Large equine blastocysts are damaged by vitrification procedures.

This study investigates how effective vitrification, a technique used to preserve embryos, is on equine blastocysts of various sizes, with findings suggesting larger blastocysts have a lower survival rate due to damaging effects of the procedure.

Introduction and Methodology

• The researchers aimed to test the effects of vitrification on equine blastocysts of differing sizes.
• Vitrification is a process used to preserve biological structures. It’s typically used for gametes and embryos in fertility treatments. In this case, it’s used on equine blastocysts – early stage equine embryos.
• The solution used for vitrification in this study is called EFS, containing 40% ethylene glycol, 18% Ficoll and 0.3 M sucrose. This solution was in the modified-phosphate-buffered saline (m-PBS).
• The blastocysts were divided into three size categories: less than 200 microns, between 200-300 microns, and larger than 300 microns. Each group contained 8 blastocysts.

Procedure

• The blastocysts were pretreated with 20% ethylene glycol in m-PBS for 20 minutes.
• Larger blastocysts responded more slowly to osmotic pressure caused by the 20% ethylene glycol compared to smaller ones.
• Each blastocyst was then exposed to the EFS solution, left for 1 minute, and then vitrified using nitrogen vapour.
• The blastocysts were then warmed by thawing for 20 seconds in water at 20 degrees Celsius.
• Following this, the researchers observed if there were any breaches in the zona pellucida, the outer layer of the blastocyst.

Observations and Conclusions

• The researchers found that a fractured zona pellucida occurred in differing subsets; 1 of smaller than 200 microns, 1 of 200-300 microns, and 2 of bigger than 300 microns.
• After culturing the blastocysts for 48 hours in TCM199 (supplemented with 10% fetal bovine serum), more smaller blastocysts survived, with 88% of those under 200 microns and 75% of those between 200-300 microns achieving re-expansion of the blastocoele.
• In contrast, only 25% of larger blastocysts managed to survive and develop, indicating that the larger the blastocysts, the less likely it is to survive vitrification. This reveals a statistically significant discrepancy in survival rates among the different sizes of blastocysts.