Thiamine ameliorates metabolic disorders induced by a long-term high-concentrate diet and promotes rumen epithelial development in goats

Several studies have demonstrated that high-concentrate (HC) diet with thiamine supplementation can alleviate rumen epithelium inflammation and protecting the barrier function in goats. This study aimed to investigate the effects of dietary supply of thiamine on mitophagy and endoplasmic reticulum stress (ERS) during high-concentrate diet feeding. Twenty-four Boer goats (35.62 ± 2.4 kg) were used in this study, goats were subsequently assigned to 3 treatment groups (8 goats in each group) as follows: a low-concentrate diet (CON; concentrate: forage 30:70), a high-concentrate diet (HC; concentrate: forage 70:30) and high-concentrate diet with 200 mg thiamine/kg DMI (HCT; concentrate: forage 70:30) for 12 weeks. Compared with the HC group, the goats of HCT group had a markedly higher final weight, net weight gain, and average daily gain (ADG). The blood physiological and biochemical results showed that the HCT group expressed a lower the content of lymphocytes and leukocytes but a higher total protein and monocytes compared with that of HC group. The rumen epithelial mitochondrial membrane potential change (ΔΨm), relative mitochondrial DNA (mtDNA) content, adenosine triphosphate (ATP) level, together with the activities of the respiratory complexes I, III, and IV were markedly higher in the HCT group relative to HC ones. Relative to the HC group, the unc-51 like autophagy activating kinase 1 (ULK1), microtubule-associated protein 1 light chain 3 (LC3), autophagy-related 5 (ATG5), autophagy related 7 (ATG7), Beclin1, PTEN induced putative kinase 1 (PINK1), PERK (PKR-Endoplasmic Reticulum Kinase), activating transcription factor 4 (ATF4), activating transcription factor 6 (ATF6), X-box binding protein 1 (XBP1), heat shock 70 kDa protein 5 (HSPA5), DNA damage-inducible transcript 3 (DDIT3) and DNA damage-inducible transcript 4 (DDIT4) mRNA levels were decreased, but the breast cancer 1 (BRCA1), ataxia telangiectasia mutated (ATM), heat shock factor binding protein 1 (HSBP1) were increased in rumen epithelium of the HCT group. The results of transmission electron micrographs showed that the cell structure of the HCT group was higher integrity than that in the HC group, and the damage degree of mitochondria as well as endoplasmic reticulum being lower than the HC group. These results demonstrated that dietary thiamine could enhance rumen epithelial integrity by suppressing the responses of ERS and mitophagy during long-term HC diet feeding. HIGHLIGHTS This study is the first to demonstrate that the reduction of inflammatory response is related to the down-regulation of autophagy and endoplasmic reticulum stress in goats. Thiamine has good anti-inflammatory and anti-oxidation properties, which can be applied in intensive industry to reduce the negative effects caused by long-term high-concentrate diet.

We investigated the effect of sodium butyrate feeding on the disruption of ruminal epithelium barrier function in goats fed a high-concentrate diet. A total of 18 male Boer goats (live weight of 31.75 ± 1.35 kg, aged 1 year) were randomly assigned to three groups, which were fed a low-concentrate diet (LC), a high-concentrate diet (HC), or a high-concentrate diet with 1% sodium butyrate by weight (SH) for 9 weeks. We found that the pH of rumen fluid in the SH and LC groups was higher than that in the HC group. The activity of protein kinase C (PKC) kinase in the rumen epithelium was higher in the HC group than that in the LC and SH groups. The mRNA expression and phosphorylated protein levels of mitogen-activated protein kinases (MAPKs) in the rumen epithelium were lower in the SH and LC groups than those in the HC group. The DNA methylation rate of occludin was higher in the HC group than that in the SH and LC groups. The mRNA and protein expression of claudin-1, claudin-4, occludin, and zona occludin-1 was greater in the SH and LC groups than that in the HC group. In addition, sodium butyrate mitigated damage to the rumen epithelium caused by the HC diet. Together, our results suggest that the supply of sodium butyrate reverses the damage of rumen epithelium tight junction by inhibiting PKC and MAPK signaling pathways and is protective to the rumen epithelium during subacute rumen acidosis.

