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Reproduction in domestic animals = Zuchthygiene2026; 61(5); e70213; doi: 10.1111/rda.70213

Nano-Encapsulated Vitamin E Enhances Cryo-Resistance of Stallion Spermatozoa: A Comprehensive Evaluation of Sperm Quality, Kinematic Parameters, Antioxidant Capacity, Microbiota and Ultrastructure.

Abstract: This experiment explores the comparative ability of vitamin E (VE) and its nano-emulsion (VEN) in enhancing cryo-resistance of stallion spermatozoa by examining semen quality, kinematic variables, acrosome integrity, apoptosis, antioxidant capacity, ultra-structural alterations and microbiota of cryopreserved stallion sperm. Semen from five stallions (8-10 years) was cryopreserved with 0.1 or 1 mM of VE or VEN; and a non-additive group served as the control. The VEN particles ranged in size from 72 to 172 nm, with a mean diameter of 287 nm. Supplementation with VE (1 mM) or VEN (0.1 or 1 mM) significantly enhanced progressive motility and viability during both the equilibration (for 30 min at 4°C) and post-thaw phases compared to the control (p < 0.05). Plasma membrane functionality was superior in all treated groups compared to control group (p < 0.001). Post-thaw sperm kinematic parameters and acrosome integrity were significantly enhanced in VEN treatment compared to VE and control groups. VEN (1 mM) notably improved antioxidant defences, specifically increasing TAC and catalase levels (p < 0.01) and a significant reduction in oxidative stress (NO, MDA and HO) in all treated semen samples (p < 0.01). VEN (1 mM) significantly reduced the proportion of apoptotic sperm (p < 0.01), increased viable sperm counts and the preservation of sperm ultrastructural integrity, as evidenced by intact acrosome and plasma membranes. VE and VEN treatments markedly lowered the total bacterial and coliform counts. Overall, supplementation with VEN (0.1 and 1 mM) effectively optimizes post-thaw stallion sperm quality by enhancing antioxidant defences and reducing the seminal microbiota.
© 2026 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.
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Publication Date: 2026-04-30 PubMed ID: 42059113DOI: 10.1111/rda.70213Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study investigates how vitamin E, particularly in a nano-encapsulated form, improves the freeze-thaw survival and overall quality of stallion sperm.
  • It compares the effects of regular vitamin E and its nano-emulsion on various sperm quality parameters, antioxidant status, sperm structure, and microbiota after cryopreservation.

Background and Purpose

  • Cryopreservation (freezing) of sperm is widely used for breeding but often causes damage to sperm cells, reducing their viability and fertility potential.
  • Oxidative stress during freezing and thawing leads to sperm membrane damage, loss of motility, and structural degradation.
  • Vitamin E (VE) is a known antioxidant that can protect sperm by neutralizing harmful free radicals.
  • The study aims to test whether nano-encapsulated vitamin E (VEN), designed for better delivery and stability, offers superior protection compared to conventional VE.

Experimental Design and Methods

  • Semen samples were collected from five mature stallions aged 8-10 years.
  • Semen aliquots were frozen using standard protocols with supplementation of either:
    • 0.1 mM vitamin E (VE),
    • 1 mM vitamin E (VE),
    • 0.1 mM nano-encapsulated vitamin E (VEN),
    • 1 mM nano-encapsulated vitamin E (VEN),
    • or no antioxidant supplement (control group).
  • Characterization of VEN particles showed sizes between 72–172 nm and a mean diameter of 287 nm.
  • Post-thaw evaluation included multiple parameters: sperm motility and viability, plasma membrane functionality, kinematic movement variables, acrosome integrity, apoptotic rates, antioxidant enzyme activities, markers of oxidative stress, ultrastructural assessment via microscopy, and the bacterial content of semen.

