TEMPERATURE-DEPENDENT EFFECTS OF DENSITY GRADIENT CENTRIFUGATION ON MULTIPLE SPERM QUALITY PARAMETERS: A COMPARATIVE ANALYSIS AT 4°C, 20°C, AND 37°C

Abstract

Percoll medium, a density gradient solution, is widely employed in reproductive biotechnologies for the efficient isolation of spermatozoa based on their specific density. This study examined the impact of temperature (4°C, 20°C, and 37°C) during and after Percoll separation on various sperm quality indicators. Semen samples from Holstein breeding bulls (n = 6) were layered onto density gradient solutions in 15 mL falcon tubes. Following centrifugation (15 min, 400 × g), sperm cells were washed to remove residual Percoll medium. Multiple parameters were assessed immediately post-separation and after 2 hours, including sperm motility, mitochondrial membrane potential, reactive oxygen species production, membrane and acrosome integrity, and DNA fragmentation. Statistical analysis employed two-way ANOVA with Tukey's multiple comparison test. The results showed a significant decrease in sperm motility after 2 h at 4°C, while mitochondrial membrane potential was least affected at 37°C. Membrane integrity remained stable at 37°C but was significantly disrupted at lower temperatures, particularly at 4°C (P<0.0001), with a corresponding increase in necrotic cells when compared to 37°C after 2h (P<0.0001). Although the lowest ROS production was recorded at 4°C in both times, the differences between temperature groups were not significant. DNA fragmentation increased significantly at higher temperatures (20°C and 37°C) compared to 4°C. In conclusion, while lower temperature (4°C) reduced ROS production and DNA fragmentation, other parameters such as motility, mitochondrial membrane potential, and membrane and acrosome integrity were best preserved at 37°C.