Effects of seminal plasma and flash-freezing on DNA structure of stallion epididymal sperm exposed to different potentiators of DNA damage.

The tolerance of sperm DNA structure to seminal plasma and freezing conditions has both clinical and basic biologic relevance. In this study, fresh (FS) or flash-frozen (FZ) stallion epididymal sperm were exposed (SP+ ) or unexposed (SP- ) to seminal plasma. Sperm were then evaluated to monitor the degree of change in DNA structure following challenge with chemical (dithiothreitol-DTT), oxidative (iron sulfate; FeSO4 ) or enzymatic (DNase I) potentiators of DNA damage. For sperm not treated with potentiators (controls), there was no effect of SP treatment (SP- vs. SP+ ) or freezing treatment (FS vs. FZ; non-significant) on measures of any DNA assays (i.e., 8-hydroxy, 2'deoxyguanosine [8OHdG], TUNEL, or sperm chromatin structure [SCSA] assays). Group FZ was more susceptible than Group FS to potentiators of DNA damage. Percent 8OHdG-positive sperm was higher in Group FZ/SP- treated with FeSO4 than all other groups (P < 0.05). Percent TUNEL-positive sperm was similar among FZ/SP- groups treated with DTT, FeSO4 , or DNase (non-significant) and was higher in these groups than all other treatments (P < 0.05). Percent COMP-αt was higher following treatment with DNase or DTT, as compared to their respective controls, regardless of prior exposure to SP (P < 0.05). Overall, sperm DNA structure was unaffected by seminal plasma or freezing treatment when samples were not exposed to potentiators of sperm DNA damage; however, marked differences were identified in DNA structure when sperm were challenged with chemical, oxidative or enzymatic treatments. These results highlight the importance of challenging DNA structure prior to analysis. The use of potentiators of DNA damage provided a model to evaluate sperm DNA structure following exposure of sperm to various experimental treatments.