Equine embryo transfer

W.K. Hendriks DVM Dipl ECAR KNMvD Erk Paardendierenarts

De Graafschap Dierenartsen / Hendriks EQ Repro Consultancy /Seldsum EQ Hendriks

CV – experience 2001 Veterinarian

Utrecht University, Faculty of Veterinary Medicine, Department of Equine Sciences, Division of Equine Reproduction, Utrecht, NL

2001-2002 Teacher

Groenhorst College, Barneveld, NL

2002-2008 Resident equine reproduction

2008-2012 Diplomate ECAR Equine Reproduction) teacher, PhD student

Utrecht University, Faculty of Veterinary Medicine, Department of Equine Sciences, division Equine Reproduction, Utrecht, NL

Maart 2012 Hendriks EQ Repro Consultancy, Ellecom, NL

DVM, Diplomate ECAR Equine Reproduction

Okt 2016 Seldsum EQ Hendriks

Equine assisted reproductive technologies

Febr 2018 PhD “Optimizing equine assisted reproductive technologies”

April 2020 Equine Clinical Director De Graafschap Dierenartsen

Application assisted reproductive technologies:

1. Genetic improvement

2. Combination sport and breeding

3. Abnormal reproductive tract

4. Abnormal health status

5. Import/export

Assisted reproductive technologies(im)possibilities:

Embryo transfer (ET)

Ovum pick up / ovum transfer

In vitro fertilisation (IVF), Intracytoplasmic sperm injection (ICSI)

Sexing semen

Superovulation

Cryopreservation embryo

Genomics (identification DNA markers)

Cloning SCNT

Embryotransfer,trend or future equine breeding industry?

Governor (Totilas x Jazz)

ET foal Jersey x Jazz

Embryo transfer

First experimental ET – 1971

Cambridge, GB (Allen and Rowson)

First ET foal 1974 (non-surgical)(Oguri and Tsutsumi)

Explosive increase ET in the 90’s (mainly

polo ponies)

Last 10 years enormous increase in NL

• The results are improved in NL, due toincrease in knowledge, experience andtechniques.

Wat is embryotransfer?

‣ The embryo is flushed out of the uterus of the donormare and transferred into the uterus of the recipient mare.

‣ The embryonic development takes place in the recipient mare and the recipient mare will nurture the foal after birth.

‣ The donor mare will deliver the genetic material (aside to the stallion), the rest is delivered by the recipient mare.

Why embryotransfer?

More offspring per year of valuable breeding mares

Combination sport and breeding (!)(?)

Mares not capable of growing a pregnancy.

Mares with delivery- or nursing problems.

Breeding a 2-year old mare!!

Mare with a “late” foal, not skipping a year

ET is also the onset of more advanced techniques (IVF, cloning, testing genetic disturbances)

start ET – fertility management

Donor and recipient mares

Stage of cycle

Synchronisation

Insemination donor (type of semen)

Recipient mares (own or lease)

Costs

Can deal with disappointments!!!!!

Cycle mare

Fertility examination

‣ Teasing

‣ Rectal exam (palpation andultrasonograhy)

‣ Evaluation:

Uterus

Ovaries

Ligamenta latae

Vagina

Synchronisation donor – recipient mare

Housed together?

Hormonal intervention:

Starting heat

Use of non-cyclic recipient

mares?

Stimulation ovulation: who

first?

Donor mare - 1

Requirements:

Fertile!

Quality (genetic, sportcapacity, breeding)

Age important factor of success (< +/- 14 years of age higher change)

Use a fertile stallion! (fresh/cooled semen higher success rate than frozen semen)

Stressfactors minimal

Donor - 2

Management

History donor mare!! Examination

donor precluding ET (swab?)

Establish exact date of ovulation

(ovulation is Day 0)

NO stress around ovulation until embryo flush

Day 7-9 after ovulation embryo flush and within thesame day embryo transfer (<24h)

Older mares and use of frozen semen no embryo flush before day 8

Recipient mare - 1

Requirements:

Healthy (mild lameness is accepted)

Fertile

Age (3 - max 14 yr)

Good character (nursing, behaviour)

Height (determines birth weight but not the final height. Preference 1:1)

Swab? , Caslick?

