Optimization of CASA-Mot Analysis of Donkey Sperm: Optimum Frame Rate and Values of Kinematic Variables for Different Counting Chamber and Fields.

In order to optimize the donkey sperm motility analysis by the CASA (Computer Assisted Sperm Analysis)-Mot system, twelve ejaculates were collected from six jackasses. Capillary loaded chamber (CLC), ISASD4C depths 10 and 20 µm, ISASD4C Leja 20 and drop displacement chamber (DDC), Spermtrack (Spk) depths 10 and 20 µm were used. Sperm kinematic variables were evaluated using each chamber and a high-resolution camera capable of capturing a maximum of 500 frames/second (fps). The optimum frame rate (OFR) (defined according to curvilinear velocity-VCL) was dependent on chamber type. The highest OFR obtained was 278.46 fps by Spk20. Values for VCL, straight-line velocity (VSL), straightness (STR), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) were high in DDC and 10 µm depth. In both DDC 10 and 20 µm, the sperm velocities (VCL, VSL, VAP) and ALH values decreased significantly from the centre to the edges, while Wobble and BCF increased. No defined behavior was observed along the CLC. However, all the kinematic variables had a higher value in a highly concentrated sample, in both chamber types. In conclusion, analyzing a minimum of nine fields at 250 fps from the centre to the edges in Spk10 chamber using a dilution of 30 × 10 sperm/mL offers the best choice for donkey computerised sperm motility analysis.