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Research Update

Vitrification of Equine Embryos

Effect of polyvinyl alcohol (PVA) in vitrification of equine embryos

J.Hudson1,T.Meredith2 J.Bailey1

1-Royal Vista Southwest, Purcell, OK, 2- Partnar Animal Health, Port Huron, MI

Techniques for cryopreserving equine embryos have advanced in recent history to the point most practitioners can reliably collect and either vitrify embryos on-site or elect to ship embryos to referral centers where equine embryos are routinely vitrified. In recent studies, it has been demonstrated that equine embryos can be vitrified, then warmed and transferred achieving comparable pregnancy rates to fresh or cooled embryos. There are endless possibilities when it comes to managing vitrified embryos. Many owners elect to simply cryopreserve valuable genetics from a particular stallion and mare combination. Others sell and purchase cryopreserved embryos and have them transferred at a specified time so a resulting foal can be born at a chosen time of the year. Many owners have aspirations to sell embryos in the international market and the technology to import or export equine embryos has been desired since the commercialization of the equine embryo transfer process. At the present time, biosecurity issues limit practitioners and owners from importing or exporting embryos vitrified in a fetal calf serum media. Therefore, a demand exists for an alternative solution which would allow for importation and exportation of vitrified embryos. The goal of this present pilot study was to compare pregnancy rates between two groups of equine embryos vitrified in either fetal calf serum or polyvinyl alcohol.
Light horse mares were chosen based on the quality of their reproductive tract. Mares were bred to a stallion with proven fertility. Ovulation was induced in thirteen (13) mares by administration of 2500 IU of human chorionic gonadotropin (hCG) or a GnRH analog once a preovulatory follicle > 35 mm was detected with good uterine edema and a relaxed cervix. A non-surgical transcervical embryo flush procedure using Emcare® flush media was performed on day 6.5 post-ovulation. Using the International Embryo Transfer Society (IETS) embryo grading method, embryos recovered were evaluated for size, grade, and morphology. Late morula or early blastocyst stage embryos with a grade of 1-2 and < 300 µm in diameter were randomly assigned to one of two treatment groups. Embryos in Group 1 (n=7) were washed four times in Emcare® holding medium and then vitrified in media containing fetal calf serum (FCS). Embryos in Group 2 (n=6) were also washed in Emcare® holding medium and then vitrified in media containing polyvinyl alcohol (PVA). Embryos were allowed to warm after randomly selecting them from the liquid nitrogen tank. The straw containing the embryo and cryoprotectants were held in room temperature air for 10 seconds followed by submerging them in a 20ºC water bath for an additional 10 seconds. The straw was held in a horizontal position for 6 to 8 minutes allowing the fluid columns to equilibrate before transferring the contents of the straw directly into an available recipient that ovulated within 48 hours of the donor mare. A non-surgical transcervical transfer technique was utilized using a Cassou gun.
A 71.4 % (5 of 7) pregnancy rate was established in the group containing fetal calf serum while an 83.3% (5 of 6) pregnancy rate was established in the group containing polyvinyl alcohol. All pregnancies were detected per rectum using ultrasonography at days 11 through 25 (heart beat). Results of this pilot study indicate that late morula and early blastocyst equine embryos (< 300 µm) can be collected and vitrified in media containing fetal calf serum or polyvinyl alcohol without losing embryo viability. This study suggests the polyvinyl alcohol solution may be a comparable option for vitrification with the added benefit of qualifying for importation and/or exportation of equine embryos.

Mare Synchronization

To be successfully vitrified, an equine embryo must developmentally be a late morula or early blastocyst. Collecting embryos 8 days after the administration of hCG to the donor mare has proven ideal. This time frame should be comparable as flushing the donor mare 6.5 days after the detection of ovulation if hCG is administered.

