Nano-Encapsulated Vitamin E Enhances Cryo-Resistance of Stallion Spermatozoa: A Comprehensive Evaluation of Sperm Quality, Kinematic Parameters, Antioxidant Capacity, Microbiota and Ultrastructure.

This experiment explores the comparative ability of vitamin E (VE) and its nano-emulsion (VEN) in enhancing cryo-resistance of stallion spermatozoa by examining semen quality, kinematic variables, acrosome integrity, apoptosis, antioxidant capacity, ultra-structural alterations and microbiota of cryopreserved stallion sperm. Semen from five stallions (8-10 years) was cryopreserved with 0.1 or 1 mM of VE or VEN; and a non-additive group served as the control. The VEN particles ranged in size from 72 to 172 nm, with a mean diameter of 287 nm. Supplementation with VE (1 mM) or VEN (0.1 or 1 mM) significantly enhanced progressive motility and viability during both the equilibration (for 30 min at 4°C) and post-thaw phases compared to the control (p < 0.05). Plasma membrane functionality was superior in all treated groups compared to control group (p < 0.001). Post-thaw sperm kinematic parameters and acrosome integrity were significantly enhanced in VEN treatment compared to VE and control groups. VEN (1 mM) notably improved antioxidant defences, specifically increasing TAC and catalase levels (p < 0.01) and a significant reduction in oxidative stress (NO, MDA and H2O2) in all treated semen samples (p < 0.01). VEN (1 mM) significantly reduced the proportion of apoptotic sperm (p < 0.01), increased viable sperm counts and the preservation of sperm ultrastructural integrity, as evidenced by intact acrosome and plasma membranes. VE and VEN treatments markedly lowered the total bacterial and coliform counts. Overall, supplementation with VEN (0.1 and 1 mM) effectively optimizes post-thaw stallion sperm quality by enhancing antioxidant defences and reducing the seminal microbiota.