CHANGES IN SPERM-BOUND AMIDASE ACTIVITY SUGGEST SUBTLE DAMAGE TO RAM SPERM ACROSOMES BY FREEZING THAWING, NOT DETECTED BY LIGHT-MICROSCOPY/

We have measured sperm-bound amidase activity in fresh, cooled and fro zen/thawed ram spermatozoa, in order to study if freezing and thawing led to some degree of acrosome damage of motile/viable spermatozoa not detected by optical methods. This assay was based on the fact that me mbrane damage would result in an increased access of the enzyme substr ate to the sperm acrosome. Semen was collected from adult Australian M erino rams, and spermatozoa were washed by centrifugation through a Fi coll solution. Sperm-bound amidase activity was measured in whole sper matozoa using the protease substrate benzoyl-arginyl-p-nitroanilide (B APNA). Acrosomal status was also assessed by light microscopy after Gi emsa staining. Most amidase activity was shown to be sperm-bound, as o nly a minor fraction of the enzyme activity was released into the medi um after induced damage. Simultaneous assessment of sperm-bound amidas e activity and the percentage of spermatozoa with microscopically evid ent acrosomal damage, after mild sonication for different times, showe d a high correlation between both parameters (r = 0.97, p < 0.001). In separate experiments, fresh, cooled and frozen/thawed semen samples w ere filtered through Sephadex G-10 to obtain a subpopulation of motile , mostly acrosome-intact spermatozoa. As controls, spermatozoa from th e same samples to which extensive acrosome damage was induced were eva luated. Slow cooling to 4 degrees C had no effect on amidase activity or percent acrosomal damage with respect to fresh samples. Freezing an d thawing resulted in a sperm population that, after filtration throug h Sephadex, had a low percentage of acrosome damage (9.4%, vs. 2.1% fo r fresh filtered controls), which was 11% of that obtained after exten sive acrosome damage (83%). However, amidase activity in these samples was markedly increased, showing values of activity that were 56% of t hose obtained in extensively damaged spermatozoa. This effect was not due to an alteration in the enzyme kinetics. We conclude that sperm-bo und amidase activity is useful to detect subtle changes, provoked by a standard freezing/thawing procedure, in the permeability of acrosomes from ram spermatozoa which are not detected by direct observation of the acrosomes after Giemsa staining.