Bovine milk-derived extracellular vesicles prevent gut inflammation by regulating lipid and amino acid metabolism.

Inflammatory bowel disease (IBD) is a global health problem in which metabolite alteration plays an important pathogenic role. Bovine milk-derived extracellular vesicles (mEVs) have been shown to regulate nutrient metabolism in healthy animal models. This study investigated the effect of oral mEVs on metabolite changes in DSS-induced murine colitis. We performed metabolomic profiling on plasma samples and measured the concentrations of lipids and amino acids in both fecal samples and colonic tissues. Plasma metabolome analysis found that mEVs significantly upregulated 148 metabolite levels and downregulated 44 metabolite concentrations (VIP > 1, and p < 0.05). In the fecal samples, mEVs significantly increased the contents of acetate and butyrate and decreased the levels of tridecanoic acid (C13:0), methyl cis-10-pentadecenoate (C15:1) and cis-11-eicosenoic acid (C20:1). Moreover, the concentrations of eicosadienoic acid (C20:2), eicosapentaenoic acid (C20:5), and docosahexaenoic acid (C22:6) were decreased in colonic tissues with mEV supplementation. In addition, compared with the DSS group, mEVs significantly increased the content of L-arginine, decreased the level of L-valine in the fecal samples, and also decreased the levels of L-serine and L-glutamate in the colonic tissues. Collectively, our findings demonstrated that mEVs could recover the metabolic abnormalities caused by inflammation and provided novel insights into mEVs as a potential modulator for metabolites to prevent and treat IBD.

Effects of combined addition of 3-nitrooxypropanol and vitamin B12 on methane and propionate production in dairy cows by in vitro-simulated fermentation.

The compound 3-nitrooxypropanol (3-NOP) is a promising methane inhibitor, which performs well in inhibiting methane emission and does not affect animal feed intake and digestibility. However, it causes a significant increase in hydrogen production while suppressing methane emission, resulting in a waste of feed energy. Vitamin B12 is a key factor in the propionate production pathway and thus plays an important role in regulating the hydrogen utilization pathway. In this study, the effects of 3-NOP combined with vitamin B12 supplementation on rumen fermentation and microbial compositional structure in dairy cattle were investigated by simulating rumen fermentation in vitro. Experiments were performed using a 2 × 2-factorial design: two 3-NOP levels (0 or 2 mg/g dry matter) and 2 vitamin B12 levels (0 or 2 mg/g dry matter). Three experiments were performed, each consisting of 4 treatments, 4 replicates, and 4 blanks containing only inoculum. The combined supplementation of 3-NOP and vitamin B12 reduced methane emission by 12% without affecting dry matter digestibility. The combined addition of 3-NOP and vitamin B12 significantly increased the concentration of propionate and reduced the concentration of acetate and the acetate to propionate ratio. At the bacterial level, 3-NOP increased the relative abundances of Christensenellaceae_R-7_group and Lachnospiraceae_NK3A20_group. Vitamin B12 increased the relative abundances of unclassified_f__Prevotellaceae and Prevotellaceae_UCG-003 and decreased the relative abundance of Lachnospiraceae_NK3A20_group. At the archaeal level, the combination of 3-NOP and vitamin B12 increased the relative abundances of Methanobrevibacter_ sp._ Abm4, OTU1125, and OTU95 and decreased the relative abundances of uncultured_methanogenic_archaeon_g__Methanobrevibacter, OTU1147, OTU1056, and OTU55. The results indicated that 3-NOP combined with vitamin B12 could alleviate rumen hydrogen emission and enhance the inhibition of methane emission compared with 3-NOP alone.

Dietary supplementation with calcium propionate could beneficially alter rectal microbial composition of early lactation dairy cows.

Dietary supplementation with calcium propionate can effectively alleviate negative energy balance and hypocalcemia of dairy cows in early lactation. The objective of this study was to investigate the effects of calcium propionate feeding levels on the immune function, liver function, and fecal microbial composition of dairy cows in early lactation. Thirty-two multiparous Holstein cows were randomly assigned to four treatments after calving. Treatments were a basal diet plus 0, 200, 350, and 500 g calcium propionate per cow per day throughout a 5-week trial period. Cows were milked three times a day, and blood was sampled to measure immune function and liver function on d 7, 21, and 35. The rectal contents were sampled and collected on d 35 to analyze the microbial composition using 16S rRNA gene sequencing. The results indicated that increasing amounts of calcium propionate did not affected the serum concentrations of total protein, IgG, IgM, and calcium, but the concentrations of albumin and IgA changed quadratically. With the increase of calcium propionate, the activity of serum alanine transaminase and aspartate aminotransferase increased linearly, in contrast, the activity of alkaline phosphatase decreased linearly. Moreover, dietary supplementation with increasing levels of calcium propionate tended to quadratically decrease the relative abundance of Firmicutes while quadratically increased the abundance of Bacteroidetes, and consequently linearly decreased the Firmicutes/Bacteroidetes ratio in the rectal microbiota. Additionally, the supplementation of calcium propionate increased the relative abundances of Ruminococcaceae_UCG-005 and Prevotellaceae_UCG-004 linearly, and Ruminococcaceae_UCG-014 quadratically, but decreased the relative abundances of Lachnospiraceae_NK3A20_group and Family_XIII_AD3011_group quadratically. Compared with the CON group, the calcium propionate supplementation significantly decreased the relative abundance of Acetitomaculum but increased the abundances of Rikenellaceae_RC9_gut_group and Alistipes. In summary, these results suggested that the supplementation of calcium propionate to dairy cows in early lactation could beneficially alter the rectal microbiota.

