Cryoprotective effect of polyols on rat embryos during two-step freezing

The cryoprotective effect of polyols on rat embryos was measured after two-step freezing, and the mechanism of action of polyols on embryo survival was examined. Rat embryos frozen in solution of polyol by two-step method at the morula stage showed higher survival than that obtained using DMSO. As the number of hydroxyl groups increased, the cryoprotective effect of the polyol increased. However, this was true only when the additive could permeate the cell membrane. Of the additives tested, four or five carbon polyols were most effective at concentrations of 0.3 or 1.0 M than two, three, six, or seven carbon polyols. The highest survival rate was obtained with adonitol, which yielded 83% embryo survival at 1.0 M and 67% even at 0.3 M. Embryos frozen in 0.3 M adonitol and transferred directly into foster mothers without any dilution of the additive after thawing developed into live young. During slow cooling below -40 degrees C, embryonic blastomeres exhibited cell fusion only in the presence of adonitol. These findings suggest that one cryoprotective action of polyols is that the hydroxyl groups act both on the cell surface and the cytoplasm to stabilize the bound water on the embryonic membrane, and that the length of the C-chain determines the permeability of the membrane to the additive.