FREEZE-INDUCED MEMBRANE DAMAGE IN RAM SPERMATOZOA IS MANIFESTED AFTER THAWING - OBSERVATIONS WITH EXPERIMENTAL CRYOMICROSCOPY

Cryoinjury in ram sperm was investigated by direct observation, using cryomicroscopy, to validate model hypotheses of freezing injury in such a specialized cell. Fluorescein diacetate was used to determine when during the freeze-thaw cycle the sperm membrane became permeable. In noncryoprotected sperm plasma membrane, integrity was maintained throughout the cooling and freezing process, but fluorescein leakage occurred during rewarming. The temperature of post-thaw permeabilization varied in relation to the minimum temperature reached during freezing; cells cooled to -10-degrees-C retained fluorescence into the post-thaw temperature range of 9-24-degrees-C (mean +/- SEM; 13.25 +/- 0.91-degrees-C), whereas cells cooled to -20-degrees-C lost fluorescence shortly after thawing (mean +/- SEM; 2.62 +/- 0.91-degrees-C). Sperm cooled to 5-degrees-C, but not frozen, retained fluorescence during rewarming up to 20-30-degrees-C. The inclusion of glycerol and egg yolk in the freezing medium significantly and independently increased the post-thaw permeabilization temperature. Maintenance of fluorescence was also correlated with ability to resume motility after thawing. Sperm reactivation experiments were undertaken to examine deleterious effects of freezing upon the flagellar microtubular assembly. No direct evidence for such effects was obtained. Instead, a highly significant correlation between minimum freezing temperature and post-thaw temperature of initial reactivation was detected.