Effects of quercetin supplementation in cooling extender on sperm quality of stallions.

Overview

• This study investigated the effects of supplementing equine semen cooling extenders with quercetin, a known antioxidant, focusing on its impact on sperm quality during cooling storage.
• The research sought to determine an optimal quercetin concentration and evaluate its influence on sperm motility, membrane integrity, and oxidative stress parameters under both normal cooling and induced oxidative stress conditions.

Background and Purpose

• Equine sperm quality declines during cooling due to oxidative stress caused by excessive reactive oxygen species (ROS).
• Quercetin is a flavonoid compound with antioxidant properties that can potentially mitigate oxidative damage.
• The study aimed to identify the best concentration of quercetin to include in semen extenders and assess its effects on sperm quality under cooling conditions.

Study Design and Methods

• Four experiments were conducted, involving ejaculates from multiple stallions:
• Experiment 1: Tested four quercetin concentrations (0, 20, 30, 45 µg/mL) at 5°C for 24 hours using 8 ejaculates from 4 stallions to select a suitable concentration.
• Experiment 2: Used the selected quercetin concentration (20 µg/mL) in a larger cohort (42 ejaculates, 21 stallions) and included detailed flow cytometry assessments to evaluate sperm function.
• Experiment 3: Examined the effects of quercetin in stallions characterized as “good coolers” or “poor coolers” based on their baseline sperm cooling tolerance, involving 20 ejaculates from 10 stallions.
• Experiment 4: Tested quercetin’s antioxidant effect under induced oxidative stress using arachidonic acid, measuring lipid peroxidation and hydrogen peroxide (H₂O₂) production in semen from 10 stallions.

Key Findings

• Optimal Dose Selection: 20 µg/mL quercetin was deemed optimal because higher concentrations (30 and 45 µg/mL) impaired sperm motility in a dose-dependent manner.
• Effect under Conventional Cooling: Quercetin supplementation at 20 µg/mL did not improve sperm motility, membrane integrity, or overall sperm function during typical cooling storage.
• Effect According to Cooling Tolerance: Both “good” and “poor” cooler stallions showed no marked improvement in sperm quality due to quercetin addition under normal cooling conditions.
• Effect under Oxidative Stress: Under severe oxidative stress induced by arachidonic acid, quercetin significantly reduced markers of oxidative damage, specifically:
• Lipid peroxidation, which reflects damage to sperm membrane lipids.
• Hydrogen peroxide production, a reactive oxygen species contributing to oxidative stress.

Conclusions and Implications

• Adding quercetin at 20 µg/mL to semen extenders does not enhance sperm quality during routine cooling storage of equine semen.
• However, quercetin effectively reduces oxidative damage markers during conditions of elevated oxidative stress, suggesting a protective antioxidant role in more challenging handling scenarios.
• These findings imply quercetin could be useful in equine breeding programs specifically when semen is exposed to heightened oxidative stress, such as during prolonged storage, transport, or freeze-thaw processes, rather than during standard cooling.
• Further research may explore its application in these specific contexts and investigate long-term fertility outcomes after quercetin supplementation.