as soon as possible after collection. A major requirement of such assays is the availability of purified and viable mononuclear leukocytes. Classically, cells should be purified and either cryopreserved and stored in liquid nitrogen or placed in culture within 24 hours of sample acquisition. The recent introduction of CPT tubes (Becton Dickinson, Franklin Lakes, NJ) has provided researchers with a novel method of obtaining mononuclear cells from peripheral blood without the need to use density-gradient techniques. Centrifugation of the CPT vacutainer tubes at 1800 g results in the mononuclear cells being permanently separated from granulocytes and red blood cells. The length of centrifugation must be determined for each species to optimize 0839_frame_C12.fm Page 242 Tuesday, May 22, 2001 11:09 AM 242 CRC Handbook of Marine Mammal Medicine cell yield and purity (i.e., horse blood is typically centrifuged for 7 min, cow blood for 30 min, whereas most cetacean and pinniped blood is centrifuged for 18 to 20 min). Immunophenotyping refers to methods that delineate multiple leukocyte subpopulations in the blood. Analysis of the density of cell-surface adhesion and activation antigens on these leukocyte populations has also become possible. Alone or in combination, these techniques are finding increasing application in identifying subtle immunological perturbations caused by infectious agents and other tissue insults. This analysis is performed using flow cytometry and requires characterized markers, usually monoclonal antibodies that are specific for unique determinants expressed on the various leukocyte populations (also commonly referred to as CD, cluster of differentiation or leukocyte differentiation antigens). Unfortunately, only a limited number of the anti-CD markers available from academic and commercial sources crossreact with marine mammal white blood cells (Romano et al., 1992; De Guise et al., 1997b). As might be expected, the ability of these reagents to cross-react usually parallels phylogenetic relationships. Consequently, it is more likely that antibovine CD reagents will work for cetacean blood and that anticanine/feline reagents will work for pinniped samples. To perform a full complement of analyses, a number of species-specific monoclonal antibodies have been developed and are currently being characterized for pinnipeds and cetaceans (De Guise et al., 1998). Future development in this area will likely see the extension of this panel of reagents. Since these reagents are usually produced in a serendipitous manner using immunizations with mixed cell populations, cloning and expression studies such as those performed for beluga (Delphinapterus leucas) CD4 (Romano et al., 1999) may be required to allow the development of some antibodies against individual cell determinants. Functional Immune Testing In Vitro The capacity of lymphocytes to proliferate in response to antigen is central to the success of the adaptive immune system. This mechanism allows small numbers of antigen-specific lymphocytes to be rapidly increased to counteract an invading pathogen. In vitro blastogenesis assays mimic this response and measure the ability of isolated blood cells to proliferate in response to broadspectrum mitogenic stimulation. These assays have been successfully adapted and optimized for use with marine mammal samples (Mumford et al., 1975; Colgrove, 1978; de Swart et al., 1993; Lahvis et al., 1993; Ross et al., 1993; De Guise et al., 1996). Differential use of mitogens such as the plant lectins (concanavalin A, phytohemagglutinin, pokeweed mitogen) and bacterial lipopolysaccharide can serve as a relative measure of T- and B-lymphocyte responsiveness. A useful alternative to these traditional blastogenesis assays is to measure the expression of the IL2 receptor on lymphocytes by flow cytometry. This technique, adapted for harbor seals and bottlenose dolphins (Tursiops truncatus), uses labeled recombinant human IL-2, which binds to upregulated IL-2 receptors expressed on activated lymphocytes (DiMolfetto-Landon et al., 1995; Erickson et al., 1995). These proliferation assays have the capacity to be modified for measuring pathogen-specific T-cell responses. Assays to assess function of additional leukocytes such as phagocytosis (De Guise et al., 1995) and NK cell function (Ross et al., 1995b; De Guise et al., 1997a) have also been described for harbor seals and belugas. In Vivo In marine mammals, challenge experiments with pathogens are rarely feasible or ethical as a means of studying immune function. The delayed-type hypersensitivity (DTH) skin test represents the only practical in vivo method of assessing cellular immune function. This procedure 0839_frame_C12.fm Page 243 Tuesday, May 22, 2001 11:09 AM Immunology 243 involves inoculating an antigen intradermally into the individual under investigation and monitoring the local immune response over the following 48 to 72 hours. The monitoring procedure can be as simple as measuring changes in skin thickness at the site of antigen inoculation. More complete information can be obtained by histopathological examination of a skin biopsy taken from the same region. A DTH response is characterized by a γ-INFassociated influx of macrophages. The γ-INF is secreted by TDTH helper cells. For this reason, this assay can be used for measuring antigen-specific immune cell responsiveness. Since animals must be housed for up to several weeks, this approach is not practicable for many field situations involving marine mammals. Decreased DTH responses to ovalbumin were measured in a Dutch study examining the effects of environmental contaminants on harbor seal immune responsiveness (Ross et al., 1995a). The results implicated an immunosuppressive effect of pollutants upon cellular components of the immune system. Humoral Immunity Immunoglobulins (antibodies) are soluble, antigen-specific effector molecules of the adaptive immune response. These proteins are produced by B-lymphocytes of the humoral immune system. Distinct classes of immunoglobulin molecules (IgG, IgM, IgA, and, of lesser importance in the peripheral circulation, IgE and IgD) have been identified in most mammalian species studied. Because of their relatively high concentration in serum, purification and characterization of these proteins are often the first tasks undertaken by comparative immunologists. To date, several studies have characterized immunoglobulin molecules with characteristic component heavy and light chains, using sera collected from a selected number of cetacean and pinniped species (Nash and Mach, 1971; Cavagnolo and Vedros, 1978; Carter et al., 1990). Binding of immunoglobulin proteins to unique determinants (epitopes) on foreign proteins is an important mechanism by which pathogens are targeted for subsequent elimination from the body. By measuring changes in the circulating levels of antigen-specific immunoglobulin, exposure to infectious agents can be documented. This can be used in epidemiological studies of infectious disease and to enhance the management and prevention of disease outbreaks by identifying naive unexposed animals. It must be emphasized, however, that pathogen-specific antibody levels do not necessarily confirm the presence of an active pathogen. Serum is the most readily obtainable and conveniently sampled source for measuring systemic humoral immune responses. Carefully collected sera can often be stored over prolonged periods at −70°C without seriously compromising their performance in diagnostic assays. Sera can also be stored for long periods at −20°C, providing the freezer is not frost-free. Serum stored in frostfree freezers will become desiccated with time and should no longer be used for the detection and measurement of antibodies. As a general rule, it is good practice to dispense sera into multiple aliquots (of appropriate volume), because this will minimize the freezing/ thawing of samples. For larger organizations, a dedicated serum bank will prove valuable for long-term monitoring of individuals and for epidemiological studies of disease in populations. Access to good quality sera is particularly important when performing retrospective serological studies. Measurement of Pathogen-Specific Antibodies (Serodiagnostics) Vast arrays of laboratory-based assays have been developed to measure pathogen-specific antibodies. Although they may vary in specificity and sensitivity, most of the approaches described below provide useful serological information when performed with appropriate controls. These controls increase the confidence of the assay data and assist with the interpretation of the results. 0839_frame_C12.fm Page 244 Tuesday, May 22, 2001 11:09 AM 244 CRC Handbook of Marine Mammal Medicine When possible, assays should be performed with established positive and negative reference sera. Unfortunately, prior exposure to pathogens, particularly for free-ranging marine mammals, is rarely documented. In these cases, designated hyperimmune sera from a closely related species can be substituted. For example, commercially available canine distemper virus (CDV) immune sera have been successfully used to validate a morbillivirus seroassay for use in harbor seals (Ham Lammé et al., 1999). To discriminate actively infected animals from those with prior exposure to the pathogen in question, it is important to use paired sera that have been collected at least 14 to 21 days apart. For many assays, a fourfold increase in antibody titer between these time points is indicative of active infection. In the absence of defined clinical signs of disease in a population, care and consideration must be taken before serological evidence alone can confirm the presence or absence of a pathogen. Since microbiology of marine mammal diseases is in its infancy, there are probably many microorganisms yet to be discovered (see Chapter 15, Viral Diseases; Chapter 16, Bacterial Diseases). Therefore, the possibility that the test used is detecting a similar agent that shares common structural domains with the agent for which it was designed should not be excluded. When possible, serological studies should be performed in concert with other independent methods such as viral/bacterial isolation or molecular identification of genomic sequences. Serum/Virus Neutralization Test The serum/virus neutralization test (SNT/VNT) is an in vitro assay that estimates the amount of pathogen-specific antibody that neutralizes the replication and subsequent cytopathic effect of a defined dose of virus. In recent years, SNTs have been successfully developed and used to monitor exposure to a number of marine mammal-specific viruses including morbilliviruses (Visser et al., 1990; Van Bressem et al., 1993), herpesviruses (Borst et al., 1986), and caliciviruses (Smith, 1987). An advantage of this test is that it is sensitive and highly specific (e.g., defining viral serotypes) for the viral pathogen being employed in the assay. However, false positives can arise as a result of the presence of serum constituents that are somewhat toxic to cells and directly inhibit virus replication. These substances are common in samples collected