4.5. Computer Assisted Semen Analysis (CASA)

The collected sperm were diluted in species-specific media and were added either to the “treated sample” containing the venom fraction, or the purified or synthetic peptide dissolved in ultrapure water, or to the “control sample” containing only ultrapure water. The ratios were 10 μL of compound together with 190 μL of semen sample. Treated and control samples were processed after 15 s. The delay between control and treated sperm analyses with the CASA system (Hamilton Thorn Research, Beverley, MA, USA) is below 1 min and the order of the analysis of samples were inverted between experiments, to avoid any time incubation artifacts. The sperm suspension was kept at 37 °C and introduced into an analysis chamber (Leja Products B.V., Nieuw-Vennep, Netherlands) of variable depth (100 μm for mouse sperm or 20 μm for human, NHP and bovine sperm). The following settings were used for analyses of human, NHP, bovine and mouse sperm, respectively: acquisition rate of 60; 60; 60; 60 Hz; number of frames recorded of 30; 20; 30; 45; a minimum contrast of 80; 80; 80; 50; minimum cell size of 3; 4; 5; 5; a low static-size gate of 0.85; 0.79; 0.1; 0.3; a high static-size gate of 4.24; 2.52; 3.4; 1.95; a low static-intensity gate of 0.39; 0.62; 0.3; 0.5; a high static-intensity gate of 1.4; 1.4; 1.7; 1.3; a minimum elongation gate of 0; 2; 8; 0; maximum elongation gate: 85; 50; 97; 87; a magnification factor of 1.89; 1.89; 1.89; 0.7. At least, forty motile spermatozoa were analyzed in each assay. The sperm were considered motile when the average path velocity (VAP) were > 1; 1; 1; 1. They were considered progressive when the VAP were >25; 25; 50; 30 and the path straightness (STR) were > 80; 80; 70; 70, for human, NHP, bovine and mouse sperm, respectively. The sperm motility parameters were measured at 37°C using a sperm analyzer. Two motility parameters were investigated in priority: VCL, the curvilinear velocity, and ALH, the amplitude of the lateral head displacement. VCL measures the real velocity of the sperm, whereas ALH measures the magnitude of the lateral displacement of the sperm head with regard to its average path. For the assays, we arbitrarily set positivity at –20% and +20% variations, below or above the mean values of these two velocities.