Effects of Temperature and Restoration of Osmotic Equilibrium during Thawing on the Induction of Plasma Membrane Damage in Cryopreserved Ram Spermatozoa1

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Abstract
The objective of this investigation was to examine the nature of freeze/thaw-induced plasma membrane damage in an effort to validate hypotheses about cryoinjury in ram spermatozoa. Spermatozoa were loaded with fluorescein diacetate (FDA), a marker for plasma membrane integrity, and cooled (15 degrees C/min) to temperatures between -10 degrees C and -30 degrees C on a cryomicroscope stage. Post-thaw fluorescence intensity measurements of individual cells indicated that freezing to temperatures between -10 degrees C and -15 degrees C did not induce significant membrane permeabilization. However, freezing below -15 degrees C was followed by membrane permeabilization immediately after thawing. A majority (> 60%) of flagellar plasma membranes of cells frozen to -10 degrees C remained ultrastructurally intact during thawing; principal-piece membranes were more robust than middle piece membranes (p = 0.001). Significant middle-piece membrane breakage was, however, induced as the post-thaw temperature increased from +10 degrees C to +30 degrees C (10 degrees C, 64 +/- 12.3% intact membranes [mean +/- SEM]; 30 degrees C, 43 +/- 12.5% intact membranes [mean +/- SEM]; p = 0.0085). Cells frozen to -30 degrees C did not exhibit this thawing effect, although the distinction between middle-piece and principal-piece plasma membranes was evident (p = 0.002). All sperm head plasma membranes were damaged by freezing and thawing to any combination of temperatures. Although acrosomes became swollen after freezing and thawing, the incidence of outer acrosomal membrane vesiculation remained at control (unfrozen) levels with all treatments used. Experimental exposure to the hyperosmotic conditions generated during freezing induced little flagellar membrane permeabilization, but significant damage was caused by restoration of osmotic equilibrium. It is suggested that membranes are initially destabilized during the freezing process, both by low temperature effects and by exposure to high salt concentrations. The resultant post-thaw degeneration of the plasma membrane is caused by a combination of temperature and osmotic effects.

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