postmortem. Further limitations of SNTs are that they can be lengthy assays to perform, requiring up to 7 to 14 days before they can be evaluated, and require cell culture expertise and equipment that is not easily adapted for field situations. SNTs require that the laboratory must have access to appropriate isolates of the virus in question and cells in which this virus can replicate, limiting its use to a small number of specialized laboratories. Although not necessarily a negative attribute, the neutralization assay is serotype-specific and will not necessarily detect antibody to closely related viruses. Precipitation/Agglutination Techniques These are traditional serodiagnostic techniques that exploit the ability of antibodies to form visible aggregates with antigen. The precipitation reaction employs soluble antigen (e.g., agar gel immunodiffusion, AGID), whereas the agglutination reaction utilizes particulate antigen (e.g., bacterial agglutination reaction) or soluble antigen bound to inert particles (e.g., latex beads). The advantage of these tests is that they are cheaper than SNT/VNT, since specialized, and therefore often expensive, equipment is not required. Furthermore, since specific reagents are not necessary, existing assays for human and domestic species are usually easily adapted for use with marine mammal sera. Examples of the use of these methods with marine mammal sera include recent studies to determine the prevalence of antibodies to Brucella (Tryland et al., 1999), as well other studies investigating the serology of Leptospira (Vedros et al., 1971). A disadvantage of these tests is that, since the formation of large immune complexes is inhibited 0839_frame_C12.fm Page 245 Tuesday, May 22, 2001 11:09 AM Immunology 245 by excess amounts of antibody or antigen (prozone effect), careful titrations must be performed to optimize the assays. Enzyme-Linked Immunosorbent Assay In recent years, enzyme-linked immunosorbent assays (ELISA) have been increasingly used in serological diagnostics. The basis for these assays is that the antigen in question is immobilized onto a solid phase, usually to a specially treated 96-well plastic plate. Antigen-specific immunoglobulin is detected by stepwise incubations with the test sera followed by an antispecies secondary reagent covalently linked to an enzyme reporter such as horseradish peroxidase or alkaline phosphatase. Although the approach described above (indirect ELISA) normally requires purified antigen, ELISA methods can be modified by the use of pathogenspecific reagents, so that antigen is captured from solution (trapping ELISA) prior to the addition of the test sera. A limited number of monoclonal and polyclonal species-specific secondary antibodies for pinniped immunoglobulin are available (Carter et al., 1990; King et al., 1993a). In the absence of species-specific reagents, staphylococcal protein A (SPA) and/or streptococcal protein G (SPG) (Ross et al., 1994; Reidarson et al., 1998) can be used. These are commercially available bacterial cell wall components that have been shown to bind the Fc portion of most mammalian immunoglobulin molecules. For most marine mammal species tested to date, SPA appears to be the preferred reagent for ELISA. In addition to these valuable reagents, development of further monoclonal antibody markers is anticipated in the near future. This should allow the subsequent establishment of new and more sensitive serological tests for these species. These exquisitely sensitive techniques are rapid to perform, adaptable to field situations, inexpensive, and can be easily applied to a large number of samples. However, assay specificity is dependent on the degree of antigen purity and is therefore easily compromised. Total Immunoglobulin Even in a hyperimmunized individual, the component of immunoglobulin that is specific for one particular antigen is usually less than 5%. Therefore, changes in the concentration of total immunoglobulin (classes and subclasses) are not usually indicative of the progression of an immune response. However, total immunoglobulin concentrations do have a diagnostic utility. Measurement of IgG concentrations in serum is performed in clinical situations to determine if passive transfer of immunoglobulin has occurred in neonates of species that are transiently hypogammaglobulinemic at birth. The clinical utility of total immunoglobulin concentrations has been demonstrated in animals with recurrent bacterial infections, suspected autoimmune disease, and lymphoproliferative disorders. In these instances, quantifying IgG is used to support a specific diagnosis, and is not used as an evaluation of specific immune function. Increases and decreases in serum levels of IgG are the consequences rather than the cause of complex events. A decrease in IgG does not necessarily mean an animal is functionally immunocompromised. A number of investigators have used a variety of techniques to quantify serum immunoglobulin concentrations of pinnipeds (Calvagnolo and Vedros, 1979; Carter et al., 1990; King et al., 1994; 1998; Marquez et al., 1998). Interestingly, serum IgG concentrations in pinnipeds appear to be significantly elevated compared with those of terrestrial carnivores. These studies provide the basis for the future establishment of clinically useful baseline values for these species. Further work in this area is still required to determine if low or abnormally elevated immunoglobulin levels are consistent with any recognized disease entities in marine mammals. 0839_frame_C12.fm Page 246 Tuesday, May 22, 2001 11:09 AM 246 CRC Handbook of Marine Mammal Medicine Clinical Approach to Suspected Marine Mammal Immunological Disorders Immunological disorders can be broadly divided into those with immunological overactivity (autoimmunity, allergic, hypersensitivity) and those with immune system deficiency. Anecdotal reports on marine mammals suggest that immunological insufficiency is a more common concern for clinicians, so this subsection will focus on a clinical approach to cases in which a functional impairment of the immune system is suspected. A number of immunodeficiency classification schemes have been developed in other species. These may be categorized by etiology (primary or secondary) or by the predominant compartment of the immune system affected (humoral, cellular, combined). Since there is little published information regarding clinical immunology in marine mammals, it is useful to borrow these classification criteria, at least until a better understanding of factors and conditions that impair immune function in marine mammals is gained. The classification scheme proposed by the World Health Organization (WHO) is broadly based on which compartment of the immune system is involved in the deficiency. Primary immunodeficiencies are those caused by intrinsic defects (congenital or acquired). This category consists of a large number of inherited defects, but also includes intrinsic defects induced by environmental insults. In secondary immunodeficiencies (Table 2), there are no intrinsic abnormalities in the development or function of B or T cells, but, instead, an external factor or condition interferes with immune function. These include viral-induced immunodeficiencies and those arising from stress, malnutrition, neoplasia, parasitic infections, or iatrogenic factors. Clinical investigations of suspected immunodeficiency should be directed at identifying which compartments of the immune system are affected. This is the first step in determining an underlying cause for the abnormality. Immunodeficiency syndromes, by definition, are characterized by an unusual susceptibility to infection. This susceptibility may include frequent infections with common or opportunistic microbes, unusually severe infections, or the failure of an infection to respond to antibiotics to which the suspect organism is susceptible. The type and extent of the infection provides the first clue to the nature of the immune dysfunction. For example, recurrent infections with pyogenic bacteria are likely caused by defects in B-lymphocytes or humoral (antibody-mediated) immunity. Severe fungal infections are more compatible with T-lymphocyte deficiencies. The repeated formation of abscesses with low-grade pathogens may suggest a neutrophil deficiency. If one or more of these scenarios is present, then it is reasonable to suspect a compromised immune system. The next tasks are to confirm this diagnosis using a stepwise clinical approach (Figure 1), to examine the possible cause of the immune compromise. In many marine mammal species, specific information regarding the immune system is difficult to obtain by physical examination, because of the difficulty in palpating external lymph nodes. However, an assessment of the size and state of the lymphoid organs (thymus, spleen, lymph nodes) can be useful, particularly in detecting primary immunodeficiency states, and may circumvent the need for extensive laboratory evaluations. If possible, this information should be acquired using radiographic or ultrasonographic imaging of these organs (see Chapters 24 through 28, Diagnostic Imaging). Information on the immune system can be obtained from routine hematological examinations (white blood cell counts with leukocyte differentials) and clinical serum chemistry analyses (see Chapter 19, Clinical Pathology). An immunological abnormality must be suspected when a persistent lymphopenia or neutropenia is observed. A marked or progressive hypo- or hyperglobulinemia, in the absence of serum albumin changes, can also indicate an immune dysfunction. It is important to emphasize that the presence of any abnormality should be confirmed by repeated sampling and comparison with healthy, age-matched individuals. 