IntroductionLong-term feeding of a high-concentrate diet can induce subacute ruminal acidosis (SARA) and hindgut acidosis in ruminants. However, at present, most studies focus on reducing rumen injury by adjusting the feed formula, adding buffers, probiotics, or enzyme preparations, and few studies pay attention to hindgut health. Sophora alopecuroides alkaloids have extensive anti-inflammatory and antioxidant effects. The purpose of this experiment was to study the effects of adding total alkaloids of Sophora alopecuroides (TASA) to a high-concentrate diet on colon barrier function, antioxidation, and the microbial flora of lambs.Methods18 Dumont lambs (26.37 ± 2.29 kg) were divided into three diet groups: medium-concentrate diet (MC, concentrate ratio 50:50), high-concentrate diet (HC, concentrate ratio 70:30), and HC diet supplemented with 121 mg/kg TASA (HCT). At the end of the experimental period, colon contents and colon epithelium were collected. These samples were used to evaluate the colon barrier, antioxidant capacity, intestinal morphology, microbial composition and short-chain fatty acid (SCFA) concentration.ResultsThe results revealed that adding TASA to the HC diet increased claudin-1 protein expression (p < 0.01), decreased the MDA concentration, and increased Glutathione peroxidase (GSH-Px), Superoxide dismutase (SOD), and Total antioxidant capacity (T-AOC) activity in the colonic epithelium (p < 0.05). The concentration of propionate and lactate in colon contents in HC group increased significantly, while the pH decreased significantly (p < 0.05). The concentration of acetate, propionate and lactate in HCT group was significantly lower than that in HC group, the concentration of butyrate in HCT group was the highest (p < 0.05). Furthermore, there was a significant increase in Bacteroidetes and a decrease in Firmicutes in the HCT group (p < 0.01). Compared with the HC group, there was a notable increase in the butyrate-producing genera Faecalibacterium, Roseburia, Lachnospiraceae_NK4A136_group, and Butyrivibrio in the HCT group (p < 0.05 or p < 0.01). Additionally, the abundances of Prevotellaceae_UCG-003 in the MC and HCT groups were significantly greater (p < 0.05 or p < 0.01).ConclusionIn conclusion, supplementing the HC diet with TASA enhances colonic barrier and antioxidant functions, and alleviates HC diet-induced colonic damage by modulating the structure and abundance of the colonic microbiota.

The aim of the present study was to evaluate how long-term high-concentrate diet feeding affected rumen epithelium (RE) of dairy cows. So, 12 mid-lactating multiparous cows were divided into two groups randomly fed either with high-concentrate diet (HC, concentrate: forage = 6: 4) or low-concentrate diet (LC, concentrate: forage = 4:6) for 20 weeks. Remarkable upregulation of lipopolysaccharide (LPS) level and depress of pH in rumen fluid were induced by HC compared with LC group. mRNA abundance of interleukin-6 (IL-6), interleukin-8 (IL-8), C-C motif chemokine ligand 5 (CCL5), caspase-3, caspase-8, and caspase-9 were elevated in RE of HC group compared with LC group. Greater protein abundance of phosphorylated NF-κB p65, IL-6, and tumor necrosis factor α (TNF-α) was observed in RE of cows fed HC than that fed LC. Abundance of protein related to proapoptotic response (cytochrome c, BAX and caspase-3) in HC group was greater than that in LC group, while the abundance of anti-apoptotic factor protein (Bcl-2) was lower in HC group than LC group. Therefore, the present study demonstrated that long-term high-concentrate diet feeding upregulated LPS level in rumen fluid and induced the proinflammatory response in the rumen epithelium and apoptosis of rumen epithelial cells.

