Cryoprotective Efficacy of Omega‐3 Nano‐Emulsion on Kinematic Parameters, Acrosome Status, Subcellular Ultrastructure, and Oxidative/Antioxidant Markers in Cryopreserved Stallion Semen

This study aims to investigate the cryoprotective effect of Omega-3 nano-emulsion (Omega-3 NE) on stallion sperm quality, kinematic parameters, acrosome status, subcellular ultrastructure, oxidative/antioxidant markers, and semen microbiota. Forty ejaculates were collected, extended, and cryopreserved from 5 fertile Pure Egyptian stallions (Equus caballus). The ejaculates were divided into five groups: a control group (without additive) and four groups supplemented with 25, 50, 100, and 200 μg of Omega-3 NE/mL. The Omega-3 NE exhibited an average particle size of 51-146 nm, a PDI of 0.58, and a zeta potential of -31 mV. Omega-3 NE (200 μg/mL) significantly improved progressive motility, viability, and membrane integrity of stallion semen (p < 0.05). Additionally, supplementation with Omega-3 NE (200 μg/mL) led to a significant enhancement in post-thawed sperm kinematic parameters, including PM, DSL, VCL, and VSL, by 40%, 21.5%, 26.7%, and 20.7%, respectively, compared to the control group. The addition of 100 or 200 μg/mL Omega-3 NE to the media resulted in a higher percentage of live sperm with intact acrosomes. Additionally, all Omega-3 NE treatments significantly decreased the percentage of dead sperm with intact acrosomes as well as microbiota load (total bacterial count and coliform bacteria count) compared to the control (p < 0.01). Significant improvements in antioxidant status (TAC and CAT) and reduction of oxidative stress markers (MDA, NO, and H₂O₂) were observed in all Omega-3 NE groups compared to the control group (p < 0.05). Omega-3 NE (200 μg/mL) significantly reduced sperm apoptosis (p < 0.01) and preserved better subcellular integrity compared to the control and other treatment groups. The results suggest that Omega-3 NE at concentrations of 100-200 μg/mL can effectively enhance sperm cryo-resistance via enhancing sperm quality and kinematic variables, reducing oxidative stress and microbiota load, and maintaining sperm subcellular ultrastructure. The study highlights the potential of Omega-3 NE as a nanotechnology-based approach to boost assisted reproductive technologies in stallion breeding programmes.