Epigenetic signature of prenatal heat stress: DNA methylation changes in whole blood of dairy calves from birth to weaning

The maternal environment during gestation influences fetal development, with long-lasting effects on postnatal health and productivity. This study evaluated the effect of prenatal heat stress (PNHS) on blood DNA methylation of dairy calves immediately after birth and whether such modifications persist into early life. Holstein calves were born to dams exposed to either PNHS (n = 36, temperature-humidity index >68, access to shade of a freestall barn) or prenatal cooling (PNTN; n = 37, access to shade and evaporative cooling) during the last 54 ± 5 d of gestation. Whole-genome enzymatic DNA methyl sequencing was performed on blood samples collected at birth (d 0; n = 3 PNHS, n = 5 PNTN) and 1 wk post-weaning (d 63 of age; n = 8 PNHS, n = 8 PNTN). From birth onward, all calves were actively cooled and managed under the same conditions. At birth, 682,898 differentially methylated cytosines (DMC) were identified genome-wide. Principal component analysis using 55,304 DMC located in genes expressed in blood cells revealed a clear clustering by prenatal treatment. However, at weaning, clear clustering by treatment was no longer observed using 23,977 treatment-associated DMC in blood-expressed genes, despite 97,289 DMC persisting genome-wide from birth to weaning. Immune cell deconvolution showed only minor differences in granulocytes (d 0) and CD4/CD8⁺ T cell proportions (d 63), suggesting methylation changes were not driven by cell-type composition. Gene ontology analysis identified more than 30 enriched pathways at each time point, mainly related to immunity, inflammation, and metabolism. Several differentially methylated genes (e.g., NMI, DUSP6, CD38, VPS37B) were common to both time points, suggesting persistent programming. Despite attenuation of global differences at weaning, a subset of 500 highly discriminant DMC remained informative for treatment separation. Blood-based methylation signatures could serve as early biomarkers of prenatal heat stress exposure and guide management and selection strategies to improve calf resilience.