Pregnancies following transfer of equine embryos cryopreserved by vitrification.

This research studied the effectiveness of a preservation method called vitrification on horse embryos. The results demonstrated both in vitro (lab environment) and in vivo (within a living organism) development potentials.

Methodology of the Study

• This study involved the collection of 28 embryos from Native pony and Thoroughbred mares 5 to 7 days after ovulation. These embryos were obtained through nonsurgical uterine flushing, a method that gently flushes the uterus to retrieve the embryos.
• The researchers employed a preservation technique known as vitrification, which uses a specialized solution to protect organisms at extremely low temperatures. The vitrification solution used consisted of 40% ethylene glycol, 18% Ficoll, and 0.3 M sucrose in PBS.
• The embryos were either immediately submerged in the vitrification solution or pre-treated with a 20% ethylene glycol solution. Those in the latter group were treated for 10 to 20 minutes before proceeding to vitrification.
• Embryos were then placed in 0.25-ml straws, carefully cooled in liquid nitrogen vapor before being immersed fully into liquid nitrogen for cryopreservation.
• Upon thawing, the content of the straws was expelled with 0.5 M sucrose in PBS, and the sucrose was diluted. This was done in either a one-step procedure for Groups 1 and 2 or using a four-step process for Group 3.
• Finally, the embryos were cultured for 120 hours in a controlled lab environment (37 degrees Celsius in 5% CO2 in air), using a specialized medium supplemented with 10% fetal bovine serum. The viability of the embryos was determined by their ability to develop to the hatching or hatched blastocyst stage.

Results and Conclusion

• The success rates for development to the hatching or hatched blastocyst stage among the three groups varied. None of the embryos in Group 1 developed to this stage. In contrast, 4 out of 7 embryos in each of Groups 2 and 3 successfully reached this stage.
• An additional 7 embryos were cryopreserved, following the methods used for Groups 2 and 3. Of those, 5 embryos were chosen after an additional 4 hours of in vitro culture and transferred into recipient mares.
• Only two transferred embryos (both from Group 2) resulted in pregnancies that developed into viable fetuses at 60 days of gestation.
• This study demonstrates that equine embryos can be cryopreserved successfully using the vitrification process. Correspondingly, such embryos have the potential to develop in vitro to a hatched blastocyst stage and in vivo to become viable fetuses.