Although high-concentrate diet feeding can temporarily increase milk production, it can cause a series of metabolic diseases, such as subacute ruminal acidosis (SARA) and milk fat depression. The main purpose of this experiment was to study the effects of a high-concentrate diet on the inflammatory response, oxidative stress, and milk fat synthesis in the mammary gland of dairy cows. Twelve Holstein cows equipped with rumen fistulas were randomly divided into 2 groups, each with 6 cows, fed a low-concentrate diet (LC) and a high-concentrate diet (HC). On d 20 and 21 of the experiment, rumen fluid was collected to measure pH, and milk samples were collected for milk component analysis and lipopolysaccharide (LPS) concentration testing. On d 21, mammary vein blood was collected to detect the LPS concentration. At the end of the 21-d experimental period, mammary gland tissue was collected, and the expression of inflammatory response-, oxidative stress-, and milk fat synthesis-related genes and proteins in the mammary gland was analyzed by real-time quantitative PCR and western blot. The pH of rumen fluid in the HC group was significantly lower than that in the LC group, and the pH of 2 time points in the HC group was lower than 5.6, indicating that a high-concentrate diet induced SARA. The LPS concentration of the peripheral blood in HC group increased significantly compared with that in the LC group. For the inflammatory response, the proinflammatory cytokines (IL-6 and IL-1α) and innate immune factors (lingual antimicrobial peptide and tracheal antimicrobial peptide) in the mammary gland of the HC group were significantly increased, and the TLR4-NF-κB signaling pathway was activated. For oxidative stress, after HC diet feeding, the content of malondialdehyde in mammary vein blood and mammary gland tissue increased, the content of glutathione in mammary vein blood decreased, the activity of superoxide dismutase and the total antioxidant capacity in mammary gland tissue and mammary vein blood decreased, and the expression of antioxidant enzymes and antioxidant transcription factor nuclear factor, erythroid 2 like 2 (NFE2L2) in mammary gland decreased. For milk fat metabolism, HC diet feeding reduced the milk fat content in milk samples and the triacylglycerol content in the mammary gland and inhibited the expression of de novo synthase (ACACA and FASN), long-chain fatty acid converting enzymes (ACSL1 and SCD), fatty acid transporters (CD36, FATP, FABP3, and LPL), triacylglycerol synthase (AGPAT6, DGAT1, and LPIN1), lipid droplet releasing enzyme (PLIN1), and transcription factors sterol regulatory element binding protein (SREBP1) and peroxisome proliferator activated receptor gamma (PPARG). In summary, a HC diet can induce SARA with increased concentration of LPS in the peripheral vein, stimulate inflammatory reactions and oxidative stress, and inhibit milk fat synthesis in the mammary gland of dairy cows.

Long-term feeding of a high-concentrate diet can induce subacute ruminal acidosis (SARA) in ruminants, which further leads to systemic inflammatory response. However, few studies have examined the effects of feeding a high-concentrate diet on the hindgut of ruminants. The purpose of this study was to investigate the effects of a high-concentrate diet on the composition of gut microbiota in colonic contents, inflammatory response, and barrier damage in the colon tissue of ruminants. A total of 12 healthy multiparous lactating Hu sheep were randomly allotted into the following 2 groups: a high-concentrate (HC) group (concentrate:forage = 7:3) and a low-concentrate (LC) group (concentrate:forage = 3:7). All sheep were fitted with ruminal fistulas. The formal feeding experiment lasted for 8 wk. After the feeding experiment, rumen fluid, portal vein blood, hepatic vein blood, colonic contents, and colon tissue samples were collected. The results showed that feeding the HC diet induced SARA in Hu sheep and significantly reduced pH in the colonic contents. The abundances of Firmicutes, Verrucomicrobiota, and Actinobacteriota decreased significantly, whereas those of Bacteroidota, Spirochaetota, and Fibrobacterota significantly increased in colonic contents. At the genus level, the relative abundances of 29 genera were significantly altered depending on the different type of diets. Analysis of the 10 bacterial genera with high relative abundance revealed that feeding the HC diet significantly reduced the abundance of UCG-005, Christensenellaceae R-7 group, UCG-010-norank, Monoglobus, [Eubacterium] coprostanoligenes group_norank, and Alistipes, whereas the abundances of Rikenellaceae RC9 gut group, Treponema, Bacteroides, and Prevotella increased. Compared with the LC group, feeding the HC diet significantly increased the concentration of LPS in rumen fluid, portal vein blood, hepatic vein blood, and colonic contents, and significantly upregulated the mRNA expression levels of proinflammatory cytokines in colon tissue, including TNF-α, IL-1β, IL-6, and IL-8, indicating the occurrence of inflammatory response in the colon tissue. In addition, the structure of colonic epithelial cells was loose, the intercellular space became larger, epithelial cells were exfoliated, and the mRNA and protein abundances of ZO-1, occludin, claudin-1, claudin-3, and claudin-4 were significantly decreased in the HC group, which was consistent with the results of immunohistochemistry. Furthermore, feeding the HC diet increased the ratios of DNA methylation and chromatin compaction in the promoter regions of occludin and claudin-1, which in turn inhibited their transcriptional expression. Therefore, the present study demonstrated that feeding an HC diet induced SARA in Hu sheep, altered the composition and structure of the microbial community in the colonic contents, induced an inflammatory response, and disrupted the intestinal mucosal barrier in the colonic tissue.

