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This study aims to evaluate the protective effects of apigenin and N-acetylcysteine amide on chilled canine spermatozoa during storage.

May 10, 2026BMC Veterinary Research0 citationsOpen Access

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

SHSoroush HeidariUrmia University ASAli SoleimanzadehUrmia University RMReza MirzaieUrmia University

Key Points

This study aims to evaluate the protective effects of apigenin and N-acetylcysteine amide on chilled canine spermatozoa during storage.
Chilled spermatozoa stored at 5 °C for 72 h were treated with apigenin, NACA, or both.
Parameters evaluated include sperm motility, membrane function, viability, DNA integrity, and mitochondrial activity at multiple time points.
Semen from five healthy mixed-breed dogs was pooled and diluted in a Tris–egg yolk extender with varying concentrations of the antioxidants.
Combined treatment with apigenin and NACA resulted in sperm motility and kinematic parameters approximately twofold higher compared to control.
Antioxidant enzyme activities significantly enhanced with both treatments, indicating strong antioxidant defense.
Mitochondrial integrity and ATP production were preserved after 72 h, reducing oxidative damage to lipids and proteins.

Abstract

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. 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. 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. 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.

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