SURVIVAL OF MOUSE BLASTOCYSTS SLOW COOLED IN PROPANEDIOL OR ETHYLENE-GLYCOL IS INFLUENCED BY THE THAWING PROCEDURE, SUCROSE AND ANTIFREEZE PROTEINS

It is known that both the cooling and warming rates are important in determining the viability of cryopreserved embryos. The effect that thawing rates have on blastocysts cryopreserved in propanediol or ethylene glycol are, however, not well documented. In this study, late blastocyst stage mouse embryos were cryopreserved using conventional slow cooling techniques in 1.5 M ethylene glycol (EG) or 1.5 M 1,2-propanediol (PG), and 4 different thawing protocols were compared. Straws were thawed by immersing them into a 37 degrees C water bath, either immediately after their removal from liquid nitrogen (Protocol 1) or after being held in air for 15 sec (Protocol 2) or 30 sec (Protocol 3). The last group of straws was held in air until the ice had melted (approx 130 sec, Protocol 4). Embryo viability was highly correlated with the thawing protocol and the cryoprotectant used. Thawing Protocol 2 resulted in uniformly high embryo viability, both in vitro (EG 93-95%, PG 90-95% plating) and in vivo (EG 33%, PG 45% fetuses). This was not significantly different from the nontreated control embryos (100% plated, 40% fetuses). By contrast, thawing Protocol 4 significantly reduced embryo survival, in particular for embryos frozen in EG (EG 13 to 35%, PG 40 to 56% plating). The damaging effects of Treatment 4 may have been due to ice crystal growth, but adding antifreeze protein Type I or III at a concentration of 0.1 or 1.0 mg/ml respectively did not increase embryo survival. We conclude that expanded and hatched blastocyst-stage mouse embryos can be frozen-thawed in either EG or PG without significant loss of viability, providing an appropriate thawing protocol is used.