The beneficial impact of pomegranate oil nanoemulsion on the quality of cryopreserved bovine sperm: antioxidant and anti-apoptotic effects.

Objective: This study was conducted to investigate the effect of pomegranate oil nanoemulsion (PO-NE) supplementation in semen extender on semen quality, redox status and apoptotic genes of cryopreserved bovine semen.

Methods: Seven proven fertility Holstein Friesian bulls (4 to 6 years) were involved, and the semen samples were collected using the artificial vagina method. Semen was pooled and cryopreserved in tris extender containing PO-NE at 0 (PO-NE0), 1 (PO-NE1), 2 (PO-NE 2) and 4 μg/mL (PO-NE4), respectively.

Results: Incorporating 2 or 4 μg/mL PO-NE into freezing media significantly increased sperm progressive motility, viability, membrane integrity, and kinematic parameters. Furthermore, the aforementioned two treated concentrations demonstrated superior antioxidative activities (total antioxidant capacity and super oxide dismutase) and higher nitric oxide levels compared to the control group (p<0.05). The levels of hydrogen peroxide, malondialdehyde, and Nuclear Factor-Kappa B were notably lower in the PO-NE4 treated group compared to the control group. The addition of 2 or 4 μg/mL of PO-NE to the freezing media significantly downregulated pro-apoptotic genes (caspase 3, Bax), while significantly induced the expression of anti-apoptotic gene Bcl2. The addition of PO-NE preserved plasma membrane and acrosome integrity and maintained the ultrastructure of sperm, contrasting with PO-NE0, which exhibited the most damage.

Conclusion: Supplementing the bovine freezing extender with 2 or 4 μg/mL of PO-NE enhanced post-thawed sperm characteristics by reducing oxidative stress, improving antioxidant indices and apoptotic genes expression, and preserving the ultrastructure integrity of frozen-thawed cattle sperm.