Artificial insemination and embryo transfer in horses

Question 1

Why use AI in stallions?

Answer 1

Competing stallions can still breed
Export to a world wide market
Physical reasons e.g. size differences
Increase stallion utilisation
Reduce risk of injury and disease
Preserve scarce bloodline
Castrate stallion and still breed from it
Frozen semen can be stored indefinitely

Question 2

Expected pregnancy rates for reasonably fertile mare and good quality semen (AI)

Answer 2

55-70% per cycle

Question 3

What are the three methods of semen storage

Answer 3

Fresh non-chilled
Chilled
Frozen

Question 4

What is the best extender for semen

Answer 4

Semen extenders should be non-spermotoxic, give an energy source, have cryoprotectant agents, and contain antibiotics to inhibit microbes. Commercial extenders (mostly Kenny-based formulas) are mostly used for non-chilled and chilled storage.

Question 5

What is the best temperature to store semen for 2-6 hrs

Answer 5

Semen should be kept in a light-proof, air-tight container at room temperature (20-25°C) in the short term (≤6 hours) but cooled slowly over 2-3 hours to 4°C for longer term storage.

Question 6

What is the equitainer

Answer 6

The Equitainer is a special container designed for cooling and transporting equine semen. It consists of a strong container with a snap-lock, housing frozen canisters, insulation, thermal ballast bags and an isothermalizer. The Equitainer is special in that it has the ability to lower the temperature of the semen package approximately 0.3°C degrees every minute until the temperature stabilizes at 4-6°C.

Question 7

What is the classic protocol for processing stallion semen for cryptopreservation?

Answer 7

Dilute the ejaculate, filter, or centrifugation
Removal of the supernatant and addition of a freezing extender that contains a cryoprotectant
Cooled and stored in liquid nitrogen

Question 8

Ages of mares used for fresh semen

Answer 8

Maiden mares from 3-20+
Lactating mares 4-20+
Barren mares 5-20+

Question 9

Age of mare used for frozen semen

Answer 9

Maiden mares 3-12
Lactating mares 4-18
Barren mares 5-16

Question 10

What is the commonly used method to induce ovulation in mares

Answer 10

IV administration of 3000iu hCG if mare has been established to be in oestrus with a soft follicle at least 3.5cm

Question 11

What % of mares will ovulate in the 24-48hr period following hCG administration

Answer 11

Approx 85%

Question 12

What is used to induce luteolysis in mares

Answer 12

Prostaglandin analogues as long as the CL is >5 days old

Question 13

What is the timing parameter for insemination post ovulation for optimal conception rates

Answer 13

Insemination no longer than 6 hrs post ovulation, irrespective of storage method

Question 14

What is the maximum interval from insemination to ovulation if insemination is to be performed pre-ovulation

Answer 14

Determined by the longevity of the sperm in the female reproductive tract and depends on storage method

Question 15

What is the maximum time period that fresh semen can be inseminated pre-ovulation

Answer 15

Up to 48hrs before

Question 16

What is the optimal time for AI with chilled semen

Answer 16

24hrs before ovulation

Question 17

What is the ideal timing for insemination for mares inseminated with frozen semen

Answer 17

Ideally no more than 12hrs before ovulation but ideally 6hrs either side

Question 18

Examination following insemination

Answer 18

Mare should be checked for ovulation 24-48 hours later (fresh semen), 12-24 hours later (chilled semen) or within 6-12 hours (frozen semen) to order a second delivery of semen if time of ovulation has been miscalculated.

Question 19

Inflammatory response to breeding

Answer 19

Normal and beneficial acute inflammatory response. Defective uterine clearance of inflammatory exudate may result in mares failing to become pregnant.

Question 20

Post ovulation insemination

Answer 20

Should be performed within 6 hours post ovulation to achieve optimal conception rates

Question 21

Per cycle conception rates with semen stored in different ways

Answer 21

Aim for 75-80% with fresh extended semen, 60-70% with cooled semen and 50-65% with frozen-thawed semen

Question 22

What is low dose videoendoscopic insemination

Answer 22

• A breeding technique where a small volume of semen is directly inseminated onto the uterotubal papilla at the tip of the uterine horn
  60-70% conception rate per cycle, can be good for sub-fertile mares

Question 23

What is embryo transfer

Answer 23

Involves the flushing and transferring of an embryo from a donor mare to a recipient mare
Increasingly popular for thoroughbreds

Question 24

What is a donor-recipient synchronisation in embryo transfer

Answer 24

The process of synchronising the ovulation of the recipient mare with the donor mare to optimise success
Recipient mares should ovulate 0 to -3 days wrt the donor mare

Question 25

What is embryo recovery in embryo transfer

Answer 25

The process of recovering embryos from the donor mare for transfer to the recipient mare.
Routinely, it is performed on day 7 (expanding blastocyst, 250-350µm diameter) or day 8 (expanded blastocyst > 400µm and visible to the naked eye) after ovulation (day 0) for routine transfer.
For embryo freezing or splitting to make identical twins, embryo recovery is performed on day 6 (morula or early blastocyst; 200-300µm).

Question 26

What is embryo transport in embryo transfer

Answer 26

Embryo transport involves transporting the embryo from the donor mare to the recipient mare.
The embryo survives well in transport medium, up to 12-24 hours at 4°C in an Equitainer.
For deep freezing in liquid nitrogen, small (young) embryos (i.e. morulae or early blastocysts of < 300µm) are required.

Question 27

What are equine-specific problems in embryo transfer

Answer 27

• Superovulation: the mare does not respond to exogenous gonadotrophin therapy for superovulation; multiple follicles inhibit each other during passage towards the ovulation fossa.
• Oviductal transport: the late arrival of the embryo into the uterus (i.e. 4-6 days post ovulation) greatly limits the recovery of embryos suitable for splitting or freezing.
• Therefore, it is necessary to maximise embryo recovery rate by other means (e.g. shorten donor cycles by PGF2α therapy; split embryos to produce twins).

Question 28

Future developments for modern breeding technologies

Answer 28

• Sex sorting of sperm
• Oocyte recovery for intracytoplasmic sperm injection (ICSI)
• Cloning