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Home / Equine Research Bank / Reprod Domest Anim / Conditional Antioxidant Protection of Stallion Spermatozoa b...
Reproduction in domestic animals = Zuchthygiene2026; 61(3); e70199; doi: 10.1111/rda.70199

Conditional Antioxidant Protection of Stallion Spermatozoa by Butylated Hydroxytoluene Under Induced Oxidative Stress.

Abstract: Despite advances in the method, equine semen cryopreservation still leads to a marked decline in sperm quality, partly attributed to oxidative stress, which motivates the search for lipophilic antioxidants capable of stabilizing the plasma membrane during cooling and freezing. This study performed three experiments to evaluate butylated hydroxytoluene (BHT) as an additive to commercial skim milk-based (Botu-Sêmen) and egg yolk-based (Botu-Crio) extenders. In Experiment 1, a dose-response curve (0.5, 1.0, and 1.5 mM) was performed using stallion semen to assess toxicity and establish a safe concentration by analysing sperm kinetics (CASA) and membrane integrity immediately after dilution and after 24 h at 5°C. In Experiment 2, BHT at 0.5 mM was tested in standardized cooling (24 h at 5°C) and freezing protocols. Stallions were classified as Good/Bad Coolers and Good/Bad Freezers, and sperm kinetics (CASA), plasma membrane integrity assessed by epifluorescence microscopy (CFDA/PI), and plasma/acrosomal integrity and oxidative stress markers assessed by flow cytometry were evaluated. In Experiment 3, spermatozoa were exposed to an oxidative challenge with arachidonic acid (50 μM for 15 min at 37°C) to elucidate the mechanism of action of BHT. BHT at 0.5 mM was pharmacologically safe; however, it did not improve cryotolerance or reduce basal oxidative stress in commercial cooling and freezing protocols (p > 0.05). Under oxidative challenge, BHT markedly reduced lipid peroxidation and intracellular reactive oxygen species production (p < 0.05), indicating a conditional protective effect dependent on oxidative imbalance and impaired antioxidant defences. These findings suggest that BHT acts as an antioxidant primarily under critically increased ROS generation, but does not enhance the performance of commercial cryopreservation when procedures already operate within optimal conditions.
© 2026 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.
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Publication Date: 2026-03-19 PubMed ID: 41854126DOI: 10.1111/rda.70199Google Scholar: Lookup
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Summary

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Overview

  • This study investigated the effectiveness of butylated hydroxytoluene (BHT), a lipophilic antioxidant, in protecting stallion sperm during cooling and freezing processes used in cryopreservation.
  • The research found that BHT provides antioxidant protection only under conditions of induced oxidative stress, but it does not improve sperm quality during standard commercial cryopreservation protocols.

Background and Purpose

  • Equine semen cryopreservation is a technique used to store stallion sperm for later use, but it often causes decreased sperm quality.
  • This decline in sperm quality is partly due to oxidative stress—damage caused by reactive oxygen species (ROS) that harm the sperm plasma membrane.
  • Scientists are interested in antioxidants that can stabilize the sperm plasma membrane and reduce oxidative damage during freezing and thawing.
  • BHT is a synthetic antioxidant that is lipophilic (fat-soluble), making it a candidate for protecting sperm membranes.
  • The study aimed to evaluate whether adding BHT to commercial semen extenders improves sperm protection under cooling and freezing conditions.

Experimental Design

  • The researchers conducted three experiments using stallion semen and two commercial extenders: Botu-Sêmen (skim milk-based) and Botu-Crio (egg yolk-based).

Experiment 1: Toxicity and Safe Dose Determination

  • Tested three different BHT concentrations: 0.5, 1.0, and 1.5 mM.
  • Evaluated sperm kinetics using computer-assisted sperm analysis (CASA) and membrane integrity immediately after dilution and after 24 hours stored at 5°C.
  • Established that 0.5 mM BHT was pharmacologically safe, meaning it did not harm sperm quality.

