Is Intracellular Ice Formation the Cause of Death of Mouse Sperm Frozen at High Cooling Rates?1

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Abstract
Mouse spermatozoa in 18% raffinose and 3.8% Oxyrase in 0.25× PBS exhibit high motilities when frozen to −70°C at 20–130°C/min and then rapidly warmed. However, survival is Lp) of the cell and for its activation energy (Ea). Using literature values for these parameters in mouse sperm, we calculated curves of water volume versus temperature for four cooling rates between 250 and 2000°C/min. The intracellular nucleation temperature was inferred to be −20°C or above based on the greatly reduced motilities of sperm that underwent rapid cooling to a minimum temperature of between −20 and −70°C. Combining that information regarding nucleation temperature with the computed dehydration curves leads to the conclusion that intracellular freezing should occur only in cells that are cooled at 2000°C/min and not in cells that are cooled at 250–1000°C/min. The calculated rate of 2000°C/min for IIF is approximately eightfold higher than the experimentally inferred value of 260°C/min. Possible reasons for the discrepancy are discussed.