Interactive Effects of Seminal Plasma and Sonchus oleraceus L. Aqueous Extract on Post-Thaw Sperm Kinematics, Oxidative Status, and In Vitro Fertilization Outcomes in Ram Semen

This study investigated the combined effects of seminal plasma (SP) and Sonchus oleraceus L. aqueous extract on post-thaw sperm quality and fertilizing ability in rams. Semen collected from seven adult rams was pooled and divided into six experimental groups according to the presence (SP+) or absence (SP−) of SP and supplementation with 0, 0.5, or 2 g/L of Sonchus oleraceus extract. Samples were cryopreserved in a Tris–egg yolk–citric acid extender. Post-thaw sperm quality using computer-assisted sperm analysis (CASA), hypo-osmotic swelling (HOS) test, and oxidative stress biomarkers, including reactive oxygen species (ROS), malondialdehyde (MDA) and total thiols as antioxidative parameter were evaluated. Removal of SP significantly enhanced several kinematic parameters (VCL, ALH, DCL) and reduced abnormal morphology (p < 0.05), whereas its presence improved motion quality indicators such as beat cross frequency (BCF), linearity (LIN), and straightness (STR) (p < 0.05). However, SP removal induced a marked oxidative imbalance, characterized by increased ROS and MDA levels and decreased total thiols (p < 0.001). Supplementation with S. oleraceus extract improved membrane integrity and restored redox balance by increasing thiols levels (p = 0.0001) and reducing lipid peroxidation (p < 0.001), with significant SP × dose interactions. Based on these results, in vitro fertilization (IVF) was performed only using SP+ groups, as the presence of SP ensured a more physiologically relevant environment and maintained oxidative balance necessary for fertilization. No significant differences were detected in cleavage or blastocyst rates among the three S. oleraceus extract doses. These findings highlight an interaction between SP and Sonchus oleraceus aqueous extract, suggesting that combining endogenous protein-rich components with exogenous plant-derived antioxidants may enhance post-thaw sperm functionality and its oxidative status in rams.