Equine embryo transfer

This book briefly reviews the history of equine embryo transfer, covering in clinically practical terms the techniques, equipment, and management protocols currently in use. Embryo transfer has become a big business, especially for breeding racing stock (horses and camels), and is therefore a very important aspect of equine practice. Ed Squires and Pat McCue have been involved with the development of embryo collection and transfer procedures since the early 60s and have both contributed important techniques and innovations to the process through their research and clinical experience. This boo Contents 1. Introduction General Introduction (i.e. what is ET) Advantages and uses of embryo transfer Changes in the ET Industry Breed regulations (include a Table of most major breeds ET, frozen embryos, etc.) Per cycle ET Success = Embryo Collection Rate (50-60%) x Embryo Transfer Pregnancy Rate (70-80%) Goals of the Manual 2. History of equine embryo transfer Livestock Horses Domestic horses as surrogates for endangered equids(?) 3. Reproductive Anatomy and Physiology of the Mare Anatomy of the mare Physiology of the estrous cycle Physiology of early embryonic development and early pregnancy 4. Management of the donor mare Selection of the donor Evaluation of the donor (BSE) Management and Day of breeding (fresh, cooled, frozen semen) Palpation/ultrasound examinations relative to flush daily vs every 6-8 hrs for frozen semen BID if goal is to collect a small embryo at day 6.5 for cryopreservation Induction of ovulation (hCG and deslorelin) Donor mare management (PMIE, fluid, etc.) Estrous cycle control (Lights, P&E, PGF, hCG, Deslorelin, Regumate) Allow a mare to carry to term by approximately 10 years of age Allowing mare to carry own every 3-4 years Effect of repeated flushing on uterine health and embryo recovery # flushes per year recommended Fertility of mares after flushing (i.e. same season) Problem mares (i.e. PMIE, etc.) Maiden mares (young vs older) Post partum mares (i.e. flushing on foal heat) 5. Superovulation History Techniques EFSH Optimal follicle size at onset... Problems - same stallion vs. goal of different stallions Not every mare responds to FSH PAF's and HAF's 6. Embryo Collection Equipment (Box Table) Facilities (stocks vs stall, etc.) Procedure (incl. clean out and wash up) Ultrasound prior to flush in problem mares (PMIE) for fluid detection Day of flush - options day 6.5, 7, 8, 9 Fluid volumes relative to maiden, open and post-foaling mares Number of lavages per flush attempt Rectal manipulation of uterus to move fluid around (massage) Direct visualization of embryos in cup Looking for embryos after each lavage Techniques (Standard vs French, Fernando Rivera) Medications (oxytocin, sedation, buscopan, etc.) Reflush option (Extra flush same day standard next-day reflush option superovulation reflush (=> 50 % embryo recovery relative to ovulation guideline) PGF after flush why (luteolysis - clean up and avoid unwanted carry-own pregnancy) what happens if you do not option to let mare carry 7. Factors affecting embryo recovery Donor age and reproductive status Day of recovery Number of flushes Stallion effects Number of ovulations (single vs. spontaneous multiple, superovulation) Effect of ovulation rate and side of multiple ovulations on recovery rate (Fernando Riera data) Synchronization of ovulations - embryo size and recovery Reflushing (same day, next day) 8. Embryo Handling Equipment - straws, dishes (size, round vs square)(Box Table) Search procedures Debris in dish (how to handle) Miscellaneous items in dish Swirling dish Embryo size expected Embryo morphology expected Hints regarding bubbles, etc. (swirl, let contents settle, then aspirate bubbles along edge) 9. Washing and holding embryos # and sizes of drops Types of holding media how long to hold a fresh embryo Types of wash dishes (flat vs round bottom) Storage vessels (dishes vs straws) 10. Evaluation of embryos Morphology Grade Size Lots of photographs and drawings ET Log (flush and transfer logs) 11. Cooled Storage and Transport of Embryos When to cool (i.e. how many hours between flush and transfer) Cooled embryo technique Time limit for holding embryos Media available (types buffer systems, etc) Ham's F-10 Equipment 12. Cryopreservation of Embryos History of embryo freezing Slow freeze vs Vitrification Selection of embryos (flush days, embryo size, etc.) Vitrification technique (supplies, method) Storage of vitrified embryos Warming and transfer Pregnancy results 13. Management of Recipient mares What makes a good recipient Selection - age, size, parity, temperament, physical health History of mares (barren, maiden, foaling) Examination schedule Examination of recipients - 5 day check pass system Housing recipient mares Synchronization options (new data from perla) general 'window' of synchrony (+1 to -3 or -4) Line up recipient with embryo characteristics (fine tune) Recipient:Donor Ration (3:1) for synchronization Individual recipient for single donor (1:1) - how to manage 'Floating' recipient herd Synchronization schemes Optimal day(s) of transfer Management after transfer (housing, hormones, etc.) Use of non-cycling, ovariectomized, XO and pregnant mares as recipients Using the donor mare as her own recipient (in the event of twin embryos) 14. Transfer Procedures Surgery (midline, flank) [Old school] vs Nonsurgical/transcervical Speculum procedure (Allen and Wilshire) Equipment for nonsurgical (Box Table) Day of transfer Medications (pre and post) Prostaglandin release during transcervical transfer (p4 Graph) Technique - details The 'art of transfer' 15. Factors affecting pregnancy rates Age and reproductive status of donor mare Embryo age, quality and size Transfer technique, technician variability Recipient factors Expected pregnancy rates (day 16 vs day 50 vs foaling) Carry to term data (AQHA data) Twins/Triplets from transfer of a single embryo 16. Pregnancy examination after transfer Days of examination (11, 12, 14, 16, 25, etc) Relationship between embryo size at transfer and first day visible on ultrasound (graph) Percentage of truly pregnant recipients with embryos visible at 11, 12, 14, 16 days (graph) 17. Disease transmission with embryos 18. International transport of embryos 19. Miscellaneous Embryo micromanipulation (splitting) Embryo sexing 20. Future directions of equine embryo transfer Superovulation Early pregnancy factor - know when to flush Improvement in reproductive management of problem mares (PGE oviduct) Assisted reproduction Embryo biopsy for genetic diagnosis Appendix 1: ET Equipment and supplies Sources Catheters Fluid types (LRS vs Complete vs old style PBS) osmolarity pH stability/shelf life protein source (FCS, albumen vs PVA) to prevent embryos from sticking ingredients (general) buffer systems (if any) Y tubing Filter cups - types (list and photos), how to use them (i.e. fill with fluid as per Fernando Rivera) direct visualization vs pour-off) Search dishes (round vs square size gridded vs plain) Microscopes and micrometer (types of scopes magnification, glass - clear vs frosted sources new vs used) Cleaning procedures (what can be re-used) autoclave enzyme cleaning gas sterilization) Front Cover; Preface; Acknowledgements; Table of Contents; CHAPTER 1 - INTRODUCTION; CHAPTER 2 - HISTORY OF EQUINE EMBRYO TRANSFER; CHAPTER 3 - REPRODUCTIVE ANATOMY AND PHYSIOLOGY; CHAPTER 4 - MANAGEMENT OF THE DONOR MARE; CHAPTER 5 - SUPEROVULATION; CHAPTER 6 - EMBRYO COLLECTION; CHAPTER 7 - FACTORS AFFECTING EMBRYO RECOVERY; CHAPTER 8 - EMBRYO HANDLING; CHAPTER 9 - EVALUATION OF EQUINE EMBRYOS; CHAPTER 10 - COOLED-TRANSPORTED EMBRYOS; CHAPTER 11 - CRYOPRESERVATION OF EQUINE EMBRYOS; CHAPTER 12 - MANAGEMENT OF RECIPIENTS; CHAPTER 13 - TRANSFER OF EQUINE EMBRYOS CHAPTER 14 - PREGNANCY EXAMINATION AFTER TRANSFERCHAPTER 15 - PREGNANCY RATES AFTER TRANSRER; CHAPTER 16 - FACTORS AFFECTING PREGNANCY RATES; CHAPTER 17 - DISEASE TRANSMISSION; CHAPTER 18 - INTERNATIONAL TRANSPORT OF EMBRYOS; CHAPTER 19 - RELATED EMBRYO TECHNOLOGIES; CHAPTER 20 - FUTURE DIRECTIONS OF EQUINE EMBRYO TRANSFER; APPENDIX 1 - EMBRYO TRANSFER SUPPLIES AND EQUIPMENT; APPENDIX 2 - FORMULARY FOR EQUINE EMBRYO TRANSFER; Back Cover