Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are the most common pathogens of mastitis, and S. aureus generally causes subclinical mastitis which is more persistent and resistant to treatment. Peptidoglycan (PGN) and lipoteichoic acid (LTA) are cell wall components of S. aureus. Although the roles of PGN and LTA in causing inflammation are well studied, the epigenetic mechanisms of the effects of PGN and LTA on the inflammation and lactation remain poorly understood. This study characterized the gene expression profiling by RNA sequencing and investigated DNA methylation and histone acetylation in relation to inflammation and lactation in the immortalized bovine mammary epithelial cell line (MAC-T). The cells were cultured for 24 h with neither PGN nor LTA (CON), PGN (30 μg/mL), LTA (30 μg/mL), and PGN (30 μg/mL) + LTA (30 μg/mL), respectively. The number of differentially expressed genes (DEGs) and the expression of proinflammatory factors including interleukin (IL)-1β, IL-6, IL-8, chemokine (C-X-C motif) ligand (CXCL)1, and CXCL6 of the treatments increased in the following order: CON < PGN < LTA < PGN + LTA, and the DEGs mainly enriched on the cytokine-cytokine receptor interaction and chemokine signaling pathway. LTA and PGN + LTA induced hypomethylation of global DNA by suppressing DNA methyltransferase (DNMT) activity. PGN and LTA, alone or combined, decreased the mRNA expression of casein genes (CSN1S1, CSN2, and CSN3) and the expression of two caseins (CSN2 and CSN3), and reduced histone H3 acetylation by suppressing histone acetyltransferase (HAT) activity and promoting histone deacetylase (HDAC) activity. Collectively, this study revealed that PGN and LTA induced inflammation probably due to decreasing DNA methylation through regulating DNMT activity, and decreased lactation possibly through reducing histone H3 acetylation by regulating HAT and HDAC activity in bovine mammary epithelial cells.

In a prior study, we observed that cows with a 0-d dry period had greater energy balance and lower milk production compared with cows with a 30- or 60-d dry period in early lactation. The objective of the current study was to evaluate the influence of dry period length on metabolic status and hepatic gene expression in cows fed a lipogenic or glucogenic diet in early lactation. Holstein-Friesian dairy cows (n=167) were assigned randomly to 3×2 factorial design with 3 dry period lengths (n=56, 55, and 56 for 0-, 30-, and 60-d dry, respectively) and 2 early lactation diets (n=84 and 83 for glucogenic and lipogenic diet, respectively). Cows were fed a glucogenic or lipogenic diet from 10d before the expected calving date and onward. The main ingredient for a glucogenic concentrate was corn, and the main ingredients for a lipogenic concentrate were sugar beet pulp, palm kernel, and rumen-protected palm oil. Blood was sampled weekly from 95 cows from wk 3 precalving to wk 8 postcalving. Liver samples were collected from 76 cows in wk -2, 2, and 4 relative to calving. Liver samples were analyzed for triacylglycerol concentrations and mRNA expression of 12 candidate genes. Precalving, cows with a 0-d dry period had greater plasma β-hydroxybutyrate, urea, and insulin concentrations compared with cows with a 30- or 60-d dry period. Postcalving, cows with a 0-d dry period had lower liver triacylglycerol and plasma nonesterified fatty acids concentrations (0.20, 0.32, and 0.36mmol/L for 0-, 30-, and 60-d dry period, respectively), greater plasma glucose, insulin-like growth factor-I, and insulin (24.38, 14.02, and 11.08µIU/mL for 0-, 30-, and 60-d dry period, respectively) concentrations, and lower hepatic mRNA expression of pyruvate carboxylase, compared with cows with a 30- or 60-d dry period. Plasma urea and β-hydroxybutyrate concentrations were greater in cows fed a lipogenic diet compared with cows fed a glucogenic diet. In conclusion, cows with a 0-d dry period had an improved metabolic status in early lactation, indicated by lower plasma concentrations of nonesterified fatty acids, greater plasma concentrations of glucose, insulin-like growth factor-I, and insulin, and lower mRNA expression of pyruvate carboxylase in the liver, compared with cows with a 30- or 60-d dry period. Independent of dry period length, the glucogenic diet also improved the metabolic status compared with the lipogenic diet.

