Methylene blue

Polystyrene nanospheres were purchased from Sigma-Aldrich (St. Louis, MO, USA), along with the other solutions necessary for the conduct of this experiment (PBS, 2′,7′-dichlorofluorescein-diacetate, DMSO, BSA, DAB). Human sperm culture and washing medium were obtained from FertilPro (Industriepark Noord, Beernem, Belgium). Eosin Y and Methylene blue and Formaldehyde were purchased from Bio-Optica (Milan, Italy). Peanut Agglutin-Fluorescein solution was obtained from Vector Laboratories (Newark, CA, USA). Fluoromount G with DAPI and Hoechst 33342 were purchased from Invitrogen (Waltham, MA, USA). A Halosperm Kit was obtained from Halotech (Madrid, Spain). HSP70 primary antibody and goat Anti-rabbit IgG pre-adsorbed Rhodamine secondary antibody were obtained from GeneTex (Irvine, CA, USA). Triton X-100 was purchased from ChemSolute (Renningen, Germany). Finally, a MiOXYS system was obtained from Medical Biological Technologies (MBT, Pretoria, South Africa).

Analysis of any DNA breaks was performed using a Halosperm Kit (Halotech, Madrid, Spain). Briefly, the sample was diluted to 20 mil/mL in PBS. Next, 50 µL of the sample was mixed with 100 µL of agar, previously dissolved for 5 min in a water bath at a temperature of 95–100 °C. An amount of 8 µL of the mixture was placed in the center of an agarized slide and covered with a coverslip of 22 × 22. The slide was transferred at 4 °C for 5 min to allow the agar to solidify. After removal of the coverslip, the sample was incubated with denaturing solution for 7 min and with lysis solution for 25 min. Next, the slide was incubated for 5 min in distilled water and then dehydrated by increasing alcohols (70% and 100% for 2 min each). Finally, staining was carried out by incubation with Eosin Y (Bio Optica, Milan, Italy) for 2 min, followed by Methylene blue (Bio Optica) for 2 min. The presence of a halo around the head was a hallmark of intact DNA. In fact, in the spermatozoa with fragmented DNA, the halo was absent.