Sperm class analyzer 5

Semen samples were preheated to 37 °C for 20 min and then 20 μL were dropwise added to a pre-warmed cell Counting-Chamber (Makler; 10 mm depth; Sefi Medical Instruments, Haifa, Israel). The sperm quality parameters were examined at 100× magnification with an Olympus BX41 microscope (Olympus Life Science Europe GMBH, Hamburg, Germany) equipped with phase-contrast optics and Sperm Class Analyzer 5 software (Microptic SL, Barcelona, Spain). A minimum of three replicates per seminal dose, with 1000 spermatozoa each, were analyzed. The motility parameters were evaluated in consecutive digitalized frames automatically acquired by Sperm Class Analyzer 5 software (Microptic SL, Barcelona, Spain) in each field. The definition of motility parameters, including VCL, VAP, VSL, ALH, LIN, STR, and WOB, was according to previous reports [67 (link),68 (link)].

After capacitation, cells were divided into two aliquots, with the concentration adjusted to 10×10⁶ sperm ml⁻¹, centrifuged (300 g, 5 min) and resuspended in BWW medium at pH 7.2 (tube 1) and pH 8.0 (tube 2). In each sample, motility was stimulated with 4-aminopyridine (4-AP; weak base that increases pH and thus stimulates CatSper) (Achikanu et al., 2018 (link)) or treated with 5 µmol l⁻¹ RU1968 (Rennhack et al., 2018 (link)). Five microliters of the sperm samples for each condition were loaded into a Spermatrack Proiser chamber at 37°C and immediately placed on the heated stage (37°C) of a Nikon model ECLIPSE Ci-L upright microscope with an attached computer-assisted sperm analysis (CASA) system equipped with a Basler Ace camera (version 5.4, Microptic) and the Sperm Class Analyzer 5 Software (López-Torres et al., 2017 (link); World Health Organization, 2010 ). The percentage motility was determined (progressive, non-progressive and static), in addition to the kinematic parameters, without the addition of stimuli and 300 s after their application. Approximately ten video photographs were collected, in which at least 300 sperm were tracked (up to 15 fields). The criteria for determining hyperactivation were curvilinear velocity (VCL)≥150 μm s⁻¹, linearity (LIN)<50% and amplitude of lateral head movement (ALH)≥3.5 μm (Mortimer and Mortimer, 2013 ).