L-Carnitine supplementation to the stallion semen extender attenuates the cryopreservation induced oxidative stress and ameliorates sperm functional attributes.

Overview

• This study investigated the effects of adding L-Carnitine to semen extenders on the quality and oxidative stress levels of stallion semen before freezing and after thawing.
• Results showed that L-Carnitine supplementation, particularly at 5 mM concentration, improved sperm function and reduced oxidative damage in semen from Marwari breed stallions.

Introduction and Purpose

• The research focused on improving cryopreservation outcomes for stallion semen by addressing oxidative stress, which damages sperm during freezing and thawing.
• L-Carnitine, known for its antioxidant properties, was tested as a supplement in semen extenders to evaluate if it could protect sperm quality during the cryopreservation process.
• The study aimed to measure sperm functional parameters and oxidative stress biomarkers at various L-Carnitine concentrations.

Methods

• Semen was collected from six adult Marwari stallions, with six ejaculates per stallion, for a total of 36 samples.
• Semen samples were treated with four different concentrations of L-Carnitine in the semen extender: 0 mM (control), 2.5 mM (T1), 5 mM (T2), and 10 mM (T3).
• Semen samples were evaluated both before freezing (pre-freeze) and after thawing (post-thaw) for various functional and oxidative parameters.

Key Semen Quality Parameters Assessed

• Progressive motility: The ability of sperm to move actively and in a straight line, critical for fertilization.
• Acrosome integrity: Integrity of the acrosome cap, essential for sperm to penetrate the egg.
• Plasma membrane integrity: Health and functionality of the sperm cell membrane, important for sperm viability.
• Mitochondrial membrane potential (MMP): Indicator of mitochondrial function and sperm energy status.

Oxidative Stress Markers Measured

• Superoxide dismutase (SOD): An antioxidant enzyme that neutralizes superoxide radicals.
• Glutathione peroxidase (GPX): An enzyme that helps reduce peroxide levels, preventing oxidative damage.
• Total antioxidant capacity (T-AOC): A measure of the cumulative antioxidant activity in the sample.
• Malondialdehyde (MDA): A marker of lipid peroxidation and oxidative damage to cell membranes.

Results

• All L-Carnitine supplemented groups showed statistically significant improvement in motility, acrosome integrity, plasma membrane integrity, and mitochondrial membrane potential compared to control.
• The 5 mM group (T2) demonstrated the most pronounced improvements, including:
  • Increase in sperm motility from approximately 32% to 44%.
  • Increase in acrosome integrity from about 59% to 67%.
  • Higher mitochondrial membrane potential values (approx. 56% to 66%).
• Regarding oxidative stress:
  • SOD and T-AOC were significantly increased (P < 0.01) at 5 mM L-Carnitine, indicating enhanced antioxidant defense.
  • GPX activity was also higher (P < 0.05) in the 5 mM group, supporting reduced oxidative damage.
  • MDA levels decreased significantly in all L-Carnitine groups, implying reduced lipid peroxidation and membrane damage.

Conclusions

• Supplementing stallion semen extenders with L-Carnitine, especially at 5 mM concentration, enhances semen quality and sperm functional attributes both before freezing and after thawing.
• L-Carnitine improves antioxidant enzyme activities, thereby reducing oxidative stress-induced damage associated with cryopreservation.
• These findings suggest L-Carnitine as an effective additive for improving stallion semen cryopreservation outcomes, potentially increasing fertility rates through improved sperm preservation.