Fig. 6

Bisulfite-pyrosequencing validations on twenty fertile and subfertile semen samples. A IGV browser views of the regions targeted for pyrosequencing in the ATG7, NPAS1, SORCS2, LBX1 and PLXNB2 genes. The red and blue bar charts represent the methylation percentages at each CpG10 position for fertile (n = 10) and subfertile (n = 10) bulls, respectively. The CpGs analyzed by pyrosequencing are numbered according to their 5′–3′ position along the genome. The CpGs identified as fertility-related DMCs are indicated in black text and red boxes, while non-DMCs are indicated in grey. B For each gene region, the average methylation percentage measured by pyrosequencing (y-axis) was calculated for the DMCs included in the region and plotted against the average methylation percentage measured by RRBS at the same DMCs (x-axis). Each dot represents one sample from the fertile (in red, n = 9 to 10) and subfertile (in blue, n = 9–10) groups. The least squares lines of best fit and Spearman’s rank R correlation coefficients are indicated. All correlations were highly significant (Spearman’s rank correlation test; p < 0.05). C Methylation percentages of individual CpGs assayed by pyrosequencing in fertile (in red, n = 10) and subfertile (in blue, n = 10) bulls. CpGs are numbered according to A and DMCs are highlighted in black. Dots show the methylation levels of individual samples, while the trends per fertility group are indicated by red and blue lines obtained using the geom_smooth function of the ggplot2 R library, together with 95% confidence intervals in light grey. Asterisks indicate that the methylation percentage measured by pyrosequencing differed significantly between fertility groups for all analyzed CpGs (Wilcoxon test, p < 0.05)