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Background <p>This study investigated DNA methylation-related changes in cows during early pregnancy and constructed gene pools associated with pregnancy.</p> Methods <p>We used peripheral blood samples from six healthy, productive, and similar cows 30 days post-insemination (Pre 30) and six non-pregnant cows (Ctrl), and performed association analysis using Reduced Representation Bisulfite Sequencing (RRBS) and transcriptomic sequencing methods. Targeted bisulfite sequencing (TBS) and quantitative real-time PCR (qRT-PCR) methods were used to identify differentially methylated genes.</p> Results <p>An obvious trend of hypomethylation was observed within the whole genome of Pre 30 dairy cows. A total of 76,530 differentially methylated sites (DMCs) were identified, comprising 32,886 hypermethylated sites and 43,644 hypomethylated sites. Additionally, there were 16,411 differentially methylated regions (DMRs), including 8,455 hypomethylated regions and 7,956 hypermethylated regions. RNA-Seq identified 44 significantly differentially expressed genes (DEGs), including 25 up and 19 down-regulated DEGs. Gene ontology (GO) analysis revealed significant enrichment in the developmental process, cell differentiation, and nervous system development in biological processes; Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated significant enrichment in cancer-related pathways, such as cancer overview and cellular and gastric cancer and immune disease signaling pathways. We validated the methylation patterns of 17 genes and showed that the overall trend was hypomethylation, consistent with sequencing results of RRBS. qRT-PCR showed that <i>HBB</i>,<i> HBA1</i> and <i>GPR4</i> were significantly increased in the Pre 30 group (<i>P</i> &lt; 0.05), whereas <i>DOK7</i> and <i>ISG15</i> were significantly decreased (<i>P</i> &lt; 0.05), consistent with transcriptome sequencing.</p> Conclusions <p>Our data encompass a diverse range of transcriptional dynamics and DNA methylation alterations observed during the early stages of pregnancy in dairy cows, offering novel insights for subsequent investigations into epigenetic changes occurring during this critical period.</p>

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Integrated analysis of DNA methylation and gene expression in dairy cows during early pregnancy

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月12日

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  • Shiyi Li,
  • Lanying Gu,
  • Qinchuan Zhang,
  • Wenyu Fan,
  • Jinliang Sheng,
  • Sihai Ou,
  • Yanbing Zhang,
  • Yanming Sun

摘要

Background

This study investigated DNA methylation-related changes in cows during early pregnancy and constructed gene pools associated with pregnancy.

Methods

We used peripheral blood samples from six healthy, productive, and similar cows 30 days post-insemination (Pre 30) and six non-pregnant cows (Ctrl), and performed association analysis using Reduced Representation Bisulfite Sequencing (RRBS) and transcriptomic sequencing methods. Targeted bisulfite sequencing (TBS) and quantitative real-time PCR (qRT-PCR) methods were used to identify differentially methylated genes.

Results

An obvious trend of hypomethylation was observed within the whole genome of Pre 30 dairy cows. A total of 76,530 differentially methylated sites (DMCs) were identified, comprising 32,886 hypermethylated sites and 43,644 hypomethylated sites. Additionally, there were 16,411 differentially methylated regions (DMRs), including 8,455 hypomethylated regions and 7,956 hypermethylated regions. RNA-Seq identified 44 significantly differentially expressed genes (DEGs), including 25 up and 19 down-regulated DEGs. Gene ontology (GO) analysis revealed significant enrichment in the developmental process, cell differentiation, and nervous system development in biological processes; Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated significant enrichment in cancer-related pathways, such as cancer overview and cellular and gastric cancer and immune disease signaling pathways. We validated the methylation patterns of 17 genes and showed that the overall trend was hypomethylation, consistent with sequencing results of RRBS. qRT-PCR showed that HBB, HBA1 and GPR4 were significantly increased in the Pre 30 group (P < 0.05), whereas DOK7 and ISG15 were significantly decreased (P < 0.05), consistent with transcriptome sequencing.

Conclusions

Our data encompass a diverse range of transcriptional dynamics and DNA methylation alterations observed during the early stages of pregnancy in dairy cows, offering novel insights for subsequent investigations into epigenetic changes occurring during this critical period.