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Veterinary research communications2025; 50(1); 62; doi: 10.1007/s11259-025-10977-5

Efficacy of butylated hydroxytoluene nanoparticles in enhancing the quality and preservation of stallion chilled semen.

Abstract: Encapsulating natural antioxidants presents a robust strategy to neutralize oxidative stress, thereby improving semen preservation. Therefore, this study aimed to evaluate the efficacy of butylated hydroxytoluene nanoparticles (BHT-NPs) in improving stallion semen preservation by investigating sperm quality, redox balance, semen bacteriology, apoptosis, ultrastructure, and acrosome status of chilled stallion semen preserved at 4 °C for 72 h. This in vitro experiment was evidenced by molecular docking analysis. Twenty-five ejaculates from five stallions were collected and extended with 0 (BHT-NP0), 0.5 (BHT-NP0.5), and 1.0 (BHT-NP1.0) mM of butylated hydroxytoluene nanoparticles. Stallions' extender fortified with 0.5 or 1 mM of BHT-NPs significantly improved progressive motility, viability, and membrane integrity while significantly reducing abnormalities after 72 h of cooling (p < 0.05). BHT-NP significantly reduced the apoptotic sperm and increased the viable sperm in cooled stallion semen (p < 0.01). Supplementation chilled stallion spermatozoa extender with 0.5 or 1 mM BHT-NPs significantly increased total antioxidant capacity (TAC) and catalase activity compared to the control extender (p < 0.001). Conversely, the levels of oxidative markers (MDA, H₂O₂, and NO) were significantly lower in all BHT-NPs -supplemented groups compared to the control extender (p < 0.001). The BHT-NP1.0 group resulted in significantly lower total bacterial count (p < 0.001), coliform bacteria count (p < 0.01), and spore-forming bacteria counts (p < 0.01) compared to other groups. The docking simulation results show that the energy of binding BHT to antioxidant-apoptosis pathways such as HSP90A, PrdX1, caspase 3, and AKAP3 were - 8.45, - 7.1, - 6.42, and - 5.99 Kcal/mol, respectively. Scanning electron microscopy revealed that BHT-NPs significantly reduced head damage, midpiece damage, and coiled tails in stallion semen after 72 h of preservation at 4 °C. This research confirms that BHT-NPs offer a promising strategy for enhancing cooled stallion sperm quality, due to their combined antioxidant, antimicrobial, and anti-apoptotic properties. Our findings enhance semen preservation in stallions using nanotechnology molecules to promote the efficiency of assisted reproductive technology protocols.
© 2025. The Author(s), under exclusive licence to Springer Nature B.V.
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Publication Date: 2025-12-03 PubMed ID: 41335269PubMed Central: 7552309DOI: 10.1007/s11259-025-10977-5Google Scholar: Lookup
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Summary

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Overview

  • This study investigated the use of butylated hydroxytoluene nanoparticles (BHT-NPs) to improve the quality and preservation of stallion semen stored at 4°C for 72 hours.
  • The researchers found that BHT-NPs enhanced sperm motility, viability, antioxidant capacity, and reduced bacterial contamination and sperm apoptosis during semen storage.

Research Objectives and Background

  • Oxidative stress during semen preservation negatively affects sperm quality and longevity.
  • Natural antioxidants can help neutralize oxidative stress but delivering them effectively is challenging.
  • Encapsulation of antioxidants into nanoparticles was proposed to enhance their stability and controlled release.
  • The objective was to evaluate the efficacy of butylated hydroxytoluene (BHT) formulated as nanoparticles (BHT-NPs) in improving chilled stallion semen quality.
  • Key quality parameters assessed included sperm motility, viability, membrane integrity, apoptosis, oxidative stress markers, bacterial contamination, ultrastructural sperm damage, and acrosome status.

Experimental Design

  • Twenty-five ejaculates were collected from five stallions to ensure sample variability and reliability.
  • Semen samples were divided into three groups based on extender supplementation:
    • Control group (BHT-NP0) with no BHT nanoparticles.
    • Low-dose group (BHT-NP0.5) with 0.5 mM BHT-NPs.
    • High-dose group (BHT-NP1.0) with 1.0 mM BHT-NPs.
  • Samples were chilled and stored at 4°C for 72 hours.
  • Molecular docking simulations were performed to explore interactions between BHT and proteins involved in antioxidant and apoptotic pathways.

