Influence of Relative Embryonic Volumes during Glycerol Equilibration on the Survival of Frozen-Thawed Equine Blastocysts.

Abstract

This study was designed to investigate the relationship between embryonic volume changes in response to addition of glycerol and the in vitro survival of frozen-thawed equine blastocysts. Unexpanded blastocysts (diameter <200 μm; n=9) shrunk rapidly when embryos were transferred in 5 and 10% glycerol solutions at 30 C in 10 min steps. At the end of equilibration in the 10% glycerol solution, the mean embryonic volumes were regained up to 59% of initial values. Expanding blastocysts (diameter 200-300 μm; n=9) shrunk with a rather slower response, and regained 41% of initial volumes. Hatched blastocysts (diameter >300 μm; n=9) shrunk gradually down to 35% of initial volumes. Following the 2-step glycerol equilibration, single embryos loaded into 0.25 ml French-straws were seeded at -6 C and cooled to -35 C at the rate of 0.3 C/min before plunging into liquid nitrogen. The straws were thawed rapidly in 37 C waterbath and glycerol was removed from embryos at 30 C in 10 min 6-steps (8.3, 6.7, 5.0, 3.3, 1.7 and 0%). The embryos were cultured for 48 h in TCM199 + 10% FBS at 37 C under 5% CO2 in air. Severe fractures of embryonic coats (zona pellucida and/or capsule) were found in 0/9 (0%; unexpanded), 2/9 (22%; expanding) and 6/9 (67%; hatched) frozen-thawed embryos. All unexpanded blasto-cysts (9/9, 100%) could develop normally in vitro. Only 5/9 (56%) expanding blastocysts and 2/9 (22%) hatched blastocysts could develop in vitro. The overall survival for embryos with intact or slightly fractured coats were 9/9 (100%), 4/9 (44%), and 2/9 (22%) in unexpanded, expanding, and hatched blastocysts, respectively. The mean values of relative embryonic volumes before freezing were related with post-thaw viability; however, the predictability of freezability of a particular embryo was low.