Sca sperm class analyzer

In the freshly collected individual ejaculates the following traits were examined: 1. volume (with automatic pipette); 2. sperm concentration—by haemocytometer method, using a 3% eosin-NaCl solution (v/V) and Thoma-type grids; 3. motility—with the use of Sperm Class Analyzer SCA®, Microptic, Barcelona, Spain); 4. the integrity and morphology of the spermatozoa—on the basis of histological smears, vital stained with nigrosin-eosin (n = 300 cells per slide), evaluated at 1250x under a light microscope (Nikon Eclipse E 100) and expressed as the percentage of particular forms of spermatozoa (300 cells = 100%) [35 (link)]; 5. the Semen Quality Factor (SQF) calculated according to the following pattern: sperm concentration (n x10⁶ mL^-1) x ejaculate volume (mL) x live normal spermatozoa (%) /100% [36 ]. To reduce error from subjective scoring, each ejaculate was evaluated by the same, well experienced person.

Seminal samples were obtained from male idiopathic patients from Instituto de Fertilidad of Palma de Mallorca, in collaboration with Genosalut and Universitat Autònoma de Barcelona. A total of 42 samples were collected, with 2–5 days of sexual abstinence. Samples were frozen immediately after liquefaction and transported to the Universitat Autònoma de Barcelona (UAB), where they were stored in liquid nitrogen at −196 °C until its analysis. Prior to cryopreservation, a basic semen analysis (sperm concentration, motility, morphology and pH) was performed with the Sperm Class Analyzer (SCA, Microptic, Barcelona, Spain), Sperm Blue (Microptic) and pH-indicator strips, according to the 2010 WHO guidelines [43 ]. Seminal viscosity was measured using 20 micron Leja chambers as previously suggested Rijnders [44 (link)], showing the results in centipoise (cps). All donors signed their informed consent and the study was approved by the Ethics Committee of Corporació Sanitaria Parc Taulí (Ref.: 2014676).

The semen was thawed in a water bath at 37°C for 30 s. The analyses were performed immediately post thaw (M0) and after the rapid thermoresistance test [RTT - incubation at 46°C for 30 min; M30; Vianna et al. (2009) (link)].
Sperm kinetics analyses were performed using a computerized system (SCA^™ - Sperm Class Analyzer, Microptic, Barcelona, Spain). The variables used were total motility (TM; %), progressive motility (PM; %), curvilinear velocity (VCL; μm s^-1), the amplitude of lateral head displacement (ALH; μm), and linearity (LIN; %), that correspond to the kinetic characteristics most closely correlated with sperm hyperactivation in a semen sample (Nassar et al., 1998 (link); Verstegen et al., 2002 (link)).
Plasma membrane integrity, acrosome integrity, plasma membrane fluidity, mitochondrial activity, and susceptibility to lipoperoxidation were assessed using flow cytometry. The supplemental data in terms of the fluorescent probes used are described in Table 1.