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.

Overview

• This study investigates how supplementing stallion semen extenders with a myo-inositol nano-emulsion (MINE) can improve the quality, antioxidant status, and microbial profile of chilled semen, potentially enhancing fertility preservation.
• Both laboratory tests on semen samples and computer-based molecular analyses were conducted to understand the effects of MINE on sperm cells’ structure, function, oxidative stress, and bacterial contamination.

Background and Objective

• Oxidative stress is a key factor impairing male fertility by damaging sperm cells.
• Assisted reproductive technologies (ART) often face challenges in preserving sperm quality during cooling and storage.
• Nano-emulsions of antioxidants like myo-inositol might better protect sperm against oxidative damage.
• The study aimed to evaluate the effect of different concentrations of MINE added to semen extenders on:
• Sperm motility and viability
• Oxidative stress markers and antioxidant status
• Sperm ultrastructure and acrosome integrity
• Semen bacteriology (bacterial contamination levels)
• Apoptotic markers and mitochondrial responses

Methods

• Chilled stallion semen samples were divided and preserved in extenders containing 0 mg/mL (MINE0), 1 mg/mL (MINE1), or 2 mg/mL (MINE2) of myo-inositol nano-emulsion.
• Sperm quality parameters including progressive motility, viability, membrane integrity, and abnormalities were analyzed after 72 hours of cooling.
• Oxidative stress was assessed by measuring markers such as malondialdehyde, nitric oxide, hydrogen peroxide, as well as total antioxidant capacity and catalase activity.
• Semen bacteriology was evaluated to quantify total bacteria, coliforms, and spore-forming bacteria in treated versus control groups.
• Molecular docking simulations explored how myo-inositol interacts with mitochondrial proteins linked to antioxidant defense and sperm function.
• Scanning electron microscopy (SEM) was used to inspect sperm ultrastructure, including mitochondria, nucleus, acrosome, and plasma membrane integrity.

Key Findings

• Sperm Quality: Both 1 mg/mL and 2 mg/mL MINE supplementation significantly improved sperm progressive motility, viability, and membrane integrity after 72 hours.
• Reduction of Abnormalities: All concentrations of MINE notably decreased the percentage of sperm abnormalities.
• Acrosome Integrity: Increased number of intact acrosomes was observed with MINE treatment, indicating better sperm functionality.
• Oxidative Stress and Antioxidant Defense:
  • Antioxidant biomarkers such as total antioxidant capacity and catalase were significantly elevated in MINE-treated groups.
  • Oxidative stress markers (malondialdehyde, nitric oxide, hydrogen peroxide) were significantly decreased, suggesting reduced lipid peroxidation and cellular damage.
• Bacteriology: MINE addition lowered total bacterial loads, coliform bacteria, and spore-forming bacteria, thus potentially increasing semen hygiene and shelf life.
• Molecular Docking Insights:
  • Myo-inositol showed strong binding affinities to mitochondrial proteins associated with oxidative defense (GPX2, ALDH1A1, UQCRC2, VDAC2, AKAP3).
  • These interactions indicate a plausible mechanism by which MINE enhances mitochondrial antioxidant functions and protects sperm cells.
• Ultrastructural Integrity: SEM images confirmed that MINE preserved critical sperm ultrastructure including the acrosome, mitochondria, nucleus, and plasma membrane.

Conclusions and Implications

• Myo-inositol nano-emulsion supplementation in semen extenders is a promising strategy to enhance stallion sperm preservation during chilling by:
• Improving motility and viability
• Reducing oxidative damage through enhanced antioxidant defense
• Supporting mitochondrial function, critical for energy and sperm motility
• Protecting structural integrity at cellular and subcellular levels
• Decreasing bacterial contamination, potentially reducing infection risks
• This dual in vivo and in silico approach provides evidence supporting the use of nanotechnology-based antioxidant delivery systems in reproductive biotechnology to improve fertility outcomes.