Influence of sampling factors on canine sperm motility parameters measured by the Sperm Class Analyzer

The aim of the present study was to investigate the effect of different technical settings and semen processing on sperm motility parameters measured by the Sperm Class Analyzer (SCA). Semen was collected from 3 dogs, pooled, and diluted in phosphate buffered saline and subsequently assessed by the SCA for the different sperm motility characteristics. The data were statistically analyzed by ANOVA and the repeatability was assessed by coefficient of variation (CV). After a principal component analysis, the reliability was determined with intra-class correlation coefficient (ICC). In experiment 1, the CV's were below 10% for all evaluated parameters. Significant differences (P < 0.05) were found between the different sperm concentrations (25, 50, and 75 x 10(6) spermatozoa/ml) in all of the motion parameters assessed, yielding the highest ICC (0.81) at 25 x 10(6) spermatozoa/ml. No significant differences (P > 0.05) in SCA read-outs were found between the number of microscopic fields captured (1, 2, 3, 4, or 5 fields), yielding the highest ICC (0.83) when 3 fields were captured. No significant differences (P > 0.05) in motility parameters were found between the number of cells analyzed in each field (20, 50, and 100 spermatozoa) with the exception of beat cross frequency. Reliability of the SCA was good (ICC = 0.71 to 0.90) for all motility measurements when 20 (ICC = 0.89) or 50 (ICC = 0.77) cells were captured in each field, but only just acceptable (ICC = 0.51 to 0.70) when 100 cells were counted (ICC = 0.67). The frame settings significantly (P < 0.05) influenced most of the measured motility characteristics. Scanning 60 frames at a frame rate of 30 Hz improved the reliability of the results (ICC = 0.92). In conclusion, we suggest that the measurements with the SCA are ideally performed at a sperm concentration of 25 x 10(6) spermatozoa/ml, counting at least 100 cells in three microscopic fields. We also propose that the SCA should analyze 60 frames at 30 Hz to yield consistent results of a set of measurements or a measuring instrument thus obtaining reliable motility results.