Riboflavin Increases Goat Sperm Motility via Enhancement of Mitochondrial β-Oxidation

Mitochondrial energy metabolism is fundamental to sperm function, and fatty acid β-oxidation is an important pathway for adenosine triphosphate (ATP) production. Riboflavin, a precursor of key flavin cofactors, plays a critical role in regulating β-oxidation and supports multiple physiological processes. This study aimed to determine whether adding riboflavin to semen dilution media could enhance goat sperm motility and to elucidate the underlying metabolic mechanisms. Goat semen was diluted in tris-citrate-glucose (TCG) medium containing 0, 5, 10, 15, and 20 μM riboflavin and incubated at 37 °C, after which sperm motility, acrosome integrity, mitochondrial membrane potential, ATP levels, malate dehydrogenase (MDH) and succinate dehydrogenase (SDH) activities, and the NADH/NAD⁺ were evaluated. The localization and expression of the β-oxidation enzymes carnitine palmitoyltransferase 1 (CPT1) and extremely long chain acyl-CoA dehydrogenase (ACADVL) were examined, and CPT1 activity was quantified. The results showed that CPT1 and ACADVL were present in goat sperm, and that 10 μM riboflavin significantly increased sperm motility, acrosome integrity, mitochondrial activity, ATP levels, and the activities of MDH, SDH, and CPT1, while also elevating NADH/NAD⁺ levels (<i>p</i> < 0.05). Notably, these enhancements were suppressed by 100 μM etomoxir, a mitochondrial β-oxidation inhibitor, which reduced total motility, ATP Levels, and CPT1 activity after riboflavin supplementation (<i>p</i> < 0.05). These findings indicate that goat sperm at least partly rely on mitochondrial β-oxidation for ATP generation and that riboflavin supplementation enhances mitochondrial metabolism, thereby improving sperm quality.