Iranian Journal of animal Science

Iranian Journal of animal Science

Effect of different levels of alpha-lipoic acid in egg yolk-based extender on the quality of frozen-thawed ram sperm

Document Type : Research Paper

Authors
Touba Nadri 1
Farhad Farrokhi Ardebily 1
Moslem Mehdizadeh 1
Saeed Zeinoaldini 2
Filipp Georgijevic Savvulidi 3
1 Department of Animal Sciences, Faculty of Agriculture, Urmia University, Urmia, Iran
2 Department of Animal Sciences, Faculty of Agriculture and Natural Resources, University of Tehran, Karaj, Iran
3 Department of Animal Science, Faculty of Agrobiology, Food and Natural Resources, Czech University, Prague, Kamýck´a, Czech Republic
10.22059/ijas.2026.408705.654117
Abstract
This study aimed to evaluate the effect of different levels of alpha-lipoic acid (ALA) (1, 2, and 4 μM; AL1, AL2, and AL4, respectively) on the quality of ram sperm after freezing and thawing. Ejaculates were collected weekly from four adult rams using an artificial vagina for six weeks. Semen was mixed and diluted with egg yolk extenders containing 1, 2, and 4 μM ALA, as well as egg yolk extender without ALA (control group). Motility, viability, and membrane functionality of sperm were evaluated after freezing. The results of this paper showed that the AL4 group showed significantly higher total motility (69.2±2.3) than the other groups. Also, AL4 had higher progressive motility (46.6±3.7) than the other treatments. Compared with the control group, the AL4 group showed significantly higher plasma membrane integrity (72.6±1.9 vs. 51.4±1.9) and plasma membrane activity (75.2±1.3 vs. 52.4±3.1). AL4 showed the highest percentage of live sperm (68.2±2.5) and the lowest percentage of dead sperm (22.3±1.9) (P<0.05). Therefore, it seems that the addition of 4 μM to the egg yolk-based extender can increase the quality and viability of ram sperm after cryopreservation.
Keywords
Freezing
Alpha-lipoic acid
Viability
Sperm
Ram
Subjects

Extended Abstract

Introduction

    Sperm cryopreservation decreases the quality of sperm, partly from oxidative stress, mainly affecting on cell membrane, sperm motility, mitochondrial activity, ATP production, and increasing DNA damage, diminishing post-thawing fertility. Small molecules like Alpha-lipoic acid are vital antioxidants and cofactors in mitochondrial activity and ROS metabolism . Artificial insemination is an effective tool for advancing the livestock industry, but structural and morphological damage to sperm during cryopreservation can reduce fertility . Among the damages that occur during the freeze-thaw process are the production of oxygen-free radicals (ROS), lipid peroxidation of the sperm membrane, DNA damage, and reduced sperm motility and viability. In this regard, the use of antioxidants in sperm extenders to reduce oxidative damage and improve sperm viability and fertility has been considered as an effective solution. Alpha-lipoic acid (ALA) is one of the most important natural antioxidants with unique properties that are active in both water-soluble and lipid-soluble forms and can directly inhibit ROS or exert its effect through the recovery of other antioxidants such as glutathione and vitamins C and E. Therefore, this study aimed to evaluate the effects of different levels of Alpha-lipoic acid on ram sperm quality.

 

Materials and methods

    Semen was collected with an artificial vagina from four mature rams once weekly for 6 wk. Ejaculates were immediately placed in a water bath (37 °C) and evaluated for color, volume, concentration, motility, and sperm morphology. The egg yolk-based extenders (EY) were prepared at concentrations of 15% (v/v). Then, the egg yolk-based extender was divided into four parts and assigned to the respective treatments, which included the control group (without ALA), egg yolk-based extender containing 1 µM ALA (AL1), egg yolk-based extender containing 2 µM ALA (AL2), and egg yolk-based extender containing 4 µM ALA (AL4).

 

Results and Discussion

    In this experiment, increasing the level of alpha lipoic acid from 1 to 4 µM increased total motility. The AL4 group showed a significantly higher total motility (69.2±2.3) than the other groups, while the control group had the lowest total motility level (47.0±1.3). Also, the AL4 level had a higher progressive motility than the other treatments, although the difference between treatments was not significant (P>0.05). For sperm viability, all the extenders containing ALA yielded a higher proportion of viable sperm than the control; AL4 had the highest percentage of Plasma membrane integrity (72.6±1.9), and there was a significant difference with AL1 (57.8±1.9) and AL2 (60.5±1.9) treatments and the control group (51.4±1.9).

 

Conclusion

    The use of 4 μM alpha-lipoic acid in semen extender for ram semen cryopreservation was found to be beneficial as a supplement for ram semen cryopreservation. Alpha-lipoic acid prevents ATP loss by maintaining sperm membrane integrity and activity and improving the efficiency of the electron transport chain, increasing total and progressive motility, and protecting cellular structures against oxidative stress.

 

Author Contributions

Moslem Mehdizade: Investigation, Data analysis; Touba Nadri: Conceptualization, Validation, Methodology, Software, Formal analysis, Writing - Original Draft, Supervision, Formal analysis, Investigation; Farhad Farrokhi: Software; Saeed Zeinoaldini: Supervision; Filipp Georgijevic Savvulidi; Methodology

 

Data Availability Statement

The data supporting the reported results are collected and used from published articles, to which we have referred in the manuscript.

 

Ethical considerations

The authors avoided data fabrication, falsification, plagiarism, and misconduct.

 

Conflict of interest

The author declares no conflict of interest.

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Iranian Journal of animal Science
Volume 57, Issue 3
Summer 2026
Pages 529-539

  • Receive Date 28 December 2025
  • Revise Date 15 March 2026
  • Accept Date 21 March 2026