Sperm DFI detected by flow cytometry. Sperm DFI was assessed by the sperm chromatin structure assay (SCSA). In brief, semen samples were treated for 30 s with 400 μl of a solution of 0.1 % Triton X-100, 0.15 mol/L NaCl, and 0.08 mol/L HCl, pH 1.2. After 30 s, 1.2 ml of staining buffer (6 μg/ml acridine orange [AO], 37 mmol/L citric acid, 126 mmol/L Na2HPO4, 1 mmol/L disodium EDTA, 0.15 mol/L NaCl, pH 6.0) was admixed to the test tube. The sample was placed into the FACS Calibur flow cytometer with the sample flowing to establish optimal sheath/sample flow, and then at exactly 3 min AO staining measurements were taken. A minimum of 5 000 cells from two aliquots of each sample were acquired and analyzed by FACS scan interfaced with a data analysis software. After completion of the sample analysis, the cytogram (red vs green fluorescence) and DFI readings were generated.