77 EFFECT OF SUGAR SUPPLEMENTATION ON THE CRYOPRESERVATION OF BOAR SPERMATOZOA

Abstract

The optimization of cryopreservation extenders is an essential issue for the successful establishment of boar sperm cryobanks. The aim of the present study was to investigate the importance of sugar supplementation to the freezing extender and/or to the thawing diluents, and the interactions between these treatments on post-thaw survival of boar spermatozoa. Pooled sperm-rich fractions from 5 mature hybrid boars (5 ejaculates per boar) were divided into 7 aliquots, centrifuged at 2300g 3 min, and diluted to a final sperm concentration of approximately 1000 × 10⁶ spermatozoa mL^–1 with the freezing extender prior to freezing in 0.5-mL plastic straws; a standard freeze–thaw procedure with computer-controlled freezing equipment was utilized. All of the freezing extenders were based on Tris-citric acid buffer supplemented with 20% egg yolk and 0 mm (TC, no glucose), 0.05 mm, 2 mm, 4 mm, 10 mm, 50 mm, or 185 mm glucose (all media adjusted to 310 mOsm kg^–1; pH 6.8). After thawing at 37°C/20 s, semen was immediately diluted 1:2 (v/v) with either TC (no glucose) or BTS (205 mm glucose, 20.39 mm NaCl, 5.4 mm KCl, 15 mm NaHCO₃, and 3.35 mm EDTA). Post-thaw sperm motility (assessed with a computer-assisted semen analysis system) and plasma membrane and acrosome integrity (viability, assessed simultaneously by flow cytometry using triple fluorescent staining) were evaluated at 0, 30, and 150 min post-thaw and used to estimate the success of cryopreservation. Data were analyzed as a split plot design using a mixed model ANOVA including cryopreservation extender, thawing diluent, incubation time, and interactions as fixed effects and replicates as a random effect. The freezing extender did not have any significant effect on the percentage of motile or viable spermatozoa at either thawing or after 150 min (P > 0.05). There was a significant effect of incubation time (P < 0.0001) and thawing diluent (P < 0.0001) on motility and viability, with a significant interaction between them on motility (P = 0.018). Motility was similar (P > 0.05) at time 0 in both thawing diluents (mean ± SEM: 49.4 ± 3.7 v. 46.2 ± 3.9% for BTS and TC, respectively), but decreased in Tris-diluted samples in a time-dependent manner (54.6 ± 1.9 v. 42.5 ± 2.6% at 30 min, and 40 ± 3.4 v. 27.1 ± 3.7% at 150 min). In contrast, viability was significantly higher (P < 0.05) in BTS-diluted samples at 0 (53.9 ± 3.7 v. 49.7 ± 3.8%), 30 (55 ± 3.1 v. 47.7 ± 3.31), and 150 min (51 ± 4.2 v. 43.7 ± 4.4%). These results indicate that, despite common beliefs, sugars are not necessary for cryopreservation but are beneficial for maintaining boar sperm metabolism for a longer time after thawing.