| In order to meet the nutritional requirements of high-yielding dairy cows,the high-concentrate diet fed to dairy cows is often prone to cause subacute rumen acidosis(SARA),decreased rumen pH,increased release of endotoxin(also known as Lipopolysaccharide,LPS),and weakened lactation capacity of dairy cows.Epigenetics is a science that studies the changes in gene expression caused by changes in DNA or chromatin morphology when DNA sequence remains unchanged.We first investigated the relationship between LPS induced decreased lactation ability of dairy cows and DNA methylation and histone acetylation,providing a reference for studying the reduced lactation ability of dairy cow mammary cells caused by LPS from the perspective of epigenetics.Experiment 1: MAC-T cells were identified by DNA barcoding,immunofluorescence assay and cell growth activity assay.The results showed that the primers designed according to the specific genes of mitochondria of cattle were amplified,and the only species of BLAST alignment in NCBI database was cattle.The presence of keratin 18 and ?-casein antigens was detected by immunofluorescence assay,indicating that the cells were epithelial and mammary cells.The cell growth curve was S-shaped.In conclusion,the cells were bovine mammary epithelial cells with great growth activity,which can meet the requirements of subsequent experiments.Experiment 2: After the MAC-T cells were treated with LPS at doses of 0,1,10,100,and 1000 EU/mL for 48 hours,Methylated DNA immunoprecipitation sequencing(MeDIP-Seq)and Massarray techniques were used to determine the methylation levels of genome and lactation related genes,and the mRNA expression level of the methylation results of lactation related genes was verified by quantitative PCR(Q-PCR)experiment,to explore the relationship between DNA methylation and mRNA expression.The results showed that as the dose of LPS gradually increased,the level of genome methylation and the number of genes methylated in the genome both showed a trend of first increase and then decrease,and the genes functional element such as genebody,upstream2 k,downstream2k and CDS also showed the same trend.The number of methylated genes in lipid metabolism,amino acid metabolism and carbohydrate metabolism pathways increased first and then decreased with the increase of LPS dose,while the number of methylated genes in immune pathways decreased with the increase of LPS dose.Compared with the control group,the number of genes with increased methylation in lipid metabolism and amino acid metabolism pathway at LPS concentration of 1 and 10 EU/mL was more than that in LPS treatment of 100 and 1000 EU/mL.while the number of genes with decreased methylation showed an opposite trend.Compared with the control group,the number of genes with significant decrease in methylation in 100 and 1000 EU/mL LPS treatment group was higher than that in 1 and 10 EU/mL LPS treatment group.Massarray detection of lactation-related gene methylation level showed that acetyl-CoA carboxylase alpha(ACACA),acyl-CoA synthetase short-chain family member 2(ACSS2)and ribosomal protein S6 kinase 1(S6K1)gene methylation increased first and then decreased with the increase of LPS concentration,and reached the highest level at 10 EU/mL,and ACSS2,ACACA and S6K1 was significantly higher than the control group(P<0.05),fatty acid synthase(FASN)gene methylation level was basically not affected by LPS concentration.At 10 EU/mL,the mRNA expression levels of ACACA,ACSS2 and S6K1 genes were significantly lower than the control group(P<0.05),and FASN showed no difference from the control group,so the mRNA expression and DNA methylation showed a high correlation.Experiment 3: The acetylation levels of histone H3 and H4 were detected after the cells were treated with 0,1,10,100 and 1000 EU/mL LPS for 48 hours,and the activity of histone deacetylase(HDAC)was detected.According to the detection results,100 EU/mL LPS concentration was selected for QPCR experiment to detect the mRNA expression of lactation related genes,and histone deacetylase inhibitor sodium butyrate(SB)was added to study its effect on lactation.The results showed that the acetylation level of histone H3 decreased after LPS treatment at different concentrations except for 10 EU/mL LPS(P<0.05).Histone H4 acetylation showed no significant difference among the groups.HDAC increased significantly with the increase of LPS dose(P<0.05).QPCR results showed that the expression of ACACA,FASN,acyl-CoA synthetase long-chain family member 1(ACSL1)and S6K1 genes in LPS group was lower than that in control group,and the expression of ACACA,FASN and S6K1 genes in LPS group was significantly lower than the control group(P<0.05).After adding SB,the expression levels of ACACA,ACSL1,ACSS2,S6K1,signal transducer and activator of transcription 5A(STAT5A),casein alpha s1(CSN1S1),casein beta(CSN2)and casein kappa(CSN3)genes were all significantly higher than control group and LPS group(P<0.01).It shows that SB has a good effect on improving lactation performance.In this experiment,it was found that low dose of LPS would increase the methylation level of the genome of cow mammary epithelial cells,while high dose of LPS would reduce the methylation level.LPS can increase the methylation level of lactation related genes and decrease the mRNA expression.LPS can also enhance the activity of HDAC,reduce H3 acetylation level,reduce the expression of lactation related genes,and SB can significantly improve the expression of lactation related genes. |
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