Effects of 4-hydroxy Tempo (Tempol) added to Tris-citric acid based extender on the preservability of buffalo bull spermatozoa

The aim of the present study is to investigate the effects of in vitro supplementation of Tempol on spermatozoa of buffalo bulls before and after freeze-thawing. A total of 25 samples from five healthy buffalo bulls (5 ejaculates from each bull on 5 different occasions) were used. Each ejaculate was diluted at 37°C in tris-citric acid based extender (without egg yolk) containing 0 (control), 0.5, 1, 4, 8 and 12 mM Tempol. Sperm progressive motility and viability were evaluated in diluted semen at 0 (T0) (immediatelyafter dilution), 60 (T1) and 120 (T2) minutes after semen dilution. In the next step, the same doses of Tempol, egg yolk and glycerol were added to the above extender and semen samples diluted, cooled to 4°C within 2 h, equilibrated for 4 hours and sperm parameters (progressive motility, viability, membrane integrity and DNA damage) estimated. The equilibrated semen was packed into 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 (TAC). Results showed that adding 0.5 and 1 mM Tempol to the semen extender preserved the sperm progressive motility in diluted and equilibrated semen compared to the control without affecting other parameters. However, in frozen-thawed semen, extenders containing 0.5 and 1 mM Tempol significantly preserved sperm progressive motility, viability, membrane integrity and semen total antioxidant capacity and also resulted in lower DNA damaged sperm counts. It was concluded that addition of 0.5 or 1mM tempol to the extender would preserve semen quality during freeze-thawing.