0839_frame_C12.fm Page 247 Tuesday, May 22, 2001 11:09 AM 247 Immunology TABLE 2 Possible Causes of Secondary Marine Mammal Immunodeficiencies Inciting Cause Possible Mechanism a Follow-Up Tests Failure of passive transfer Immunoglobulin deficiency Serum immunoglobulin levels Malnutrition (protein, caloric, and/or micronutrient) (Chandra, 1997) Multifactorial and complex: impaired antibody production, cell-mediated immunity, phagocyte function, and complement activity Response to nutritional supplementation Lymphocyte proliferation Trauma/surgery (Page and Ben-Elihau, 2000) Acute-phase response Cytokine imbalance Pain (neuroendocrine) Response to analgesics Acute-phase proteins Viral infection (see Chapter 15) Varies with etiological agent (e.g. lymphoid depletion, suppression of lymphocyte proliferation, downregulation of MHC expression) Identification of viral agent Lymph node biopsy Lymphocyte proliferation Flow cytometry Hormonal (e.g., endocrine imbalance, pregnancy) (Mellor and Munn, 2000) (see Chapter 10) Varies with hormone (e.g., pregnancy can invoke a cytokine imbalance) Detection of pregnancy Flow cytometry Lymphocyte proliferation Bacterial infection (Song et al., 2000) (see Chapter 16) Cytokine imbalance Identification of pathogen Acute-phase proteins Lymphocyte imbalance Stress (Elenkov et al., 1999) (see Chapter 13) Hormonally induced changes in cytokines, lymphocyte function, and expression of cell-surface proteins Flow cytometry Lymphocyte proliferation Neoplasia/malignancy (see Chapter 23) Quantitative and qualitative alterations in humoral and cell-mediated immunity Flow cytometry Serum immunoglobulin levels Lymph node/bone marrow biopsy Drug-induced (e.g., corticosteroids) Varies with drug; corticosteroids affect cytokine production Flow cytometry Lymphocyte proliferation a Tests with abnormal results should be repeated and results compared with those of age-matched control animals. Although these simple hematological and serum protein changes are clearly not pathognomonic for immune deficient states, they do provide strong justification for pursuing more specific and reliable immunological testing. Furthermore, there are a large number of immunological disorders that will not be detected by changes in absolute leukocyte numbers or serum globulin levels. Many of the species-specific assays for reliably examining the immune systems of marine mammals are not available through routine diagnostic laboratories. However, there are a number of research laboratories that are able to provide advice and to perform these services. A broad evaluation of the cellular immune system can be obtained by immunophenotypic analysis and lymphocyte function tests. A detailed serum immunoglobulin profile will provide useful information concerning the antibody-producing capabilities of the immune system. In most cases, these tests will identify the nature and extent of an immune dysfunction. On occasion, the abnormality is more subtle, and its identification and characterization may require 0839_frame_C12.fm Page 248 Tuesday, May 22, 2001 11:09 AM 248 CRC Handbook of Marine Mammal Medicine FIGURE 1 Clinical evaluation of the immune system. the inoculation of an exogenous antigen and the measurement of the responses induced by the inoculation. Conclusion In summary, there is a need to expand the knowledge and understanding of immunological disorders in marine mammals. By adopting a systematic approach to examining the immune system, it is possible to determine the nature and extent and, possibly, the etiology of immune dysfunction in an individual. This information will be vital in designing management and preventative strategies in susceptible populations. Acknowledgments The authors thank Tracy Romano for her peer-review of this chapter. 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Visser, I.K.G., Grachev, M.A., Orvell, C., DeVries, P., Broeders, J., van de Bildt, M.W.G., Groen, J., Teppema, J.S., Burger, M.C., Uyt de Haag, F.G.C.M., and Osterhaus, A.D.M.E., 1990, Comparison of two morbilliviruses from seals during outbreaks of distemper in northwest Europe and Siberia, Arch. Virol., 111: 149–164. Zhong, J.F., Harvey, J.T., and Boothby, J.T., 1999, Characterization of a harbor seal class I major histocompatability complex cDNA clone, Immunogenetics, 48: 422–424. 0839_frame_13fm Page 253 Tuesday, May 22, 2001 11:10 AM 13 Stress and Marine Mammals David J. St. Aubin and Leslie A. Dierauf Introduction As early as 450 B.C., Hippocrates considered health to be a state of harmonious balance and disease a state of disharmony (Chrousos, 1988). The Oxford English Dictionary notes that the word stress first appeared in the literature in 1303, but did not occur in the context of biological science until 1936 (OED, 1999). In that year, the journal Nature published a short article entitled “A Syndrome Produced by Diverse Nocuous Agents” by Hans Selye (Selye, 1936). This article laid the groundwork for current stress research by describing a three-stage syndrome of (1) alarm and adaptation, (2) hormonal events, and (3) resistance, exhaustion, and death, where “the symptoms ... are independent of the nature of the damaging agent or the pharmacological type of drug employed” (Neylan, 1998). Moberg (1985; 1987a) further defined Selye’s three stages of stress as (1) recognition of the stressful stimulus, (2) the body’s actual response to the stimulus, and (3) the resulting consequences to the body. Is stress harmful? The answer is no and yes. When an individual can predict and control the threatening stressor, a coping mechanism can be established. It might even be argued that periodic activation of the stress response is beneficial to maintaining health in the same way that physically demanding exercise promotes fitness. However, when the responses to stress are uncontrolled, excessive, and prolonged, a state of distress results. Distress is not always deleterious, although it is unpleasant and uncomfortable (Goldstein, 1995). This chapter considers the diverse mammalian responses to stressors and examines manifestations of the stress response in marine mammals. The chapter addresses clinical approaches and indicators for assessing stress in these species and concludes by identifying needs for future research to sharpen diagnostic abilities and to allow better prediction of the long-term consequences of stress. Stressors Stressors are not equally stressful to all individuals or species. The response to a given stressor depends on how an animal’s sensory systems receive and interpret information about the surrounding environment, the reaction to this information, and the degree of positive and negative feedback that occurs during the response (Lovallo, 1997a). Experience and acclimation will blunt the response to potentially stressful procedures; for example, bottlenose dolphins 0-8493-0839-9/01/$0.00+$1.50 © 2001 by CRC Press LLC 253 0839_frame_13fm Page 254 Tuesday, May 22, 2001 11:10 AM 254 CRC Handbook of Marine Mammal Medicine (Tursiops truncatus) can become quite tolerant to transportation in a stretcher. The introduction of novel stimuli into an animal’s environment constitutes a stress for some, but necessary enrichment for others. A new arrival in a pinniped colony can either enhance the social framework or precipitate stressful aggression. In a captive setting, where it is desirable to eliminate, or at least manage, potential stressors in an animal’s environment to optimize health, it is important to evaluate each case in the context of the species and individuals involved. In the wild, marine mammals encounter natural stressors daily. Predators, demanding meteorological and oceanographic conditions, intraspecific aggression, and even aspects of their normal activities, such as prolonged fasts or extended dives, are significant challenges to homeostasis and may elicit stress responses. Of greater concern is the impact of unnatural or anthropogenic stressors on the health of marine mammals, particularly species that are threatened or endangered. Increasingly, biologists and medical professionals are called upon to evaluate and provide opinions that might lead to important management decisions. For example, in 1997, an amendment to the U.S. Marine Mammal Protection Act, directed the National Marine Fisheries Service to conduct a review of the scientific literature on stress to provide a context for future research concerning the effects of stress on dolphins (Curry, 1999). Human activities such as vessel traffic, fishing, petroleum and mineral exploration and development, low-frequency sounds for ocean thermometry, and sonar systems are highly controversial, in terms of the degree to which they elicit damaging stress responses in marine mammals. Oil spills (Geraci and St. Aubin, 1990) and other environmental contaminants can be directly harmful, but more often the impact must be measured through subtle physiological changes considered indicators of stress. There are few experimental data to address these points, largely because it is difficult to identify “control” populations in the wild or to isolate the effects of one particular stressor in the midst of a substantially degraded habitat. Stress Response and Regulation The literature on the stress response in mammals identifies four broad categories of interest: physiology, endocrinology, immunology, and neurology. There is considerable overlap among these, particularly since hormones alter physiological processes and immune responses, neurological stimulation elicits certain endocrine and physiological responses and is also linked to the immune system, and mediators of inflammation activate some endocrine pathways (Figure 1). Within each category, there is the important consideration of whether the response is acute or chronic, and whether the associated perturbations are beneficial or more damaging than the original stressor. Survival for the organism depends on feedback regulation of many of these systems, and when unchecked or stimulated to exhaustion, the result is distress and possibly death (Breazile, 1987). A significant challenge to studying stress in marine mammals, or any wild species, is to obtain baseline data representing an unstressed state. Chase, capture, restraint, and sampling procedures are recognized stressors that can influence analytes, sometimes within minutes. In captivity, cetaceans and pinnipeds can be trained to allow specimen collection with minimal disturbance, yielding data that are as close to resting as can be expected. At the very least, the slight deviations that might be encountered under such circumstances serve as controls for the same procedures that must be employed to assess stress in free-ranging individuals or in captive animals suspected of stress-related abnormalities. For those working in the field, rapid and efficient capture strategies can sometimes be designed to allow specimen collection of baseline quality. 0839_frame_13fm Page 255 Tuesday, May 22, 2001 11:10 AM Stress and Marine Mammals 255 FIGURE 1 Major changes to body systems under stress. Neurological Factors The acute stress response begins with recognition of a stressor, and is initially orchestrated by the limbic and hypothalamic centers of the brain. Perception of a stressful stimulus produces fear and anxiety, which feed back to the limbic system of the brain. Corticotropinreleasing factor (CRF) is secreted from the hypothalamus (paraventricular nucleus) and the limbic system, and is the main neuropeptide regulator activating the hypothalamic–pituitary–adrenal (HPA) axis (Rivier, 1991). It also acts as a neurotransmitter, helping integrate the animal’s sensory, behavioral, and endocrinological responses to stimuli (Lovallo, 1997a). Direct innervation of the adrenal medulla results in the release of catecholamines to adjust physiological processes; neurological connections to lymph nodes serve to link the central nervous and immune systems. These elements of the stress response in marine mammals are examined below. 