The integrity of equine sperm is compromised by various environmental and biochemical factors. Reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), induce oxidative stress, leading to DNA damage. There are many DNA damage assays available, and the relative prognostic value of these has not yet been compared in the horse. Therefore, the objective of the present study was to compare the sensitivity and specificity of two popular sperm DNA damage assays, the sperm chromatin structure assay (SCSA) and the single cell gel electrophoresis (comet) assay in identifying sperm DNA damage in the horse.
A total of 4 ejaculates from individual stallions were diluted (2:1, extender: semen) and processed via single-layer colloidal centrifugation to isolate the high-quality sperm fraction, which was resuspended in Biggers, Whitten and Whittingham (BWW) medium for treatment. Samples were treated with four different concentrations of H₂O₂ (control, 0.125 mM, 1.25 mM, 2.5 mM and 5mM), followed by incubation at 37°C for 1h, after which aliquots were snap-frozen for SCSA and COMET assay, and stored at -80°C until analysis.
Data was analyzed by PROC GLIMMIX of SAS and normality of the data was checked through UNIVARIATE procedure and Shapiro-Wilk test, and results are expressed as mean ± S.E.M. There was no significant difference between treatments for DNA fragmentation index (DFI) as measured by SCSA. In contrast, the comet assay was better able to detect DNA damage, with a significant, dose-dependent effect of H₂O₂ on Tail Intensity (TI) (P≤ 0.05) even at the lowest H2O2 dose. The TI was significantly higher (P< 0.05) at 5mM (69.50 ± 3.65) of H₂O₂ compared to control (20.28 ± 4.01). In conclusion, COMET assay is more sensitive to detect DNA damage compared to SCSA in stallion sperm and should be the preferred method of sperm DNA damage assessment on horses where feasible.