Liquid storage of ram semen for 96 hours: effects on kinematic parameters, membranes and DNA integrity, and ROS production

A complete assessment of morphological and functional characteristics of ram semen during refrigeration is necessary to optimize the process of semen manipulation and storage. The aim of this study was to describe changes in main predictive parameters of ram semen diluted in a commercial soy lecithin-based extender (OVIXcell), during long term liquid storage at 4 °C. Ejaculates of 5 Sarda rams were collected, pooled and diluted in OVIXcell. Samples were cooled at 4 °C and stored at this temperature until 96 h. At 0-24-48-72-96 h semen samples were analysed for the following parameters: motility [computer assisted sperm analysis (CASA)]; integrity of cytoplasm membrane and acrosome (PI/PSA staining); DNA fragmentation index [DFI(%); sperm chromatin structure assay (SCSA)]; levels of reactive oxygen species (ROS; H2DCFDA staining). Effect of time of storage on main parameters and correlations among them were assessed respectively by ANOVA and Pearson's correlation coefficient. Primary and secondary motility parameters were significantly affected by time of storage (P < 0.05) decreasing throughout 96 h. Integrity of cytoplasm membranes was preserved for the first 24 h and decreased by around 10% from 48 to 96 h (P < 0.05). No effects on acrosomes were observed (P > 0.05). Long term storage did not affect the levels of DFI(%) (P > 0.05) while ROS production significantly increased from 48 to 96 h (P < 0.05). Weak negative correlations were found among DFI(%), ROS, and kinematic parameters. In conclusion, long term liquid storage of ram semen at 4 °C induces progressive detrimental effects on the main predictive quality parameters of sperm, especially from 24 h onwards, but does not compromise DNA integrity.