Improving Assisted Reproductive Technologies in the Endangered Black-Footed Ferret: Artificial Insemination and Sperm Cryopreservation Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Tara Leigh Strickler, B.S. Graduate Program in Animal Sciences The Ohio State University 2010 Thesis Committee: Joseph S. Ottobre, Ph.D., Advisor Douglas R. Danforth, Ph.D. Mandi M. Vick, Ph.D. Copyright by Tara Leigh Strickler 2010 Abstract The black-footed ferret (Mustela nigripes) is an endangered mustelid native to the grasslands of the North American western Great Plains. Recovery efforts for the species warranted the capture of the last remaining 18 black-footed ferrets for ex situ propagation. Despite vigorous attempts to equalize genetic representation, reproductive inefficiency has been documented. Black-footed ferret spermatozoa suffer significant cryodamage and a decline in percentages of normal sperm in fresh ejaculates has been observed. The decline in reproductive fitness has been attributed to the low number of founders (n = 7) that comprise the current population. The first aim of this project was to improve genetic diversity in the black-footed ferret population by using cryopreserved spermatozoa from a genome resource bank. Black-footed ferret females (n = 4) chosen for laparoscopic artificial insemination were evaluated for estrus. Ovulation was induced with luteinizing hormone. Sires from the genome resource bank were selected based on genetic representation. Two of four (50%) females inseminated became pregnant and produced kits (mean litter size, 1.0). The second aim of this project was to investigate three commercially available density gradient products (PureSperm, ISolate, Enhance-S Plus) to develop a technique to improve the quality of black-footed ferret frozen-thawed sperm fractions for artificial insemination. Domestic ferret ejaculates (used as a model for this study) were collected via electroejaculation and frozen according to standard procedures. Frozen- ii thawed spermatozoa were divided into 4 groups and either received a simple washing (control) or were subjected to one of three gradient treatments. Results indicated that PureSperm filtration produced the best sperm fractions with the highest percentages of structurally normal and motile sperm. The third aim of this project was to investigate cholesterol-loaded cyclodextrin (CLC) technology as a means to develop a pre-freeze treatment to improve sperm cryosurvival. Domestic ferret ejaculates (chosen as the model for this study) were collected via electroejaculation, evaluated, and were divided among three groups: 1) control, 2) low dose CLC (1.5 mg CLC/ 120 x106 sperm/ml), and 3) high dose CLC (3.0 mg CLC/ 120 x106 sperm/ml). After receiving treatment, ejaculates were subjected to standard cryopreservation methods. Results indicated significant improvement in acrosomal integrity due to treatment, but there was no significant difference in this regard between low and high doses. CLC treatment appeared to have no significant effect on sperm motility or forward progression. Offspring that resulted from this study (n = 2) are currently considered the most genetically valuable animals in the black-footed ferret population. However, further studies are warranted in these individuals and subsequent generations to determine whether increased genetic fitness improves sperm viability in this species. Moreover, future artificial inseminations using cryopreserved sperm from the black-footed ferret genome resource bank may benefit from PureSperm density gradient processing since application of this product enhanced cryopreserved sperm fractions in the domestic ferret. Although use of CLC as a pre-freeze treatment did not appear to affect sperm longevity, iii further investigations are warranted to confirm the degree of membrane manipulation that occurs with CLC application prior to cryopreservation, which is shown to improve acrosomal integrity in domestic ferret frozen-thawed sperm. iv Acknowledgements I am grateful for having been granted such an incredible opportunity. Initially inspired by the groundbreaking births of Andalas, a Sumatran rhinoceros, and later Tai Shan, a giant panda, I set off for graduate school in 2006 to study reproduction. At that time, my lifelong career goal was to one day help propagate an endangered species. There are many people who played critical roles in my journey to graduate school and I would like to acknowledge them at this time. First, I would like to acknowledge my friends and co-workers at the Bodine Center for Cancer Treatment at Thomas Jefferson University in Philadelphia where I was employed prior to attending graduate school. Dr. Phyllis Wachsberger and Dr. Randy Burd