Key Findings

  • Sperm Motility and Viability:
    • Both 1 mM VE and VEN at 0.1 and 1 mM increased progressive sperm motility and viability during equilibration at 4°C and after thawing.
    • VEN groups showed greater improvement compared to VE and control groups.
  • Plasma Membrane Functionality: All antioxidant-treated groups had significantly better membrane integrity than controls, indicating reduced cryodamage.
  • Sperm Kinematic Parameters: Notable enhancements were observed only in VEN-treated samples, suggesting improved sperm movement quality after freezing.
  • Acrosome Integrity: Hairpin-shaped cap structure on sperm head was better preserved in VEN groups, essential for fertilization capacity.
  • Antioxidant Defense:
    • VEN at 1 mM significantly increased total antioxidant capacity (TAC) and catalase enzyme activities.
    • Markers of oxidative stress like nitric oxide (NO), malondialdehyde (MDA), and hypochlorous acid (HO) were significantly reduced with VEN supplementation.
  • Apoptosis and Viable Cell Counts: VEN (1 mM) lowered the proportion of apoptotic sperm and increased the number of viable sperm compared to VE and control.
  • Ultrastructural Integrity: Electron microscopy showed VEN helped maintain intact acrosome and plasma membranes, crucial for sperm fertilizing ability.
  • Microbiota Reduction: Both VE and VEN groups had significantly decreased total bacterial and coliform counts in semen, which could aid sperm health and storage.

Conclusions and Implications

  • Nano-encapsulated vitamin E (VEN), particularly at 0.1 and 1 mM concentrations, provides superior cryo-protective effects compared to traditional vitamin E.
  • VEN enhances stallion sperm quality post-thaw by improving motility, membrane and acrosome integrity, reducing oxidative stress and apoptosis, and preserving ultrastructure.
  • VEN also helps control microbial contamination in semen, possibly improving preservation outcomes.
  • This suggests nano-formulated antioxidants could be a valuable addition to cryopreservation media to maximize stallion sperm longevity and fertilizing potential.
  • These findings may guide improved protocols for equine breeding programs and potentially benefit cryostorage in other species.

Cite This Article

APA
Khalil WA, Mostafa HE, Derbala MK, Hassan MAE, Abdelnour SA, El-Harairy MA. (2026). Nano-Encapsulated Vitamin E Enhances Cryo-Resistance of Stallion Spermatozoa: A Comprehensive Evaluation of Sperm Quality, Kinematic Parameters, Antioxidant Capacity, Microbiota and Ultrastructure. Reprod Domest Anim, 61(5), e70213. https://doi.org/10.1111/rda.70213

Publication

Reproduction in domestic animals = Zuchthygiene
ISSN: 1439-0531
NlmUniqueID: 9015668
Country: Germany
Language: English
Volume: 61
Issue: 5
Pages: e70213

Researcher Affiliations

Khalil, Wael A
  • Department of Animal Production, Faculty of Agriculture, Mansoura University, Mansoura, Egypt.
Mostafa, Hesham E
  • Department of Animal Production, Faculty of Agriculture, Mansoura University, Mansoura, Egypt.
Derbala, Mohamed K
  • Diagnostic Imaging and Endoscopy Unit, Animal Reproduction Research Institute (ARRI), Agriculture Research Center (ARC), Giza, Egypt.
Hassan, Mahmoud A E
  • Animal Production Research Institute, Agriculture Research Centre, Ministry of Agriculture, Giza, Egypt.
Abdelnour, Sameh A
  • Department of Animal Production, Faculty of Agriculture, Zagazig University, Zagazig, Egypt.
El-Harairy, Mostafa A
  • Department of Animal Production, Faculty of Agriculture, Mansoura University, Mansoura, Egypt.

MeSH Terms

  • Animals
  • Male
  • Horses
  • Cryopreservation / veterinary
  • Semen Preservation / veterinary
  • Semen Preservation / methods
  • Spermatozoa / drug effects
  • Spermatozoa / ultrastructure
  • Spermatozoa / physiology
  • Semen Analysis / veterinary
  • Antioxidants / pharmacology
  • Vitamin E / pharmacology
  • Vitamin E / administration & dosage
  • Sperm Motility / drug effects
  • Microbiota / drug effects
  • Cryoprotective Agents / pharmacology
  • Acrosome / drug effects
  • Apoptosis / drug effects

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