Potential antioxidant effect of creatine on the functionality of intratesticular spermatozoa of coho salmon (Oncorhynchus kisutch)

Quality control of intratesticular sperm samples obtained through testicular maceration faces significant challenges in salmon aquaculture. Mechanical damage during maceration and tissue contamination induces oxidative stress owing to reactive oxygen species (ROS). Due to the ability of antioxidants to reduce oxidative stress, this study evaluated the antioxidant effect of creatine on intratesticular sperm functionality and quality in fifteen males of coho salmon (Oncorhynchus kisutch). Two experiments were conducted under short-term storage (4 °C). In experiment 1, four creatine concentrations were tested: group 1 (0 mM, control), group 2 (2.5 mM), group 3 (5.0 mM), and group 4 (7.5 mM), with measurements at 0 h and 24 h. Experiment 2 involved lower concentrations: group 1 (0 mM), group 2 (0.1 mM), group 3 (0.2 mM), and group 4 (0.4 mM), assessed after 1-h incubation. Both experiments evaluated sperm kinetics (motility, curvilinear velocity [VCL], straight-line velocity [VSL], average path velocity [VAP]) using Computer-Assisted Sperm Analysis (CASA) and sperm functionality (membrane integrity, mitochondrial potential, anion superoxide levels, and DNA fragmentation) via flow cytometry. In experiment 1, group 2 showed higher motility, and group 4 displayed reduced mitochondrial ROS at 0 h. In experiment 2, group 4 had significantly lower cytoplasmic and mitochondrial ROS levels, indicating enhanced antioxidant protection. Overall, creatine monohydrate demonstrated antioxidant properties, reducing oxidative stress, lowering ROS levels and DNA fragmentation in group 4 (0.4 mM). These findings highlight creatine's potential to enhance sperm quality in aquaculture, particularly for short-term storage of intratesticular sperm.