P-142: Effects of A Synthetic Antioxidant (4-Hydroxy Tempo) Additive to The Semen Extender on the Ejaculated Spermatozoa Characteristics before and after Freezing in Water Buffaloes (Bubalus Bubalis)

Background: Semen cryopreservation is the most important section of artificial insemination programs; it allows preservation of semen fertility for a long time. Materials and Methods: The aim of the present study was to detect the effect of in vitro supplementation of 4-hydroxy Tempo on fresh and frozen spermatozoa quality of buffalo bulls. Five healthy buffalo bulls (5 ejaculates from each bull) were used. Each ejaculate was diluted at 37°C with tris-based extender containing 0 (control), 0.5, 1, 4, 8 and 12 mM 4-hydroxy Tempo and the sperm motility and viability were evaluated at 0(T0) (immediately after dilution), 60 (T1) and 120 (T2) minutes after diluting semen. In the second step, semen samples were diluted with tris-egg yolk-glycerol extender containing the same amounts of 4-hydroxy Tempo, cooled to 4°C, equilibrated and semen parameters (motility, viability, membrane integrity and DNA fragmentation) were estimated. Then, the semen was packed in 0.5 mL French straws and frozen in liquid nitrogen. Later, the semen was thawed and analyzed for the same parameters, as well as total antioxidant capacity. Results: Results showed that addition of 0.5 and 1mM 4-hydroxy Tempo to the semen extender significantly increased the sperm motility of fresh and equilibrated semen compared to the control without affecting other parameters. However, in frozen-thawed semen, extenders containing 0.5 and 1mM 4-hydroxy Tempo significantly improved sperm motility, viability, membrane integrity and semen total antioxidant capacity and also resulted in lower DNA fragmented sperms. In this study 4-hydroxy Tempo supplementation of semen extender of 8 and 12 mM had deleterious effects on sperm parameters as early as the samples were prepared for freezing. Conclusion: This study indicates that Tempo supplementation may help to ameliorate negative effects of semen storage on characteristics of sperm quality; improved sperm motility, viability, plasma membrane integrity and DNA fragmentation, with higher values obtained after adding 0.5 mM 4-hydroxy Tempo to the extender.