Membrane and mitochondrial antioxidants improve post-thaw functional quality of stallion spermatozoa.

Membrane- and mitochondria-targeted antioxidants were tested to see if they improve the quality and function of stallion sperm after freezing and thawing, with positive effects on motility, viability, and oxidative stress markers observed.

Background and Rationale

• Cryopreservation (freezing and thawing) of stallion semen causes oxidative stress that damages sperm cells.
• Damage occurs in key sperm components, including motility structures, cell membranes, and mitochondria, which are essential for energy production.
• Equine spermatozoa heavily depend on mitochondrial function and are sensitive to reactive oxygen species (ROS), which harm sperm quality.
• Antioxidants targeting specific sperm compartments (membranes or mitochondria) may reduce oxidative damage and improve sperm function after thawing.

Aims of the Study

• To evaluate whether astaxanthin, a membrane-targeted antioxidant, and MitoTEMPO, a mitochondria-targeted antioxidant, can improve functional quality of frozen-thawed stallion spermatozoa.
• To determine which concentrations of these antioxidants provide optimal protective effects.
• To assess multiple sperm functional parameters and oxidative stress indicators following cryopreservation with these antioxidants.

Methods

• Semen samples were collected from nine stallions and cryopreserved using a standard extender (BotuCrio®) supplemented with different concentrations of:
  • Astaxanthin (10, 25, 50 µM)
  • MitoTEMPO (5, 10, 25 µM)
• Post-thaw sperm quality was evaluated using:
  • Computer-assisted sperm analysis (CASA) for motility and kinematics
  • Multiparametric flow cytometry assays for:
    • Viability (ability of sperm to live)
    • Apoptosis-like changes (early signs of cell death)
    • Acrosomal integrity (important for fertilization capability)
    • Mitochondrial activity (energy-producing capacity)
    • Levels of reactive oxygen species (ROS) inside the sperm
    • Intracellular calcium levels (linked to sperm activation and function)

Key Findings

• Both astaxanthin and MitoTEMPO supplementation led to significant improvements in several sperm functional parameters after thawing compared to control samples without antioxidants.
• Improvements included:
  • Higher total and progressive motility—meaning sperm moved more and moved forward better
  • Better kinematic parameters (such as speed and trajectory)
  • Greater viability, indicating healthier sperm cells
  • Improved acrosomal integrity, important for the fertilization process
• Both antioxidants were effective at reducing intracellular and mitochondrial ROS levels, thus lessening oxidative damage.
• Astaxanthin mainly enhanced plasma membrane stability, suggesting it protects the outer sperm cell structure.
• MitoTEMPO primarily improved mitochondrial activity, supporting the sperm’s energy production capacity.
• The best effects were seen at intermediate concentrations:
  • 25 µM astaxanthin
  • 10 µM MitoTEMPO

Implications and Future Directions

• Membrane- and mitochondrial-targeted antioxidants provide complementary protective effects during semen cryopreservation in stallions.
• Using these antioxidants could enhance post-thaw sperm function and potentially improve fertility outcomes in equine artificial insemination.
• Further research is needed to:
  • Confirm the effects on actual fertilization and pregnancy rates
  • Fine-tune antioxidant dosages and combinations for optimal semen preservation protocols
  • Understand long-term impacts on stallion fertility and offspring quality