Received January 26, 2026
Accepted February 10, 2026

Objective: Density gradient centrifugation is widely used for sperm preparation, although centrifugation-induced oxidative stress may cause DNA damage. This study compared the rates of γH2AX-positive spermatozoa (a surrogate marker associated with DNA double-strand break–related damage signaling) among sperm prepared by density gradient centrifugation, ZyMōt, and Felix, and evaluated sperm recovery, total motility, and processing time. Methods: Fifteen fresh semen samples collected from January to June 2025 were processed in parallel using density gradient centrifugation, ZyMōt, and Felix. Following sperm preparation, γH2AX immunostaining was performed, and at least 200 spermatozoa per sample were scored to determine the γH2AX-positive rate. Sperm recovery, total motility, and workflow-defined processing time were also recorded. Statistical analyses were performed using the Friedman test followed by Wilcoxon signed-rank tests with Bonferroni correction, and data were expressed as medians with interquartile ranges. Results: The γH2AX-positive rate was significantly lower in the ZyMōt (11.8%) and Felix (10.0%) groups compared with the density gradient centrifugation group (16.0%; p < 0.01). Density gradient centrifugation yielded the highest sperm recovery, ZyMōt achieved the highest total motility, and Felix required the shortest processing time, indicating that the three methods exhibit distinct performance characteristics. Conclusion: The non-centrifugal systems ZyMōt and Felix reduced the proportion of γH2AX-positive spermatozoa compared with density gradient centrifugation under our laboratory conditions while maintaining comparable overall performance. Whether these laboratory differences translate into improved assisted reproductive technology outcomes should be evaluated in future prospective studies.