Sperm analyzer ivos version 12.2l

Sperm motility was evaluated using the CASA system (Hamilton Thorne Sperm Analyzer IVOS version 12.2l; Hamilton Thorne Biosciences, MA, USA). Sperm samples were diluted 1 : 100 (fresh) and 1 : 5 (fresh diluted sperm, frozen-thawed) in phosphate-buffered saline (PBS) to a concentration of around 30–50 × 10⁶ cells/mL. A 5 μL aliquot of the sample was placed in a pre-warmed Makler counting chamber (Sefi-Medical Instruments Ltd., Haifa, Israel) and evaluated at 37 °C. In each sample, spermatozoa were analyzed in five fields of view selected randomly by the computer. The analyses included the determination of: percentage of total motile sperm (TMOT), percentage of progressive motile sperm (PMOT), and parameters characterizing sperm movement: VAP, VSL, VCL, ALH, BCF, LIN (VSL/VCL ratio × 100 %), and straightness (STR, VSL/VAP ratio × 100 %).
The CASA system settings recommended by Hamilton Thorne for gazelle/deer [26 (link)] were used in the analyses. Progressive motility was defined as the percentage of spermatozoa with a VAP > 75.0 μm/s and an STR > 80%.

Sperm motility was evaluated using computer assisted semen analyzer (CASA) Hamilton Thorne Sperm Analyzer IVOS version 12.2l (Hamilton Thorne Biosciences, MA, USA) under 1.89 × 10 magnification. Semen was diluted in 1 : 100 (fresh) and 1 : 20 (frozen-thawed) in DMEM (Dulbecco modified medium low glucose) [35 (link)] to obtain sperm concentration of about 50 × 10⁶/mL. Three μL aliquot of semen was placed in Leja4 analysis chamber (Leja, Nieuw-Vannep, Netherlands) at 35°C and evaluated. Five fields randomly selected by the computer were analyzed for each semen sample. The parameters measured were the percentage of motile sperm (MOT), the percentage of progressively motile spermatozoa (PROG), path velocity (VAP, average velocity/smoothed average position of the spermatozoa), progressive velocity (VSL, straight-line distance between the beginning and the end of the track), curvilinear line velocity (VCL, average velocity measured over the actual point-to-point track followed by the cell), straightness (STR, a measure of VCL side to side movement determined by the ratio VSL/VAP × 100), linearity (LIN, a measure of the departure of the cell track from a straight line; the ratio VSL/VCL × 100), and percentage of rapid spermatozoa (RAPID).