Effect of different antioxidant additives in semen diluent on cryopreservability (-196 degrees C) of buffalo semen

Aim: The aim of this study was to evaluate the effect of different antioxidant additives in standard tris-fructose-egg yolk-glycerol (TFYG) extender on the cryopreservability of buffalo semen. Materials and Methods: Semen collection using artificial vagina, twice weekly for 5 weeks from three pedigreed health breeding bulls of Mehsani breed, aged between 6 and 8 years. Immediately after initial evaluation all 30 qualifying ejaculates (10/bull) were split into three aliquots and diluted at 34 degrees C keeping the concentration of 100 million spermatozoa/ml with standard TFYG extender as control and TFYG having two antioxidant additives - Cysteine HCl at 1 mg/ml and ascorbic acid at 0.2 mg/ml to study their comparative performance. Semen filled in French Mini straws using IS-4 system and gradually cooled to 4 degrees C and equilibrated for 4 h in cold handing cabinet. After completion of equilibration, straws were cryopreserved in LN2 by Programmable Bio-freezer. Semen was examined at post-dilution, post-equilibration, and post-thaw stages for sperm quality parameters, and at each stage plasma was separated for enzymatic analysis of aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and alkaline phosphatase (AKP). Results: The mean percentage of sperms in TFYG, TFYG + cysteine HCl and TFYG + ascorbic acid diluents at post-thaw stage in terms of progressive motility (52.83+/-0.52, 57.83+/-0.52, 57.83+/-0.52), livability (78.70+/-0.21, 82.33+/-0.23, 81.73+/-0.22), and abnormality (5.43+/-0.21, 5.03+/-0.17, 5.23+/-0.18) varied significantly (p<0.05) between control TFYG and TFYG having antioxidant additives. The mean U/L activities of AST (78.70+/-0.47, 72.80+/-0.48, 73.30+/-0.54), LDH (172.70+/-0.41, 155.78+/-0.42, 156.33+/-0.41), and AKP (103.61+/-0.34, 90.20+/-0.34, 91.03+/-0.34) in semen diluted with TFYG, TFYG + cysteine HCl and TFYG + ascorbic acid diluents at post-thaw stage, respectively, which showed significantly (p<0.05) higher leakage of enzymes in control TFYG than TFYG incorporated with additives. Conclusion: Incorporation of antioxidant additives such as cysteine HCl and ascorbic acid in standard TFYG diluents improves sperm quality parameters, reduces enzyme leakage, and ultimately advances cryopreservability of buffalo semen.