# Targeted metabolomics reveals the impact of glucose and pyruvate on energy metabolism and storage potential of stallion spermatozoa

## TL;DR

This study shows how glucose and pyruvate affect energy metabolism and storage in stallion sperm, highlighting the role of glycolysis and the TCA cycle.

## Contribution

The study reveals the specific impact of pyruvate on glycolysis and TCA cycle efficiency in stallion sperm metabolism.

## Key findings

- 10 mM pyruvate improves glycolysis efficiency in 40 mM glucose media.
- 10 mM pyruvate enhances TCA cycle efficiency in 67 mM glucose media.
- Low glucose media with 10 mM pyruvate increases kinematic efficiency and reduces methylglyoxal.

## Abstract

Oxidative phosphorylation is the main source of ATP for the stallion spermatozoa. Consequently, metabolites that favor mitochondrial function are receiving increased interest. However, glycolysis itself may be the major source of pyruvate and acetyl-CoA.

To determine the contribution of glycolysis to feed the tricarboxylic acid cycle to generate the reducing equivalents for the electron transport chain.

We stored stallion spermatozoa in the presence of different concentrations of glucose and pyruvate (1mM glucose /1mM pyruvate, 1mM glucose /10 mM pyruvate, 40 mM glucose / 1 mM pyruvate, 40 mM glucose /10 mM pyruvate, 67 Mm glucose / 1 mM pyruvate and 67 mM glucose /10 mM pyruvate). We performed targeted metabolomics using UHPLC-MS/MS, as well as several flow cytometry and computer-assisted motility assays, to investigate sperm function during storage.

Pyruvate 10 mM improved the efficiency of glycolysis in the 40 mM glucose media. This improvement may be related to the action of lactate dehydrogenases as revealed by relative changes in lactate and pyruvate in this group. Interestingly, the TCA cycle is fed through glutamine and glutamate, and 10 mM pyruvate improves the efficiency of TCA in a 67 mM glucose extender. Lower methylglyoxal (P < 0.05) and higher levels of GSH (P < 0.01) were observed when the 1 mM glucose extender was supplemented with 10 mM pyruvate. The kinematic efficiency (P < 0.05) was higher in the low glucose media.

Glucose probably contributes to stallion sperm metabolism feeding the TCA cycle, and aerobic glycolysis may play a major role in sperm functionality.

The online version contains supplementary material available at 10.1007/s11306-025-02393-2.