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

## TL;DR

Adding riboflavin to goat semen improves sperm motility by boosting mitochondrial energy production through fatty acid β-oxidation.

## Contribution

This study demonstrates that riboflavin enhances goat sperm quality by promoting mitochondrial β-oxidation and energy metabolism.

## Key findings

- Riboflavin increased sperm motility, ATP levels, and key metabolic enzyme activities in goat sperm.
- The β-oxidation inhibitor etomoxir reversed the positive effects of riboflavin, confirming the pathway's role.
- CPT1 and ACADVL enzymes were identified in goat sperm, and their activity was enhanced by riboflavin.

## Abstract

Artificial insemination (AI) is a widely used assisted breeding technique in goats, and improving sperm quality is key to enhancing AI efficiency. Sperm motility and function depend on a continuous supply of energy, and the fatty acid β-oxidation pathway regulated by riboflavin-derived cofactors is one of the energy sources for sperm. In this study, goat semen was treated with different concentrations of riboflavin, and sperm quality was assessed after incubation. Riboflavin supplementation significantly increased sperm motility, ATP levels, and the activity of key metabolic enzymes, whereas the addition of β-oxidation inhibitors reversed these improvements. These results indicate that riboflavin enhances sperm energy metabolism through the fatty acid β-oxidation pathway, thereby improving overall sperm quality. This finding could help increase the success rate of AI in goats, improve reproductive efficiency, and provide economic benefits for animal production.

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 (p < 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 (p < 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.