Idebenone improves post-thaw motility and mitochondrial function of buck sperm

Objective: This study was performed to evaluate the effects of idebenone (ID) supplemented freezing extender on the post-thaw quality of buck sperm, with particular focus on sperm kinematic parameters, viability, acrosome integrity, mitochondrial function, and antioxidant-related gene expression.Methods: Semen samples collected from five sexually active bucks were diluted in freezing extender supplemented with different concentrations of ID (0, 1, 5, and 10 μM).Post-thaw sperm kinematic parameters were analyzed using computer-assisted sperm analysis (CASA), whereas sperm viability, acrosome integrity, mitochondrial activity, and mitochondrial membrane integrity were assessed by flow cytometry using SYBR-14/DRAQ7, FITC-PNA/DRAQ7, MitoTracker Green/DRAQ7, and Rhodamine 123/DRAQ7 staining, respectively.Relative mRNA expression levels of glutathione peroxidase (GPX) and superoxide dismutase (SOD) were measured by real-time PCR using β-actin as a reference gene.Results: Among the tested concentrations, the ID5 group showed improved total and progressive motility compared with the control group.Based on these findings, ID5 was selected as optimal concentration for cryoprocessing and used for further functional evaluation.Sperm viability was not significantly different between the control and ID5 groups.In contrast, sperm acrosomal integrity and mitochondrial activity were significantly improved in the ID5 group compared with the control. Mitochondrial membrane integrity was not significantly different between the control and ID5 groups.In gene expression analysis, GPX expression was significantly lower in the ID5 group, whereas SOD2 expression was not significantly changed.Conclusion: These results suggest that freezing extender supplemented with optimum level of ID (5 µM) can improve the post-thaw quality of buck sperm.The beneficial effects of ID5 appear to be associated with improved motility, acrosomal status, and mitochondrial function, indicating its potential as a useful additive for buck sperm cryoprocessing.