Sperm class analyzer software

FT sperm kinematics was assessed immediately after thawing (AT) and post-incubation for 120 min using Sperm Class Analyzer (SCA) software (Version 5.1; Microptic, Barcelona, Spain). Briefly, the samples were thawed at 38 °C for 25 s and then diluted in BTS (1:4 v/v). Then, 2 μL of diluted semen were loaded into a pre-warmed counting chamber (Leja, Nieuw Vennep, The Netherlands). Samples were kept at 24 °C for 2 h before being analyzed again. For each analysis, at least 1500 spermatozoa were tested using standard settings (38 °C, 60 frames/s). The percentages of total motile spermatozoa (TM, %), progressive motile spermatozoa (PM, %), and mucus penetration (MP, %) were determined. The kinematic parameters measured for each sperm included curvilinear velocity (VCL, μm/s), straight-line velocity (VSL, μm/s), average path velocity (VAP, μm/s), and amplitude of lateral head displacement (ALH, μm). This experiment was repeated six times.

Sperm motility and kinetic parameters were measured in ejaculates using the Sperm Class Analyzer (SCA) software (version 4.0, Microptic S.L., Barcelona, Spain). A small aliquot (50 μL) of fresh ejaculates was further diluted in refrigerated 0.9% NaCl to 50 × 10⁶ sperm/mL, incubated for 20 min at room temperature and motility assessed at room temperature placing 10 μL of semen on a Makler chamber (Sefi Medical Instruments, Haifa, Israel) under a microscope. The following motility parameters were recorded: total motile sperm (TMS), progressive motile sperm (PMS) and some kinetic parameters (VCL, VSL, VAP, ALH, BCF, LIN, STR and WOB), previously described in detail by Mosca et al. [31 (link)]. In each sample, a minimum of three fields and 500 sperm tracks at 100× magnification were analyzed; 25 frames per second (Hz) and 25 frames per field were set. The following software settings were used: range cell size from 5 to 190 µm²; sperm classified as motile if VCL ≥ 13 µm/s; sperm classified as progressive if STR ≥ 70%.