High survival rate of one-cell mouse embryos cooled rapidly to -196 degrees C after exposure to a propylene glycol-dimethylsulfoxide-sucrose solution.

We investigated the effect of duration of exposure at 22 degrees C to a solution containing dimethylsulfoxide (Me2SO), propylene glycol (PROH), and sucrose on the survival rate of one-cell mouse embryos cooled rapidly to -196 degrees C. The cryoprotectant solutions were made up on a molar basis and six different times of exposure were tested (1, 2.5, 5, 10, 15, and 20 min). After rapid thawing and dilution in a 1 M sucrose solution the survival rate was calculated as the number of hatching or hatched blastocysts formed per frozen-thawed and recovered one-cell embryo. We demonstrated that the optimum time of exposure depends on the type of cryoprotectant solution used. The optimum time of exposure was 1 min when a 4.5 M PROH-0.25 M sucrose solution was used, 2.5 min when a 2.25 M PROH-2.25 M Me2SO-0.25 M sucrose solution was used, and 5 min when a 4.5 M Me2SO-0.25 M sucrose solution was used. For prolonged exposure times beyond the optimum, survival rates were the highest when solutions containing 2.25 M PROH-2.25 M Me2SO-0.25 M sucrose were used. To identify factors that may influence the survival rates, we carried out further experiments on one-cell embryos in the different cryoprotectant solutions. We evaluated (i) the survival rates after exposure without freezing and (ii) the volume changes during exposure. It is suggested that the optimum time of exposure to a cryoprotectant solution depends on cryoprotectant permeation and that detrimental effects after prolonged exposure are a consequence of chemical toxicity. When one-cell mouse embryos were exposed for 3 min to a solution containing 2.25 M PROH-2.25 M Me2SO-0.25 M sucrose before rapid cooling to -196 degrees C, 98% of the zygotes were morphologically intact after rapid thawing. There was no further loss in viability during in vitro culture on after transfer in vivo as compared to unfrozen control embryos.