Large equine blastocysts are damaged by vitrification procedures

Abstract

Viability following vitrification of equine blastocysts with different sizes was investigated in vitro. Twenty-four blastocysts were classified into three groups according to their diameters (< 200 microns, 200-300 microns and > 300 microns; n = 8 each). The solution used for vitrification was defined as EFS and contained 40% ethylene glycol, 18% Ficoll and 0.3 M sucrose in modified-phosphate-buffered saline (m-PBS). During pretreatment with 20% ethylene glycol in m-PBS for 20 min, the larger blastocysts responded to the osmotic pressure caused by 20% ethylene glycol more slowly than the smaller blastocysts. Single blastocysts were loaded into the EFS in 0.25-mL straws, left to stand for 1 min and vitrified in nitrogen vapour. After thawing for 20 s in water (20 degrees C), a fractured zona pellucida or capsule was seen in: 1 of 8 blastocysts < 200 microns in diameter; 1 of 8 blastocysts 200-300 microns in diameter; and 2 of 8 blastocysts > 300 microns in diameter. When the blastocysts were cultured for 48 h in TCM199 supplemented with 10% fetal bovine serum at 37 degrees C in 5% CO2 in air, 7 of 8 (88%) blastocysts < 200 microns in diameter and 6 of 8 (75%) blastocysts 200-300 microns in diameter developed with re-expansion of the blastocoele. However, the developmental ability of blastocysts > 300 microns in diameter (2 of 8, 25%) was significantly lower than that of blastocysts < 200 microns in diameter (P < 0.05).