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Targeted metabolomics reveals the impact of glucose and pyruvate on energy metabolism and storage potential of stallion spermatozoa.
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. Objective: To determine the contribution of glycolysis to feed the tricarboxylic acid cycle to generate the reducing equivalents for the electron transport chain. Methods: 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. Results: 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. Conclusions: Glucose probably contributes to stallion sperm metabolism feeding the TCA cycle, and aerobic glycolysis may play a major role in sperm functionality.
© 2026. The Author(s).
Publication Date: 2026-03-28 PubMed ID: 41903002PubMed Central: PMC13032957DOI: 10.1007/s11306-025-02393-2Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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Overview
- This research investigates how different concentrations of glucose and pyruvate affect energy metabolism and functional traits of stallion spermatozoa during storage.
- The study focuses on understanding the metabolic pathways, especially glycolysis and the tricarboxylic acid (TCA) cycle, that provide ATP necessary for sperm activity.
Background and Objective
- Stallion sperm primarily generate ATP through oxidative phosphorylation within mitochondria to meet their energy demands.
- Metabolites influencing mitochondrial function, such as pyruvate, are gaining attention for their role in sperm metabolism.
- Although oxidative phosphorylation is key, glycolysis may serve as an important source of pyruvate and acetyl-CoA, feeding into the TCA cycle.
- The study aims to elucidate the contribution of glycolysis to energizing the TCA cycle, which produces reducing equivalents for the electron transport chain critical for ATP synthesis.
Methods
- Stallion sperm samples were stored in extenders containing various combinations of glucose and pyruvate concentrations:
- 1 mM glucose / 1 mM pyruvate
- 1 mM glucose / 10 mM pyruvate
- 40 mM glucose / 1 mM pyruvate
- 40 mM glucose / 10 mM pyruvate
- 67 mM glucose / 1 mM pyruvate
- 67 mM glucose / 10 mM pyruvate
- Targeted metabolomics analyses were performed using Ultra-High-Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (UHPLC-MS/MS) to detect metabolite changes.
- Flow cytometry and computer-assisted motility assays were used to assess sperm functional parameters during the storage period.
Key Findings
- Pyruvate at 10 mM concentration enhanced glycolytic efficiency in the medium with 40 mM glucose.
- The observed improvement was linked to the role of lactate dehydrogenase enzymes, as indicated by relative changes in lactate and pyruvate levels in this experimental group.
- The TCA cycle primarily received metabolic input from glutamine and glutamate, rather than directly from glucose-derived products.
- A 10 mM pyruvate concentration improved the efficiency of the TCA cycle when sperm were stored in 67 mM glucose medium.
- Lower levels of methylglyoxal, a toxic by-product of glycolysis, were detected when 1 mM glucose medium was supplemented with 10 mM pyruvate (statistically significant with P < 0.05).
- Higher concentrations of glutathione (GSH), an important antioxidant, were observed in this same group (P < 0.01), suggesting reduced oxidative stress.
- Sperm kinematic efficiency, representing motility and movement quality, was higher in low glucose media, indicating that excessive glucose may not be favorable for sperm movement.
Conclusions and Implications
- Glucose appears to support stallion sperm metabolism by feeding into the TCA cycle indirectly, possibly via amino acid substrates like glutamine and glutamate.
- Aerobic glycolysis, including pyruvate metabolism, plays a substantial role in maintaining sperm functionality during storage.
- Optimizing glucose and pyruvate concentrations in sperm storage media can improve energy metabolism efficiency and sperm quality by balancing pathways involved in ATP production and oxidative stress control.
- This knowledge has practical applications in equine reproduction, particularly in the development of better extenders for sperm preservation and artificial insemination programs.
Cite This Article
APA
Becerro-Rey L, Martín-Cano FE, Silva-Rodríguez A, Ortega-Ferrusola C, da Silva-Álvarez E, Gil C, Peña FJ.
(2026).
Targeted metabolomics reveals the impact of glucose and pyruvate on energy metabolism and storage potential of stallion spermatozoa.
Metabolomics, 22(2), 45.
https://doi.org/10.1007/s11306-025-02393-2 Publication
Researcher Affiliations
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, Universidad de Extremadura, Avenida de la Universidad s/n, 10003, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, Universidad de Extremadura, Avenida de la Universidad s/n, 10003, Cáceres, Spain.
- Facility of Innovation and Analysis in Animal Source Foodstuffs, Universidad de Extremadura, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, Universidad de Extremadura, Avenida de la Universidad s/n, 10003, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, Universidad de Extremadura, Avenida de la Universidad s/n, 10003, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, Universidad de Extremadura, Avenida de la Universidad s/n, 10003, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, Universidad de Extremadura, Avenida de la Universidad s/n, 10003, Cáceres, Spain. fjuanpvega@unex.es.
MeSH Terms
- Animals
- Male
- Horses
- Pyruvic Acid / metabolism
- Pyruvic Acid / pharmacology
- Glucose / metabolism
- Glucose / pharmacology
- Spermatozoa / metabolism
- Spermatozoa / drug effects
- Metabolomics / methods
- Energy Metabolism / drug effects
- Glycolysis
- Citric Acid Cycle
- Semen Preservation / methods
- Sperm Motility / drug effects
- Tandem Mass Spectrometry
Conflict of Interest Statement
Declarations. Conflict of interest: The authors have no competing interests to declare that are relevant to the content of this article.
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