Cryoprotective efficacy of kappa-carrageenan on camel epididymal spermatozoa: Assessments of sperm quality, kinematics, oxidative stress, ultrastructure, and microbial load.

This study aimed to evaluate for the first time the protective effects of kappa-carrageenan (κ-carrageenan; CAR) on cryopreserved camel epididymal spermatozoa. To achieve this, we assessed post-thaw sperm motility, kinematic parameters, oxidative stress biomarkers, subcellular ultrastructure, apoptotic profiles, microbial load, and molecular docking interactions. Epididymal spermatozoa were harvested from 40 pairs of dromedary camel testes. The samples were cryopreserved using a freezing tris-SHOTOR extender alone (CAR0) or supplemented with κ-carrageenan (CAR) 0.25 (CAR0.25), or 0.5 (CAR0.5) mg/mL prior to cryopreservation via a standard protocol. Supplementation of the cryopreservation medium with CAR markedly preserved camel epididymal sperm viability, plasma membrane integrity, and motion kinetics post-equilibration, immediately after thawing, and following 2 h of incubation (P < 0.05). Notably, the CAR0.5 treatment improved key kinematic parameters specifically progressive motility, average path velocity (VAP), amplitude of lateral head displacement (ALH), and beat-cross frequency (BCF) while yielding lower wobble (WOB), linearity (LIN), and straightness (STR) values compared to the untreated control (P < 0.05). Additionally, CAR supplementation significantly increased the percentage of viable spermatozoa and reduced necrotic populations, with the CAR0.5 group demonstrating the most favorable outcomes (P < 0.05). Treatment with CAR also enhanced acrosome integrity and antioxidant defense mechanisms (TAC and catalase), while decreasing acrosomal damage and oxidative stress biomarkers (NO and malondialdehyde; P < 0.05). Transmission electron microscopy confirmed these protective effects on the acrosome, plasma membrane, and overall subcellular ultrastructure. Furthermore, all CAR-treated samples exhibited a significant reduction in total bacterial and coliform counts relative to the control (P < 0.05). In silico docking revealed weak-to-modest interactions between CAR and three target proteins ferritin, calmodulin-2 B, and the EF-hand calcium-binding domain-containing protein providing structural insights into potential binding mechanisms. Overall, κ-carrageenan improved post-thaw sperm quality, boosted antioxidant capacity, attenuated oxidative damage and declined microbial counts, and warrants further evaluation in fertility trials.