Xenogenous Intrafallopian Transfer of Horse (Equus Caballus) Gametes
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Xenogenous Intrafallopian Transfer of Horse (Equus caballus) Gametes By Gemechu Wirtu Thesis submitted to the Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of Master of Science In Veterinary Medical Sciences Approved: ___________________ Thomas L. Bailey, Chair ___________________ ___________________ William B. Ley Francis C. Gwazdauskas ___________________ ___________________ Nikola A. Parker John J. Dascanio July 29,1999 Blacksburg, Virginia Keywords: horse, oocyte recovery, in vitro, maturation, xenogenous, fertilization Xenogenous Intrafallopian Transfer of Horse (Equus caballus) Gametes By Gemechu Wirtu Committee Chair: Thomas L. Bailey Department of Veterinary Medical Sciences ABSTRACT This study was undertaken to evaluate fertilization and early embryo development of in vitro matured (IVM) horse oocytes following transfer with homologous sperm to the oviduct of estrous ewes. A total of 1023 follicles (5.1 per ovary) were found after processing 202 slaughterhouse ovaries by aspiration and subsequent slicing. Most follicles (79%) were less than 20-mm in diameter. Six hundred sixty-seven oocytes were recovered (3.3 per ovary; recovery rate, 65%). About two-thirds of oocytes were recovered by slicing, which yielded twice the number of oocytes as aspiration. Sixty four percent cumulus oocyte complexes (COCs) recovered by each method were grade A and the overall distribution of oocytes by grade was not affected by the method of recovery. Oocytes underwent IVM for an average of 41-h and were subjected to either in vitro fertilization (IVF) or xenogenous gamete intrafallopian transfer (XGIFT). At the onset of IVM, 83% COCs had compact cumulus investment. At the end of IVM, 78% COCs showed cumulus expansion. The expansion score was not improved with increasing the IVM duration from 32.3 to 50.3 h. Five (15%) IVF oocytes showed changes indicative of fertilization and two cleaved to 3 and 4-cell stages. Oviducts of 16 ewes were use for XGIFT, which involved surgical transfer of an average of 13 oocytes with 40x103 capacitated spermatozoa per oocyte. Of 259 oocytes transferred, 36 (14%) were recovered between 2 to 7 d post XGIFT and 13 (36%) showed cleavage ranging from the 2-cell to hatching blastocyst stage. The ovarian status of ewes and ligation of the uterotubal junction (UTJ) at the time of XGIFT, or the duration gametes were allowed to reside in the uterine tube, did not affect the recovery and cleavage rate. However, the most advanced stage embryos were recovered from ewes ovulating shortly after XGIFT. Fertilization following XGIFT was further demonstrated by the detection of ZFY loci in one embryo. This study demonstrated, for the first time, that horse embryos could be produced in a non-equine species. However, further studies focusing on the establishment of pregnancy in the mare using such embryos and improvement of the recovery and fertilization rates following XGIFT are recommended for use of XGIFT in horse assisted reproduction. ACKNOWLEDGEMENTS First of all, I would like to extend my deepest gratitude to the USIA/Fulbright program for supporting me to come to the United States for graduate study. Dr. Bailey: thanks for the idea that led to this project and for your active involvement in the project even on weekends, holidays and after-hours. Thank you very much also for supporting me in many aspects of my stay at Virginia Tech starting from the first day of my arrival in Blacksburg. I am grateful to you for your understanding and the continued confidence you had in me. Drs. Ley, Gwazdauskas, Parker and Dascanio: thanks for serving as members of my advisory committee. Dr Ley, thanks a lot for ensuring the availability of stallion semen even after short notices. I have also been impressed by your quick responses to my proposals and thesis documents. Dr. Parker, thank you very much for offering to help with surgery whenever Dr. Bailey was unable to make it. Dr. Gwazdauskas, thank you very much for all the support, for allowing me to use your facilities and for bringing Dr. Chauhan to your lab. Dr. Manmohan Chauhan, many thanks for your unreserved assistance, direct participation in the project and also for teaching me IVF and related technologies. I wouldn’t imagine myself at this phase if you weren’t there for me. I received the generous support of many other individuals who directly or indirectly contributed to the successful completion of my program at Virginia Tech. Lisa Knepshield was helpful by offering advice and by timely actions on administrative issues at IIE, New York office. Stephanie Milburn, Dr. Sher Nadir, John Strauss, Taryn Brandt, Shawne Spencer and Erick Spencer, thank you for all the help with sheep and for the fun. Sexing of embryos using PCR could have not been successful without the excellent assistance received from Dr. Stephen Boyle, Julie Bard, Sherry Poff and Steve Butler. Dr. Ludeman Eng, Dr. John Lee and Dee Shephard offered administrative