Alterations in the Milk Metabolome of Dairy Cows Supplemented with Different Levels of Calcium Propionate in Early Lactation.

This study aimed to investigate the effects of dietary supplementation with different levels of calcium propionate on the lactation performance, blood energy metabolite parameters, and milk metabolites of dairy cows in early lactation. Thirty-two multiparous Holstein cows were randomly divided into 4 groups, which were orally drenched with 0, 200, 350, and 500 g/d calcium propionate per cow supplemented to a basal diet for 5 weeks from calving. The milk and blood of the dairy cows were sampled and measured every week. The milk samples from the last week were used for the metabolomic analysis via liquid chromatography-mass spectrometry (LC-MS). The results showed that the calcium propionate supplementation quadratically increased the dry matter intake, energy-corrected milk yield, and 4% fat-corrected milk yield; linearly reduced the milk protein and milk lactose concentrations; and quadratically decreased the somatic cell count in the milk. With the increase in calcium propionate, the serum glucose content showed a linear increase, while the serum insulin content showed a quadratic increase. The diets supplemented with calcium propionate quadratically decreased the ß-hydroxybutyric acid and linearly decreased the non-esterified fatty acid content in the serum. The metabolomic analysis revealed that eighteen different metabolites were identified in the milk samples of the dairy cows supplemented with calcium propionate at 350 g/d, which decreased the abundance of genistein and uridine 5-monophosphate and increased the abundance of adenosine, uracil, protoporphyrin IX, and sphingomyelin (d 18:1/18:0) compared with the control group. The milk metabolic analysis indicated that the calcium propionate effectively improved the milk synthesis and alleviated the mobilization of adipose tissue and bone calcium. In summary, the calcium propionate could improve the lactation performance and energy status and promote the milk metabolic profile of dairy cows in early lactation. Calcium propionate (350 g/d) is a well-recommended supplement for dairy cows for alleviating negative energy balance and hypocalcemia in early lactation.

Supplementation with Milk-Derived Extracellular Vesicles Shapes the Gut Microbiota and Regulates the Transcriptomic Landscape in Experimental Colitis.

Harboring various proteins, lipids, and RNAs, the extracellular vesicles (EVs) in milk exert vital tissue-specific immune-protective functions in neonates via these bioactive cargos. This study aims to explore the anti-inflammatory effects of bovine milk-derived EVs on a dextran sulfate sodium (DSS)-induced colitis model and to determine the underlying molecular mechanisms. Sixty C57BL/6 mice were divided into the NC group (normal control), DSS group (DSS + PBS), DSS + LOW group (DSS + 1.5 × 108 p/g EVs), DSS + MID group (DSS + 1.5 × 109 p/g EVs), and DSS + HIG group (DSS + 1.0 × 1010 p/g EVs). Histopathological sections, the gut microbiota, and intestinal tissue RNA-Seq were used to comprehensively evaluate the beneficial functions in mitigating colitis. The morphology exhibited that the milk-derived EVs contributed to the integrity of the superficial epithelial structure in the intestine. Additionally, the concentrations of IL-6 and TNF-α in the colon tissues were significantly decreased in the EVs-treated mice. The abundances of the Dubosiella, Bifidobacterium, UCG-007, Lachnoclostridium, and Lachnospiraceae genera were increased in the gut after treatment with the milk-derived EVs. Additionally, the butyrate and acetate production were enriched in feces. In addition, 1659 genes were significantly down-regulated and 1981 genes were significantly up-regulated in the EVs-treated group. Meanwhile, 82 lncRNAs and 6 circRNAs were also differentially expressed. Overall, the milk-derived EVs could attenuate colitis through optimizing gut microbiota abundance and by manipulating intestinal gene expression, implying their application potential for colitis prevention.

Consumption of Supplementary Inulin Modulates Milk Microbiota and Metabolites in Dairy Cows with Subclinical Mastitis.

