Veterinary Research Forum

Abstract The present study showed riociguat supplementation modulates in a Tris–egg yolk extender on post-thaw ram semen quality. Semen samples were collected from eight healthy, sexually mature Tuj rams (n = 48 ejaculates) during the breeding season and cryopreserved after supplementation with riociguat at concentrations of 0.00 (control), 0.50, 5.00, 50.00, and 250 µM. Post-thaw sperm quality was evaluated using computer-assisted sperm analysis for motility and kinematic parameters, alongside assessments of plasma and acrosomal membrane integrity, mitochondrial membrane potential, DNA integrity and oxidative stress markers including malondialdehyde and glutathione. Riociguat supplementation (5.00 - 250 µM) significantly modulated mitochondrial membrane potential and intracellular antioxidant capacity as evidenced by reduced malondialdehyde and increased glutathione levels. Moreover, all riociguat-treated groups exhibited significantly improved DNA integrity compared with the control. Moreover, acrosomal damage was declined at the highest concentrations (5.00, 50.00 and 250 µM). These findings suggest that riociguat exerted a protective effect on acrosomal membranes during cryopreservation. In conclusion, riociguat modulated post-thaw ram sperm function in a dose-dependent manner, exerting both beneficial and detrimental effects depending on concentration. Although fertility outcomes were not assessed in the present study, the observed biochemical and functional changes provided preliminary evidence that riociguat might influence key pathways associated with sperm resilience to cryoinjury, thereby, justifying further investigations incorporating in vivo fertility and reproductive performance assessments.