SIRT3 Mediates Coordination Between Energy Metabolism and SOD Activity in Melatonin-Enhanced Boar Sperm Motility

Previous studies have demonstrated that melatonin (MLT) enhances boar sperm motility by modulating energy metabolism status, yet the underlying mechanisms remain incompletely understood. This study aims to investigate whether sirtuin 3 (SIRT3), a key mitochondrial deacetylase, mediates MLT's effects. Herein, the semen of six Landrace boars (16-18 months of age) was treated with 1.0 μM MLT with/without the SIRT3 inhibitor 3-TYP, preserved at 17 °C for 3 days, and subsequently maintained at 37 °C for a duration of 10 min. We demonstrated that MLT upregulated SIRT3 protein expression and reduced the acetylation level in mitochondrial proteins. MLT significantly increased glucose uptake and suppressed lactate release in the sperm, while elevating levels of pyruvate and acetyl-CoA, the substrates of pyruvate dehydrogenase (PDH) and the tricarboxylic acid (TCA) cycle, respectively, and the protein expression of PDH, indicating enhanced metabolic flux. Notably, inhibition of SIRT3 reversed MLT's effects: it blocked the increases in SIRT3 expression, glucose consumption, PDH expression, complex I activity, ATP content, and superoxide dismutase (SOD) activity, and prevented the decreases in the levels of acetylation and lactate, as well as pyruvate kinase (PK) activity, confirming the essential role of SIRT3. Functionally, the MLT-induced improvements in sperm motility parameters (total, progressive, fast motility, immotile) were also reversed by 3-TYP. Collectively, these findings demonstrate that the SIRT3-mediated pathway is essential for MLT to enhance boar sperm energy metabolism and antioxidant defense, thereby increasing ATP production and enhancing sperm motility. Targeting SIRT3 represents a promising therapeutic strategy for improving boar fertility and may also provide insights for research into human male infertility.