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

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 (H2O2/FeSO4•7H2O) 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 H2O2/800 µM FeSO4•7H2O) 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.

Keywords: 8-OHdG; DNA; SCSA®; flow cytometry; oxidative stress; ram sperm.

Figure 1

Experimental design of the study to assess the accuracy of ram sperm DNA oxidation quantification using an 8-OHdG immunodetection assay and chromatin stability using Sperm Chromatin Structure Assay (SCSA^®).

Figure 2

DNA oxidation quantification using an 8-OHdG immunodetection assay. Mean of Alexa Fluor 488 intensity of fluorescence ± standard error of the mean (MIF ± SEM) differences from 8-OHdG immunodetection assay. OS1 = 1 mM H₂O₂/0.1 µM FeSO₄•7H₂O; OS2 = 10 mM H₂O₂/1 µM FeSO₄•7H₂O; OS3 = 100 mM H₂O₂/10 µM FeSO₄•7H₂O; OS4 = 1 M H₂O₂/100 µM FeSO₄•7H₂O; and OS5 = 8 M H₂O₂/800 µM FeSO₄•7H₂O.

Figure 3

Histograms of DNA oxidation quantification using an 8-OHdG immunodetection assay. Basal fluorescence of evaluated subjects (control) and samples submitted to 1 mM H₂O₂/0.1 µM FeSO₄•7H₂O (OS1); samples submitted to 10 mM H₂O₂/1 µM FeSO₄•7H₂O (OS2); samples submitted to 100 mM H₂O₂/10 µM FeSO₄•7H₂O (OS3); samples submitted to 1 M H₂O₂/100 µM FeSO₄•7H₂O (OS4); and samples submitted to 8 M H₂O₂/800 µM FeSO₄•7H₂O (OS5).

Figure 4

Effects of oxidant treatments on Sperm Chromatin Structure Assay (SCSA^®) results. Mean of DNA fragmentation index (DFI %) ± standard error of the mean (mean ± SEM). a, b and c indicate significant differences (p < 0.05). OS1 = 1 mM H₂O₂/0.1 µM FeSO₄•7H₂O; OS2 = 10 mM H₂O₂/1 µM FeSO₄•7H₂O; OS3 = 100 mM H₂O₂/10 µM FeSO₄•7H₂O; OS4 = 1 M H₂O₂/100 µM FeSO₄•7H₂O; and OS5 = 8 M H₂O₂/800 µM FeSO₄•7H₂O.

Figure 5

Effects of oxidant treatments on Sperm Chromatin Structure Assay (SCSA^®) results. Mean of high DNA stainability (HDS %) ± standard error of the mean (mean ± SEM). a, b and c indicate significant differences (p < 0.05). OS1 = 1 mM H₂O₂/0.1 µM FeSO₄•7H₂O; OS2 = 10 mM H₂O₂/1 µM FeSO₄•7H₂O; OS3 = 100 mM H₂O₂/10 µM FeSO₄•7H₂O; OS4 = 1 M H₂O₂/100 µM FeSO₄•7H₂O; and OS5 = 8 M H₂O₂/800 µM FeSO₄•7H₂O.

Figure 6

Assessment of sperm chromatin status. Cytogram obtained after carrying out the SCSA^® protocol. Green dots correspond to a sample that has not been treated with oxidant solution, and red dots correspond to a sample submitted to high exogenous oxidative stress. Dots plotted to the right of the diagonal line have increased DNA fragmentation index (DFI), and dots plotted above to the horizontal line have increased high DNA stainability (HDS).