Example:

  1. 1) Mare is administered hCG at 1 pm Friday the 1st of May after ultrasonography reveals a 38 mm diameter preovulatory follicle with good associated uterine edema and a relaxed cervix
  2. 2) Mare is artificially inseminated with good quality fresh or cooled semen 24 hours post hCG administration on Saturday the 2nd of May
  3. 3) Mare is ultrasounded on Sunday the 3rd of May and ovulation is detected. Human chorionic gonadotropin should induce a mare to ovulate 36 to 42 hours post-injection or 1.5 days after administration
  4. 4) Mare is flushed on Saturday the 9th of May at 1 pm to retrieve a late morula or early blastocyst to be vitrified

If a GnRH analog is utilized to induce an ovulation in the mare, the time frame should be adjusted appropriately to assure a late morula or early blastocyst is retrieved after flushing the donor mare.Remember, in most cases the GnRH analog should induce an ovulation between 42 to 48 hours post administration.

Vitrification Procedures

Identifying and utilizing the proper size and grade of embryo is imperative to the success of vitrification. A late morula equine embryo should measure around 160 µm in contrast to an early blastocyst which measures around 190 to 300 µm. Recovered embryos can easily be searched for and discovered utilizing a dissecting microscope. A linear eye piece micrometer is used to measure the diameter of each embryo. Morphological grading should be consistent with the size of each embryo. The IETS grading method is a common classification system. Figure 1

Required Equipment (Figure 1):

1) Dissection Microscope
2) 200 µl Micropipettor
3) Sterile Embryo Search Dish
4) NON-gamma irradiated 0.25 ml PVA embryo straws
5) EmCare Holding Media
6) Partnar Vitrification Kit Solutions
7) Heat Sealer
8) Insulated Container
9) Liquid Nitrogen
10) Digital Timer
11) 10 mm goblet and canes

Figure 2 Organized drops of media should be placed in a Petri dish at room temperature (Figure 2). This organization of drops allows for rapid media changes in increasing concentrations of cryoprotectants and in loading straws.

Procedure:
1) Embryo is removed from Emcare® holding media (Figure 3) and transferred into the first vitrification solution (S1)
a. Embryo should be transferred with minimal residual amount of holding media into VS1.
b. Hold embryo in S1 (200 µl) for precisely 5 minutes

2) Embryo is removed from S1 and placed into the second vitrification solution (S2) using minimal amount of S1 solution

Figure 3

a. Hold embryo in S2 (200 µl) for precisely 5 minutes

3) Embryo is removed from S2 and placed into the third or final vitrification solution using minimal amount of S2 solution
a. Hold embryo in S3 (30 µl) for no longer than 45 to 60 seconds and this time includes loading the straw before exposing it to liquid nitrogen vapor.

4) Loading the 0.25 ml non-irradiated, polyvinyl chloride straw includes (Figure 4):
a. Drawing up 90 µl of dilution solution
b. Followed by an air bubble (5 to 10 µl)
c. Followed by the embryo in 30 µl of S3
d. Followed by another air bubble (5 to 10 µl)
e. Finally followed by a second 90 µl of dilution solution

Figure 4

5) Heat seal the open end of the straw with two heat seals. This takes about 5 seconds (Figure 5).

6) Placing the heat sealed straw into a pre-cooled plastic goblet held vertically in liquid nitrogen within an insulated container is the second to last step (Figure 6).
a. The straw is held vertically inside the goblet where only vapor surrounds the straw
b. The straw is held for 1 minute in vapor before being plunged into the liquid nitrogen (Figure 7)
c. The vitrified embryo can then be transferred into a holding tank
Figure 5 Figure 6 Figure 6

Warming Procedures

Warming the embryos is just as critical as vitrifying them. Precise warming time and identification of the embryo within the straw are critical

Procedure:

1) Straws are removed from the liquid nitrogen tank
a. Held in room temperature air for 10 seconds
b. Place straw into a 20 to 22ºC water bath for 10 seconds
c. Mix contents of the straw by flicking the straw like a thermometer 5 times

2) Straw is held horizontally under the dissecting scope taking care not to expose it too long over the light source
a. The embryo is identified and subjectively graded and measured
b. After identifying the embryo it is then laid on a horizontal surface for 6 minutes at room temperature
3) After 6 minutes, the heat-sealed end of the straw is cut with scissors
a. The straw is loaded into a Cassou gun and transferred non-surgically into an available recipient mare

All steps should be completed within 8 minutes