assistance and support. Dr. Gregory Lewis donated three ewes and Meghan Wulster has been of great help in facilitating this. Chris Wakley, Mary Nickle, Suzan Anderson, Steve Miller, Barbara Dryman and Kevin Weaver had to fall, get wounded etc. while assisting with the sheep that often went crazy. Ralph Roop, Chuck Randel, Cara Talbert and Dr. Wynne Digrassie offered technical support and Daniel Ward, Lire Ersado and Dr. Francoise Elvinger assisted with statistics. Connie and Don Heindel have been very nice family in Blacksburg. Belhu, Lire and Yonael, you have been wonderful roommates. THANK YOU ALL. iii TABLE OF CONTENTS ABSTRACT ii ACKNOWLEDGEMENTS iii LIST OF FIGURES vi LIST OF TABLES vii LIST OF ABBREVIATIONS viii 1. INTRODUCTION 1 2. LITERATURE REVIEW 3 2.1. Gametogenesis, Sperm-Oocyte Interaction and Fertilization 3 2.2. Early Embryo Development 7 2.3. Assisted Reproductive Technologies (ART) 7 2.3.1. Why ART in the Horse? 7 2.3.2. Impediments to the Development of ART in the Horse 9 2.3.3. Oocyte Collection, Determination of Quality and In Vitro Maturation 10 2.3.4. In Vitro Fertilization and Related ART 12 2.3.5. Gamete Intrafallopian Transfer 16 2.3.6. Other ART in the Horse 16 3. MATERIALS AND METHODS 19 3.1. Media, Reagents, Drugs and Other Supplies 19 3.2. Acquisition of Ovaries, Oocyte Recovery and In Vitro Maturation 21 3.3. Semen Collection, Sperm Capacitation and In Vitro Fertilization 22 3.4. Preparation of Ewes and Xenogenous Gamete Intrafallopian Transfer 23 iv 3.5. Oocyte/Embryo Recovery from Ewes, and Evaluation 25 3.6. Determination of Embryo Sex 26 3.7. Statistical Analysis 28 4. RESULTS 29 4.1. Ovarian Status and Oocyte Recovery 29 4.2. In Vitro Maturation and In Vitro Fertilization 33 4.3. Xenogenous Gamete Intrafallopian Transfer and the Recovery and Evaluation of Oocytes/Embryos 35 5. DISCUSSION 43 6. CONCLUSIONS 49 7. LITERATURE CITED 50 8. VITA 58 v LIST OF FIGURES Figure 1. Distribution of the number of follicles from slaughterhouse horse ovaries subjected to two methods of oocyte recovery 28 Figure 2. Embryonic development of horse oocytes following xenogenous gamete intrafallopian transfer in sheep 34 Figure 3. Horse oocytes/embryos recovered from the uterine tubes of sheep following xenogenous gamete transfer 37 Figure 4. Determination of the sex of horse embryos recovered at 7-d following xenogenous gamete intrafallopian transfer in the sheep 40 vi LIST OF TABLES Table 1. Summary of xenogenous gamete intrafallopian transfer studies undertaken in different species 6 Table 2. Methods of horse oocyte recovery and success rates from slaughterhouse ovaries and live mares as reported in different studies 15 Table 3. Distribution of follicles by size (mm) on slaughterhouse horse ovaries as estimated during oocyte recovery by two methods 31 Table 4. Distribution by grade of horse oocytes recovered from slaughterhouse ovaries by using aspiration and slicing 32 Table 5. Degree of expansion of cumulus investment of horse oocytes at the beginning and end of in vitro maturation. 34 Table 6. Effect of the duration of in vitro maturation on cumulus expansion of horse oocytes recovered from slaughterhouse ovaries 34 Table 7. Effect ovarian state of ewes at the time of transfer on the recovery rate and development of horse oocytes in sheep 37 Table 8. Effect of time of recovery and ligation of the uterotubal junction on the recovery and development rate of horse oocytes subjected to xenogenous intrafallopian transfer in sheep40 vii LIST OF ABBREVIATIONS AI: Artificial insemination ART: Assisted reproductive technology BO: Brackett and Oliphant (medium) BSA: Bovine serum albumin CH: Corpus hemorrhagicum COC: Cumulus-oocyte complex D: Day(s) ET: Embryo transfer FCS: Fetal calf serum FSH: Follicle stimulating hormone GIFT: Gamete intrafallopian transfer H: Hour (hours) ICSI: Intracytoplasmic sperm injection IFOT: Intrafollicular oocyte transfer IM: Intramuscular (injection) IV: Intravenous (injection) IVC: In vitro culture (of embryos) IVF: In vitro fertilization IVM: In vitro maturation of (in vitro matured) oocytes LH: Luteinizing hormone Min: Minute(s) PBS: phosphate buffered saline PCR: Polymerase chain reaction TCM: Tissue culture medium TVFA: Transvaginal follicular aspiration UTJ: Uterotubal junction XGIFT: Xenogenous gamete intrafallopian transfer ZF: Zinc finger (protein) ZP: Zona pellucida viii 1 . INTRODUCTION Domestic animals, including the horse, suffer from infertility that lowers their lifetime productivity and reduces the number of offspring that could be obtained from a sire or a dam. The prevalence of this problem coupled with the desire of people to understand and subsequently control the reproductive process has led to the development of novel techniques such as artificial insemination (AI) and embryo transfer (ET). In vitro processing of oocytes is an emerging assisted reproductive technology (ART) which is well developed in the human and bovine for use in routine reproductive management. Researchers working with the horse have yearned to develop and use these technologies.