The milk microbiota and mediated metabolites directly affect the health of the udder in dairy cows. Inulin, a dietary prebiotic, can modulate the profile of gastrointestinal microbiota. However, whether the inulin intake affects the milk microbial population and metabolites remains unknown. In this study, 40 subclinical mastitis (SCM) cows were randomly divided into 5 groups. Five inulin addition doses, 0, 100, 200, 300, and 400 g/day per cow, based on the same basal diet, were supplemented. The experiments lasted for 8 weeks. The results showed lower relative abundance of mastitis-causing and proinflammation microbes in milk (i.e., Escherichia-Shigella, Pseudomonas, Rhodococcus, Burkholderia-Caballeronia-Paraburkholderia, etc.) and higher abundances of probiotics and commensal bacteria, such as Lactobacillus, Bifidobacterium, etc., in the cows fed 300 g/day inulin compared to that in the control group. Meanwhile, the levels of arachidonic acid proinflammatory mediators (leukotriene E3, 20-carboxy-leukotriene B4, and 12-Oxo-c-LTB3) and phospholipid metabolites were reduced, and the levels of compounds with antibacterial and anti-inflammatory potential (prostaglandin A1, 8-iso-15-keto-prostaglandin E2 [PGE2], etc.) and participating energy metabolism (citric acid, l-carnitine, etc.) were elevated. These data suggested that inulin intake might modulate the microflora and metabolite level in extraintestinal tissue, such as mammary gland, which provided an alternative for the regulation and mitigation of SCM. IMPORTANCE The profile of the microbial community and metabolic activity in milk are the main determinants of udder health status and milk quality. Recent studies have demonstrated that diet could directly modulate the mammary gland microbiome. Inulin is a probiotic dietary fiber which can improve the microbiota population in the gastrointestinal tract. However, whether inulin intake can further regulate the profile of the microbiota and metabolic activities in milk remains unclear. In subclinical mastitic cows, we found that inulin supplementation could reduce the abundance of Escherichia-Shigella, Pseudomonas, Rhodococcus, and Burkholderia-Caballeronia-Paraburkholderia and the levels of (±)12, 13-DiHOME, leukotriene E3 and 20-carboxy-leukotriene B4 etc., while it elevated the abundance of Lactobacillus, Bifidobacterium, and Muribaculaceae, as well as the levels of prostaglandin A1 (PGA1), 8-iso-15-keto-PGE2, benzoic acid, etc. in milk. These data suggest that inulin intake affects the profile of microorganisms and metabolites in milk, which provides an alternative for the regulation of mastitis.

Dietary Supplementation of Inulin Ameliorates Subclinical Mastitis via Regulation of Rumen Microbial Community and Metabolites in Dairy Cows.

Subclinical mastitis (SCM) is one of the highly infectious diseases in dairy cows with the characteristics of high incidence and nonvisible clinical symptoms. The gastrointestinal microbiota is closely related to mastitis. Inulin is a prebiotic fiber with functions in improving intestinal microbial communities and enhancing the host's immunity. However, the impact of dietary inulin on the rumen inner environment remains unknown. The current study investigated whether inulin could relieve SCM by affecting the profiles of ruminal bacterial and metabolites in dairy cows. Inulin inclusion rates were 0, 100, 200, 300, and 400 g/day per cow, respectively. Inulin increased milk yield, milk protein, and lactose and reduced the somatic cell counts (SCC) in milk. In serum, the concentration of proinflammatory cytokines, such as interleukin-6 (IL-6), IL-8, tumor necrosis factor α (TNF-α), and malondialdehyde (MDA) were decreased, and IL-4 and superoxide dismutase (SOD) were increased. Meanwhile, inulin increased the concentration of propionate, butyrate, and lactic acid (LA), while it decreased NH3-N in rumen. The propionate- and butyrate-producing bacteria (e.g., Prevotella and Butyrivibrio) and several beneficial commensal bacteria (e.g., Muribaculaceae and Bifidobacterium) as well as metabolites related to energy and amino acid metabolism (e.g., melibiose and l-glutamate) were increased. However, several proinflammatory bacteria (e.g., Clostridia UCG-014, Streptococcus, and Escherichia-Shigella) were decreased, accompanied by the downregulation of lipid proinflammatory metabolites, for example, ceramide(d18:0/15:0) [Cer(d18:0/15:0)] and 17-phenyl-18,19,20-trinor-prostaglandin E2. In the current study, the above indicators showed the best response in the 300 g/day inulin group. Overall, dietary supplementation of inulin could alleviate inflammatory responses in cows with SCM through improving the rumen inner environment. IMPORTANCE The correlation between mastitis and the gastrointestinal microbiome in dairy cows has been demonstrated. Regulating the profile of rumen microorganisms may contribute to remission of subclinical mastitis (SCM). Supplementation of inulin in the diets of cows with SCM could increase the abundance of short-chain fatty acid (SCFA)-producing bacteria and beneficial commensal bacteria in rumen and meanwhile the levels of amino acids and energy metabolism. Conversely, the abundance of ruminal bacteria and metabolites with proinflammatory effects were decreased. Our study suggests that the improvement of the rumen internal environment by inulin supplementation could ameliorate inflammatory responses during SCM in dairy cows and thus improve lactation performance and milk quality. Our results provide a theoretical basis for regulation measures of SCM in dairy cows.

