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Objectives <p>Semen preservation is a critical aspect of assisted reproductive technologies in canine breeding programs; however, oxidative stress during chilled storage negatively affects sperm quality by impairing motility, membrane integrity, and mitochondrial function. This study investigated the protective effects of apigenin and N-acetylcysteine amide (NACA), individually and in combination, on the quality, antioxidant defense system, and mitochondrial activity of canine spermatozoa stored at 5&#xa0;°C for 72&#xa0;h.</p> Methods <p>Semen samples collected from five healthy mixed-breed dogs with proven fertility were pooled and diluted in a Tris–egg yolk extender supplemented with apigenin (0.2 or 0.4 mM), NACA (0.5 or 1 mM), or a combination of both (0.4 mM apigenin + 1 mM NACA). Sperm motility, membrane functionality, viability, DNA integrity, oxidative/antioxidant biomarkers, and mitochondrial parameters were evaluated at 0, 24, 48, and 72&#xa0;h.</p> Results <p>Both antioxidants significantly improved all parameters compared with the control, with the combined treatment exhibiting the most pronounced and synergistic effects: motility and kinematic parameters approximately twofold higher, antioxidant enzyme activities markedly enhanced, lipid/protein oxidation reduced, and mitochondrial integrity and ATP production preserved after 72&#xa0;h.</p> Conclusions <p>In conclusion, the synergistic combination of apigenin and N-acetylcysteine amide significantly improves the quality and longevity of chilled canine spermatozoa, offering a promising strategy for optimizing semen extenders in canine breeding programs.</p>

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Synergistic protection of chilled canine spermatozoa by apigenin and N-acetylcysteine amide through enhanced antioxidant defense and mitochondrial function

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月8日

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  • Soroush Heidari,
  • Ali Soleimanzadeh,
  • Reza Mirzaie

摘要

Objectives

Semen preservation is a critical aspect of assisted reproductive technologies in canine breeding programs; however, oxidative stress during chilled storage negatively affects sperm quality by impairing motility, membrane integrity, and mitochondrial function. This study investigated the protective effects of apigenin and N-acetylcysteine amide (NACA), individually and in combination, on the quality, antioxidant defense system, and mitochondrial activity of canine spermatozoa stored at 5 °C for 72 h.

Methods

Semen samples collected from five healthy mixed-breed dogs with proven fertility were pooled and diluted in a Tris–egg yolk extender supplemented with apigenin (0.2 or 0.4 mM), NACA (0.5 or 1 mM), or a combination of both (0.4 mM apigenin + 1 mM NACA). Sperm motility, membrane functionality, viability, DNA integrity, oxidative/antioxidant biomarkers, and mitochondrial parameters were evaluated at 0, 24, 48, and 72 h.

Results

Both antioxidants significantly improved all parameters compared with the control, with the combined treatment exhibiting the most pronounced and synergistic effects: motility and kinematic parameters approximately twofold higher, antioxidant enzyme activities markedly enhanced, lipid/protein oxidation reduced, and mitochondrial integrity and ATP production preserved after 72 h.

Conclusions

In conclusion, the synergistic combination of apigenin and N-acetylcysteine amide significantly improves the quality and longevity of chilled canine spermatozoa, offering a promising strategy for optimizing semen extenders in canine breeding programs.