Recipient mare - 2

Management

Synchronisation with donor mare (bringing into heat; PG, Chorulon)

Establish exact date of ovulation by daily exams (ovulation is D0)

The ovulation can occur 24h prior to 72h after ovulation of donor mare (D+1 - D-3)

Scan day after ovulation for 2nd ovulation and free fluid in uterus

Preference 2-3 recipient mares to start up, gives higher chance of suitable recipient mare. But 1 tot 1 synchronisation is possible Start in December with light

Day of embryo flushing

Time of arrival embryo into uterus is depending on:

Moment of flushing

Season

Type of semen

Embryo flushing

D6 – D9, common is D8

Stock (environmental temperature)

Examine donormare (ultrasonography: CL (s),fluid, oedema, bladder)

Sedation donormare?

Materials: bivonacatheter, 3-weg flushing system, embryo

filter, flushing solution Dulbecco’s PBS and FCS or Ringer or

EmCare (37˚ C; volume)

Performing the flush (0,5 – 2 litre; 3-6x) (hygiëne!)

After flushing bringing donor mare back into heat?

Washing the embryo

Embryo flushing - success

Fresh/cooled semen: 52%

Frozen semen: 33%

Influence age donor mare:

≤ 15jr 57%

> 15jr 39% (McCue et al. 2010)

Synchroniation donor – recipient mare

2 days prior until 6 days after ovulation: pregnancy rate 60-100% (Wilsher et al. 2010)

Morula Early blastocyst Expanded blastocyst

Grade 2 expanded blastocyst Grade 4 embryo Unfertilized oocyte(Remains in the oviduct)

Embryotransfer

Direct embryo transfer into recipient mare. Transfer of embryo in straw via a pistolet into uterus of recipient mare (sterile!!!)

Embryo shipment to recipient mare, cooled. Slow cooling of embryo in holding medium during transport (HippoXpress) and transfer within 12 / 24 h

Transplantation technique

1. Examination recipient mare – suitable?(reproductive tract/health)

2. Sedation recipient mare.3. Intense cleaning vulva/perineum – HYGIENE!!!!4. Preparing embryo for transfer (in straw in

pistolet)5. Embryo transfer (Wilsher forceps?)6. After transfer controlling pistolet under

microscope for remaining the embryo.7. Maybe medication (hormons, NSAID’s, AB)

air airembryo

Pregnancy examination

Visible from D9-10, optimal D14-16

Palpation, ultrasonography (circular), location?

Pregnancy rate after embryo transfer

Embryo quality

Experience veterinarian

Technique (surgical vs. non-surgical)

Hygiëne

fresh embryos >75% at D12-14

Cooling and transport until 24 h at 5oC after

embryo flushing doesn’t have a negative effect

on pregnancy rate (Squires et al, 2003)

Statistics

Ca. 50-60 % chance of an embryo after flushing* fresh semen: 70%* cooled semen: 60 %* frozen semen: 40 %

Average 80-90 % chance of pregnancy after transfer into recipient mare

Average 70 % live foaling rate

Concluding: 3 embryo flushes for 1 live foal!!

Costs ET

Average costs per embryo:

-Breeding fee: 1500-Vet (fertility management and flushing): 1200

-Own recipient mare (fertility management and transfer): 1000-Leasing a recipient mare: 2500-3000

-additional costs (studbook, DNA): 200-10 % calculated loss: 800

Conclusion: ET-foal cost at least 3200 euro (own mare) or 4700Euro (lease mare)! excl breeding fee and housing

Critical note on ET

ET cost a lot of money, so the offspring has to bevaluable during gestation or after birth!

Intensive management of donor and recipient mare is necessary, and a lot of factors are influencing thesuccess rate.

Combination (top)sport and embryotransfer is possible, but is not always easy to perform physicaland technical, with fluctuating success rate.

Trend or future?

For valuable mares it is a trend, but is not the future for“common” mares

Embryotransfer offers extra chances for good breeding