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.

BACKGROUND

Oxidative stress is a major contributor to male infertility. Therefore, fortifying assisted reproductive technology with nanotechnology could enhance sperm preservation.

AIMS/

OBJECTIVES

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.

CONCLUSION

MINE's potent antioxidants and anti-apoptotic properties effectively mitigate oxidative stress and boost mitochondrial function in stallion sperm cells.