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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. "... a well-written book. It covers everything involved in equine embryo transfer work from start to finish. The book is written in a manner that will provide valuable information to anyone interested in learning embryo transfer or someone who is already doing some embryo transfer but would like more information. ... I particularly liked the pictures and charts, because they nicely complimented and augmented the text. This book also contains a lot of descriptive statistics for a large number of embryo transfers. It is a good value and will be a nice addition to the library of anyone interested in equine embryo transfer."
-Shawn Jensen, DVM, Bluff Country Equine, Winona, Minnesota, USA, from Vet Med Today: Book Reviews, JAVMA, Vol 247, No. 6, September 15, 2015

Equine Embryo Transfer

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)