In practical dairy production, cows are frequently subjected to inflammatory diseases, such as high-grain diet-induced subacute ruminal acidosis (SARA) as well as mastitis and metritis. Under the circumstances, lipopolysaccharide (LPS) induces oxidative stress within the cow and in the mammary epithelial cells. It has implications in practical production to alleviate oxidative stress and to optimize the lactational function of the mammary epithelial cells. This study thus aimed to investigate the antioxidative effects of dandelion aqueous extract (DAE) on LPS-induced oxidative stress and the mechanism of DAE as an antioxidant to alleviate oxidative stress through the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in the bovine mammary epithelial cell line MAC-T cells. The cells were cultured for 48 h in six treatments including control (without LPS and DAE), LPS (100 ng/mL), DAE10 (100 ng/mL LPS and 10 μg/mL DAE), DAE50 (100 ng/mL LPS and 50 μg/mL DAE), DAE100 (100 ng/mL LPS and 100 μg/mL DAE), and DAE200 (100 ng/mL LPS and 200 μg/mL DAE), respectively. The results showed that cell viability was reduced by LPS, and the adverse effect of LPS was suppressed with the supplementation of DAE. Lipopolysaccharide-induced oxidative stress through enhancing reactive oxygen species (ROS) production, resulted in increases in oxidative damage marker concentrations, while 10 and 50 μg/mL DAE alleviated the LPS-induced oxidative stress via scavenging cellular ROS and improving antioxidant enzyme activity. The upregulation of antioxidative gene expression in DAE treatments was promoted through activating the Nrf2 signaling pathway, with DAE at a concentration of 50 μg/mL exhibiting the highest effect. Overall, DAE acted as an effective antioxidant to inhibit LPS-induced oxidative stress and as a potential inducer of the Nrf2 signaling pathway.

Heat stress (HS) is one of the most serious factors to negatively affect the lactation performance of dairy cows. Bovine mammary epithelial cells are important for lactation. It was demonstrated that HS decreases the lactation performance of dairy cows, partly through altering gene expression within bovine mammary epithelial tissue. However, the cellular metabolism mechanisms under HS remains largely unknown. The objective of this study was to determine whether HS induced changes in intracellular metabolites and gene transcription related to amino acid metabolism, amino acid transportation and the mTOR signaling pathway. Immortalized bovine mammary epithelial cell lines (MAC-T cells, n = 5 replicates/treatment) were incubated for 12 h at 37 °C (Control group) and 42 °C (HS group). Relative to the control group, HS led to a greater mRNA expression of heat shock protein genes HSF1, HSPB8, HSPA5, HSP90AB1 and HSPA1A. Compared with the control group, metabolomics using liquid chromatography tandem–mass spectrometry identified 417 differential metabolites with p < 0.05 and a variable importance in projection (VIP) score >1.0 in the HS group. HS resulted in significant changes to the intracellular amino acid metabolism of glutathione, phenylalanine, tyrosine, tryptophan, valine, leucine, isoleucine, arginine, proline, cysteine, methionine, alanine, aspartate and glutamate. HS led to a greater mRNA expression of the amino acid transporter genes SLC43A1, SLC38A9, SLC36A1, and SLC3A2 but a lower mRNA expression of SLC7A5 and SLC38A2. Additionally, HS influenced the expression of genes associated with the mTOR signaling pathway and significantly upregulated the mRNA expression of mTOR, AKT, RHEB, eIF4E and eEF2K but decreased the mRNA expression of TSC1, TSC2 and eEF2 relative to the control group. Compared with the control group, HS also led to greater mRNA expression of the CSN1S2 gene. Overall, our study indicates that bovine mammary epithelial cells may have the ability to resist HS damage and continue milk protein synthesis partly through enhanced intracellular amino acid absorption and metabolism and by activating the mTOR signaling pathway during HS.

Mastitis is usually caused by a variety of pathogenic bacteria that include both Gram-positive and Gram-negative bacteria. Lipopolysaccharide (LPS) is the pathogen-associated molecular pattern (PAMP) of Gram-negative bacteria, and peptidoglycan (PGN) and lipoteichoic acid (LTA) are those of Gram-positive bacteria. The effects of LPS, PGN and/or LTA on inflammatory response and lactation in bovine mammary epithelial cells (BMECs) are well studied, but the epigenetic mechanisms of their effects received less attention. Furthermore, since the three PAMPs are often simultaneously present in the udder of cows with mastitis, it has implications in practice to study their additive effects. The results show that co-stimulation of bovine mammary epithelial cells with PGN, LTA, and LPS induced a higher number of differentially expressed genes (DEGs) and greater expressions of inflammatory factors including interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-α (TNF-α), chemokine (C-X-C motif) ligand (CXCL)1, and CXCL6. In addition, co-stimulation further increased DNA hypomethylation compared with sole LPS stimulation. Co-stimulation greatly decreased casein expression but did not further decrease histone acetylation levels and affect the activity of histone acetyltransferase (HAT) and histone deacetylase (HDAC), compared with sole LPS stimulation. Collectively, this study demonstrated that PGN, LTA, and LPS had an additive effect on inducing transcriptome changes and inflammatory responses in BMECs, probably through inducing a greater decrease in DNA methylation. Co-stimulation with PGN, LTA, and LPS decreased casein expression to a greater degree, but it might not be linked to histone acetylation and HAT and HDAC activity.