Sephadex filtration in conjunction with preputial washing: A strategy to minimize bacterial load and enhance cryo-survival in Murrah buffalo bull semen.

The presence of bacteria poses a significant challenge to the quality of bovine semen used in artificial insemination. This study was conducted based on the hypothesis that Sephadex reduces the bacterial content of semen, while also separating dead or immotile spermatozoa from the motile ones. The potential of Sephadex filtration to decrease bacterial load has not been well documented so far.The present study was conducted to standardize the optimal height of Sephadex G-75 and G-100 slurry columns for semen filtration, assess the bacterial load in raw and filtered semen, and evaluate the functional quality of semen post-filtration. In Experiment I, three different column height combinations of Sephadex G-75 and G-100 (1 ml: 2 ml, 1.5 ml: 1.5 ml and 2 ml: 1 ml) were evaluated using ejaculates from Murrah buffalo bulls. In Experiment II, a total of 36 ejaculates were collected biweekly over one month after preputial washing with sterile normal saline (200 ml). Each ejaculate was divided into two aliquots: one served as a control (unfiltered), while the other was filtered through the best selected Sephadex column from experiment I. Bacterial load was determined by calculating colony-forming units (CFU/ml) in both control and filtered samples. Filtered semen was cryopreserved and evaluated post-thaw for sperm motility, viability, membrane integrity, acrosomal integrity and post-thaw sperm kinetic parameters. The study demonstrated that Sephadex slurry columns G-75 (1 ml) and G-100 (2 ml) significantly improved semen quality parameters like post-thaw motility, viability, membrane integrity and acrosomal integrity. Post-thaw sperm kinetic parameters were significantly higher in filtered samples than control. Moreover, a significant reduction in bacterial load after filtration through sephadex slurry also indicates potential application of filtration methods in enhancing semen processing protocols for buffalo bulls.