Developing Assisted Reproductive Technologies for Endangered North American Amphibians
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Mississippi State University Scholars Junction Theses and Dissertations Theses and Dissertations 1-1-2016 Developing Assisted Reproductive Technologies for Endangered North American Amphibians Cecilia Jane Langhorne Follow this and additional works at: https://scholarsjunction.msstate.edu/td Recommended Citation Langhorne, Cecilia Jane, "Developing Assisted Reproductive Technologies for Endangered North American Amphibians" (2016). Theses and Dissertations. 1373. https://scholarsjunction.msstate.edu/td/1373 This Dissertation - Open Access is brought to you for free and open access by the Theses and Dissertations at Scholars Junction. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholars Junction. For more information, please contact [email protected]. Template A v3.0 (beta): Created by J. Nail 06/2015 Developing assisted reproductive technologies for endangered North American amphibians By TITLE PAGE Cecilia Jane Langhorne A Dissertation Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Animal Physiology in the Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology Mississippi State, Mississippi May 2016 Copyright by COPYRIGHT PAGE Cecilia Jane Langhorne 2016 Developing assisted reproductive technologies for endangered North American amphibians By APPROVAL PAGE Cecilia Jane Langhorne Approved: ____________________________________ Scott T Willard (Major Professor/Graduate Coordinator) ____________________________________ Andrew J Kouba (Committee Member) ____________________________________ Peter L Ryan (Committee Member) ____________________________________ Jean M Feugang (Committee Member) ____________________________________ William V Holt (Committee Member) ____________________________________ George M Hopper Dean College of Agricultural and Life Sciences Name: Cecilia Jane Langhorne ABSTRACT Date of Degree: May 6, 2016 Institution: Mississippi State University Major Field: Animal Physiology Major Professor: Scott T Willard Title of Study: Developing assisted reproductive technologies for endangered North American amphibians Pages in Study 280 Candidate for Degree of Doctor of Philosophy An alarming number of anuran (frog and toad) species are facing the threat of extinction in the wild. In efforts to address this conservation crisis, captive breeding programs are rapidly being established at zoos and research institutions worldwide. However, the captive management of anurans can be challenging, as their reproduction is a tightly regulated hormonal response to environmental stimuli, often unknown or absent in captivity. Consequently, ex-situ breeding efforts tend to be greatly hindered by a paucity of knowledge in anuran reproductive physiology and, for many species on the brink of extinction, time is running out. Assisted reproductive technologies (ARTs), such as exogenous hormone induction of gamete release, artificial fertilization for population augmentation, and cryopreservation for the long-term storage of genetics, have the potential to greatly enhance captive breeding efforts in lieu of natural breeding. Broadly, research aims were to develop assisted reproductive technologies for captive populations of the declining Southern Rocky Mountain boreal toad (Anaxyrus boreas boreas), the critically endangered Mississippi Gopher Frog (Lithobates captio sevosa) and Puerto Rican crested toad (Peltophryne lemur). Specific objectives were to a) trial the efficacy of exogenous hormone treatments on sperm release in male target species by characterizing spermiation response across time; b) investigate methods for increasing sperm longevity through cold-storage and cryopreservation techniques; c) ascertain motility recovery rates and functional capacity of cold-stored and frozen-thawed spermatozoa through artificial fertilization techniques, and; d) apply successfully developed ARTs to determine the feasibility of genetically linking in-situ and ex-situ populations of A. b. boreas, through artificial fertilization of male and female gametes from wild and captive toads, respectively. Research outcomes from this study include the successful development of exogenous hormone protocols, spermiation profiles and sperm cryopreservation techniques for all target species. Additionally, these studies enabled validation of an alternative method for increasing genetic diversity in captive anurans through in-situ-ex-situ gamete linkage. Overall, this research emphasizes the potential value of assisted reproductive technologies as conservation tools for supporting the recovery of endangered frog and toad species worldwide. DEDICATION This manuscript is dedicated to my family and friends for their unwavering support and encouragement in my quest to Save the Frogs, and to the following people: Prof. Roger Downie, who opened up the world of frogs to me all those years ago; Prof. David Houston who introduced me to the concept of cryopreservation for conservation; and Sarah Johnson and the NIMR crew for providing me with five wonderful years of ART training. Finally, but most significantly (P<0.05), I dedicate this research to the frogs, for their inspiring tenacity and for making our world a better place. ‘….the natural world is the greatest source of excitement; the greatest source of visual beauty; the greatest source of intellectual interest. It is the greatest source of so much in life that makes life worth living’ – Sir David Attenborough ii ACKNOWLEDGEMENTS I am expressly grateful to Scott Willard and Andy Kouba for providing me the opportunity to pursue my passion through this PhD, and for the many other opportunities that accompanied it. I would like to extend my sincerest gratitude to my whole committee: Scott Willard, Andy Kouba, Peter Ryan, Jean Feugang and Bill Holt, for guidance and support over the last five years (a special thanks to Bill for negotiating time-zones to serve on my committee). I would also like to thank all the staff at the Colorado Parks and Wildlife’s Native Aquatic Species Restoration Facility in Alamosa, CO, especially Ted Smith, Harry Crockett and Tom Mix, for all their help and hospitality over three wonderful Rocky Mountain summers. I am also grateful to the following CPW staff for their assistance with coordinating our fieldwork (and putting up the tent): Kevin Thompson, Scott Schneider, and Bradley Lambert. Many thanks also to the staff and keepers at the Memphis Zoo, Fort Worth Zoo, and Omaha’s Henry Doorly Zoo for their generosity and assistance. Finally, I would like to acknowledge my wonderful colleagues Natalie Calatayud and Katie Graham for not only being such outstanding lab mates but also delightful friends, Candace Williams (local tour guide/chauffeur/real estate agent) for helping me settle in, GB for getting me through that final stretch, and to the many other people who passed through during my time here, especially our wonderful student workers who so attentively cared for our colony of frogs and toads over the years: iii Michael Robinson, Lindsay Bullock, Christy Steadman, Johnny May, Amanda Mullen, Andrew House and Mathew Christiansen and Allison Julien. iv TABLE OF CONTENTS DEDICATION .................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF TABLES .............................................................................................................. x LIST OF FIGURES ........................................................................................................... xi CHAPTER I. INTRODUCTION: AMPHIBIANS IN PERIL ....................................................1 II. LITERATURE REVIEW .....................................................................................6 Frogs: Evolution’s great success story ..................................................................6 Life history and ecology ..................................................................................7 Hibernation and Estivation ........................................................................8 Reproductive strategies .................................................................................10 Amphibian reproductive system ..........................................................................14 The Hypothalamic-Gonadal-Pituitary (HPG) axis ........................................14 The gonadotropin-releasing hormone systems ........................................17 Modulation of the GnRH system by dopamine .......................................17 Gonadotropin hormones ..........................................................................18 Testes and spermatogenesis ...........................................................................20 Ovaries and ovulation ....................................................................................21 Anuran fertilization .......................................................................................22 The Amphibian Extinction Crisis ........................................................................23 Global forces of destruction ..........................................................................25 The importance of frogs ................................................................................25 The ecological role of frogs .....................................................................25