Coenzyme Q10 supplementation enhances HSP70 gene expression and preserves mitochondrial function in cryopreserved...

Cryopreservation is essential for the long-term preservation and dissemination of superior goat genetics but induces oxidative, osmotic, and thermal stress that compromises sperm quality and fertilizing potential. Mitochondrial dysfunction, lipid peroxidation, and impaired cellular stress responses are major contributors to cryoinjury. Coenzyme Q10 (CoQ10), a mitochondrial electron carrier and potent lipid-soluble antioxidant, has been proposed as a cryoprotective supplement; however, its effects on heat shock protein 70 (HSP70) gene expression and mitochondrial function in cryopreserved Peranakan Ettawa (PE) goat spermatozoa remain insufficiently characterized. This study evaluated the effects of graded CoQ10 supplementation in Extender A on post-thaw HSP70 gene expression, mitochondrial membrane potential (MMP), lipid peroxidation, and sperm viability.

Semen was collected from one clinically healthy 4-year-old PE buck, and eight independent ejaculates were processed using a split-ejaculate design. Each ejaculate was divided into five treatment groups supplemented with CoQ10 at 0 (C0), 5 (C1), 10 (C2), 20 (C3), and 40 mg/dL (C4). Following cryopreservation and thawing, HSP70 gene expression was quantified using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. MMP was assessed using rhodamine 123 staining and confocal microscopy, lipid peroxidation was determined by measuring malondialdehyde (MDA) concentrations, and sperm viability was evaluated using eosin-nigrosine staining. Data were analyzed using one-way analysis of variance followed by Tukey or Games-Howell post hoc tests, with statistical significance set at p < 0.05.

CoQ10 supplementation significantly improved all evaluated post-thaw parameters (p < 0.001). The C4 group exhibited the highest HSP70 expression (3.19 ± 0.08), representing approximately a 6.5-fold increase over the control, together with the highest MMP (56.91 ± 4.21%) and sperm viability (64.37 ± 6.58%). In contrast, the lowest MDA concentration was observed in C2 (8.20 ± 3.27 ng/µL), whereas the control group showed the highest lipid peroxidation (28.80 ± 1.38 ng/µL). These findings indicate a dose-dependent but non-linear antioxidant response, with higher CoQ10 concentrations preferentially supporting mitochondrial integrity and cellular stress adaptation rather than maximally suppressing lipid peroxidation.

Supplementation of Extender A with CoQ10 effectively enhanced post-thaw quality of PE goat spermatozoa by preserving mitochondrial function, improving sperm viability, and upregulating HSP70 gene expression. Although 10 mg/dL most effectively reduced lipid peroxidation, 40 mg/dL provided the most favorable overall cryoprotective profile and represents a promising candidate dose for further optimization of goat semen cryopreservation protocols. Future studies involving multiple bucks, fertility trials, and mechanistic investigations at the protein level are warranted to validate these findings and facilitate the practical application of CoQ10 in artificial insemination programs.