Equine Embryo Transfer

Description

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 60’s 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.

Key features

• Clinically relevant step-by-step coverage of the techniques of embryo collection, storage and transport procedures
• Detailed guidelines for the selection of breeding stock and management of the pregnancy are included.
• World renowned expert authors within the equine practitioner community.

Published by Teton New Media in the USA and distributed by Manson Publishing outside of North America.

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)

Patrick M. McCue, DVM, PhD, DACT
Director, Equine Reproduction Laboratory, Colorado State University, College of Veterinary Medicine

Edward L. Squires, PhD
Gluck Equine Research Center, Embryo Transfer Laboratory, Lexington, Kentucky