Cryopreservation procedures for Day 7-8 equine embryos.

The research investigates the effectiveness of three cryopreservation methods for preserving equine embryos on day 7 or 8 after ovulation. The study found that Treatment 2, which involved a step-down equilibration method, was the most successful in terms of embryo survival and growth post-thaw.

Methodology

The researchers used larger grade 1 or 2 equine embryos, with diameters ranging from 300 to 680 micrometers. These were obtained from mares on Day 7 or 8 following ovulation.

• They were subjected to three types of cryopreservation treatments:
• Treatment 1: cooling from -6°C to -35°C at 0.5°C per minute, then plunged into liquid nitrogen, with a one-step addition and a 4-step removal of 1.0 M glycerol.
• Treatment 2: a step-down equilibration that involved a two-step addition of glycerol to 4.0 M, then decreased to 2.0 M prior to freezing, with galactose introduced during the last step and all glycerol removal stages. This treatment followed the same cooling procedure as Treatment 1.
• Treatment 3: a standard vitrification protocol involving step-by-step addition of ethylene glycol up to 11.9 M, with a stepwise removal of the cryoprotectant and decreasing concentrations of galactose in the dilution medium.
• Next, cryoprotectants were removed at 20-21°C and the embryos were cultured in 5% CO2-in-air for 36 hours at 38.5°C on equine oviductal epithelial cell monolayers. Finally, the morphology of the embryos was assessed; their protective capsules removed, and the embryos themselves were fixed and stained to study their nuclei and cytoplasm’s morphology.

Findings

• All the embryos in Treatment 1 degenerated either during thawing or culture.
• Of the six embryos in Treatment 2, four were graded 1, one was graded 2, and the remaining one was graded 3 after in vitro culture.
• For Treatment 3, one embryo was graded 2, another graded 3, and the remaining five were degenerated. Statistically, the results significantly differed among treatments (P<0.01).
• The average changes in the initial diameter displayed by the frozen and thawed embryos during post-thaw culture were -91 micrometers for Treatment 1, +179 micrometers for Treatment 2, and +20 micrometers for Treatment 3 (P<0.05).
• Additionally, two 26-day pregnancies were established following the transfer of six step-down equilibrated (Treatment 2) embryos to recipient mares, suggesting the real-world application and potential success of this method.

Conclusions

• The study concluded that Treatment 2 (step-down equilibration) was the most effective method for cryopreserving equine embryos, outperforming the other two treatments both in terms of embryo survival and growth after thawing.
• The successful outcome of this method indicates its potential for practical application in equine breeding and embryo transfer programs. Nevertheless, more research is needed to further validate the efficacy of this method and to refine the protocols for the best results.