Determination of Ram (Ovis aries) Sperm DNA Damage Due to Oxidative Stress: 8-OHdG Immunodetection Assay vs. SCSA(®)

SIMPLE SUMMARY: The particular architecture and biology of spermatozoa make them highly susceptible to oxidative stress, which can lead to DNA decondensation and fragmentation. It might also induce the formation of 8-OHdG, which is an early marker of DNA damage caused by oxidative stress. Because ruminant sperm DNA is highly compacted, it is rare to detect damage in ram DNA by conventional techniques. In this study, our aim was to evaluate the efficacy of detecting oxidative DNA damage in ram sperm samples using SCSA(®) vs. an 8-OHdG immunodetection assay. Our results showed that SCSA(®) and an oxidative-stress-specific 8-OHdG immunodetection assay can detect DNA damage caused by oxidative stress in ram sperm cells under high oxidative conditions; therefore, it is not necessary to use an oxidative-stress-specific technique to detect DNA damage in ovine spermatozoa. ABSTRACT: Conventional DNA analysis techniques can hardly detect DNA damage in ruminant spermatozoa due to high DNA compaction in these cells. Furthermore, these techniques cannot discriminate whether the damage is due to oxidative stress. The main purpose of this study was to evaluate the efficacy of two techniques for determining DNA damage in ovine sperm when the source of that damage is oxidative stress. Semen samples from twenty Manchega rams (Ovis aries) were collected and cryopreserved. After thawing, the samples were subjected to different levels of oxidative stress, and DNA oxidation was quantified using an 8-hydroxy-2′-deoxyguanosine (8-OHdG) immunodetection assay and Sperm Chromatin Structure Assay (SCSA(®)). For this purpose, we evaluated five different concentrations of an oxidation solution (H(2)O(2)/FeSO(4)•7H(2)O) on ram sperm DNA. Our study with the 8-OHdG immunodetection assay shows that there are higher values for DNA oxidation in samples that were subjected to the highest oxidative stress (8 M H(2)O(2)/800 µM FeSO(4)•7H(2)O) and those that were not exposed to high oxidative stress, but these differences were not significant (p ≥ 0.05). The two SCSA(®) parameters considered, DNA fragmentation index (DFI %) and high DNA stainability (HDS %), showed significant differences between samples that were subjected to high concentrations of the oxidation agent and those that were not (p < 0.05). We can conclude that the 8-OHdG immunodetection assay and SCSA(®) detect DNA damage caused by oxidative stress in ovine sperm under high oxidative conditions; SCSA(®) is a more straightforward method with more accurate results. For these reasons, an oxidative-stress-specific assay such as 8-OHdG immunodetection is not needed to measure DNA damage caused by oxidative stress in ram sperm samples.