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 nano-emulsion (PO-NE) supplementation in semen extender on semen quality, redox status and apoptotic genes of cryopreserved bovine semen.

Methods: Seven proven fertility Holstein cattle bulls (4-6 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 anti-oxidative 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 - 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.