In vitro, the aging of stallion spermatozoa at 22 °C is linked to alteration in Ca2+ and redox homeostasis and may be slowed by regulating metabolism.
Abstract: Conservation of equine semen in the liquid state is a central procedure in horse breeding and constitutes the basis of associated reproductive technologies. The intense mitochondrial activity of the stallion spermatozoa increases oxidative stress along the storage period, leading to sperm demise within 24-48 h of storage, particularly when maintained at room temperature. Recently, the relationship between metabolism and oxidative stress has been revealed. The study aimed to extend the period of conservation of equine semen, at room temperature through modification of the metabolites present in the media. Methods: Processed ejaculates (n = 9) by single-layer colloid centrifugation were split in different aliquots and extended in Tyrode's basal media, or modified Tyrode's consisting of 1 mM glucose, 1 mM glucose 10 mM pyruvate, 40 mM glucose, 40 mM Glucose 10 mM pyruvate, 67 mM glucose and 67 mM glucose 10 mM pyruvate. At time 0h, and after 24 and 96 h of storage, motility was evaluated by CASA, while mitochondrial production of Reactive oxygen species (ROS), and intracellular Ca+ concentrations were determined via flow cytometry using Mitosox Red and Fluo-4 respectively. ROS and Ca were estimated as Relative Fluorescence Units (RFU) in compensated, arcsin-transformed data in the live sperm population. Results: After 48 h of incubation, motility was greater in all the 10 mM pyruvate-based media, with the poorest result in the 40 mM glucose (41 ± 1.1 %) while the highest motility was yielded in the 40 mM glucose 10 mM pyruvate aliquot (60.3 ± 3.5 %; P < 0.001); after 96 h of storage highest motility values were observed in the 40 mM glucose 10 mM pyruvate media (23.0 ± 6.2 %) while the lowest was observed in the 1 mM glucose media was 9.2 ± 2.0 % (P < 0.05). Mitochondrial ROS was lower in the 40 mM glucose 10 mM pyruvate group compared to the 40 mM glucose (P < 0.01). Over time Ca increased in all treatment groups compared to time 0h. Conclusions: Viable spermatozoa may experience oxidative stress and alterations in Ca2+ homeostasis during prolonged storage, however, these effects can be reduced by regulating metabolism. The 40 mM glucose- 10 mM pyruvate group yielded the highest sperm quality parameters.
Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.
Publication Date: 2024-08-22 PubMed ID: 39178614DOI: 10.1016/j.theriogenology.2024.08.021Google Scholar: Lookup
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Summary
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The study examines how altering the metabolism of stallion spermatozoa through modification of metabolites present in the storage media can decrease oxidative stress and extend the lifespan of the semen conserved at room temperature. The researchers found that the modification presented the highest sperm quality.
Background and Objective of the Research
- The aim of the study was to extend the period of conservation of stallion semen at room temperature. This is of crucial importance in horse breeding, where conservation of semen in a liquid state forms the basis of associated reproductive technologies.
- Stallion spermatozoa have high mitochondrial activity, which increases oxidative stress over the course of the storage period, leading to the deterioration of sperm within 24 to 48 hours of storage.
Research Methodology
- Researchers used processed ejaculates and extended them in different media: Tyrode’s basal media (a commonly used buffer) and modified Tyrode’s with various concentrations of glucose and pyruvate.
- They then analysed motility after 0, 24, and 96 hours of storage, as well as the mitochondrial production of Reactive Oxygen Species (ROS) and intracellular calcium concentrations. These latter two factors were determined via flow cytometry with specific fluorescent dyes (Mitosox Red and Fluo-4).
- The researchers also evaluated changes over time in all treatment groups.
Research Findings
- After 48 hours of incubation, all the groups extended with 10 mM pyruvate-based media had greater motility. The least motility was observed in the 40 mM glucose group and the highest in the 40 mM glucose plus 10 mM pyruvate media group.
- After 96 hours of storage, the highest motility was again observed in the 40 mM glucose plus 10 mM pyruvate media group, while the lowest was found in the 1 mM glucose media group.
- Regarding the production of mitochondrial ROS, the 40 mM glucose plus 10 mM pyruvate group had lower levels than the 40 mM glucose group.
- Over time, calcium increased in all treatment groups in comparison with the initial time.
Conclusion of the Research
- The study concluded that, during prolonged storage, viable spermatozoa may experience oxidative stress and alterations in calcium homeostasis. However, these effects can be mitigated by regulating metabolism. The group with 40 mM glucose and 10 mM pyruvate yielded the highest sperm quality parameters.
Cite This Article
APA
Becerro-Rey L, Martín-Cano FE, Fabres Robaina Sancler-Silva Y, Gil MC, Ortega-Ferrusola C, Aparicio IM, Gaitskell-Phillips G, da Silva-Álvarez E, Peña FJ.
(2024).
In vitro, the aging of stallion spermatozoa at 22 °C is linked to alteration in Ca2+ and redox homeostasis and may be slowed by regulating metabolism.
Theriogenology, 229, 127-137.
https://doi.org/10.1016/j.theriogenology.2024.08.021 Publication
Researcher Affiliations
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain; Department of Animal Science, Universidade Federal de Viçosa, Viçosa, 36570-900, Brazil.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
- Department of Anatomy, University of Extremadura, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain. Electronic address: fjuanpvega@unex.es.
Conflict of Interest Statement
Declaration of competing interest All authors declare no potential conflict of interest.
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