Response of Cryopreserved Nili Ravi Buffalo Bull Semen to Gallic Acid Inclusion in Semen Extender

During cryopreservation, the spermatozoa faces osmo-chemical, mechanical and thermal stresses, which are predominant at dilution, cooling, equilibration, freezing and thawing stages. They damage functional and morphological characteristics of sperms. Beside these exogenous stresses there is an oxidative stress which damages the spermatozoa endogenously. These stresses can be controlled with the inclusion of antioxidants in semen extender at the time of cryopreservation. In the current study, semen from four (n=4) healthy Nili Ravi buffalo bulls was collected through artificial vagina and Gallic acid was added to the semen at 1 µM, 15 µM, 30 µM, 45 µM, 60 µM, and 100 µM. A total of six groups were prepared. One group was kept as control where no Gallic acid was added. After the addition of extender, semen was cooled to 4°C, filled in 0.5mL straws for 4 h and frozen in liquid nitrogen at -196 °C. The parameters evaluated were percentage motility, plasma membrane integrity (HOST assay), acrosomal integrity (NAR), viability (Live/Dead), DNA integrity (Acridine orange assay) and oxidative stress (TBARS assay). Five straws from each Gallic acid group were thawed individually in water bath at 37°C for 30 seconds. The addition of 15 µM GA to semen extender improved marginally the buffalo bull spermatozoa motility, viability and membrane integrity but still not sufficient to reach the statistical significance, while it has no protective effects on other parameters like Acrosomal integrity, DNA status and oxidative stress. However further studies are needed to assess the role of Gallic acid in different concentrations and in other animals.