Semen Preparation
The semen sample was divided, with 0,5 mL being used for cryopreservation. Another 0.5 mL was centrifuged at 2000 rpm for 10 minutes, to separate seminal plasma and the pellet. Furthermore, 1 mL of the sample was stratified using a 80% / 40% of a solution of Pure sperm 100 Nidacon in Sperm Washing Medium (Irvine Scientific, Santa Ana, CA) to separate round cells from the remaining semen. The resulting fraction was then centrifuged at 1600 rpm for 15 minutes, and the pellet containing spermatozoa was resuspended in TRIZOL (Thermo Scientific, Waltham, MA) and stored at -80C. The layer between the gradients, which contained round cells, was recoverd, centrifuged at 1800 rpm for 10 minutes, and resuspended in 500 mL of FBS10% DMSO.