47 Influence of olive extracts on buffalo semen quality following cryopreservation

Buffalo sperm are highly sensitive to cryopreservation-induced oxidative damages (Longobardi et al. 2017 Theriogenology 88, 1–8). We hypothesised that the enrichment of semen extender with olive extracts (OE) before cryopreservation would improve quality of buffalo sperm, due to its antioxidant action. The aim of this study was to evaluate the effects of OE supplementation in the semen extender on post-thaw viability, motility, and kinetic parameters of buffalo spermatozoa. The olive extracts were obtained by hot extraction with acidified water and the hydroxytyrosol content of 10.8% was obtained by high-performance liquid chromatography analysis with a calibration line. A preliminary dose response trial (0, 50, 100 and 150 µL of OE) was carried out to identify the most effective concentration to be used in the present trial. Ejaculates from 16 buffalo bulls were collected by electroejaculation and each ejaculate was split in 2 aliquots and diluted at 37°C with Triladyl extender, containing 0 (control) and 50 µL OE, to a final concentration of 30 × 10⁶ spermatozoa/mL. The diluted semen was cooled from 37°C to 5°C (cooling rate 2°C/3 min), equilibrated at 5°C for 6 h, exposed to the liquid by nitrogen vapor for 10 min and then plunged into liquid nitrogen. At thawing, sperm membrane integrity and viability were assessed by hyposmotic swelling test and Trypan Blue staining. Sperm motility (total and progressive motility) and semen kinetic parameters such as curvilinear velocity (VCL), average path velocity (VAP), straight-line velocity (VSL), straightness (STR), linearity (LIN), amplitude lateral head (ALH), beat cross frequency (BCF) and wobble were assessed by sperm class analyzer (SCA) system. Differences between groups were analysed by Student’s t-test. Results (mean ± standard deviation) are reported in Table 1. The enrichment of the extender with 50 µL OE improved (P ≤ 0.05) post-thaw sperm membrane integrity, as well as the percentages of total live and acrosome-intact live sperm. Likewise, the OE treatment increased both total and progressive sperm motility, whereas kinetic parameters were not affected. In conclusion, these findings demonstrated the beneficial effects of OE supplementation in the extender on buffalo sperm qualitative traits following cryopreservation. It is likely that the positive effects are related to the antioxidant properties of OE. However, further studies are needed to assess oxidative stress markers, as well as in vivo and in vitro fertilizing ability.