Project description:DNA methylation is an essential epigenetic mechanism involved in cell differentiation, proliferation, apoptosis, and metabolism. However, the DNA methylation changes of related genes during early pregnancy in dairy cows remain unclear . The purpose of this study was to study DNA methylation-related changes in early pregnancy cows and construct gene pools associated with pregnancy. In this study, we used peripheral blood samples from 15 healthy, productive and similar cows at 30 days post-breeding (Pre30) and 10 non-pregnant cows (Ctrl) and association analysis was performed using Reduced Representation Bisulfite Sequencing (RRBS) and transcriptomic sequencing methods. Targeted bisulfite sequencing (TBS) and Quantitative Real-time PCR (RT-qPCR) method are used to verify the Methylation differential genes. Our findings showed that DNA methylation majorly occurred in the cytosine-guanine (CG) range, and the amount of hypomethylation was higher than that of hypermethylation in 3Utrs, introns and promoters. A total of 76530 differentially methylated sites (DMCs) and 16411 differentially methylated regions (DMRs) were identified. RNA-seq identified 8858 differentially expressed genes (DEGs), with 4499 down-DEGs and 4359 up-DEGs. However, there were 44 significant differentially expressed genes, including 25 up-DEGs and 19 down-DEGs. DMC gene ontology (GO) analysis revealed significant enrichment in developmental process, cell differentitaion and nevous system development in biological processes (BP)；Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated significant enrichment in cancer overview and cellular and Gastric cancer and Immune disease signaling pathways. DMRs GO analysis highlighted significant enrichment in the developmental process, cell differentiation and nervous system development; KEGG the results showed that significant enrichment in Signaling pathways regulating pluripotency of stem cells, Systemic lupus erythematosus，Transcriptional misregulation in cancer，Spliceosome，Herpes simplex virus 1 infection, Maturity onset diabetes of the young. DEG GO analysis results show that the main enrichment in BP differentiation of bone marrow cells, red blood cell differentiation and erythrocyte steady-state and anion during transportation and transshipment; KEGG analysis mainly concentrated in retinol metabolism, purine metabolism, cAMP signaling pathway and Gap junction pathway. Through a comprehensive analysis of RRBS and transcriptomics, 12 genes, including HBB, HBA 1, GPR 4, CPXM 2, SLC20A2, AFAP 1 and DOK 7, were different both in the methylome and the transcriptome. Early pregnancy changes in DNA methylation were significant and mainly down-regulated. These results can be the epigenetic changes in early pregnancy for cows provide new insights.

Project description:DNA methylation is an essential epigenetic mechanism involved in cell differentiation, proliferation, apoptosis, and metabolism. However, the DNA methylation changes of related genes during early pregnancy in dairy cows remain unclear . The purpose of this study was to study DNA methylation-related changes in early pregnancy cows and construct gene pools associated with pregnancy. In this study, we used peripheral blood samples from 15 healthy, productive and similar cows at 30 days post-breeding (Pre30) and 10 non-pregnant cows (Ctrl) and association analysis was performed using Reduced Representation Bisulfite Sequencing (RRBS) and transcriptomic sequencing methods. Targeted bisulfite sequencing (TBS) and Quantitative Real-time PCR (RT-qPCR) method are used to verify the Methylation differential genes. Our findings showed that DNA methylation majorly occurred in the cytosine-guanine (CG) range, and the amount of hypomethylation was higher than that of hypermethylation in 3Utrs, introns and promoters. A total of 76530 differentially methylated sites (DMCs) and 16411 differentially methylated regions (DMRs) were identified. RNA-seq identified 8858 differentially expressed genes (DEGs), with 4499 down-DEGs and 4359 up-DEGs. However, there were 44 significant differentially expressed genes, including 25 up-DEGs and 19 down-DEGs. DMC gene ontology (GO) analysis revealed significant enrichment in developmental process, cell differentitaion and nevous system development in biological processes (BP);Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated significant enrichment in cancer overview and cellular and Gastric cancer and Immune disease signaling pathways. DMRs GO analysis highlighted significant enrichment in the developmental process, cell differentiation and nervous system development; KEGG the results showed that significant enrichment in Signaling pathways regulating pluripotency of stem cells, Systemic lupus erythematosus，Transcriptional misregulation in cancer，Spliceosome，Herpes simplex virus 1 infection, Maturity onset diabetes of the young. DEG GO analysis results show that the main enrichment in BP differentiation of bone marrow cells, red blood cell differentiation and erythrocyte steady-state and anion during transportation and transshipment; KEGG analysis mainly concentrated in retinol metabolism, purine metabolism, cAMP signaling pathway and Gap junction pathway. Through a comprehensive analysis of RRBS and transcriptomics, 12 genes, including HBB, HBA 1, GPR 4, CPXM 2, SLC20A2, AFAP 1 and DOK 7, were different both in the methylome and the transcriptome. Early pregnancy changes in DNA methylation were significant and mainly down-regulated. These results can be the epigenetic changes in early pregnancy for cows provide new insights.