Simple SummaryIn order to increase the growth performance and make up for the lack of high-quality roughage, farmers are accustomed to adopting high-concentration diets during the fattening period of ruminants. The long-term consumption of high-concentration diets induces a series of nutritional metabolic diseases. In production practice, the major mutton sheep fattening region (e.g., Jiangsu, China) generally uses diets with a concentrate ratio of up to 80% to 90% during the fattening period, and reports of cases of nutritional metabolic diseases are also rare. This seems to indicate that sheep and goats are more adaptable to high-concentration diets than cattle. Our work shows that high-concentrate diets can significantly promote the digestion of nutrients; the liver enhances the adaptability of goats to high-concentration diets by regulating the expression of genes involved in nutrient metabolism and toxin clearance. Therefore, the liver plays a vital role in the adaptation of ruminants to high-concentration diets. The results here can lay the foundation for the rational application of high-concentration diets in production.The objective of this study was to determine the effect of high-concentrate diets on the blood parameters and liver transcriptome of goats. Eighteen goats were allocated into three dietary treatments: the high level of concentrate (HC) group, the medium level of concentrate (MC) group, and the low level of concentrate (LC) group. The blood parameters and pathological damage of the gastrointestinal tract and liver tissues were measured. In hepatic portal vein blood, HC showed higher LPS, VFAs, and LA; in jugular vein blood, no significant differences in LPS, VFAs, and LA were recorded among groups (p > 0.05). Compared to the LC and MC groups, the HC group showed significantly increased interleukin (IL)-1β, IL-10, TNF-α, and diamine oxidase in jugular vein blood (p < 0.05). Liver transcriptome analysis discovered a total of 1269 differentially expressed genes (DEGs) among the three groups and most of them came from the HC vs. LC group. There were 333 DEGs up-regulated and 608 down-regulated in the HC group compared to the LC group. The gene ontology enrichment analysis showed that these DEGs were mainly focused on the regulation of triacylglycerol catabolism, lipoprotein particle remodeling, and cholesterol transport. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the liver of the HC group enhanced the metabolism of nutrients such as VFAs through the activation of AMPK and other signaling pathways and enhanced the clearance and detoxification of LPS by activating the toll-like receptor signaling pathway. A high-concentrate diet (HCD) can significantly promote the digestion of nutrients; the liver enhances the adaptability of goats to an HCD by regulating the expression of genes involved in nutrient metabolism and toxin clearance.

High-concentrate diets are continually used in ruminants to meet the needs of milk yield, which can lead to the occurrence of subacute rumen acidosis in ruminants. This study investigated the protective effects of dietary thiamine supplementation on the damage of the ruminal epithelium barrier function in goats fed a high-concentrate diet. Twenty-four healthy Boer goats (live weight of 35.62 ± 2.4 kg; age, 1 year) were randomly assigned into three treatments, with eight goats in each treatment, consuming one of three diets: a low-concentrate diet (CON; concentrate/forage, 30:70), a high-concentrate diet (HC; concentrate/forage, 70:30), or a high-concentrate diet with 200 mg of thiamine/kg of dry matter intake (HCT; concentrate/forage, 70:30) for 12 weeks. The additional dose of thiamine was based on our previous study wherein thiamine ameliorates inflammation. Compared with HC treatment, the HCT treatment had markedly higher concentrations of glutathione, superoxide dismutase, and glutathione peroxidase and total antioxidant capacity (P < 0.05) in plasma and rumen epithelium. The results showed that the apoptosis index was lower (P < 0.05) in the HCT treatment than in that of the HC treatment. Compared with the HC treatment, permeability and the electrophysiology parameter short circuit current for ruminal epithelial tissue were significantly decreased (P < 0.05) in the HCT treatment. The immunohistochemical results showed that the expression distribution of tight junctions including claudin-1, claudin-4, occludin, and zonula occludin-1 (ZO-1) was greater (P < 0.05) in the HCT treatments than in the HC treatment. The mRNA expression in the rumen epithelium of ZO-1, occludin, claudin-1, B-cell lymphoma/leukemia 2, nuclear factor erythroid-2 related factor 2 (Nrf2), superoxide dismutase 2 (SOD2), glutathione peroxidase 1, and the phase II metabolizing enzymes quinone oxidoreductase and heme oxygenase in the HCT group was significantly increased in comparison with the HC diet treatment (P < 0.05), whereas the mRNA expression of caspase 3, caspase 8, caspase 9, bcl-2 associated X protein, lipopolysaccharide binding protein, toll-like receptor 4, nuclear factor kappa-B (NFκB), tumor necrosis factor alpha, interleukin-1β, interleukin, and tumor necrosis factor receptor-associated factor 6 decreased significantly in the HCT treatment (P < 0.05). Compared with the HC treatment, the HCT diet significantly increased the protein expression of ZO-1, occludin, claudin-1, NQO1, HO-1, SOD2, serine/threonine kinase, p-Akt, Nrf2, and p-Nrf2; conversely, the expression of NFκB-related proteins p65 and pp65 was significantly decreased (P < 0.05). In addition, thiamine relieved the damage on the ruminal epithelium caused by the HC diet. The results show that dietary thiamine supplementation improves the rumen epithelial barrier function by regulating Nrf2–NFκB signaling pathways during high-concentrate-diet feeding.

