We have previously reported high survival in mouse sperm frozen at 21°C/min to −70°C in a solution containing 18% raffinose in 0.25× PBS (400 mOsm) and then warmed rapidly at approximately 2000°C/min, especially under lowered oxygen tensions induced by Oxyrase, a bacterial membrane preparation. The best survival rates were obtained in the absence of glycerol. The first concern of the present study was to determine the effects of the cooling rate on the survival of sperm suspended in this medium. The sperm were cooled to −70°C at rates ranging from 0.3 to 530°C/min. The survival curve was an inverted “U” shape, with the highest motility occurring between 27 and 130°C/min. Survival decreased precipitously at higher cooling rates. Decreasing the warming rate, however, decreased survivals at all cooling rates. The motility depression with slow warming was especially evident in sperm cooled at the optimal rates. This fact is consistent with our current view that the frozen medium surrounding sperm cells is in a metastable state, perhaps partly vitrified as a result of the high concentrations of sugar. The decimation of sperm cooled more rapidly than optimum (>130°C/min), even with rapid warming, is consistent with the induction of considerable quantities of intracellular ice at these rates. When glycerol was added to the above medium, motilities were also dependent on the cooling rate, but they tended to be substantially lower than those obtained in the absence of glycerol. The minimum temperature in the above experiments was −70°C. When sperm were frozen to −70°C at optimum rates, lowering the temperature to −196°C had no adverse effect.
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