Long-term high-concentrate diet (HCD) feeding can cause subacute ruminal acidosis in cows and subsequently trigger systemic inflammatory and immune responses. Therefore, we conducted the present study in which twelve lactating cows installed with ruminal fistula were randomly assigned to the HCD group (forage:concentrate = 4:6, n = 6) or the low-concentrate diet (LCD) group (forage:concentrate = 6:4, n = 6) and were fed for 20 weeks. Ruminal fluid, plasma and mammary gland tissue samples were collected at week 20 for analysing lipopolysaccharide (LPS), pro-inflammatory cytokines, and immune relevant gene expression. The aim of this study was to investigate the effect of rumen-derived LPS on lingual antimicrobial peptide (LAP) synthesis and immune responses in mammary glands of lactating cows fed a HCD. Compared with the LCD group, the ruminal pH was lower in the HCD group, while LPS concentrations in the rumen, lacteal artery and vein were higher. The expression of LAP, BNBD5, IL-1β, IL-6, IL-8, and TNF–α was enhanced in the HCD group. LAP protein expression was higher in the HCD group than that in the LCD group. The expression of nuclear factor kappa B (NF-кB) did not change, but was activated, as the amounts of phosphorylated NF-kB and phosphorylated inhibitory kBα increased in the HCD group compared with that in the LCD group. After long-term HCD feeding, rumen-derived LPS translocated to the blood stream, triggered inflammatory and immune responses and enhanced LAP synthesis via the NF-kB signalling pathway in mammary glands of lactating cows.

Lipopolysaccharide (LPS) is the dominating endotoxin of Gram-negative bacteria, which can cause mastitis. Bovine mammary epithelial cells (BMECs), as major components of the mammary gland, usually suffer LPS challenge. Cis-9, trans-11 conjugated linoleic acid (CLA) has been reported to have anti-inflammatory characteristics, while its anti-oxidative ability to maintain cellular homeostasis in BMECs under LPS challenge is limited. Therefore, we studied whether cis-9, trans-11 CLA can restore the disturbance of cellular homeostasis indicated by the redox status and autophagy level caused by LPS and have an effect on cellular function- milk fat metabolism. For oxidative stress, LPS challenge promoted the formation of reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) and decreased the concentration of glutathione. Anti-oxidative signaling regulated by transcription factor nuclear factor, erythroid 2 like 2 (Nrf2) was also depressed by LPS at the mRNA and protein level. However, cis-9, trans-11 CLA pretreatment downregulated the formation of ROS and TBARS and upregulated the expression of antioxidative enzymes. As a part of innate immunity, autophagy was also motivated by LPS challenge, while CLA decreased the autophagy level. LPS and H₂O₂ inhibited milk fat synthesis-related transcription factor sterol regulatory element binding protein (SREBP1), peroxisome proliferator activated receptor gamma (PPARG) and their downstream enzymes. Furthermore, 50 uM cis-9, trans-11 CLA promoted the mRNA and protein abundance of milk fat synthesis-related genes and lipid droplet formation in BMECs. In conclusion, LPS challenge disturbed the cellular homeostasis and depressed milk fat synthesis in BMECs; while cis-9, trans-11 CLA alleviated oxidative stress and decreased autophagy level, thus promoting milk fat synthesis, which offers a natural therapeutic strategy for mastitis.