Post thaw treatment of frozen buffalo semen with antioxidants vitamin C and 2-mercaptoethanol

Abstract

The aim of this study was to evaluate the effect of ascorbic acid (Vitamin C) and 2-mercaptoethanol (ME) added to buffalo semen samples after thawing for 1 h and 2 h incubation. Ejaculates (n = 20) from eight Bulgarian Murrah buffaloes were frozen in pellets and straws at a final sperm concentration of 80 × 106 mL. We investigated the effect of supplementations on the motility, morphology of spermatozoa at 1 h and 2 h after thawing, sperm plasma membrane status and mitochondrial trans-membrane potential. The comparative CASA analysis of the motility and velocity parameters of spermatozoa showed significant differences in the progressive motility and in the VCL values in favour of control vs. samples with additives (p < 0.05). The molecular changes in PM phospholipid asymmetry were higher after thawing (p < 0.05). The semen treated with Vit C showed significant (p < 0.05) lower number of apoptotic spermatozoa (6CF+/Ann V Cy3+) in comparison with samples incubated with ME. The numbers of dead spermatozoa (6CF-/Ann V Cy3+) in samples with ME were significantly higher than other groups. The percentage of spermatozoa with well preserved and functioning mitochondria in control semen was higher (p < 0.05) than that in the experimental group with ME. The results obtained revealed that addition of Vit C (5 µmol/L) and ME (50 µmol/L) has no positive effect on post-thaw semen quality of buffalo sperm, because of the impaired integrity of PM and mitochondrial trans-membrane potential.