Seasonal variation in the protective effect of seminal plasma on frozen-thawed ram spermatozoa.

The response of ram spermatozoa to seminal plasma is highly variable, in part due to the presence of both stimulatory and inhibitory factors. The aim of this study was to assess variation in the protection of ram spermatozoa during freezing by seminal plasma. The seminal plasma variables studied were season of collection, fractionation and method of supplementation. Spermatozoa were supplemented before freezing with 4 mg of seminal plasma proteins (SPPs) per 10(8) cells, and their motility and viability were assessed during post-thaw incubation (37 degrees C). In Experiment 1, semen was (a) frozen with no supplementation (Control) (b) extended with a Tris-based diluent (C + TRIS), or (c) supplemented with seminal plasma collected throughout the year (in the Southern Hemisphere) and pooled for January-March, April-June, July-August and October-December, and either fractionated to produce a concentrated >10 kDa seminal plasma protein retentate (>10 kDa SPP), or kept as crude seminal plasma (CP). There was no effect of season or seminal plasma type (CP or >10 kDa SPP) on motility of spermatozoa. CP and >10 kDa SPP improved the viability of spermatozoa when collected from January-September compared to Control. Supplementation with >10 kDa SPP increased viability of spermatozoa, compared to CP, when collected from January to July. In Experiment 2, >10 kDa SPP were either added directly to the spermatozoa or included in the cryodiluent or >10 kDa SPP were not supplemented (Control). Both supplementation methods improved the motility and the proportion of viable, acrosome-intact spermatozoa but direct supplementation resulted in more viable, acrosome-intact spermatozoa compared with supplementation of the cryodiluent. These results show that supplementation of ram spermatozoa with CP, or its protein component (>10 kDa SPP), before freezing protects them from freeze-thaw damage. The protective effect is greatest when seminal plasma is collected during the breeding season, fractionated with >10 kDa filters and added directly to the spermatozoa.