Project description:In dairy cows, administration of high dosages of niacin (NA) was found to cause anti-lipolytic effects, which are mediated by the NA receptor hydroxyl-carboxylic acid receptor 2 (HCAR2) in white adipose tissue (WAT), and thereby to an altered hepatic lipid metabolism. However, almost no attention has been paid to possible direct effects of NA in cattle liver, despite showing that HCAR2 is expressed also in the liver of cattle and is even more abundant than in WAT. Due to this, we hypothesized that feeding of rumen-protected NA to dairy cows influences critical metabolic and/or signaling pathways in the liver through inducing changes in the hepatic transcriptome. In order to identify these pathways, we applied genome-wide transcript profiling in liver biopsies obtained at 1 wk postpartum (p.p.) from dairy cows of a recent study (Zeitz et al., 2018) which were fed a total mixed ration without (control group) or with rumen-protected NA from 21 d before calving until 3 wk p.p. Hepatic transcript profiling revealed that a total of 487 transcripts were differentially expressed [filter criteria fold change (FC) > 1.2 or FC < -1.2 and P < 0.05] in the liver at 1 wk p.p. between cows fed NA and control cows. Substantially more transcripts were down-regulated (n = 338), while only 149 transcripts were up-regulated by NA in the liver of cows. Gene set enrichment analysis (GSEA) for the up-regulated transcripts revealed that the most enriched gene ontology (GO) biological process terms were exclusively related to immune processes, such as leukocyte differentiation, immune system process, leukocyte differentiation, activation of immune response and acute inflammatory response. In line with this, the plasma concentration of the acute phase protein haptoglobin tended to be increased in dairy cows fed rumen-protected NA compared to control cows (P < 0.1). GSEA of the down-regulated transcripts showed that the most enriched biological process terms were related to metabolic processes, such as cellular metabolic process, small molecule metabolic process, lipid catabolic process, organic cyclic compound metabolic process, small molecule biosynthetic process and cellular lipid catabolic process. In conclusion, hepatic transcriptome analysis shows that rumen-protected NA induces genes which are involved mainly in immune processes including acute phase response and stress response in dairy cows at wk 1 p.p. These findings indicate that supplementation of rumen-protected NA to dairy cows in the periparturient period may induce or amplify the systemic inflammation-like condition which is typically observed in the liver of high-yielding dairy cows in the p.p. period.

