Effect of Coumestrol Supplementation on Ovine Semen Cryopreservation

Background: Antioxidant supplementation promotes the fertilizing capacity of post-thawed ram spermatozoa. Objectives: This study was designed to validate the effect of different levels of coumestrol as an antioxidant on post-thawing parameters of cryopreserved spermatozoa in rams. Methods: A total of 60 ejaculates were collected from six sexually mature Barki rams. The accepted semen samples were extended, pooled, cooled, and then divided into five aliquots: A control group (tris-based egg yolk extender) without coumestrol addition, and 4 aliquots with concentrations of 0.1, 1, 10, and 100 µM of coumestrol. These aliquots were then subjected to the cryopreservation process. The control and treated frozen semen were thawed and assessed for motility using computer-assisted sperm analysis (CASA), total antioxidant capacity (TAC), malondialdehyde (MDA) levels, mitochondrial activity, comet assay for DNA integrity, acrosome integrity, and casein kinase 2 alpha 2 (CK2α2) gene expression. Results: The addition of 1 µM of coumestrol significantly improved progressive motility. Metabolic activity was significantly higher in semen treated with 1, 10, and 100 µM coumestrol compared to control and 0.1 µM coumestrol groups. Furthermore, acrosome integrity was significantly higher in the 0.1 and 1 µM of coumestrol groups. In the 1, 10, and 100 µM coumestrol groups, TAC was significantly higher than in controls. Furthermore, MDA levels were significantly lower in all coumestrol groups compared to the controls. The comet assay exhibited a significant reduction in fragmented DNA in semen treated with coumestrol, especially with the addition of 1 µM coumestrol. The expression of CK2α2 showed a significant fold decline in semen supplemented with 10 and 100 µM coumestrol compared to the control group. Conclusion: The addition of 1 µM of coumestrol could ameliorate the deleterious impacts of cryo-damage by improving the sperm antioxidant capacity, mitochondrial activity, and both acrosome and DNA integrity.