Use Punica Granatum Seed Extract as a Botanical Antioxidant for Enhancing Cold-Preserved (5°c) Sheep Epididymal Spermatozoa Quality

One of the most significant challenges in reproductive biotechnology is preserving male gametes during hypothermic storage due to oxidative stress-induced cellular injury during storage. The goal of this study was to investigate whether a natural antioxidant such as pomegranate (Punica granatum) seed extract could preserve sheep epididymal spermatozoa at 5 degrees Celsius when added to a Tris-Citrate-Fructose yolk (TCFY) extender. Spermatozoa were collected from freshly processed goat testis and diluted in TCFY extender containing different concentrations of pomegranate juice: 0% (control), 5%, 10%, 15%, and 20%. Sample were analysed at 24, 48, 72, 96, and 120 hours post-storage for sperm progressive motility, sperm plasma membrane integrity (by HOST testing), sperm viability (by eosin-nigrosin staining) and morphological normalcy. In comparison to the controls and higher concentrations of PJ 10% PJ concentrations provided significant preservation of spermatozoa quality parameters. Spermatozoa in the 10% PJ exhibited the highest level of progressive motility (87.39 at 24h) and had the highest level of plasma membrane integrity (84.90) and viability (81.04) and had the highest level of Morphological normality (89.78) . On the other hand, the 20% concentration of PJ demonstrated a deleterious effect on spermatozoa with the highest degree of damage to plasma membrane integrity (17.12 at 120h). Collectively, these findings indicate that moderate JP concentrations (10%) have capabilities of counteracting oxidative damage to spermatozoa produced during cold storage due to their high polyphenolic content, while excess concentrations of PJ result in pro-oxidative damage to spermatozoa. Therefore, from this study, optimal protocols for pomegranate extract provide for improved preservation of sheep epididymal spermatozoa in sheep artificial insemination programs