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This research investigates how different levels of glutathione peroxidase affect ram semen quality after freezing and thawing.

May 9, 2026Open Access

Supplementation of the freezing extender with glutathione peroxidase improves the post-thaw quality of ram semen by enhancing antioxidant capacity, regulating metabolic enzyme activity, and modulating proteome expression.

Key Points

This research investigates how different levels of glutathione peroxidase affect ram semen quality after freezing and thawing.
Semen samples collected from six Dorper rams and pooled.
Four groups were created with varying concentrations of glutathione peroxidase (0, 0.8, 1.6, and 3.2 U/L).
Post-thaw assessments included motility, viability, structural integrity, antioxidant status, and proteomic profiles.
1.6 U/L GSH-Px significantly improved sperm motility and viability after thawing.
Total antioxidant capacity, SOD, and CAT activities increased with reduced malondialdehyde levels.
Proteomic analysis identified 150 differentially expressed proteins, with notable upregulation of GSPT1 and RPL11.

Abstract

Objective: To study the effects of different concentrations of glutathione peroxidase (GSH-Px) on sperm motility and viability, structural integrity, antioxidant capacity and metabolic enzyme activity after frozen semen of sheep, and to evaluate the expression of differential proteins.Methods: Semen samples were collected from six Dorper rams, pooled, and subsequently divided into four groups.GSH-PxGlutathione peroxidase was added to the cryoprotectant solution at concentrations of 0, 0.8, 1.6, and 3.2 units per liter, respectively.Following thawing, sperm motility, viability, and kinematic parameters were assessed, along with acrosomal and plasma membrane integrity, DNA integrity, mitochondrial activity, antioxidant status, metabolic enzyme activity, and proteomic profiles.Results: Adding 1.6 U/L GSH-Px to the freezing extender significantly enhanced the motility and viability of sperm after freeze-thaw, as well as the integrity of the acrosome, plasma membrane and DNA.The activities of total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and catalase (CAT) were significantly increased.The oxidative stress marker malondialdehyde (MDA) was significantly reduced, and the activities of metabolic enzymes including lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were also decreased.Proteomic analysis identified 150 differentially expressed proteins (DEPs).Western blotting verification confirmed the upregulation of G1 to S Phase Transition 1 (GSPT1), ribosomal protein L11 (RPL11), as well as the downregulation of annexin A2 (ANXA2) expression. Conclusion:Supplementation of the cryopreservation extender with 1.6 U/L GSH-Px

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