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

SIMPLE SUMMARY: A reliable sperm motility exam is important for semen analysis and breeding soundness examination. Different parameters can affect the Computer Assisted Sperm Analysis (CASA) motility results. Today, new high-resolution cameras and different chambers are introduced to CASA systems, and protocol optimization is required to render the estimation results for donkey sperm. The objective of this study is the optimization of the conditions used for donkey semen motility analysis with CASA-Mot by defining the optimum frame rate for different chamber types. Additionally, to study the effect of different chamber types, chamber field and sperm dilution on the sperm kinematic parameters with higher frame rates are examined. ABSTRACT: 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), ISAS(®)D4C depths 10 and 20 µm, ISAS(®)D4C 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(6) sperm/mL offers the best choice for donkey computerised sperm motility analysis.