Increasing the proportion of concentrate in diets can effectively improve ruminant production, and is therefore widely used. However, high-concentrate diets (HCD) enriched with rapidly fermentable carbohydrates can accelerate the production of lactate and short-chain fatty acids (SCFAs). The accumulation of lactate and SCFAs in the rumen leads to a reduction in rumen fluid pH, potentially resulting in subacute rumen acidosis (SARA), which can decrease dry matter intake (DMI), induce local and systemic inflammation, and cause other negative impacts on the host. The substantial prevalence of SARA attributable to long-term HCD causes considerable economic losses, as it can decrease DMI by up to 20%. Understanding its mechanisms and pathogenesis is essential. The rumen epithelium (RE), which is in direct contact with rumen fluid, is an important tissue in the rumen due to its roles in absorption, transport, and barrier functions. The changes that occur in RE under HCD and the subsequent impacts of these changes are worth exploring. In the short term, HCD feeding promotes RE cell proliferation and upregulates the activity of various transporter proteins, enhancing RE absorption and metabolism. However, with prolonged feeding, these functions of RE are negatively affected, accompanied by the development of inflammation. This review elucidates the structure, the functions, and the responses of RE under HCD, providing a detailed analysis of SARA pathogenesis at the cellular and molecular levels.

Thiamine ameliorates inflammation of the ruminal epithelium of Saanen goats suffering from subacute ruminal acidosis

Dietary β-hydroxybutyrate sodium alters rumen microbiome and nutrient metabolism in the rumen epithelium of young goats

Transcriptional regulators transforming growth factor-β1 and estrogen-related receptor-α identified as putative mediators of calf rumen epithelial tissue development and function during weaning

1. Thirty Ayrshire bull calves were used in an experiment to study the effects of diets containing different proportions of concentrates to hay on rumen development at 12 weeks of age. In addition, six milk-fed calves were slaughtered at 3 weeks of age for comparison. 2. Restriction of the maximum daily allowance of concentrates to the lower levels was accompanied by an increase in the voluntary intake of hay, but the performance of calves from 3 to 12 weeks of age was significantly better in calves given the high-concentrate diets. 3. The weight of contents of the reticulo-rumen increased from 0.7 kg at 3 weeks of age in the milk-fed calf to 7.7 kg and 10.9 kg at 12 weeks in calves given the high-concentrate and high-roughage diets respectively. Between 68 and 79% of the total contents of the alimentary tract was contained in the reticulo-rumen of the ruminant calf. The weight of contents of the omasum was significantly greater in calves given 1.36 kg concentrates/day than in those given either 0.45 or 2.27 kg concentrates/day. Equations were developed from the results by which the weight of contents of the various parts of the alimentary tract, and hence empty body weight in the live animal, can be determined from a knowledge of the live weight and daily consumption of concentrates and hay, 4. Volume displacement of the reticulo-rumen tended to increase with increase in the proportion of hay in the diet. The volume displacement of the omasum increased in response to concentrate intake up to a maximum of 1.36 kg/day and thereafter declined, but the nature of the diet had no significant effect on the volume displacement of the abomasum. 5. Calves given the high-concentrate diets had a significantly greater weight of reticulo-rumen tissue at 12 weeks than those given the high-roughage diets. 6. Whereas the thickness of the muscular wall of the rumen did not differ significantly between treatments, there was an increase in the length and density of papillas, particularly in the anterior dorsal and ventral sacs of the rumen, as the intake of concentrates was increased. The results show the marked relationship between the performance of the animal and an advanced stage of development of the rumen papillas.

