The Development of Assisted Reproductive Techniques for Managing Maned Wolves (Chrysocyon brachyurus) Ex situ A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at George Mason University by Amy E.M. Johnson Bachelor of Science in Agriculture University of Guelph, 2005 Director: Elizabeth Freeman, Assistant Professor New Century College Fall Semester 2012 George Mason University Fairfax, VA This work is licensed under a creative commons attribution-noderivs 3.0 unported license. ii DEDICATION This thesis is dedicated to my remarkable parents, Moya and Rick Johnson, who have never failed to tell me or show me how proud they are. For my mother, your feistiness and boundless energy towards making a difference, both locally and provincially, has taught me that even little voices can make big changes. You accept every challenge with a smile and you never turn down an opportunity to learn new things. I will undoubtedly take the examples you have set for me to make changes for conservation. For my father, your passion for animals and the outdoors has inspired me to enjoy the simple things in nature, and to make sure it is there for future generations. Furthermore, your extreme patience and unparalleled work ethic has shown me that hard work always pays off, as long as you do what you love. I will never go into work without a smile because of you. Oh, and there’s no crying in baseball… iii ACKNOWLEDGEMENTS First and foremost, I’d like to express my sincere gratitude to my advisors, Drs. Nucharin Songsasen and Elizabeth Freeman, for looking past my transcripts and taking a chance on me. You were the most encouraging, accommodating and optimistic advisors I could have asked for. Together, you have set the bar high and I look forward to the day when I can follow in your footsteps and mentor others the way you have me. Dr. David Luther, thank you for your guidance and direction in school and life. I look forward to our future birding adventures. Dr. Tom Lovejoy, thank you for your support. Your conservation efforts inspire me more than you know and I hope that I can make a fraction of the accomplishments you have made towards preserving the Earth’s biodiversity. Dr. Wynne Collins, look what you got me into! Thank you for everything. Melissa Rodden and the MWSSP, thank you for being so supportive of the project and encouraging so many zoos to participate; this project would have been impossible without you. To everyone at SCBI who’s helped me along the way – Keeper staff Julie McLaughlin, Ken Lang, Jessica Kordell, thank you for your patience and your assistance in procedures, transporting animals and sample collections. Veterinary staff Lisa Ware, Dr. Padilla, Dr. Hope and Veronica Acosta, thank you for making procedures run so smoothly. SCBI scientists Dr. Wildt, Dr. Comizzoli and Dr. Pukazhenthi, thank you for your advice and contributions to the project. Laboratory staff Jenny Santiestevan, Niki Presley and Sarah Putman, thank you for your guidance in the lab. Interns Lara Mouttham, Tathiana Matheo and Ainjil Bills, thank you for joining me on my travels and for your endless hours helping me with procedures and lab work. To all the staff at participating institutions - Houston Zoological Park, White Oak Conservation Center, Fossil Rim Wildlife Center, Sedgwick County Zoo, Dickerson Park Zoo, Natural Science Center of Greensboro, Louisville Zoo, Connecticut’s Beardsley Zoo, thank you for all of your hard work and contributions to the project. We would be nowhere without your support. An enormous thank you to the Morris Animal Foundation for providing project funding and to Mark Colgin of AspenBio Pharma Inc., for donating reLH for the project. Finally, to Eric. Thank you for being my home away from home. iv TABLE OF CONTENTS Page List of Tables .................................................................................................................... vii List of Figures .................................................................................................................. viii List of Abbreviations ......................................................................................................... ix Abstract ............................................................................................................................... x Chapter 1: Literature Review ............................................................................................ 12 The Maned Wolf ........................................................................................................... 12 Assisted Reproduction for Ex situ Species Management .............................................. 14 Reproductive Biology of the Maned Wolf .................................................................... 15 Endocrinology of Reproductive Function ..................................................................... 16 Manipulation of Ovarian Activity ................................................................................. 19 Induction of Ovulation .................................................................................................. 21 Spermatozoal Response to Changes in Temperature and Osmotic Stress .................... 22 Hypotheses and Objectives ........................................................................................... 26 Chapter 2: Maned Wolf (Chrysocyon brachyurus) Spermatozoa are Highly Senstive to Exposure to Hypertonic Solutions of Non-permeating Compounds, but not to Permeating Cryoprotectants ................................................................................................................. 28 Abstract ......................................................................................................................... 28 Introduction ................................................................................................................... 29 Materials and Methods .................................................................................................. 32 Animals ...................................................................................................................... 32 Semen Collection, Evaluation and Processing .......................................................... 33 Evaluation of Sperm Morphology ............................................................................. 35 Evaluation of Sperm Plasma Membrane Integrity .................................................... 36 Experimental Design ................................................................................................. 36 Statistical Analysis .................................................................................................... 37 Results ........................................................................................................................... 38 v Discussion ..................................................................................................................... 41 Chapter 3: Interactions of Cryoprotectant, Cooling Rate and Warming Rate on the Cryosurvival of Maned Wolf (Chrysocyon brachyurus) Spermatozoa ............................ 47 Abstract ......................................................................................................................... 47 Introduction ................................................................................................................... 49 Materials and Methods .................................................................................................. 53 Animals ...................................................................................................................... 53 Semen Collection, Evaluation and Processing .......................................................... 54 Evaluation of Sperm Morphology ............................................................................. 56 Evaluation of Acrosomal Integrity ............................................................................ 57 Evaluation of Sperm Plasma Membrane Integrity .................................................... 58 Experimental Design ................................................................................................. 58 Statistical Analysis .................................................................................................... 61 Results ........................................................................................................................... 61 Motility ...................................................................................................................... 62 Membrane Integrity ................................................................................................... 64 Acrosomal Integrity ................................................................................................... 66 Discussion ..................................................................................................................... 67 Chapter 4: Induction of Ovarian Activity and Ovulation in an Induced Ovulator, the Maned Wolf (Chrysocyon brachyurus), using GnRH Agonist and Recombinant LH ..... 73 Abstract ......................................................................................................................... 73 Introduction ................................................................................................................... 74 Material and Methods...................................................................................................