Experiment 2: Effects on Cooling and Freezing Protocols

  • Applied 0.5 mM BHT to semen samples undergoing standard cooling (24 hours at 5°C) and freezing protocols.
  • Classified stallions into groups based on their semen’s cryotolerance: Good/Bad Coolers and Good/Bad Freezers.
  • Assessed sperm quality through multiple parameters:
    • Sperm motility and kinetics via CASA.
    • Plasma membrane integrity by epifluorescence microscopy using CFDA/PI staining.
    • Oxidative stress markers and plasma/acrosomal membrane integrity through flow cytometry.
  • Found no significant improvement in sperm quality or reduction in basal oxidative stress caused by BHT supplementation under these commercial freezing and cooling conditions.

Experiment 3: Oxidative Challenge Test

  • Challenged spermatozoa with arachidonic acid (50 µM for 15 minutes at 37°C) to simulate oxidative stress and increase ROS production.
  • Monitored lipid peroxidation and intracellular ROS levels.
  • Observed that BHT significantly reduced levels of lipid peroxidation and ROS, demonstrating effective antioxidant protection under oxidative stress conditions.

Conclusions and Implications

  • BHT at 0.5 mM concentration is safe for stallion sperm, without toxic effects during cooling and freezing.
  • However, under standard commercial cryopreservation conditions (which presumably maintain relatively low oxidative stress), BHT does not enhance sperm preservation or reduce oxidative damage.
  • BHT shows a conditional antioxidant effect that becomes apparent only when sperm are subjected to elevated ROS levels beyond typical baseline conditions, such as the induced oxidative challenge in the experiment.
  • This suggests BHT and similar antioxidants might be useful in specific scenarios where oxidative stress is abnormally high, but routine use in commercial extenders may not provide additional benefits.
  • Further research could explore targeted antioxidant supplementation tailored to scenarios of critical oxidative imbalance rather than routine cryopreservation protocols.

Cite This Article

APA
de Araujo EAB, Papa FO, de Oliveira Baldini PH, de Paula Freitas-Dell'Aqua C, de Oliveira SN, Silva LFMC, Junior LRPA, Rodrigues LT, Monteiro GA. (2026). Conditional Antioxidant Protection of Stallion Spermatozoa by Butylated Hydroxytoluene Under Induced Oxidative Stress. Reprod Domest Anim, 61(3), e70199. https://doi.org/10.1111/rda.70199

Publication

Reproduction in domestic animals = Zuchthygiene
ISSN: 1439-0531
NlmUniqueID: 9015668
Country: Germany
Language: English
Volume: 61
Issue: 3
Pages: e70199

Researcher Affiliations

de Araujo, Endrigo Adonis Braga
  • School of Veterinary Medicine and Animal Science, Federal University of Bahia, Salvador, Bahia, Brazil.
Papa, Frederico Ozanam
  • Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.
de Oliveira Baldini, Pedro Henrique
  • Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.
de Paula Freitas-Dell'Aqua, Camila
  • Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.
de Oliveira, Sidnei Nunes
  • Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.
Silva, Luis Fernando Mercês Chaves
  • Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.
Junior, Luiz Roberto Pena Andrade
  • Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.
Rodrigues, Lucas Troncarelli
  • Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.
Monteiro, Gabriel Augusto
  • Department of Veterinary Surgery and Animal Reproduction, School of Veterinary Medicine and Animal Science, São Paulo State University (UNESP), Botucatu, São Paulo, Brazil.

MeSH Terms

  • Animals
  • Male
  • Horses
  • Butylated Hydroxytoluene / pharmacology
  • Oxidative Stress / drug effects
  • Antioxidants / pharmacology
  • Spermatozoa / drug effects
  • Semen Preservation / veterinary
  • Semen Preservation / methods
  • Cryopreservation / veterinary
  • Cryopreservation / methods
  • Cryoprotective Agents / pharmacology
  • Cell Membrane / drug effects
  • Semen Analysis / veterinary

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