Abstract

Background and Aim: Cryopreservation is commonly used in canine reproductive biotechnology to preserve genetic material for long-term storage. However, the freeze–thaw process induces oxidative stress, mitochondrial dysfunction, and structural damage in spermatozoa, thereby reducing post-thaw sperm quality and fertility potential. Mitoquinone (MitoQ), a mitochondria-targeted antioxidant, has been suggested as a promising cryoprotective additive that mitigates mitochondrial reactive oxygen species and enhances cellular integrity. This study aimed to assess the dose-dependent effects of MitoQ supplementation on mitochondrial function, oxidative stress, and the post-thaw quality of cryopreserved canine spermatozoa to identify an optimal concentration for semen cryopreservation.

Materials and Methods: Semen samples were collected from 10 healthy and fertile male dogs of various breeds. Each ejaculate was divided into four equal parts and cryopreserved with a Tris–fructose–egg yolk extender supplemented with different MitoQ concentrations (0, 100, 200, or 400 nM). After four weeks of storage in liquid nitrogen, the samples were thawed and evaluated in vitro. Post-thaw sperm quality was measured using computer-assisted sperm analysis to assess motility and kinematic parameters. Additional evaluations included sperm viability, plasma membrane integrity, acrosome integrity, lipid peroxidation levels, apoptotic status via flow cytometry, mitochondrial membrane potential, and intracellular hydrogen peroxide production. Data were analyzed using a mixed-effects model with dog as a random effect, and significance was set at p < 0.05.

Results: MitoQ supplementation had concentration-dependent effects on several sperm quality parameters. The 200 nM MitoQ group showed the most consistent improvements in post-thaw sperm quality. Total motility and curvilinear velocity were significantly higher at this concentration compared to the control and other treatment groups. Additionally, sperm treated with 200 nM MitoQ displayed improved plasma membrane and acrosome integrity, reduced lipid peroxidation, and a lower proportion of dead sperm cells. Trends toward increased viability and enhanced mitochondrial activity were also seen at this concentration. In contrast, 400 nM MitoQ supplementation was linked to decreased membrane integrity and increased oxidative stress markers, suggesting possible pro-oxidant effects at higher doses. Overall, the results indicate that moderate MitoQ supplementation supports mitochondrial redox balance and partially reduces cryo-induced damage in canine spermatozoa.

Conclusion: MitoQ supplementation at 200 nM modestly but consistently enhanced several in vitro indicators of post-thaw sperm quality in dogs, such as motility, membrane stability, and oxidative stress markers. These findings highlight the narrow therapeutic window of mitochondria-targeted antioxidants and stress the importance of dose optimization during semen cryopreservation. Although the observed improvements suggest potential benefits for reproductive biotechnology, further research with fertility-related functional outcomes and larger sample sizes is needed to confirm the practical reproductive impact of MitoQ supplementation in canine semen cryopreservation.

Keywords: canine sperm cryopreservation, mitoquinone, mitochondrial function, oxidative stress, post-thaw sperm quality, reactive oxygen species, sperm motility, sperm viability.