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 book captures the clinical experience, so far, and applies it directly to equine practice. The book is of great value to general equine practitioners for reference, equine reproduction specialists, animal science at the graduate level (equine track), and breeders.

Contents1. IntroductionGeneral Introduction (i.e. what is ET) Advantages and uses of embryo transferChanges in the ET IndustryBreed 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 Manual2. History of equine embryo transfer LivestockHorsesDomestic horses as surrogates for endangered equids(?)3. Reproductive Anatomy and Physiology of the MareAnatomy of the marePhysiology of the estrous cycle Physiology of early embryonic development and early pregnancy4. Management of the donor mareSelection of the donorEvaluation 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 cryopreservationInduction 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 ageAllowing mare to carry own every 3-4 yearsEffect of repeated flushing on uterine health and embryo recovery # flushes per year recommendedFertility 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. SuperovulationHistoryTechniquesEFSHOptimal follicle size at onset…Problems – same stallion vs. goal of different stallionsNot every mare responds to FSHPAF’s and HAF’s6. 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 detectionDay of flush – options day 6.5, 7, 8, 9Fluid volumes relative to maiden, open and post-foaling maresNumber of lavages per flush attemptRectal manipulation of uterus to move fluid around (massage)Direct visualization of embryos in cupLooking for embryos after each lavageTechniques (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 carry7. Factors affecting embryo recovery Donor age and reproductive statusDay of recovery Number of flushes Stallion effectsNumber 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 recoveryReflushing (same day, next day)8. Embryo HandlingEquipment – straws, dishes (size, round vs square)(Box Table)Search proceduresDebris in dish (how to handle)Miscellaneous items in dishSwirling dish Embryo size expectedEmbryo morphology expectedHints regarding bubbles, etc. (swirl, let contents settle, then aspirate bubbles along edge)9. Washing and holding embryos# and sizes of dropsTypes of holding media; how long to hold a fresh embryoTypes of wash dishes (flat vs round bottom)Storage vessels (dishes vs straws)10. Evaluation of embryosMorphologyGradeSizeLots of photographs and drawingsET Log (flush and transfer logs)11. Cooled Storage and Transport of EmbryosWhen to cool (i.e. how many hours between flush and transfer)Cooled embryo techniqueTime limit for holding embryosMedia available (types; buffer systems, etc) Ham’s F-10Equipment 12. Cryopreservation of EmbryosHistory of embryo freezingSlow freeze vs VitrificationSelection of embryos (flush days, embryo size, etc.)Vitrification technique (supplies, method)Storage of vitrified embryosWarming and transferPregnancy results13. Management of Recipient maresWhat makes a good recipientSelection – age, size, parity, temperament, physical healthHistory of mares (barren, maiden, foaling)Examination scheduleExamination of recipients – 5 day check; pass systemHousing recipient maresSynchronization 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 synchronizationIndividual recipient for single donor (1:1) – how to manage ‘Floating’ recipient herdSynchronization schemesOptimal day(s) of transferManagement after transfer (housing, hormones, etc.)Use of non-cycling, ovariectomized, XO and pregnant mares as recipientsUsing the donor mare as her own recipient (in the event of twin embryos)14. Transfer ProceduresSurgery (midline, flank) [Old school] vs Nonsurgical/transcervicalSpeculum procedure (Allen and Wilshire)Equipment for nonsurgical (Box Table)Day of transferMedications (pre and post)Prostaglandin release during transcervical transfer (p4 Graph)Technique – detailsThe ‘art of transfer’15. Factors affecting pregnancy rates Age and reproductive status of donor mare Embryo age, quality and sizeTransfer 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 embryo16. Pregnancy examination after transferDays 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 embryos18. International transport of embryos 19. MiscellaneousEmbryo micromanipulation (splitting)Embryo sexing20. Future directions of equine embryo transferSuperovulationEarly pregnancy factor – know when to flushImprovement in reproductive management of problem mares (PGE oviduct)Assisted reproduction Embryo biopsy for genetic diagnosis Appendix 1: ET Equipment and suppliesSourcesCathetersFluid 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 tubingFilter 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)