Automated Sperm Motility and Concentration Analysis

Five microliters of concentrated spermatozoa cloud was collected and placed on a Leja slide (Leja Products BV, Nieuw Vennep, Netherlands). The Leja slide was placed onto a temperature-controlled stage of the Nikon E200 microscope (37°C). A 4x negative phase-contrast objective in conjunction with a phase-contrast condenser was used to determine sperm motility and concentration via the motility/concentration module of the Sperm Class Analyzer® version 5.4.0.1 software (Microptic SL, Barcelona, Spain) at 50 frames/s. Data were collected by capturing images with a digital camera (Basler, A78075gc, Germany). For motility analysis, eight fields were captured with the SCA system until 200 motile spermatozoa were analyzed, as recommended by WHO (1999) [46 (link)–48 (link), 51 (link)].