The current study aimed to investigate the protective effects of dietary thiamine supplementation on the regulation of colonic integrity and mucosal inflammation in goats fed a high-concentrate (HC) diet. Twenty-four Boer goats (live weight of 35·62 (sem 2·4) kg) were allocated to three groups (CON: concentrate/forage = 30:70; HC; concentrate/forage = 70:30 and HCT: concentrate/forage = 70:30 with 200 mg thiamine/kg DMI) for 12 weeks. Results showed that compared with the HC treatment, the HCT group had a significantly higher ruminal pH value from 0 to 12 h after the feeding. The haematoxylin-eosin staining showed that desquamation and severe cellular damage were observed in the colon epithelium of the HC group, whereas the HCT group exhibited more structural integrity of the epithelial cell morphology. Compared with the HC treatment, the HCT group showed a markedly increase in pyruvate dehydrogenase and α-ketoglutarate dehydrogenase enzymes activity.The mRNA expressions in the colonic epithelium of SLC19A2, SLC19A3, SLC25A19, Bcl-2, occludin, claudin-1, claudin-4 and ZO-1 in the HCT group were significantly increased in comparison with the HC diet treatment. Compared with the HC treatment, the HCT diet significantly increased the protein expression of claudin-1 and significantly decreased the protein expression of NF-κB-related proteins p65. The results show that dietary thiamine supplementation could improve the colon epithelial barrier function and alleviate mucosal inflammation injury in goats after lipopolysaccharide and low pH challenge.

We investigated the relationship between ruminal pH and transcriptomic adaptation of the rumen epithelium (RE) of calves fed calf starter with and without forage during the weaning transition. Holstein calves were assigned to groups fed calf starter either with forage (HAY group, n = 3) or without forage (CON group, n = 4). Ruminal pH was measured continuously, and rumen fluid and epithelium were collected 3 wk after weaning. mRNA expression profiles of the RE were examined by one-color microarray. Differentially expressed genes (DEGs) were investigated using the Ingenuity Pathway Analysis (IPA). Mean and maximum ruminal pH were significantly (P < 0.05) higher, and the duration of pH < 5.8 during 1 day was significantly (P < 0.05) shorter, in the HAY group. The proportion of ruminal acetate and the acetate-to-propionate ratio were significantly (P < 0.05) lower in the CON group. DEGs encoding transcription regulators (SREBP1), insulin-like growth factor binding proteins (IGFBP7 and CTGF), ketogenic enzymes (HMGCL, BDH1, and BDH2), and a transporter (SLC16A3) were identified (P < 0.05) between the two groups. A growth factor (TGFB1) and signaling pathway (EGF and EGFR) were activated as upstream regulators. These results suggest that dietary forage alleviates ruminal acidosis, and the decrease in ruminal pH may damage the RE, leading to changes in gene expression to repair the damage. Furthermore, rumen development may be regulated by growth factor (TGFB1) and signaling pathways (EGF and IGFBP) for adaptation to feeding on calf starter with and without forage during the weaning transition.

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.

Rumen-derived lipopolysaccharide induced ruminal epithelium barrier damage in goats fed a high-concentrate diet