0839_frame_13fm Page 256 Tuesday, May 22, 2001 11:10 AM 256 CRC Handbook of Marine Mammal Medicine Endocrine Factors The primary endocrine components of the stress response are derived from the autonomic nervous system (norepinephrine, or NEpi), the adrenal medulla (epinephrine, or Epi, and NEpi), the hypothalamus (CRF), the pituitary (adrenocorticotropic hormone, or ACTH), the adrenal gland (cortisol, corticosterone, and aldosterone), and the brain (NEpi and β-endorphins) (Dunn, 1995; 1996; Lovallo, 1997b). Secondarily, enkephalins, substance P, neuropeptide Y, prolactin, growth hormone, thyroid hormones, vasopressin, angiotensin II, vasoactive intestinal peptides, and other pituitary hormones become involved in a cascading fashion (Dunn, 1995; 1996; Breazile, 1988). For many of these, there are no specific data from marine mammals. Nevertheless, there has been significant progress in the last three decades in the understanding of how some of these hormones participate in the stress response in these animals (Table 1). Information on the normal function of marine mammal endocrine systems is presented in Chapter 10, Endocrinology. Catecholamines Catecholamines (Epi and NEpi) are the first line of defense in an animal’s response to stress—the “fight or flight” reaction. Their effects are induced rapidly, and circulating levels can be altered by the mere anticipation of a stressful event. Unlike many other hormones, the changes that they elicit subside quickly. The physiological systems affected by catecholamines are many, but principally involve the cardiovascular system and energy metabolism, to prepare the organism for immediate action. Thomas et al. (1990) examined changes in catecholamine levels in captive belugas (Delphinapterus leucas) exposed to playbacks of high-amplitude noise from oil-drilling rigs. Although the animals’ initial response was to flee, there was little or no consistent effect on circulating levels of catecholamines (Epi: 0 to 101 pg/ml; NEpi: 160 to 604 pg/ml). St. Aubin and Geraci (unpubl. data) compared Epi and NEpi concentrations in 29 belugas sampled immediately after a 5- to 15-min pursuit, with those in 10 whales captured and held for repeated sampling over 5 days. Epi levels averaged 634 pg/ml at the time of capture, but only 76 pg/ml in 95 samples collected during the 5-day holding period. Average NEpi concentrations of 1423 pg/ml after capture declined only slightly to a mean of 1042 pg/ml. The latter hormone is generally more reflective of muscular activity and discharge from the sympathetic nervous system than anxiety or alarm. Recent investigations on stranded cetaceans have revealed a pattern of lesions suggestive of massive release of endogenous catecholamines (Turnbull and Cowan, 1998; Cowan, 2000). Contraction band necrosis in cardiac and skeletal muscle, along with injuries of ischemia and reperfusion in gut and kidney, are manifestations of an excessive and prolonged alarm response, with fatal consequences. These observations were thought to account for the abrupt deaths during handling of highly stressed, stranded marine mammals. The acute deaths of three ringed seals (Phoca hispida) exposed experimentally to an oil spill (Geraci and Smith, 1976) were less a function of the toxicity of the petroleum than of cardiac tissue hypersensitized to certain volatile hydrocarbons by the stress of the situation (St. Aubin, 1990). Glucocorticoids Glucocorticoids (cortisol, corticosterone) have three functions in stress. They (1) alter carbohydrate metabolism to increase circulating substrates for energy; (2) permit catecholamines to act on metabolic pathways and blood vasculature; and (3) provide protective adaptations to distress by limiting immunological reactions, including inflammation, thus minimizing cell and tissue damage (Munck et al., 1984; Breazile, 1988). Cortisol is the dominant circulating Increase Neutrophils Capture and handling Capture and handling Immune Capture and handling Capture and handling Capture and handling Noise playback Capture and handling Noise playback Capture and handling Glucocorticoid administration No change Increase No change No change Increase No change Increase Increase Increase Capture and handling Capture and handling Capture and handling Decrease No change Decrease Leukocytes Insulin Norepinephrine Epinephrine Reverse triidothyronine Triiodothyronine Capture and handling Capture and handling No change Increase Aldosterone No change No change Herpesvirus infection Stranding Capture and handling Capture and handling Corticosterone Arginine vasopressin Thyroxine Capture and handling Endocrine Stressor Increase Effect Cortisol Factor Beluga Ringed seal (P. hispida) Beluga Bottlenose dolphin Beluga Bottlenose dolphin Beluga Bottlenose dolphin Bottlenose dolphin Beluga Bottlenose dolphin Beluga Beluga Beluga Beluga Bottlenose dolphin Beluga Bottlenose dolphin Bottlenose dolphin Beluga (Delphinapterus leucas) Bottlenose dolphin (Tursiops truncatus) Harbor seal (Phoca vitulina) Pilot whale (Globicephala melas) Bottlenose dolphin Bottlenose dolphin Species TABLE 1 Stress Indicators in Marine Mammals (effects noted are in blood, unless otherwise indicated) Stress and Marine Mammals continued St. Aubin and Geraci, 1989 Geraci and Smith, 1975 St. Aubin and Geraci, 1989 Medway and Geraci, 1964 St. Aubin and Geraci, 1988; 1992 St. Aubin et al., 1996 St. Aubin and Geraci, 1988; 1992 Orlov et al., 1988 St. Aubin et al., 1996 St. Aubin and Geraci, 1988; 1992 St. Aubin et al., 1996 Thomas et al., 1990 St. Aubin and Geraci, unpubl. Thomas et al., 1990 St. Aubin and Geraci, unpubl. Reiderson and McBain, 1999 St. Aubin and Geraci, 1989; 1992 Thomson and Geraci, 1986; St. Aubin et al., 1996 Gulland et al., 1999 Geraci and St. Aubin, 1987 Ortiz and Worthy, 2000 Thomson and Geraci, 1986; St. Aubin et al., 1996 St. Aubin and Geraci, 1989 Ortiz and Worthy, 2000 Ortiz and Worthy, 2000 Reference 0839_frame_13fm Page 257 Tuesday, May 22, 2001 11:10 AM 257 Bottlenose dolphin Handling Seven species of cetaceans Disease, entrapment, habitat degradation Increase Decrease Present (in tissue) Increase (in tissue) Creatine kinase Haptoglobins Alkaline phosphatase Contraction band necrosis Stress responsive proteins Disease Stranding Disease Disease Capture and handling Handling No change Increase Sodium Dugong (Dugong dugon) Bottlenose dolphin Harp seal (Pagophilus groenlandicus) Ringed seal Bottlenose dolphin Harp seal Beluga Steller sea lion (Eumetopias jubatus) and harbor seal Bottlenose dolphin Various cetacean species Capture Capture and handling Nutritional stress Increase No change Decrease Miscellaneous Diagnostics Gray seal (Halichoerus grypus) Ringed seal Bottlenose dolphin Beluga Bottlenose dolphin Ringed seal Bottlenose dolphin Ringed seal Bottlenose dolphin Beluga Bottlenose dolphin Species Intradermal PHA injection Glucocorticoid administration Glucocorticoid administration Capture and handling Glucocorticoid administration Capture and handling Stressor Fothergill et al., 1991 Turnbull and Cowan, 1998; Cowan, 2000 Southern, 2000 Marsh and Anderson, 1983 Ortiz and Worthy, 2000 Geraci 1972, Engelhardt and Geraci, 1978 Geraci et al., 1979 Ortiz and Worthy, 2000 St. Aubin et al., 1979 St. Aubin and Geraci, 1989 Zenteno-Savin et al., 1997 Medway and Geraci, 1964 Geraci and Smith, 1975 Medway et al., 1970 St. Aubin and Geraci, 1989 Medway and Geraci, 1964; Thomson and Geraci, 1986 Geraci and Smith, 1975 Medway et al., 1970 St. Aubin and Geraci, 1989 Thomson and Geraci, 1986 Geraci and Smith, 1975 Medway et al., 1970; Reidarson and McBain, 1999 Hall et al., 1999 Reference 258 Potassium Decrease Lymphocytes Effect Decreased proliferation (in tissue) No change Decrease Eosinophils Factor TABLE 1 (CONTINUED) Stress Indicators in Marine Mammals (effects noted are in blood, unless otherwise indicated) 0839_frame_13fm Page 258 Tuesday, May 22, 2001 11:10 AM CRC Handbook of Marine Mammal Medicine 0839_frame_13fm Page 259 Tuesday, May 22, 2001 11:10 AM Stress and Marine Mammals 259 glucocorticoid in all marine mammals studied to date, although corticosterone levels vary in parallel with those of cortisol in bottlenose dolphins (Ortiz and Worthy, 2000). Exogenous ACTH has been used to alter circulating levels of cortisol in some odontocetes and pinnipeds, providing a standard against which stress-induced changes can be measured (see Chapter 10, Endocrinology). No dose–response studies have been attempted, reflecting the cautious experimental approach that must often be used with marine mammals, especially cetaceans. Despite this caution, two bottlenose dolphins tested with ACTH died 2 and 5 days later, possibly as a cumulative effect of preexisting stress (Thomson and Geraci, 1986). Thus, it is difficult to define in absolute terms what the maximum potential is for glucocorticoid secretion in marine mammals. Even with such information, it is misleading to use the degree of corticosteroid elevation as a direct measure of the intensity of the stressor (Rushen, 1986). Capture and handling is a stressor that is of particular interest to those who must manipulate animals in captivity or in the wild. Thomson and Geraci (1986) compared cortisol levels in bottlenose dolphins calmly captured and sampled within 10 min, with those in dolphins subjected to 3 hours of pursuit prior to sampling. The cortisol levels of the former group averaged approximately 1.25 µ g/dl, whereas the latter showed concentrations of 2.5 µ g/dl. During the next 7 hours, when the animals were held in stretchers to simulate transport and allow the collection of serial samples, cortisol levels for the most part did not rise above 4.7 µ g/dl, with no clear differences seen based on the earlier treatment of the dolphins. In the calmly captured animals, cortisol levels rose steadily during the first 90 min to reach concentrations similar to those in the first samples obtained from the chased dolphins. The changes as a result of handling and restraint were comparable to those following ACTH administration. Overall, the elevations in cortisol were modest, compared with those in other species. Wild bottlenose dolphins unaccustomed to capture or, in the case of the population in Sarasota Bay, Florida, infrequently captured might be expected to exhibit a stronger glucocorticoid response to this stress, but no such difference was noted (St. Aubin et al., 1996; Ortiz and Worthy, 2000). The narrow range of cortisol concentrations in most cetaceans limits its utility as a stress indicator. Although Thomson and Geraci (1986) concluded that it was a good measure of adrenal activity in bottlenose dolphins, Ortiz and Worthy (2000) found that cortisol levels were no higher in free-ranging dolphins sampled more than 41 min after capture than in those sampled within 27 min. Either the animals were undisturbed by the procedure in the latter study or the specimens were drawn before changes in cortisol occurred. In ACTH stimulation studies in this species, cortisol levels rose only slightly during the first hour postinjection (Thomson and Geraci, 1986). Belugas sampled at capture and after a 3- to 5-hour transport to field holding facilities showed rising levels of cortisol, from 3.2 to 5.8 µ g/dl (St. Aubin and Geraci, 1989). When they were next sampled, 2 to 4 days later, concentrations were comparable to the lower values found immediately following capture. The dynamics of the cortisol response to handling stress were examined in more detail in belugas serially sampled every 6.5 hours over a 5-day period after capture (St. Aubin and Geraci, 1992). Following the hour-long process of lowering the water twice daily to access the whales, blood cortisol levels averaged 3.9 µ g/dl, whereas samples