LARGE EQUINE BLASTOCYSTS ARE DAMAGED BY VITRIFICATION PROCEDURES

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 mu m, 200-300 mu m and > 300 mu m; 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 mu m in diameter; 1 of 8 blastocysts 200-300 mu m in diameter; and 2 of 8 blastocysts > 300 mu m 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 mu m in diameter and 6 of 8 (75%) blastocysts 200-300 mu m in diameter developed with re-expansion of the blastocoele. However, the developmental ability of blastocysts >300 mu m in diameter (2 of 8, 25%) was significantly lower than that of blastocysts (200 mu m in diameter (P < 0.05).