2.6. SDF Assessment

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL assay) was used to assess SDF as described by Mitchell et al. [18]. Briefly, the sperm cells were incubated with 2 mM dithiothreitol (DTT, Sigma-Aldrich, Overijse, Belgium) for 45 min. After washing with phosphate-buffered saline (PBS, GIBCO Life technologies, Paisley, UK) the samples were fixed in 3.7% formaldehyde (Sigma-Aldrich, Belgium) for 20 min at 4 °C. The semen analysis and SDF assay were carried out at three time-points: directly on fresh semen samples without storage, after cryopreservation and after processing with density gradient centrifugation (post-thaw). For the assay, the spermatozoa were resuspended in 500 µL of fresh permeabilization solution (100 mg Sodium citrate, 100 µL Triton X–100 in 100 mL dH2O). The positive control samples were treated with 5 µL of DNase I (Qiagen, Hilden, Germany) 1500 Kunitz Units for 30 min at room temperature. The assay was performed using the fluorescein In Situ Cell Death Detection.

Kit (Roche Diagnostics, Mannheim, Germany) using an Accuri C6 flow cytometer (BD Sciences, Erembodegem, Belgium) recorded 5000–10,000 events for each sample at a flow rate of 35 µL/min. The method has been standardized and cut-off values were defined [19,20].