Document Type : Original Articles
Authors
1
Department of Biological chemistry, Ryazan State Medical University; Ryazan, Russia
2
Department of Biological Chemistry of Ryazan State Medical University, Ryazan. Russia
3
head of the laboratory, leading researcher of Cryobiology Laboratory of the All-Russian Research Institute of Horse Breeding named after Academy Member V.V. Kalashnikov, Divovo, Russia
4
junior Researcher of Cryobiology Laboratory of the All-Russian Research Institute of Horse Breeding named after Academy Member V.V. Kalashnikov, Divovo, Russia
5
Department of Biological Chemistry at Ryazan State Medical University Ryazan, Russia
Abstract
Background: Age-related changes in stallion sperm cryotolerance and the underlying biochemical mechanisms are poorly understood. Objectives: To determine the effect of stallion age on post-thaw sperm quality by assessing acrosomal integrity, cathepsin activity, and energy metabolism efficiency. Methods: Cryopreserved semen from 62 purebred Arabian stallions, aged 3–24 years, was analyzed. Stallions were categorized into young (3-5 yrs), mature (6-15 yrs), and older (16-24 yrs) groups. Post-thaw assessments included computer-assisted sperm analysis (CASA) for motility, imaging flow cytometry for acrosomal integrity and mitochondrial membrane potential (MMP), and biochemical assays for key metabolic enzymes (Cytochrome c oxidase, ATP synthase, SDH, LDH), lactate, and cathepsins B,L,H activities. Results: Sperm of mature stallions had the highest total and progressive motility combined with increased LDH activity and effective lactate use. Older stallions showed clear signs of mitochondrial dysfunction, including decreased MMP, decreased cytochrome c oxidase and ATP synthase activity, a higher frequency of spontaneous acrosome reactions, and increased activity of cathepsin H. Young stallions, while maintaining high activity of mitochondrial enzymes, had significantly lower LDH activity and sperm motility. LDH activity was strongly correlated with MMP and intact acrosomes, while lactate levels were negatively correlated with sperm longevity and viability. Conclusions: Optimal sperm cryotolerance in mature stallions is provided by a synergistic combination of high mitochondrial efficiency and active LDH-mediated metabolic flexibility. In contrast, cryodamage in older stallions is caused by mitochondrial dysfunction, while young stallions lack metabolic adaptability. Activity of LDH and lactate levels are promising biomarkers for predicting stallion sperm cryotolerance.
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