Long-chain fatty acids promote ATP production in post-thaw boar sperm through mitochondrial β-oxidation

Due to the current limitations of boar semen cryopreservation systems, the effective restoration of sperm quality following thawing remains a significant challenge. This study investigates whether post-thaw boar sperm can uptake exogenous long-chain fatty acids (LCFAs) and utilize them for ATP generation, thereby sustaining linear motility and enhancing sperm vitality. Boar semen was diluted in extender solutions supplemented with varying concentrations of a lipid mixture (0, 0.01 %, 0.1 %, and 1 % LM). Following cryopreservation and subsequent thawing, key sperm parameters were assessed, including motility, viability, acrosome integrity, mitochondrial membrane potential (MMP), and ATP levels, as well as the enzymatic activities of malate dehydrogenase (MDH), succinate dehydrogenase (SDH), and carnitine palmitoyltransferase-1 (CPT-1). Compared to the control group, supplementation with 0.1 % LM significantly enhanced post-thaw sperm motility and improved viability and acrosomal integrity (P < 0.05). This concentration also led to increased MMP, elevated ATP levels, as well as enhanced activities of MDH, SDH, and CPT1 (P < 0.05). Furthermore, to further verify that post-thaw boar sperm utilize LCFAs to generate ATP through mitochondrial β-oxidation. It was found that adding 100 μM mitochondrial β-oxidation inhibitor etomoxir to the extender, significantly reduced post-thaw boar sperm progressive motility, MMP, ATP levels, and CPT1 activity (P < 0.05), thereby attenuating the beneficial effects of exogenous LCFAs supplementation. These findings suggest that post-thaw boar sperm are capable of assimilating exogenous LFCAs, which subsequently promote mitochondrial β-oxidation, increasing TCA cycle activity and ATP production, therefore improving the quality of post-thaw boar sperm.