provided the support and encouragement that I needed to seek graduate schooling and I will always honor them with fond memories. After arriving at The Ohio State University to pursue a doctorate degree, it soon became apparent that my path was not quite going as planned. Consequently, I soon realized that I needed to pursue another direction. Dr. Steve Rissing in the Department of Ecology, Evolution, and Organismal Biology was there to help me figure out the next step. His guidance and unwavering backing as a temporary graduate advisor in E.E.O.B. kept me afloat during uncertain times. His advice to pursue other projects within the university and to accept the internship at Smithsonian National Park‟s v Conservation and Research Center was key to my success. I cannot positively say that I‟d be where I am today without his support. This project would not have gotten off the ground without Dr. JoGayle Howard, theriogenologist at Smithsonian National Zoological Park. Striving for excellence in everything she does, Dr. Howard has inspired me more than she will ever know. I am truly grateful for her incredible mentorship and steadfast dedication to this project. She may be known around the world for her scientific works, but one of her unsung successes is changing my life, opening doors that perhaps would have remained closed. Although a project was in hand, obtaining an actual degree would not be possible without educational support from the Department of Animal Sciences at The Ohio State University. I will always be grateful to Dr. Joseph Ottobre, my graduate advisor and professor. His willingness to work with me on this project has made this manuscript and my degree possible. The kindness and support that I‟ve received from Dr. Ottobre and from the entire Department has not gone unnoticed and it truly makes this Department great. I would also like to thank Dr. James Kinder, departmental Chair, as well as my entire graduate committee, Dr. Joseph Ottobre, Dr. Douglas Danforth, and Dr. Mandi Vick, for all their efforts toward this manuscript and myself. Additional support I've received includes two very important friends. My dear friends Dr. Mandi Vick, Smithsonian Conservation and Research Center post-doctoral fellow, and Dr. Joel Piperburg, professor at Millersville University, have been like family members to me over the past few years. Dr. Vick is an outstanding person who started out as a mentor to me during my internships at The Wilds and later at Conservation and vi Research Center. Dr. Vick coached me through the process of proposal writing, research ideas, and provided statistical assessments for this manuscript. Over the years we've become great friends and I am truly blessed to have met her. Dr. Joel Piperberg has also been incredibly instrumental to my success as a student, offering valuable insight to my professional goals. He is the one faculty member who has known me the longest and has always encouraged me to pursue professional schooling. I highly respect his opinions and have sought his advice for every turning point in my career. It is not often that we meet truly incredible people and I wish both of them every happiness. Finally, I would like to thank my family for their love, especially my sister and brother-in-law, Tanya and Tim Hopkins, for lending emotional and financial support during a time when I was not receiving funding. My family, friends, peers, and co- workers that are scattered throughout the tri-state area of Ohio, Pennsylvania, and Virginia have all contributed a critical piece to this puzzle and I find myself humbled for all of the friendship, support, and kindness. vii Vita June 1999 ....................................................Conestoga Valley Senior High School July 1999 to April 2006 ..............................Catalogue Bindery Employee R.R. Donnelley & Sons May 2001 ....................................................A.S. Veterinary Technology Harcum College July 2001 – September 2001 ......................Intern Veterinary Technician Cincinnati Zoo & Botanical Gardens May 2004 ....................................................B.S. Biology Millersville University June 2004 – August 2006 ...........................Research Technician Bodine Center for Cancer Treatment Thomas Jefferson University August 2006 – December 2006 ..................Graduate Research Assistant Department of Evolution, Ecology, and Organismal Biology The Ohio State University January 2007 – June 2007 ...........................Graduate Teaching Assistant Department of Evolution, Ecology, and Organismal Biology The Ohio State University July 2007 – December 2007 .......................Intern Department of Reproductive Sciences Smithsonian National