view - Department of Reproduction, Obstetrics and Herd Health

CHAPTER 3.2
92
Preliminary findings indicated that loading a chamber with capillary force might have an
influence on CONC. Therefore, it was decided not to use a classical haemocytometer as gold
standard for CONC. The NucleoCounter has been shown to be highly repeatable and to have very
good correlation with sperm concentration (Hansen et al., 2006). As such, the concentration as
determined with the NucleoCounter was used as gold standard. Motility outcomes obtained with
the WHO prepared slide were used as gold standard for motility.
3.2.3.5. Statistical Analysis
The data obtained for CONC showed a left skewed distribution. A normal distribution was
achieved using the natural log transformation (lnCONC). Progressive motility (PM) and percentage
rapid sperm (Rapid) were transformed by dividing them by 100 followed by an arcsine of the square
root of that value, in order to obtain a normal distribution. The other motility parameters, VAP, VSL,
VCL, ALH, BCF, STR, and LIN were normally distributed.
First, it was analyzed whether the averages obtained with the different chambers were
different. Mixed models (including stallion as a random effect) were fit with chamber and capillarity
(1 or 0) as fixed effects, respectively, to analyze chamber effect and effect of loading [filled with
capillarity (=1) vs. drop filled with cover slide (=0)]. LSD was used as post hoc tests to compare the
different chambers with the gold standard.
Secondly, scatter plots were produced for CONC and PM, where the value obtained with the
gold standard was plotted against the corresponding value obtained with the different chambers
(Microsoft Office Excel 2007). Ideally, this should result in the line of perfect agreement, which is the
hatched line at 45° and intercept zero. In each plot, this line of perfect agreement was drawn in
comparison to the regression line fit to the data (Arunvipas et al., 2003).
Third, to assess agreement between the values for CONC and PM obtained using the gold
standard with the values obtained using different chambers, Bland-Altman plots were generated.
This method plots the difference of the paired measurements (y-axis) against their mean (x-axis)
(Bland and Altman, 1986). The 95% limits of agreement were computed as the mean difference plus
or minus 1.96 times the standard deviation (SD) of the difference.