collected 6 hours after acclimation to shallow water showed a mean cortisol level of 2.7 µ g/dl. As noted following ACTH administration, a cortisol response to stress is expected after 1 to 2 hours, with a return to baseline levels 4 to 5 hours later, in the absence of continued stimulation (St. Aubin and Geraci, 1990). Extreme elevations in cortisol have been noted in marine mammals in distress. Stranded pilot whales (Globicephala melas) on the shore for more than 6 hours showed levels up to 16 µ g/dl, 0839_frame_13fm Page 260 Tuesday, May 22, 2001 11:10 AM 260 CRC Handbook of Marine Mammal Medicine far in excess of any values recorded after ACTH stimulation or other handling (Geraci and St. Aubin, 1987). It is likely that these supraphysiological concentrations were the result of reduced hepatic clearance in animals in shock. Gulland et al. (1999) found that harbor seals (P. vitulina) infected with an adrenotropic herpesvirus showed elevated baseline cortisol levels that peaked at an average of 38.7 ± 16 µ g/dl within 2 hours of death. Mineralocorticoids The mineralocorticoid aldosterone is not customarily considered as part of the stress response in most mammals. However, a series of studies and other fortuitous observations have revealed its particular role in stress in marine mammals. It has been postulated that the role of aldosterone in water conservation is beneficial to stressed marine mammals, especially those that may not soon have an opportunity to acquire water through feeding (see Chapter 10, Endocrinology). Stimulation by ACTH elicits a proportionally larger elevation in aldosterone in bottlenose dolphins (Thomson and Geraci, 1986), belugas (St. Aubin and Geraci, 1990), ringed and harp seals (Pagophilus groenlandicus) (St. Aubin and Geraci, 1986), and northern fur seals (Callorhinus ursinus) (St. Aubin et al., unpubl. data) than it does in terrestrial mammals. Consistent with these findings, capture and handling stress produce the same changes in belugas (St. Aubin and Geraci, 1989) and bottlenose dolphins (Thomson and Geraci, 1986; St. Aubin et al., 1996), although Ortiz and Worthy (2000) found no aldosterone release in the latter species during the time frame of their sampling. Aldosterone elevations, when they do occur, are highly variable, peaking in less than 1 hour in some cases and at 3 hours in others; still other animals show no residual elevation after 3 hours of continuous handling (Thomson and Geraci, 1986). The sensitivity of aldosterone to central stimulation from the pituitary and higher neurological centers in phocid seals provides a mechanism that is subject to exhaustion and failure during chronic stress. The result is hyponatremia (Geraci, 1972a), which can occur not only in salt-restricted environments, as might be expected, but also as a consequence of a variety of nonspecific stresses such as vitamin deficiency (Geraci, 1972b; Engelhardt and Geraci, 1978). In the wild, ringed seals in poor body condition from undetermined causes also exhibit hyponatremia, suggesting that they were chronically stressed (Geraci et al., 1979). Thyroid Hormones The activity of the thyroid gland is modulated during stress to conserve resources for more urgent survival needs. Thyroid hormone (TH)-mediated mobilization of energy stores could be adaptive at such times, but not the thermogenic catabolism that accompanies this process. Ridgway and Patton (1971) recognized that capture stress profoundly affects TH balance in some cetaceans. In belugas, St. Aubin and Geraci (1988; 1992) noted decreased levels of triiodothyronine (T3) approximately 6 to 8 hours after capture, whereas changes in thyroxine (T4) did not occur until more than 20 hours later. There was no recovery in whales monitored for as long as 10 weeks. During the acute phase of the response, levels of reverse T3 (rT3) rose, suggesting a diversion of the metabolism of T4 to the inactive rT3 rather than to the physiologically potent T3. Administration of ACTH depressed T3 levels even farther. These changes are consistent with glucocorticoid-mediated effects in other mammals (Larsen et al., 1998). Cortisol is capable of inhibiting thyrotropin secretion from the anterior pituitary, and also the tissue monodeiodinating enzyme that is responsible for converting much of the T4 in the circulation to T3. Because of its short half-life in circulation, T3 declines relatively rapidly, whereas T4 shows a more gradual decrease from a larger pool of circulating hormone that is not being replenished from the thyroid gland. A similar pattern of change in T3, but not T4, 0839_frame_13fm Page 261 Tuesday, May 22, 2001 11:10 AM Stress and Marine Mammals 261 was observed in an abbreviated study on bottlenose dolphins (Orlov et al., 1988). The dramatic changes in TH in belugas may have been exaggerated by the coincident annual stimulation of thyroid activity at the time when the studies were performed (see Chapter 10, Endocrinology). Other Hormones There is little information on the role of other hormones in the stress response of marine mammals. The dynamics of growth hormone, prolactin, insulin, and glucagon, among others, bear investigation, considering their importance in producing metabolic adjustments that are advantageous during stress. Reidarson and McBain (1999) noted an increase in insulin levels in two dolphins given glucocorticoids to stimulate appetite. Arginine vasopressin (AVP) was examined for its possible influence on ACTH, and concomitantly adrenocortical hormones, in captured bottlenose dolphins, but no relationship was found (Ortiz and Worthy, 2000). Immunological Factors For many years, the potent anti-inflammatory and immunosuppressive properties of glucocorticoids were not readily reconciled with a general impression that the stress response better equips the organism to meet potentially threatening conditions (Munck et al., 1984). A fully charged immune system would seem to be the best defense against opportunistic pathogens. Yet, it is widely recognized that stress can render individuals more, rather than less, susceptible to disease (Levine, 1993; Leonard and Miller, 1995). The suppressive action of glucocorticoids on the immune system is necessary to keep in check a powerful complement of cells and cell mediators that eventually would be detrimental (Keller et al., 1991; McEwen et al., 1997). Some of the mediators released during inflammation stimulate CRF secretion from the hypothalamus and, consequently, increase ACTH and cortisol levels to abate the immune response (Lovallo, 1997a). The general organization of the immune system in marine mammals is considered in Chapter 12, Immunology. Assessment of the various cellular and biochemical components of this system is a rapidly expanding discipline, and has grown quickly from the long-standing reliance on leukocyte differential counts to lymphocyte phenotyping, cytokine analysis, and blastogenesis studies (see Chapter 12, Immunology) (DiMolfetto-Landon et al., 1995; Erickson et al., 1995; Nielsen, 1995; Blanchard et al., 1999). Leukocyte counts are a convenient, albeit “low-tech,” approach to recognizing stress in these animals. The classic stress leukogram (leukocytosis, neutrophilia, eosinopenia, lymphopenia) attributable to the action of glucocorticoids on the various cell lines was described to varying degrees in bottlenose dolphins subjected to transportation stress (Medway and Geraci, 1964), treated with glucocorticoids (Medway et al., 1970; Reidarson and McBain, 1999) or following ACTH administration (Thomson and Geraci, 1986), and in belugas after capture (St. Aubin and Geraci, 1989) or ACTH (St. Aubin and Geraci, 1990). Ringed seals stressed by capture in nets showed similar changes (Geraci and Smith, 1975). Dexamethasone suppressed lymphocyte proliferation in gray seals (Halichoerus grypus) injected intradermally with the mitogen phytohemagluttinin (PHA) (Hall et al., 1999). Taken together, these observations demonstrate that the immune systems of marine mammals display the same sensitivity as other species to stress-related hormonal changes, and that stress may compromise their ability to resist infection. The immune system is also subject to direct regulation by the central nervous system. In belugas, as in other mammals, lymphoid organs are innervated by noradrenergic and peptidergic fibers (Romano et al., 1994). Activation of central structures during the stress response therefore has the potential to affect immunological activity. 0839_frame_13fm Page 262 Tuesday, May 22, 2001 11:10 AM 262 CRC Handbook of Marine Mammal Medicine Indicators of Acute and Chronic Stress To help diagnose and treat stress in marine mammals, interdisciplinary teams are working to develop clinically useful laboratory tests to quantify better acute, prepathological, and chronic stress reactions (Figure 2). Because the stress response is a series of complex interrelated events, differing from species to species and from individual to individual within each species, this is a daunting task. Where in the stress response should one look for valid indicators—at the start (i.e., early warning systems), midway, or at the end (end-point measurements)? Are negative results as valuable as positive results in testing for stress indicators? Should one be looking for direct or indirect measurements of stressful events? What is the best way to induce stress to study it? These are questions that must be answered by those engaged in stress research, prior to designing any study. Acute Response Behavioral assessments are commonly used to recognize acute stress. Anxiety is often the first outward sign of an animal under stress. Chrousos and Gold (1992) and Dunn (1995) suggest this anxiety results from the release of norepinephrine from the noradrenergic neurons in the brain stem locus ceruleus. Dolphins disturbed by the presence of, and noise from, a large ship, positioned themselves as far away from it as possible, and showed “agitation, stress and fear” by tail-slapping, head-slapping, hyperactive swimming, bunching about, and thrashing (Norris et al., 1978; Norris and Dohl, 1980). In some situations, passivity rather than hyperactivity might signal stress, as noted in spinner (Stenella longirostris) and spotted dolphins (S. attenuata) that were encircled as part of the tuna fishery (Norris et al., 1978). The acute stages of the stress response are most often examined through analysis of blood constituents. In addition to, and as a consequence of, the hormonal changes described earlier, one typically sees ketosis, hyperlipemia, hyperglycemia, hyperaminoacidemia, and metabolic acidosis signaling increased hepatic gluconeogenesis, and lipid and protein catabolism; hematological changes follow the expected pattern. Exertional stress during capture and handling can lead to muscle damage and the release of diagnostically useful indicators such as creatine kinase, aminotransferases, and potassium (St. Aubin et al., 1979; Marsh and Anderson, 1983) (see Chapter 19, Clinical Pathology). Capture myopathy, and its pathognomonic signs (Spraker, 1993), should be considered following any procedure involving wildlife, including marine mammals. FIGURE 2 Results of acute vs. chronic stress. 