Factors Affecting Computer-Assisted Sperm Analysis (CASA) Measurements for Porcine Spermatozoa.

The effect of analysis time; dilution; and slide location on motility (MOT); progressive motility (PMOT); curvilinear (VCL) and straight line (VSL) velocities; beat frequency (BCF); and lateral head displacement (ALH) was determined for porcine spermatozoa in 3 experiments. For each experiment, the split ejaculate technique was used with 18 boars being collected weekly for 5 to 6 weeks (n=100 to 120 observations per treatment per experiment). For analysis time, CASA measurements were obtained at 0, 30, 60, 90 and 120 s after sample loading. For dilution, the neat sample was analyzed along with subsamples diluted with BTS extender in ratios of 1:1, 1:2, and 1:3. For slide location, the center of the counting chamber was divided into 3, 3 x 5 mm sections, Central Lower (LOW), Central Middle (MID) and Central Upper (UP), and measurements were taken randomly within each section. All CASA parameters decreased during the first 60 s after loading the slide chamber (p<0.001) with the highest measurements occurring at time 0 (p<0.05). The only exception was for VCL for which times 0 and 30 s were similar (p>0.05). Increasing the dilution of neat semen had variable effects on CASA variables. However, measurements for the 1:1 rate were consistently equal to or higher (p<0.05) than those recorded for neat semen and their counterparts with higher dilutions. The location within the chamber at which measurements were taken had no effect on VCL (p=0.12); VSL (p=0.31); and BCF (p=0.17). For MOT, PMOT, and ALH all locations were similar (p>0.05) with the exception of MID in which the lowest (p<0.05) readings were recorded. Collectively, results from these studies indicate that neat samples should be diluted 1:1 and readings should be taken from any location except central middle slide area within 30 s of loading the sample in order to obtain the highest measurements.