The major population of buffalo in the world (more than 75%) is located in Asia and 14% in Pakistan, where buffaloes play an important role in rural livestock production providing milk, meat and work draft force. Genetically the ratio of proven buffalo bulls is lesser than cattle bulls, and additionally the volume of semen and spermatozoa concentration is also lower than cattle semen. The success of inseminating buffalos with cryopreserved semen is also low, which account for major cause of fertility problems.
During cryopreservation, the spermatozoa faces osmochemical, mechanical and thermal stresses, which are predominant at dilution, cooling, equilibration, freezing and thawing stages which lead to damage spermatozoa functional and morphological characteristics. Beside these exogenous stresses, oxidative stress damages the spermatozoa endogenously. Keeping in mind all these problems, the reduction of these stresses through inclusion of antioxidants in semen may improve its quality and ultimately the fertility of buffalo bulls. This may be obtained from antioxidant addition to extender at the time of cryopreservation. Gallic acid possess good antioxidative properties, the use of this polyphenolic compound may reduce oxidation in buffalo bull semen.
In the current study, semen from four (n=4) healthy Nili Ravi buffalo bulls was collected by artificial vagina and GA was added to the semen @ 1 µM, 15 µM, 30 µM, 45 µM, 60 µM, and 100 µM and a total of six groups were prepared. One group was kept control and no GA was added to that group. The routine quality evaluation of semen for motility and concentration was made, extender was added, then semen cooled to 4°C filled in 0.5mL straws for 4 hours and frozen in liquid nitrogen at -196 °C.
The semen was then transported to the Physiology Laboratory of UVAS for further evaluations. 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 GA group were thawed individually in water bath at 37°C for 30 seconds and evaluated for quality parameters. The data collected was presented as cells ± SE and treatment groups were compared using one way analysis of variance. The group differences were compared by using the Duncan Multiple Range Test. The results revealed that GA improved (P<0.05) the spermatozoa viability and plasma membrane integrity.
In conclusion, 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 GA in different concentrations and other animals.