0839_frame_13fm Page 263 Tuesday, May 22, 2001 11:10 AM Stress and Marine Mammals 263 Chronic Response Chronic stress may occur if stressors are frequent, intermittent, and/or repetitive. Chronic stress can produce one of three responses: (1) habituation, in which the stress response decreases with each episode; (2) sensitization, where the stress response increases with each episode; or (3) desensitization, when there is no change (Dantzer and Mormede, 1995). In chronic stress, there is sustained activation of the HPA axis, producing repetitive, pulsatile secretions of glucocorticoids. The chronic effects of stress are difficult to diagnose, and even more difficult to relate back to specific stressful events. Nevertheless, it is a task commonly presented to medical professionals and biologists. In reality, chronic stress is probably of greater significance in terms of an animal’s well-being than short-term responses to transient stimuli. Impaired growth and reproduction, frequent infection, and pathological changes in organs are among the many consequences that can be linked to chronic stress. Stress can disrupt reproductive functions in many mammalian species. CRF, ACTH, glucocorticoids, and β-endorphins secreted in response to stressful stimuli can inhibit reproductive processes (Moberg, 1987b). Stress-induced elevations of glucocorticoids may affect the reproductive system by inhibiting hypothalamic secretion of gonadotropin-releasing hormone, blocking the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and altering the gonadal response to LH and FSH secretion from the anterior pituitary (Rivier and Rivest, 1991). At present, there is no specific information on these pathways in marine mammals. Furthermore, one can only speculate about the long-term consequences of lowered TH levels on growth and development in species such as belugas, in which TH can be substantially altered by stress (St. Aubin and Geraci, 1988; 1992). The immune system has many cellular components useful in measuring chronic stress. In vitro, mitogens such as PHA, concanavalin A, and pokeweed mitogen act as nonspecific stimulators of immune function, causing lymphocyte proliferation and activation. Such tests have been used in killer whales (Orcinus orca), bottlenose dolphins, harbor seals, and gray seals, to gauge health (DiMolfetto-Landon et al., 1995; Erickson et al., 1995; Nielsen, 1995; Blanchard et al., 1999; Hall et al., 1999) (see Chapter 12, Immunology). Failure of lymphocytes to respond to mitogens can be an indicator of severe immune system deficiency, possibly as a result of stress. A young gray seal with elevated cortisol levels showed no response to intradermal PHA, and died 12 hours later of a respiratory infection (Hall et al., 1999). Additional research is needed to determine how reliable or sensitive such indicators might be in marine mammals. Cowan and Walker (1979) suggested that a variety of pathological changes in spinner and spotted dolphins killed in dolphin–fishery interactions were related to stress. They noted massive cardiac response to stress in some of the dolphins, and described the microscopic pathological lesions as consistent with those in laboratory animals injected with catecholamines and in humans with stress cardiomyopathy. Adrenal glands are an obvious site to examine for morphological evidence of chronic stimulation. Several cetacean species necropsied after stranding, including Atlantic white-sided dolphins (Lagenorhynchus acutus), harbor porpoises (Phocoena phocoena), belugas, and a common dolphin (Delphinus delphis), had adrenocortical cysts on necropsy exam (Geraci et al., 1978; Kuiken et al., 1993; Cartee et al., 1995) (see Chapter 23, Noninfectious Diseases). The adrenal glands from 95% of 90 spinner dolphins and 172 spotted dolphins chased during capture showed darkened adrenal cortices, which were interpreted as a consequence of continuous acute stress and/or vasogenic shock leading to death (Myrick and Perkins, 1995). Belugas from the St. Lawrence River have a high prevalence of adrenal lesions, including cortical hyperplasia, cortical and medullary nodular hyperplasia, and serous cysts, which were 0839_frame_13fm Page 264 Tuesday, May 22, 2001 11:10 AM 264 CRC Handbook of Marine Mammal Medicine increasingly common in older whales (Lair et al., 1997). Chronic exposure to organohalogens was suggested as an underlying cause of adrenal hyperfunction in this species (De Guise et al., 1994) (see Chapter 22, Toxicology). These compounds are highly toxic in vitro to adrenal mitochondria from gray seals, inhibiting glucocorticoid-synthesizing enzymes and leading to adrenal hyperplasia (Lund, 1994). Associations are frequently made among overwhelming, but nonspecific, pathological changes in free-ranging marine mammals and the stresses imposed by a contaminated environment. Bergman (1999) described adrenocortical hyperplasia in gray and ringed seals found dead along the shores of the Baltic Sea. The animals also exhibited a variety of lesions, including claw and digit deformities, bone lesions, particularly around the teeth, overburdens of acanthocephalans (Corynosoma spp.) in the proximal colon, intestinal ulcers, arteriosclerosis of the aorta and its bifurcations, and uterine leiomyomas, stenosis, and occlusion. The adrenal changes may have been a consequence of exposure to endocrine-disrupting compounds and the stress of multisystemic disease. At the same time, adrenal hyperactivity might have further compromised an immune system already suppressed by environmental contaminants (de Swart et al., 1994; Ross et al., 1996). Zenteno-Savin et al. (1997) examined circulating levels of haptoglobins (Hp) as potential indicators of chronic stress in harbor seals and Steller sea lions (Eumetopias jubatus) from declining populations in Prince William Sound, Alaska. Elevated levels of these proteins had been demonstrated in river otters (Lutra canadensis) 1 year after the Exxon Valdez oil spill there, and were felt to be linked to that event (Duffy et al., 1993; 1994). Levels in the Prince William Sound harbor seals and Steller sea lions were higher than those from the more stable populations of southeast Alaska, and were associated with infection, inflammation, trauma, and tumors in the former groups. Recently, Southern (2000) identified a group of 30 stress-responsive proteins (SRP) with recognized roles in oxidative cell response, active cell death, cell growth and differentiation, cell adhesion, and immunological and neurological signaling. By using a multitarget antibody cocktail, the suite of SRPs can be simultaneously detected in tissues, including readily available epidermal biopsies. In a survey of seven species of cetaceans, tenfold or greater increases in SRP levels were noted in animals stressed by conditions such as ice entrapment, chronic illness, starvation, net capture, and coastal pollution (Southern, 2000). The SRP assay system shows great potential for monitoring the impacts of conservation and management strategies on marine mammals. Future Research Marine mammal stress research has advanced considerably in recent years. The goals of stress research are twofold: First, to conduct interdisciplinary studies of the interactions among endocrine, immune, and neurological systems that maintain homeostasis, control acute stress, and respond to distress; and, second, to develop a broad database for indicators that will improve the ability to recognize and manage stress in the animals both in captivity and in the wild. To this end, further research is needed in the following areas: • • • • • • Glucocorticoid metabolism; The effects of age and gender on the stress response; Differences among species with varying sensitivities to stress; New and creative diagnostic tests that can reliably detect stress; Rational prophylaxis and treatment for stressed marine mammals; The ways environmental pollutants act as stressors or interfere with the stress response; 0839_frame_13fm Page 265 Tuesday, May 22, 2001 11:10 AM Stress and Marine Mammals 265 • Reproductive physiology and stress; • Marine mammal population dynamics in relation to environmental stressors; • Correlations between pathological conditions and stressors. Conclusion In virtually every clinical situation, stress and its consequences must be addressed, since disease itself is a stressor, and stress may be at the root of the illness in question. Nevertheless, the term is too often applied indiscriminately as a convenient “catch-all” when efforts to reach some other diagnosis fall short. Advancement of understanding of this important determinant of marine mammal health will depend on a focused, scientific approach to stress and the stress response. Acknowledgments The authors thank Mona Haebler and Barbara Curry for reviewing an earlier version of this chapter and offering helpful suggestions for improving its content. Special thanks are due from the primary author (St. Aubin) to Joseph Geraci for the many long discussions about stress and what it means in marine mammals. Funding for studies on catecholamine research in belugas was provided by the Office of Naval Research to the primary author (St. Aubin) and to Joseph Geraci. This is contribution number 125 of the Sea Research Foundation. 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Thomson, C.A., and Geraci, J.R., 1986, Cortisol, aldosterone, and leucocytes in the stress response of bottlenose dolphins, Tursiops truncatus, Can. J. Fish. Aquat. Sci., 43: 1010–1016. Turnbull, B.S., and Cowan, D.F., 1998, Myocardial contraction band necrosis in stranded cetaceans, J. Comp. Pathol., 118(4): 317–327. Zenteno-Savin, T., Castellini, M.A., Rea, L.D., and Fadely, B.S., 1997, Plasma haptoglobin levels in threatened Alaskan pinniped populations, J. Wildl. Dis., 3(1): 64–71. 