Differentially methylated regions identified in bovine embryos are not observed in adulthood

**Background:** DNA methylation undergoes extensive reprogramming during early embryogenesis, making the embryonic methylome highly dynamic and potentially transient. A previous study (Nochi et al., 2022) found that low levels of sulfur and cobalt in the diet of oocyte donor heifers altered the DNA methylome of bovine blastocysts, identifying 2,320 differentially methylated regions (DMRs). However, due to the second wave of epigenetic reprogramming that occurs during embryogenesis and fetal development, it remained unclear whether these embryonic DMRs persist into adult tissues. This study tests the hypothesis that DMRs identified in embryos are not maintained in adult somatic or germline tissues.

**Methods:** The study used six Nellore bulls (Bos taurus indicus) — two from the control group and four from the low S/Co group — derived from heifers fed experimental diets for six months during pre- and periconceptional periods. Blood and sperm samples were collected from these adult bulls. Genomic DNA was isolated from white blood cells using the DNeasy Blood & Tissue Kit and from sperm using a salting-out protocol. After sodium bisulfite treatment (EZ DNA Methylation-Lightning kit), bisulfite PCR was performed to amplify DMRs in four genes involved in histone methylation machinery: KDM2A (28 CpG sites, 387 bp amplicon), KDM5A (13 CpG sites, 334 bp), KMT2D (11 CpG sites, 333 bp), and DOT1L (19 CpG sites, 335 bp). Amplicons were cloned into TOPO TA Cloning vectors, sequenced, and analyzed using QUMA. Only sequences with ≥95% identity and ≥97% cytosine conversion were included (n=684 clones). Statistical comparisons used Mann-Whitney test (two groups), Kruskal-Wallis with Dunn's post-hoc (multiple groups), and Fisher's exact test (individual CpG sites), with significance set at p<0.05.

**Key Results:** Overall, 688 clones were analyzed. All four genes showed a hypermethylated pattern in both blood and sperm across all animals and groups. Specific methylation levels included: KDM2A/sperm/low S/Co/Bull 4 at 57.7%; KMT2D/blood/low S/Co/Bull 6 at 60.2%; and KMT2D/sperm/control/Bull 2 at 54.5%. Some sperm samples showed greater allelic variation (e.g., KDM5A/sperm/low S/Co/Bull 3 at 75.8%; DOT1L/sperm/control/Bull 2 at 86%). When comparing individual CpG sites, some differentially methylated CpGs were found between groups: KDM2A CpG 17 (blood) and CpG 25 (sperm), KDM5A CpG 4 (sperm), and DOT1L (sperm). However, when comparing all CpG sites collectively, there were no statistically significant differences in DNA methylation patterns between control and low S/Co groups for any gene in either blood or sperm. A tissue-specific difference was found only for KDM2A in the control group (blood vs. sperm), though both tissues remained hypermethylated.

**Clinical Implications:** These results confirm that DMRs identified in blastocysts are not necessarily maintained in adult tissues, likely due to extensive epigenetic reprogramming during embryogenesis and fetal development. The findings suggest that embryonic methylome analysis can serve as an initial screening tool but requires adult validation before drawing conclusions about intergenerational epigenetic inheritance. The study also highlights that the timing and duration of dietary stimuli are critical — effects during gametogenesis alone may not produce lasting epigenetic changes if reprogramming occurs later. Notably, the greater variation observed in sperm DNA methylation patterns warrants further investigation into whether maternal diet can influence offspring sperm epigenome and potentially affect fertility or disease susceptibility. The authors recommend that future studies validate embryonic DMRs in adult tissues before concluding epigenetic inheritance.