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Home / Equine Research Bank / Reprod Domest Anim / Cryoprotective Efficacy of Omega-3 Nano-Emulsion on Kinemati...
Reproduction in domestic animals = Zuchthygiene2026; 61(1); e70169; doi: 10.1111/rda.70169

Cryoprotective Efficacy of Omega-3 Nano-Emulsion on Kinematic Parameters, Acrosome Status, Subcellular Ultrastructure, and Oxidative/Antioxidant Markers in Cryopreserved Stallion Semen.

Abstract: This study aims to investigate the cryoprotective effect of Omega-3 nano-emulsion (Omega-3 NE) on stallion sperm quality, kinematic parameters, acrosome status, subcellular ultrastructure, oxidative/antioxidant markers, and semen microbiota. Forty ejaculates were collected, extended, and cryopreserved from 5 fertile Pure Egyptian stallions (Equus caballus). The ejaculates were divided into five groups: a control group (without additive) and four groups supplemented with 25, 50, 100, and 200 μg of Omega-3 NE/mL. The Omega-3 NE exhibited an average particle size of 51-146 nm, a PDI of 0.58, and a zeta potential of -31 mV. Omega-3 NE (200 μg/mL) significantly improved progressive motility, viability, and membrane integrity of stallion semen (p < 0.05). Additionally, supplementation with Omega-3 NE (200 μg/mL) led to a significant enhancement in post-thawed sperm kinematic parameters, including PM, DSL, VCL, and VSL, by 40%, 21.5%, 26.7%, and 20.7%, respectively, compared to the control group. The addition of 100 or 200 μg/mL Omega-3 NE to the media resulted in a higher percentage of live sperm with intact acrosomes. Additionally, all Omega-3 NE treatments significantly decreased the percentage of dead sperm with intact acrosomes as well as microbiota load (total bacterial count and coliform bacteria count) compared to the control (p < 0.01). Significant improvements in antioxidant status (TAC and CAT) and reduction of oxidative stress markers (MDA, NO, and HO) were observed in all Omega-3 NE groups compared to the control group (p < 0.05). Omega-3 NE (200 μg/mL) significantly reduced sperm apoptosis (p < 0.01) and preserved better subcellular integrity compared to the control and other treatment groups. The results suggest that Omega-3 NE at concentrations of 100-200 μg/mL can effectively enhance sperm cryo-resistance via enhancing sperm quality and kinematic variables, reducing oxidative stress and microbiota load, and maintaining sperm subcellular ultrastructure. The study highlights the potential of Omega-3 NE as a nanotechnology-based approach to boost assisted reproductive technologies in stallion breeding programmes.
© 2026 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.
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Publication Date: 2026-01-09 PubMed ID: 41508714DOI: 10.1111/rda.70169Google 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.

Research Overview

  • This study evaluates the protective effects of Omega-3 nano-emulsion (Omega-3 NE) during the cryopreservation of stallion semen.
  • The research explores how different concentrations of Omega-3 NE influence sperm quality, movement, cellular structure, oxidative stress markers, and bacterial contamination after freezing and thawing.

Background and Objectives

  • Cryopreservation of semen is critical for stallion breeding but often damages sperm due to ice crystal formation, oxidative stress, and microbial contamination.
  • Omega-3 fatty acids are known for antioxidant and membrane-stabilizing properties, making them promising cryoprotective agents.
  • The study aimed to test a nano-emulsified form of Omega-3 to improve its bioavailability and protective effect on stallion sperm parameters during cryopreservation.

Methodology

  • Forty ejaculates collected from 5 fertile Pure Egyptian stallions were used.
  • Ejaculates were split into five groups: one control (no additive) and four treated groups with Omega-3 NE at concentrations of 25, 50, 100, and 200 μg/mL.
  • Various assessments were conducted post-thawing including:
    • Sperm kinematic parameters: progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL), and distance straight line (DSL)
    • Viability, membrane integrity, and acrosome status
    • Subcellular ultrastructure examined via microscopy
    • Oxidative stress markers like MDA (lipid peroxidation), nitric oxide (NO), and hypochlorous acid (HO)
    • Antioxidant markers such as total antioxidant capacity (TAC) and catalase (CAT) activity
    • Sperm apoptosis levels
    • Bacterial counts to evaluate semen microbiota
  • The nano-emulsion characteristics of Omega-3 included:
    • Particle size between 51-146 nm
    • Polydispersity index (PDI) of 0.58 indicating moderate uniformity
    • Zeta potential of -31 mV suggesting stable dispersion

Key Findings

  • Omega-3 NE at 200 μg/mL significantly improved sperm progressive motility, viability, and membrane integrity compared to the control group.
  • Kinematic improvements for the 200 μg/mL group included increases of:
    • Progressive motility by 40%
    • Distance straight line by 21.5%
    • Curvilinear velocity by 26.7%
    • Straight line velocity by 20.7%
  • The 100 and 200 μg/mL concentrations significantly increased the percentage of live sperm with intact acrosomes, indicating better functional integrity.
  • All doses notably reduced:
    • Dead sperm with intact acrosomes
    • Total bacterial load including coliform bacteria
  • Antioxidant capacity (TAC) and catalase activity were enhanced across all Omega-3 NE treated groups, reflecting improved defense against oxidative damage.
  • Oxidative stress markers (MDA, NO, HO) were significantly lowered in the treated groups, showing decreased cellular damage.
  • The highest Omega-3 NE dose (200 μg/mL) reduced sperm apoptosis and better preserved subcellular ultrastructure compared to control and lower doses, indicating enhanced cell survival and membrane stabilization.

Implications and Conclusions

  • Omega-3 NE, particularly at concentrations of 100–200 μg/mL, provides effective cryoprotection for stallion semen by:
    • Improving sperm motility and viability
    • Maintaining membrane and acrosome integrity
    • Reducing oxidative stress and reactive oxygen species
    • Suppressing sperm apoptosis
    • Decreasing microbial contamination in semen samples
    • Preserving critical ultrastructural features of sperm cells
  • The nano-emulsion form enhances delivery and efficacy of Omega-3 fatty acids in the cryopreservation medium.
  • This approach demonstrates promise as a nanotechnology-based tool to improve fertility outcomes in stallion assisted reproductive techniques, optimizing breeding programs.

Cite This Article

APA
Khalil WA, Sharf MI, Derbala MK, Hassan MAE, Abdelnour SA, El-Harairy MA. (2026). Cryoprotective Efficacy of Omega-3 Nano-Emulsion on Kinematic Parameters, Acrosome Status, Subcellular Ultrastructure, and Oxidative/Antioxidant Markers in Cryopreserved Stallion Semen. Reprod Domest Anim, 61(1), e70169. https://doi.org/10.1111/rda.70169

Publication

Reproduction in domestic animals = Zuchthygiene
ISSN: 1439-0531
NlmUniqueID: 9015668
Country: Germany
Language: English
Volume: 61
Issue: 1
Pages: e70169

Researcher Affiliations

Khalil, Wael A
  • Department of Animal Production, Faculty of Agriculture, Mansoura University, Mansoura, Egypt.
Sharf, Mohamed I
  • 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
  • Fatty Acids, Omega-3 / pharmacology
  • Cryoprotective Agents / pharmacology
  • Semen Analysis / veterinary
  • Acrosome / drug effects
  • Sperm Motility / drug effects
  • Antioxidants / metabolism
  • Semen / drug effects
  • Spermatozoa / drug effects
  • Spermatozoa / ultrastructure
  • Oxidative Stress / drug effects
  • Emulsions

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