Key Findings

  • Sperm Motility and Viability
    • BHT-NP supplementation at both 0.5 mM and 1.0 mM significantly improved sperm progressive motility and viability post-chilling.
    • Membrane integrity was better preserved in treated groups compared to controls.
    • There was a notable reduction in sperm morphological abnormalities.
  • Apoptosis Reduction
    • The proportion of apoptotic sperm decreased significantly in BHT-NP supplemented groups.
    • The number of viable sperm increased accordingly, indicating protective anti-apoptotic effects.
  • Oxidative Stress and Antioxidant Status
    • Total antioxidant capacity (TAC) and catalase enzyme activity increased in treated samples, indicating enhanced defense against oxidative damage.
    • Oxidative stress markers like malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and nitric oxide (NO) were significantly reduced with BHT-NP treatment.
  • Microbial Control
    • The 1.0 mM BHT-NP group showed markedly lower total bacterial counts, including coliform and spore-forming bacteria, suggesting antimicrobial properties.
  • Ultrastructural Integrity
    • Scanning electron microscopy revealed that BHT-NP treatment reduced sperm head and midpiece damage as well as tail abnormalities after storage.
  • Molecular Docking Insights
    • Computational simulations demonstrated that BHT binds strongly to key proteins involved in antioxidant defense and apoptosis regulation, including HSP90A, Prdx1, caspase-3, and AKAP3.
    • Binding energies indicated favorable interactions that may underpin the observed biological effects.

Significance and Implications

  • This study confirms that encapsulating the antioxidant BHT into nanoparticles enhances its protective effects on chilled stallion semen.
  • The combined antioxidant, antimicrobial, and anti-apoptotic properties of BHT-NPs help maintain sperm quality during cold storage.
  • Improving semen preservation directly supports assisted reproductive technologies by increasing the viability and fertilizing potential of stored sperm.
  • Nanotechnology offers a promising avenue to improve the delivery and efficacy of semen extenders in animal breeding.

Conclusions

  • BHT nanoparticles at concentrations of 0.5 mM and 1.0 mM effectively enhance the preservation of stallion semen stored at 4°C for 72 hours.
  • Benefits include improved sperm motility and viability, reduced oxidative damage and apoptosis, lower bacterial contamination, and preserved ultrastructural integrity.
  • The findings suggest that BHT-NPs can be integrated into semen preservation protocols to boost the success of equine assisted reproduction.

Cite This Article

APA
Khalil WA, Mostafa HE, Derbala MK, Alfattah MA, Alhujaili W, Hassan MAE, El-Harairy MA, Abdelnour SA. (2025). Efficacy of butylated hydroxytoluene nanoparticles in enhancing the quality and preservation of stallion chilled semen. Vet Res Commun, 50(1), 62. https://doi.org/10.1007/s11259-025-10977-5

Publication

Veterinary research communications
ISSN: 1573-7446
NlmUniqueID: 8100520
Country: Switzerland
Language: English
Volume: 50
Issue: 1
Pages: 62

Researcher Affiliations

Khalil, Wael A
  • Department of Animal Production, Faculty of Agriculture, Mansoura University, Mansoura, 35516, Egypt. w-khalil@mans.edu.eg.
Mostafa, Hesham E
  • Department of Animal Production, Faculty of Agriculture, Mansoura University, Mansoura, 35516, Egypt.
Derbala, Mohamed K
  • Diagnostic Imaging and Endoscopy Unit, Animal Reproduction Research Institute (ARRI), Agriculture Research Center (ARC), Giza, Egypt.
Alfattah, Mohammed A
  • Department of Biology, College of Science, Jazan University, Jazan, Saudi Arabia.
Alhujaili, Waleed
  • Department of Biology, College of Science, Taibah University, Al Madinah Al Munawwarah, 41321, Saudi Arabia.
Hassan, Mahmoud A E
  • Animal Production Research Institute, Agriculture Research Centre, Ministry of Agriculture, Dokki, Giza, 12619, Egypt.
El-Harairy, Mostafa A
  • Department of Animal Production, Faculty of Agriculture, Mansoura University, Mansoura, 35516, Egypt.
Abdelnour, Sameh A
  • Department of Animal Production, Faculty of Agriculture, Zagazig University, Zagazig, 44511, Egypt. samehtimor86@gmail.com.

MeSH Terms

  • Animals
  • Horses / physiology
  • Male
  • Semen Preservation / veterinary
  • Semen Preservation / methods
  • Butylated Hydroxytoluene / pharmacology
  • Butylated Hydroxytoluene / chemistry
  • Nanoparticles / chemistry
  • Semen Analysis / veterinary
  • Antioxidants / pharmacology
  • Spermatozoa / drug effects
  • Spermatozoa / physiology
  • Semen / drug effects
  • Semen / microbiology
  • Semen / physiology
  • Molecular Docking Simulation
  • Apoptosis / drug effects

Conflict of Interest Statement

Declarations. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests. Animal Ethics: This research was approved by the Mansoura University, Animal Care and Use Committee, Code number: MU-ACUC (MU-ACUC, AGR.MS.24.12.5) and in compliance with the ARRIVE guidelines.

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