The aim of this study was to evaluate the effect of antioxidant agents and freezing methods on the ability of ram sperm to preserve its post-thaw quality characteristics. Six Chios rams were subjected to 52 weekly semen collections. Each ram was used as semen donor for freezing experiments once every 2 weeks. Equal number of good quality spermatozoa from each ejaculate (concentration ≥1 × 109 spermatozoa/ml, motility ≥70%, motility score ≥3.5) were pooled. Three equal aliquots of the pooled sample were diluted using three different fractions of a milk-based and glycerol extender (control, quercetin-enriched, α-tocopherol-enriched). Three freezing methods were applied (slow and fast freezing rate in a programmable freezer, vapors of liquid nitrogen) in every aliquot. Sperm aliquots were tested before freezing, immediately after thawing and after 3 h of incubation at 37 °C. Sperm motility (%) was evaluated microscopically. The percentage of membrane and acrosome-intact spermatozoa (IL%) as well as the percentage of membrane-intact and acrosome-reacted spermatozoa (ARL%) were determined by eosin-nigrosin stain. Furthermore, the percentage of hypo-osmotic swelling (HOS) test-positive spermatozoa was estimated. The results revealed no beneficial effect of the antioxidant treatment on the parameters of post-thaw semen (P > 0.05). However, the slow freezing rate method was more beneficial regarding motility, IL, ARL and HOS-positive spermatozoa compared to the other methods. In conclusion, the antioxidant agents used in this study failed to protect sperm against cryopreservation stress; however, the choice of the appropriate freezing method could contribute to the improvement of post-thaw ram sperm quality.
Keywords: Antioxidants; Chios sheep; Cryopreservation; Freezing method; Frozen semen; Ram semen.