Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 3-13-2019 Can Assisted Reproductive Technologies Help Conserve 300 Million Years of Evolution? A First Attempt at Developing These Technologies for Male Reptiles Sean M. Perry Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Other Veterinary Medicine Commons, Reproductive and Urinary Physiology Commons, and the Veterinary Physiology Commons Recommended Citation Perry, Sean M., "Can Assisted Reproductive Technologies Help Conserve 300 Million Years of Evolution? A First Attempt at Developing These Technologies for Male Reptiles" (2019). LSU Doctoral Dissertations. 5184. https://digitalcommons.lsu.edu/gradschool_dissertations/5184 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. CAN ASSISTED REPRODUCTIVE TECHNOLOGIES HELP CONSERVE 300 MILLION YEARS OF EVOLUTION? A FIRST ATTEMPT AT DEVELOPING THESE TECHNOLOGIES FOR MALE REPTILES. A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and School of Veterinary Medicine in partial fulfillment of the requirement for the degree of Doctor of Philosophy in Biomedical and Veterinary Medical Sciences Veterinary Clinical Sciences by Sean Michael Perry B.S. University of California Santa Cruz, 2009 D.V.M. Western University of Health Sciences, 2013 May 2020 ACKNOWLEDGMENTS I am grateful to the numerous individuals whose expertise and support were instrumental in helping me complete my dissertation research and realize this vision, which was once a dream. First, I would like to thank my funding sources for financial support with these projects, the Audubon Center for Endangered Species Research grants, the Louisiana State University Department of Veterinary Clinical Sciences Corp grant, and the Association of Reptile and Amphibian Veterinarian Research and Conservation Grant. Next, I would like to express my appreciation for my advisor, Dr. Mark Mitchell, and all the support he has provided me during this process, including the bickering (self-induced) and challenges. If it were not for your support, enthusiasm, and guidance, I would not be the clinician scientist that I am today. I would also like to thank the other members of my thesis committee, Javier Nevarez, Dale Paccamonti, Michael Líerz, David Kersey, Chris Austin, and the graduate school representative Victor Rivera-Monroy, for their encouragement, insightful comments, and guidance through the completion of my program. Thank you for committing so much time to help me in making this a reality. Many thanks go to the individuals at Fluker’s Cricket Farm. David, Waco, Casey, and Sam- your support was instrumental and I wouldn’t have been able to complete these projects without your help. I also want to express my gratitude to the undergraduate students (Marilynn Bailey, Joshua Doss, and Jennifer Membleb), veterinary students (Ian Konsker and Sarah Camlic), and staff (Janelle Allen) of the Division of Laboratory Animal Medicine who helped with sample collection and animal care. These individuals help make these projects a reality by ii managing the day to day operations of our animal colonies. Additionally, thanks go to Ty Park and Sonny White for helping acquire animals for these projects. I would like to thank my fellow lab mates and team members within the Zoological Medicine and Theriogenology departments at Louisiana State University, including Kim Boykin, Laure Molitor, Christine Higbie, Alyssa Scagnelli, Kelly Rockwell, Charlie Cummings, Anke Stöhr, Gabriel Davoli, Victor Medina, Eric Mak, Viviane Gomes, Luis Aguiar, Carlos Pinto, Jennifer Sones, Clare Scully, and Chance Armstrong. Additionally, my thanks goes to the other house officers that have helped me along the way, including Jennifer West, Cole Sandow, Carlos Aguilar, Joe Cacioppo, and Alexandra Mason. I want to especially thank the team at the Aquatic Germplasm and Genetic Resources Center, including Terrence Tiersch, Yue Liu, Amy Guitreau, Bill Childress, and Hamed Shamkhalichenar, for allowing me to use their facilities for some of my experimentation and to expose me to a different way of thinking, new technologies, novel concepts, and evolve my thought process during my time at Louisiana State University. Additionally, I want to thank my family at the Audubon Nature Institute, including Robert MacLean, Jamie Torres, Adrienne Atkins, Robert Mendyk, Melissa Tomingas, Amanda Atkins, Amanda Franklin, and Melanie Litton, for helping me grow as a clinician. Second to last, but not least, are my colleagues and friends that have supported me through these past several years by providing me professional/life advice or just listening during this journey, including Charlie Innis, Shane Boylan, Tres Clarke, Matt Allender, Doug Mader, La’toya Latney, Jöreg Mayer, Zach Goodnow, Jonathan Ho, and Dana Lindemann. iii Finally, my greatest thanks are for my parents, Kathie and Tim Perry. They have supported and loved me in all that I have accomplished in my life, despite not fully grasping the logic to go and do a second doctorate, especially on reptiles……Perry Out. iv TABLE OF CONTENTS ACKNOWLEGEMENTS…………………………………………………………………………ii ABSTRACT……………………………………………………………………………….