THE EFFECT OF EQUILIBRATION TEMPERATURE AND TIME ON THE OUTCOME OF ULTRARAPID FREEZING OF 1-CELL MOUSE EMBRYOS

Mouse 1-cell embryos were frozen ultrarapidly at a rate of similar to 2500 degrees C/min in solutions containing 0.25 M sucrose, 0.5% (w/v) bovine serum albumin (BSA) and 3 or 4,5 M dimethyl sulphoxide (DMSO) or 3 or 4.5 M 1,2-propanediol (PROH) in HEPES-buffered modified Earle's medium. We investigated the effect of pre-freeze equilibration for 1, 3, 5 or 10 min at 22 degrees C and for 1, 3, 5, 10, 15 or 20 min at 4 degrees C. After thawing in a 22 degrees C water bath at a rate of similar to 2500 degrees C/min and dilution in 1 M sucrose in HEPES-buffered modified Earle's medium, embryos were cultured in vitro in bicarbonate-buffered modified Earle's medium with 0.5% (w/v) crystalline BSA. Embryo viability was expressed as the percentage of hatching or hatched bIastocysts resulting from the initial number of frozen-thawed 1-cell embryos. To determine the toxicity of the freezing solutions, embryo viabiIity was evaluated after equilibration without freezing. Our results demonstrated that the concentration, the equilibration temperature and time are very important factors in ultrarapid freezing of mouse 1-cell embryos. Optimal viability was found when equilibration was done in 4.5 M DMSO for 3-5 min at 22 degrees C and in 4.5 M PROH for 3-5 min at 4 degrees C. The results with regard to exposure to the freezing solutions indicated that the loss of viability beyond an optimum is not due solely to cryoprotectant toxicity, in particular not at 4 degrees C and not for DMSO. It is suggested that the temperature and time of equilibration influence the degree of cryoprotectant permeation and subsequent rehydration, which play a role in determining freezing susceptibility in terms of ice formation. We conclude that both DMSO and, in contradiction to previous reports, PROH can be used perfectly adequately for ultrarapid freezing on condition that concentration, and the temperature and time of equilibration are controlled.