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<p>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&#xa0;°C for 72&#xa0;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&#xa0;h of cooling (<i>p</i> &lt; 0.05). BHT-NP significantly reduced the apoptotic sperm and increased the viable sperm in cooled stallion semen (<i>p</i> &lt; 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 (<i>p</i> &lt; 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 (<i>p</i> &lt; 0.001). The BHT-NP1.0 group resulted in significantly lower total bacterial count (<i>p</i> &lt; 0.001), <i>coliform</i> bacteria count (<i>p</i> &lt; 0.01), and spore-forming bacteria counts (<i>p</i> &lt; 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&#xa0;h of preservation at 4&#xa0;°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.</p>

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

  • 期刊论文

  • 发表日期：

    2025年12月3日

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  • Wael A. Khalil,
  • Hesham E. Mostafa,
  • Mohamed K. Derbala,
  • Mohammed A. Alfattah,
  • Waleed Alhujaili,
  • Mahmoud A. E. Hassan,
  • Mostafa A. El-Harairy,
  • Sameh A. Abdelnour

摘要

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.