…….vii CHAPTER 1. INTRODUCTION…………………………………………………………………1 CHAPTER 2. LITERATURE REVIEW………………………………………………………….5 2.1. Is the Sixth Mass Extinction a Good Enough Reason to Develop Assisted Reproductive Technologies in Reptiles?.............................................................................5 2.2. Squamate and Chelonian Reproductive Anatomy and Physiology………………....21 2.3. Current Status of Assisted Reproductive Technologies (ART) in Reptiles………..105 CHAPTER 3. DETERMINING THE SAFETY OF REPEATED ELECTROEJACULATIONS IN VEILED CHAMELEONS CHAMAELEO CALYPTRATUS…………………………………………………………………………….......140 3.1 Introduction…………………………………………………………………………140 3.2 Materials and Methods……………………………………………………………...143 3.3 Results………………………………………………………………………………150 3.4 Discussion ………………………………………………………………………….156 CHAPTER 4. CHARACTERIZATION OF THE ANNUAL REPRODUCTIVE CYCLE IN THE VEIELD CHAMELEON CHAMAELO CALYPTRATUS AND THE PANTHER CHAMELEON FURCIFER PARDALIS…………………………………………………………………….…..163 4.1 Introduction…………………………………………………………………………163 4.2 Materials and Methods……………………………………………..…………….....166 4.3 Results………………………………………………………………………………173 4.4 Discussion ………………………………………………………………………….188 CHAPTER 5. DOSE DETERMINATION AND EFFECTS OF EXOGENOUS HUMAN CHORONIC GONADOTROPIN (hCG) ADMINISTRATION ON PLASMA TESTOSTERONE CONCENTRATIONS AND SEMEN PRODUCTION IN THE VEIELD CHAMELEON CHAMAELO CALYPTRATUS ………………………………………………………………....201 5.1 Introduction…………………………………………………………………………201 5.2 Materials and Methods………………………………………………………….…..204 5.3 Results………………………………………………………………………………214 5.4 Discussion ………………………………………………………………………….220 CHAPTER 6. MEASURING THE LEVEL OF AGREEMENT BETWEEN OSMOMETER AND CALCULATED PLASMA OSMOLALITIES IN TWO SPECIES OF CHAMELEONS, FURCIFER PARDALIS AND CHAMAELEO CALYPTRATUS………………………………………...............................................................227 6.1 Introduction…………………………………………………………………………227 6.2 Materials and Methods………………………………………………………….…..229 6.3 Results………………………………………………………………………………232 v 6.4 Discussion ………………………………………………………………………….235 CHAPTER 7. EVALUATION OF SEMEN EXTENDERS FOR SHORT-TERM REFRIGERATED STORAGE OF EPIDIDYMAL SPERMATOZOA IN TWO SPECIES OF SQUAMATES……………………………………………………………………………….…248 7.1 Introduction…………………………………………………………………………248 7.2 Materials and Methods……………………………………………………….……..251 7.3 Results………………………………………………………………………………255 7.4 Discussion ………………………………………………………………………….256 CHAPTER 8. EVALUATING DIFFERENT METHODS OF SPERMATOZOA STORAGE IN RED-EARED SLIDERS TRACHEMYS SCRIPTA ELEGANS 8.1 Introduction…………………………………………………………………………279 8.2 Materials and Methods…………………………………………………….………..283 8.3 Results………………………………………………………………………………292 8.4 Discussion ………………………………………………………………………….299 CHAPTER 9. CONCLUSIONS AND FUTURE DIRECTIONS……………………………...316 9.1 Conclusions…………………………………………………………………………316 9.2 Future Direction…………………………………………………………………….319 APPENDIX. COPYRIGHT INFORMATION.………………………………………………...320 REFERENCES…………………………………………………………………………………323 VITA……………………………………………………………………………………………387 vi ABSTRACT Biodiversity loss is the most critical environmental problem threatening ecosystem, animal, and human health today. Increases in extinction rates have been observed over the past 50 years, with reptile losses occurring twelve times faster than traditional extinction rates. This demonstrated biodiversity loss is secondary to climate change, habitat destruction, infectious disease, invasive species, poaching, and unsustainable trade. Approximately 20% of all reptiles are threatened with extinction and population declines are approaching rates similar to the current amphibian extinction crisis. Preventing the extinction of reptiles will require humans to acknowledge these losses and develop plans to preserve these evolutionary sentinel species. Assisted reproductive technologies (ART) are well developed in a handful of species, and these technologies have become integral parts of conservation programs for threatened and endangered species. The creation of functional and sustainable reproductive assistance programs