0839_frame_13fm Page 270 Tuesday, May 22, 2001 11:10 AM 0839_frame_C14.fm Page 271 Tuesday, May 22, 2001 11:11 AM 14 Genetic Analyses Deborah A. Duffield and William Amos Introduction This chapter explores how genetic techniques can contribute to understanding of marine mammals and their problems, with special emphasis on marine mammal strandings and maintenance and breeding of marine mammals in captivity. The chapter outlines genetic methodologies available, attempting to concentrate on those methods used most often. Also included are brief descriptions of the processes for sampling animals that strand. Genetic Techniques The literature contains references to a wide and even bewildering range of genetic techniques, reflecting a restless search for greater resolution, robustness, comparability, and ease of use. A good review of the various techniques, including DNA analysis, is given in Hillis et al. (1996). It was as late as 1960 that starch gel electrophoresis was first used to reveal and quantify genetic variability in the form of protein polymorphisms. Bypassing the need to look at the genes themselves or their products, data were also collected from differences among chromosomes revealed by various staining techniques. In about 1970, with the discovery of restriction enzymes that recognize and cut particular DNA motifs, the world of DNA analysis opened. In its most basic form, the presence or absence of a cutting site yields either one long or two shorter fragments, known as restriction fragment length polymorphisms (RFLP). RFLP analysis is a generic method for revealing polymorphism that is still used widely in one form or another. More recently, two primary approaches have come to dominate the scene, and these appear to be poised to stay for most applications. DNA Sequencing The first technique is DNA sequencing. Although once laborious and expensive, DNA sequencing is now rapid, accessible, and cheap. The most commonly sequenced genes are those with particularly attractive features, and at top of the list are two or three genes found on the DNA in mitochondria (Moritz, 1994). Mitochondria are the modern descendants of ancient bacteria that came to live inside the cells of higher organisms, and each mitochondrion carries its own degenerate, circular chromosome. Mitochondrial DNA (mtDNA) is a popular target for sequencing because of several unusual properties. First, mtDNA evolves very rapidly and, hence, even populations and closely related species tend to carry diagnostic differences. Second, every cells carries hundreds or even thousands of mitochondria, and therefore there are many more 0-8493-0839-9/01/$0.00+$1.50 © 2001 by CRC Press LLC 271 0839_frame_C14.fm Page 272 Tuesday, May 22, 2001 11:11 AM 272 CRC Handbook of Marine Mammal Medicine copies of an mtDNA gene compared with an equivalent gene present as a single copy in the cell nucleus. This high copy number can allow genetic tests even when most of the DNA has been degraded by putrefaction or antiquity (Hagelberg, 1994; Hagelberg et al., 1994). Third, in most higher organisms mitochondria are inherited strictly through the female line, giving a simple pattern of inheritance, which tends to reveal differences between populations more strongly than most other genetic markers. Fourth, even within the tiny mitochondrial genome there are regions that evolve at different rates. The fastest-evolving sequences are found in a region with little clear function known as the D-loop or control region. Evolving some five to ten times slower, are any one of a number of genes coding for mitochondrial proteins, of which a common target is called cytochrome oxidase, or CO1; another is cytochrome b. “Tandem Repeats” and DNA Fingerprinting The second main technique involves an unusual class of DNA sequences called tandem repeats that show extreme levels of polymorphism. The term tandem repeat embraces any short DNA motif repeated head to tail from a few to hundreds or thousands of times, (e.g., ACCACCACCACCACCACC). Most exciting was the discovery in 1985 of medium-sized repeats called “minisatellites,” which show the greatest variability of all, and form the basis of the technique known popularly as DNA fingerprinting (Jeffreys et al., 1985a). DNA fingerprinting is a remarkably powerful technique, able to identify individuals uniquely and able to assign unambiguous parentage (Jeffreys et al., 1985b; Amos et al., 1993). Unfortunately, the technique also proved technically difficult to apply and has since been almost completely replaced by an alternative approach based on the smaller repeats, christened with simple logic microsatellites. Since its potential was discovered in 1989 (Litt and Luty, 1989; Tautz, 1989), microsatellite analysis has grown to a dominant position in the literature, being both accessible and powerful. Microsatellites are the shortest possible tandem repeats, with the repeating unit usually two to five DNA letters long, for example, ACACACACAC (see reviews in Bruford and Wayne, 1993; Bruford et al., 1996; Goldstein and Schlötterer, 1999). Microsatellites are attractive markers for several reasons. First, they are highly polymorphic, typically carrying 5 to 10 alleles/locus. By combining information from several loci, this is sufficient to allow a range of analyses, from the identification of individuals (Jeffreys et al., 1992) and parentage testing (Worthington Wilmer et al., 1999) to the detection of differences between populations and species (Paetkau et al., 1995). Second, microsatellites are assayed by the polymerase chain reaction (PCR). In PCR, an enzyme is used repeatedly to make copies of a target piece of DNA, identified by its sequence. The result is an immensely powerful tool that can analyze as little as a single molecule, making the technique ideal for dealing with the sort of DNA one can extract from decaying or degraded tissue (Reed et al., 1997; Taberlet et al., 1997). Third, microsatellite analysis is easy to use and yields data that are ideally suited to inclusion in databases (each allele is recorded as a fragment with a discrete length). A slight drawback is that some preparatory work is needed to develop microsatellite markers for each new species, although with more and more studies appearing and with many markers working on related species (Valsecchi and Amos, 1996; Gemmell et al., 1997), this problem is resolving. Genetic Analyses Applied to Stranded Marine Mammals A stranded marine mammal can provide material for genetic analysis that can elucidate many aspects of a species biology. For marine mammals, many of which live in inaccessible regions and spend much of their lives out of view below the sea surface, information gained through genetics can play an even greater role than for more easily studied terrestrial species. The sorts 0839_frame_C14.fm Page 273 Tuesday, May 22, 2001 11:11 AM Genetic Analyses 273 of questions that can be addressed include identification, from species through population down to individual identity; studies of social organization, based on determining the relationships among individuals in a group; and diagnosing the cause of death by, for example, using gene sequencing to identify a particular pathogen. Species Identification Genetic analysis is most obviously useful for determining the species from which a sample has been collected, and the primary technique used here is gene sequencing. Much progress has been made, largely as a result of pioneering studies by Baker and others aimed at identifying the origin of whale products sold in food markets in Japan and Korea (Baker et al., 1996). Results showed that much of the meat was from minke whales (Balaenoptera acutorostrata) taken under license for scientific whaling, but that significant numbers of samples could be attributed to protected species, including at least two different individual blue whales (B. musculus). There is now an almost complete catalog of mtDNA sequences available, with all but a handful of extant species represented, along with most of the major populations. Any new, unidentified specimen can be matched with great precision, essentially always to species, and often to the ocean basin it came from. Many fresh strandings provide material that can be identified with high confidence based on morphological traits. In such cases, DNA sequencing can have two functions. First, it provides a useful double-check for field misidentification. Cetacean coloration can change rapidly after death, making identification difficult. Even when a nominal species has been accurately determined, several instances have emerged where genetic analysis has revealed the presence of cryptic species, subspecies, or races. Second, the more sequences that can be added to the database, the more complete the database becomes, thereby facilitating future matches. This is particularly important for rarer species whose distribution may be poorly understood, and for species with strong population structure, where a more complete database can be used to pinpoint an animal’s origin. It is important not to forget that a dead marine mammal may contain more than one species. Parasites, bacteria, and viruses also contain DNA, which can be used for their identification. In 1988, large numbers of harbor seals (Phoca vitulina) were found washed up dead and dying, first around Denmark and then spreading up around the North Sea coasts to Scotland and Ireland (Swinton et al., 1998). In some areas, more than 50% of all seals died (see Chapter 15, Viral Diseases). The cause was initially a mystery, although the acute respiratory distress and secondary infections were suggestive of canine distemper. DNA sequences obtained from viral isolates later proved to be from a new pathogen known as phocine distemper virus. Similar, although less spectacular, mortality events in porpoises, dolphins, and (possibly) monk seals yielded further members of this viral family (Barrett et al., 1993); morbilliviruses are now prime suspects when marine mammals start dying in large numbers (see Chapter 2, Emerging Diseases). Population Identification Below the level of species, one is interested in identification of the population or stock from which a given individual derives. Such questions are an ongoing concern with marine mammals because of their great capacity for movement (Dizon et al., 1997); threats posed in one area can exert strong influences elsewhere. Until the patterns of movement of each species, and how to recognize where one population ends and the next one begins, are better understood, any attempts at management or conservation will be difficult. 0839_frame_C14.fm Page 274 Tuesday, May 22, 2001 11:11 AM 274 CRC Handbook of Marine Mammal Medicine Population studies usually involve either mtDNA sequencing (mainly of the fast-evolving D-loop or control region) or microsatellite analysis (Allen et al., 1995), although protein polymorphisms historically played an important part. Together these techniques have helped to elucidate patterns of movement of many species, from great whales (Baker et al., 1990; Palsbøll et al., 1995) and belugas (Delphinapterus leucas) (O’Corry-Crowe et al., 1997) to manatees, seals (Burg et al., 1999), and sea otters (Cronin et al., 1996). Size is no predictor of where divisions will exist. Thus, while sperm whales (Physeter macrocephalus) show little evidence of population structure throughout the world oceans (Lyrholm and Gyllensten, 1998), humpback whales (Megaptera novaeangliae) exhibit very strong structure, because of the way offspring learn their mothers’ patterns of movements (Baker et al., 1990). Other interesting examples include the harbor seal, in which great individual mobility belies strong genetic isolation between most breeding colonies (Goodman, 1998), and killer whales (Orcinus orca), in which two behaviorally and genetically distinct groups of the same nominal species coexist in the same area off the Washington coast (Hoelzel and Dover, 1991). Although most studies looking for evidence of population substructure use either mtDNA or microsatellites, a few use both mitochondrial and nuclear markers (Burg et al., 1999). The advantage of using both markers together is that their contrasting modes of inheritance can indicate sex-specific patterns of gene flow. In many mammals, females tend to stay to breed near their natal site, whereas males disperse to avoid inbreeding. Here, mtDNA sequences, inherited solely through the female line, will show a pattern