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

ABSTRACT 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 H 2 O 2 ) 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.