Project description:Over-conditioning is a risk factor for upregulated pre- and postpartum fat mobilization. Therefore, we hypothesized that over-conditioning at the end of pregnancy leads to the accumulation of lipids in the liver and modifications of the hepatic gene expression pattern. The aim of this study was to evaluate the effect of over- versus normal-conditioning on the hepatic transcriptomic profile of dairy cows at the end of pregnancy. Ten dry multiparous Holstein cows were euthanized two weeks before expected calving. Body condition score (BCS) and backfat thickness (BFT) were evaluated, and blood samples for nonesterified fatty acids (NEFA) were taken before euthanasia. After euthanasia, liver biopsy samples were collected for further assessment of total lipids and RNA sequencing. Five cows were classified as normal-conditioned (BCS = 2.5–3.5) and five as over-conditioned (BCS = 3.75–5). Regression models confirmed that normal-conditioned cows had lower BCS [3.17 ± 0.10 (least square mean ± SE)], BFT (1.29 ± 0.29 cm), and serum NEFA (0.16 ± 0.04 mmol/L) in comparison to over-conditioned cows (4.35 ± 0.21, 3.14 ± 0.43 cm, and 0.38 ± 0.07 mmol/L for BCS, BFT, and NEFA, respectively). Total liver lipid percentage tended to be lower in normal- than over-conditioned cows (4.63 ± 0.40% and 6.06 ± 0.44%, respectively). In comparison to the mean liver lipid percentage of the normal and over conditioned cows, one over-conditioned cow had a relatively low (5.21%) and one normal-conditioned cow had a relatively high (6.07%) liver lipid percentage. Differentially expressed genes (DEGs) analysis (edgeR quasi-likelihood method) showed that normal-conditioned presented two upregulated and five downregulated genes in comparison to over-conditioned cows. Linear discriminant analysis effects size revealed 23 DEG between normal- versus over-conditioned cows. Notably, the liver of normal-conditioned cows had upregulated genes associated with liver functionality (albumin, selenoprotein P, and insulin-like growth factor binding protein 1 and 2). On the other hand, over-conditioned cows had upregulated genes associated with the acute phase response (complement C3, hemopexin, and lipopolysaccharide-binding protein). High basal lipolysis in over-conditioned cows at the end of pregnancy increases liver lipid content and alters the hepatic gene expression pattern to a pro-inflammatory state.

Project description:Co-ordinated regulation of endometrial gene expression is essential for successful pregnancy establishment. A non-receptive uterine environment may be a key contributor to pregnancy loss, as the majority of pregnancy losses occur prior to embryo implantation. DNA methylation has been highlighted as a potential contributor in regulating early pregnancy events in the uterus. It was hypothesized that DNA methylation regulates expression of key genes in the uterus during pregnancy. To gain support for this hypothesis the correlation between DNA methylation and gene expression was tested. Endometrial samples from fertile and sub-fertile dairy cow strains were obtained at day 17 of pregnancy or the reproductive cycle. Microarrays were used to characterize genome-wide DNA methylation profiles and data compared with transcription profiles which have been previously reported. 39% of DNA methylation probes assayed mapped to RefSeq genes with transcription measurements. The 1,000 most significant correlations were used for subsequent analysis. Of these, 52% percent were negatively correlated with gene expression. When this gene list was compared with previously reported gene expression studies on the same tissues, 42% were differentially expressed when comparing pregnant and cycling animals and 11% were differentially expressed comparing pregnant fertile and sub-fertile animals. DNA methylation status was correlated with gene expression in several pathways implicated in early pregnancy events. Although these data do not provide direct evidence of a causative association between DNA methylation and gene expression, this study provides critical support for an effect of DNA methylation in early pregnancy events and highlights candidate genes for future studies. The estrous cycles of 24 lactating dairy cows were synchronized (at 58.8 (SEM 3.77) and 60.2 (SEM 1.51) days post calving in dairy cows of sub-fertile and fertile strains, respectively) and 14 received a single embryo transferred on day 7 of the estrous cycle. Animals were slaughtered at day 17 of the reproductive cycle and endometrial tissues (both caruncular and intercaruncular) were sampled. Selection criteria for the study included strain and calving date, and health postcalving was an exclusion criterion (cows with severe uterine infections or mastitis were excluded before being enrolled in the embryo transfer round). Cows in each strain were matched for calving number and age. A total of 10 cycling and 12 pregnant animals enrolled in the study were utilized, due to the associated costs of slaughtering the cows. These animals represented fertile (six pregnant and five cycling Holstein-Friesian cows with New Zealand ancestry/M-bM-^IM-$30% North American genetics, n=11, NZ) and sub-fertile (six pregnant and five cycling Holstein-Friesian cows with >87% North American ancestry, n=11, NA) phenotypes of Holstein-Friesian dairy cows

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Characterization of DNA methylation and transcriptomics during early pregnancy in dairy cows [RNA-seq]

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