of strong substructure, reflecting the lack of movement by females between sites. However, microsatellites are nuclear markers and alleles are inherited from both parents. Consequently, even though only males move between sites, this will provide sufficient mixing to reduce or even eliminate evidence of substructure. By using the two markers together, it becomes possible to deduce these sex-based differences in dispersal behavior; whenever mtDNA shows strong substructure while microsatellites do not, this is good evidence that females return to breed where they were born, whereas males tend to disperse (Palumbi and Baker, 1994). Social Organization The primary tool for examining questions about relatedness and social organization is microsatellite analysis. These markers are eminently suitable for identifying individuals, calculating indices of relatedness, and conducting parentage analysis, and they have the particular advantage that they can be genotyped in older, more degraded samples, including museum specimens. Genetic identity can be used to track individual movements in just the same way that early workers implanted “discovery tags,” large metal projectiles that lodged inside a whale’s body and were later recovered and recorded when that whale was subsequently killed (Palsbøll et al., 1997). With the advent of biopsy darting, genetic “tagging” is now a practical way to follow an individual throughout its life. As the number of studies increases, the chances improve that a sample from a meat market, stranding, or net entanglement will provide an informative last data point. Studies of parentage and relatedness using genetic analysis provide vital information that allows reconstruction of the social organization and breeding behavior of any organism, but they are particularly important for inaccessible marine mammals. Individual projects can be based on as few as two or three animals that strand together, but range in size to long-term studies with directed sampling, where databases of thousands of individuals now exist. Many revelations are emerging. These include the relative lack of polygynous behavior in gray seals (Halichoerus grypus) (Ambs et al., 1999; Worthington Wilmer et al., 1999), with some individuals even showing partner fidelity (Amos et al., 1995) and the dissection of social groups of cetaceans, such as pilot whales (Amos et al., 1993). 0839_frame_C14.fm Page 275 Tuesday, May 22, 2001 11:11 AM Genetic Analyses 275 There is a further use of microsatellites that is of particular interest to veterinarians. Since every individual inherits one allele from each parent, the similarity of alleles at a locus provides a measure of the degree of parental similarity. Thus, highly inbred individuals will tend to carry pairs of alleles that are very similar to each other, whereas animals born to genetically dissimilar parents will tend to carry dissimilar alleles. By using this logic, studies on red deer (Cervus elaphus) (Coulson et al., 1998), harbor seals (Coltman et al., 1998), and Soay sheep (Aries aries) (Coltman et al., 1999) have used molecular estimates of parental similarity to show that the level of inbreeding has a significant impact on fitness. Juvenile survival is greater in individuals born to more genetically dissimilar parents. It has even been shown that individuals born to dissimilar parents tend to carry lower parasite burdens as adults (Coltman et al., 1999). The possibility of a genetic explanation for at least some of the observed variation in susceptibility to infection is an exciting one that may well blossom in the near future. Genetic Analysis Applied to Captive Maintenance and Breeding Programs Zoos and aquaria play an important role in species conservation and propagation. As wild populations dwindle, it often falls on captive breeding programs, not only to maintain captive populations, but also to reintroduce individuals to the wild (Kleiman et al., 1996). For marine mammals, successful captive breeding has been well documented with births reported in 17 species (Asper et al., 1990), including cetaceans, pinnipeds, sea otters, and manatees (see Chapter 11, Reproduction). In commonly held species, such as bottlenose dolphins (Tursiops truncatus), California sea lions (Zalophus californianus), and harbor seals, breeding groups have had second- and third-generation offspring. Paternity Testing Maintaining genetic diversity is a primary population goal for long-term management of captive populations (Ballou and Foose, 1996). Genetic variation is important to the ability of a captive population to adapt to changing environments, as well as to help prevent loss of individual fitness due to the deleterious effects of inbreeding (Ralls et al., 1988; Lacy et al., 1993). Tracking parentage in captive propagation programs by genetic monitoring ensures that a balanced gene pool is maintained and that breeding programs avoid inbreeding. Documentation of the relationships between individuals provides valuable information for use when setting up breeding colonies and when exchanging animals or sperm for breeding or artificial insemination purposes. In most instances, a mother–offspring relationship is known, so that the evaluation of parentage usually rests on determination of paternity. Among group-living animals, paternity cannot always be reliably assigned based on social dominance or observed copulatory behavior, hence, the importance of genetic discrimination of paternity in colonies with multiple males. In the past decade, there have been significant technological advances influencing the range of molecular genetic analyses that are being used to aid breeding programs in zoos (Bruford et al., 1996; Ryder and Fleischer, 1996). Methodologies currently in use with marine mammals include protein electrophoresis; in particular, hemoglobin electrophoresis; fluorescent R-band chromosome analysis and DNA microsatellite analysis. Hemoglobin electrophoresis is inexpensive and has been useful for establishing paternity in cases where the potential sires were of different hemoglobin types (Duffield and Chamber-Lea, 1990). Similarly, cetacean chromosomes are excellent discriminators for paternity testing, because they have numerous variable regions, referred to as heteromorphisms, in their karyotypes. These are readily visualized by fluorescent R-banding (Duffield 0839_frame_C14.fm Page 276 Tuesday, May 22, 2001 11:11 AM 276 CRC Handbook of Marine Mammal Medicine FIGURE 1 Fluorescent R-band chromosome heteromorphism analysis for bottlenose dolphin chromosome pair 19. An example of the use of fluorescent R-band chromosome heteromorphism analysis for paternity testing in bottlenose dolphins. (A) Calf. The karyotype of the offspring is screened for chromosome pairs with heteromorphic variants. (B) Mother. The heteromorphic pairs of the offspring are compared with those same pairs in the female to establish which variants were inherited from the mother. This defines the “required paternal match.” (C) Potential fathers. The karyotypes of all possible sires are compared with the offspring to determine which male has the paternal match. Given the number of heteromorphic chromosome pairs in cetacean karyotypes, each paternal discrimination is made on the basis of matching several such variants. and Chamberlin-Lea, 1990; Duffield and Wells, 1991; Duffield et al., 1991). In contrast, the chromosomes of pinnipeds, the sea otter, and the manatee do not exhibit the degree of chromosomal heteromorphism seen in cetaceans. An example of how chromosome heteromorphism analysis is used in paternity testing in cetaceans is presented in Figure 1. With the advent of microsatellite analysis, this latter technique is becoming the DNA methodology of choice for paternity testing in captive breeding colonies. Microsatellite primer sequences have now been reported for a broad range of cetacean and pinniped species (Buchanan et al., 1996; 1998; Valsecchi and Amos, 1996; Gemmell et al., 1997; Shinohara et al., 1997). An example of paternal assignment using the microsatellite primer EV-37 (Valsecchi and Amos, 1996) in bottlenose dolphins is given in Figure 2. More than 20 different alleles for this single locus have been identified in the North American captive bottlenose dolphin population, and this amount of variability has made paternal discrimination very effective in these breeding groups. Hybrid Detection Genetic analysis is also useful for identifying interspecies hybrids. For odontocete cetaceans, hybrids have occurred between Tursiops truncatus and Grampus griseus, T. truncatus and Steno 0839_frame_C14.fm Page 277 Tuesday, May 22, 2001 11:11 AM Genetic Analyses 277 FIGURE 2 Example of microsatellite paternity testing in bottlenose dolphins. An example of assigning paternity with microsatellites, using primer EV-37 (Valsecchi and Amos, 1996). Alleles are represented by a top darker band, followed by fainter stutter (shadow) bands, which decrease in intensity. Microsatellite alleles in calves (C) are compared with those in the dam (D) to identify the allele given by the sire (arrows). The photograph was cropped to eliminate lanes that were not pertinent to this assignment. The sire (S) for both calves (C1 and C2) is in lane three from the left. No other animal shares his top allele (given to C1), and the animals sharing his bottom allele (given to C2) are an unrelated female and a male that was not present at the time of conception. bredanensis, T. truncatus and Globicephala macrorhynchus, T. truncatus and Pseudorca crassidens, T. truncatus and Delphinus delphis, Phocoena phocoena and Phocoenoides dalli, and possibly between D. capensis and Lagenorhynchus obscurus (Sylvestre and Tanaka, 1985; Reyes, 1996; Baird et al., 1998; Sea World, pers. comm.). One T. truncatus and Pseudorca crassidens hybrid has had two live-born offspring, sired by bottlenose dolphin males. One of these secondgeneration hybrids survived for nearly 8 years (North American Bottlenose Dolphin Studbook). The ability to have offspring proves that these particular hybrids are fertile, an important confirmation for evaluation of naturally occurring hybrids. Live-born pinniped hybrids have been reported between Zalophus californianus and Callorhinus ursinus, Z. californianus and Arctocephalus pusillus, Z. californianus and Eumetopius jubatus, and Phoca kurilensis and P. largha (King, 1983; Kamogawa SeaWorld, pers. comm.; DeLong, pers. comm.). One of the crosses between Z. californianus and C. ursinus gave birth to two pups, sired by California sea lions, again indicating the fertility of this hybrid. One pup was live-born, but died within a few days; the second pup was stillborn. Breeding and recognition of cetacean and pinniped hybrids in captivity affords a rare opportunity to develop anatomical and genetic profiles for these hybrids, which one hopes will further recognition of interspecific crosses in the wild. Sampling To perform genetic analysis, it is first necessary to obtain tissue samples from which to extract DNA. For live animals, there are several possible sampling routes, including blood sampling, direct tissue sampling with a biopsy device, and collection of b
