RAPID FREEZING OF THE MOUSE BLASTOCYST - EFFECTS OF CRYOPROTECTANTS AND OF TIME AND TEMPERATURE OF EXPOSURE TO CRYOPROTECTANT BEFORE DIRECT PLUNGING INTO LIQUID-NITROGEN

This study investigates the effects of time and temperature of exposure to a high concentration (4.5 M) of dimethyl sulfoxide (DMSO), glycerol, 1,2-propanediol (PROH), or a mixture of DMSO and glycerol (DG) in a solution containing 0.25 M sucrose, on the survival and development of rapidly frozen mouse blastocysts. Embryos had significantly (P < 0.01) higher rates of survival and development when exposed to cryoprotectant at 0-degrees-C compared with room temperature. The time of exposure to cryoprotectant at either 0-degrees-C or room temperature before being plunged into liquid nitrogen significantly (P < 0.01) affected the survival and development of frozen-thawed embryos. Survival and development of blastocysts in vitro and in vivo was significantly (P < 0.05) higher when exposed at 0-degrees-C for 10 min to DG, DMSO and glycerol than to PROH. It is concluded that, unlike early-cleavage stage embryos, blastocysts need to be equilibrated at a low temperature (0-degrees-C) with high concentrations of cryoprotectant before rapid freezing. Exposure of blastocysts to 4.5 M cryoprotectant and 0.25 M sucrose at room temperature either was toxic or else markedly reduced their viability after freezing and thawing, depending on the duration of the initial exposure.