Myo-Inositol Nano-Emulsion for Enhancing Stallion Chilled Semen: Insights from In Vivo and In Silico Analyses of Sperm Quality, Oxidative Stress, Ultrastructure, Mitochondrial Response, and Semen Bacteriology.

Abstract: Oxidative stress is a major contributor to male infertility. Therefore, fortifying assisted reproductive technology with nanotechnology could enhance sperm preservation. Objective: This study aimed to examine the impact of myo-inositol nano-emulsion (MINE) supplementation in semen extender on sperm quality, redox balance, semen bacteriology, apoptosis, ultrastructure, and acrosome status of chilled stallion semen. Methods: Semen samples were collected and preserved with 0 (MINE0), 1 (MINE1), and 2 (MINE2) mg of MINE/mL of extender. Results: Results revealed that extender fortified with 1 or 2 mg of MINE significantly improved sperm progressive motility, viability, and membrane integrity after 72 hours of cooling (p < 0.05). All MINE levels significantly reduced the sperm abnormalities of chilled stallions' semen (p < 0.01) and significantly improved the number of intact acrosomes (p < 0.05). Fortified chilled stallion semen with MINE significantly boosted antioxidant defenses (total antioxidant capacity and catalase), while significantly lowering oxidative stress markers (malondialdehyde, nitric oxide, and hydrogen peroxide) (p < 0.05). MINE significantly reduced the populations of total bacteria, coliform, and spore-forming bacteria in the treated groups (p < 0.01). The molecular docking study showed that myo-inositol has a strong binding affinity to key mitochondrial proteins (GPX2, ALDH1A1, UQCRC2, VDAC2, and AKAP3), with calculated binding energies of -4.0, -3.62, -2.94, -2.72, and -2.31 kcal/mol indicating antioxidant defense mechanisms. The inclusion of MINE preserved the sperm ultrastructure, as evidenced by normal acrosome, mitochondria, nucleus, and plasma membrane integrity when observed using SEM. Conclusions: MINE's potent antioxidants and anti-apoptotic properties effectively mitigate oxidative stress and boost mitochondrial function in stallion sperm cells.