Ruminal Degradation of Rumen-Protected Glucose Influences the Ruminal Microbiota and Metabolites in Early-Lactation Dairy Cows.

Rumen-protected glucose (RPG) plays an important role in alleviating the negative energy balance of dairy cows. This study used a combination of rumen microbes 16S and metabolomics to elucidate the changes of rumen microbial composition and rumen metabolites of different doses of RPG's rumen degradation part in early-lactation dairy cows. Twenty-four multiparous Holstein cows in early lactation were randomly allocated to control (CON), low-RPG (LRPG), medium-RPG (MRPG), or high-RPG (HRPG) groups in a randomized block design. The cows were fed a basal total mixed ration diet with 0, 200, 350, and 500 g of RPG per cow per day, respectively. Rumen fluid samples were analyzed using Illumina MiSeq sequencing and ultrahigh-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. MRPG supplementation increased bacterial richness and diversity, including increasing the relative abundance of cellulolytic bacteria, such as Ruminococcus, Lachnospiraceae_NK3A20_group, Ruminiclostridium, and Lachnospiraceae_UCG-008 MRPG significantly increased the concentrations of acetate, propionate, butyrate, and total volatile fatty acid in the rumen. Ruminal fluid metabolomics analysis showed that RPG supplementation could significantly regulate the synthesis of amino acids digested by protozoa in the rumen. Correlation analysis of the ruminal microbiome and metabolome revealed some potential relationships between major bacterial abundance and metabolite concentrations. Our analysis found that RPG supplementation of different doses can change the diversity of microorganisms in the rumen and affect the rumen fermentation pattern and microbial metabolism and that a daily supplement of 350 g of RPG might be the ideal dose.IMPORTANCE Dairy cows in early lactation are prone to a negative energy balance because their dry matter intake cannot meet the energy requirements of lactation. Rumen-protected glucose is used as an effective feed additive to alleviate the negative energy balance of dairy cows in early lactation. However, one thing that is overlooked is that people often think that rumen-protected glucose is not degraded in the rumen, thus ignoring its impact on the microorganisms in the rumen environment. Our investigation and previous experiments have found that rumen-protected glucose is partially degraded in the rumen. However, there are few reports on this subject. Therefore, we conducted research on this problem and found that rumen-protected glucose supplementation at 350 g/day can promote the development and metabolism of rumen flora. This provides a theoretical basis for the extensive application of rumen bypass glucose at a later stage.

Ruminal Methanogenic Responses to the Thiamine Supplementation in High-Concentrate Diets.

BACKGROUND: Thiamine supplementation in high-concentrate diets (HC) was confirmed to attenuate ruminal subacute acidosis through promoting carbohydrate metabolism, however, whether thiamine supplementation in HC impacts methane metabolism is still unclear. Therefore, in the present study, thiamine was supplemented in the high-concentrate diets to investigate its effects on ruminal methanogens and methanogenesis process. METHODS: an in vitro fermentation experiment which included three treatments: control diet (CON, concentrate/forage = 4:6; DM basis), high-concentrate diet (HC, concentrate/forage = 6:4; DM basis) and high-concentrate diet supplemented with thiamine (HCT, concentrate/forage = 6:4, DM basis; thiamine supplementation content = 180 mg/kg DM) was conducted. Each treatment concluded with four repeats, with three bottles in each repeat. The in vitro fermentation was sustained for 48h each time and repeated three times. At the end of fermentation, fermentable parameters, ruminal bacteria and methanogens community were measured. RESULTS: HC significantly decreased ruminal pH, thiamine and acetate content, while significantly increasing propionate content compared with CON (p < 0.05). Conversely, thiamine supplementation significantly increased ruminal pH, acetate while significantly decreasing propionate content compared with HC treatment (p < 0.05). No significant difference of ruminal methanogens abundances among three treatments was observed. Thiamine supplementation significantly decreased methane production compared with CON, while no significant change was found in HCT compared with HC. CONCLUSION: thiamine supplementation in the high-concentrate diet (HC) could efficiently reduce CH4 emissions compared with high-forage diets while without causing ruminal metabolic disorders compared with HC treatment. This study demonstrated that supplementation of proper thiamine in concentrate diets could be an effective nutritional strategy to decrease CH4 production in dairy cows.

Metagenomic Insights into Effects of Thiamine Supplementation on Carbohydrate-Active Enzymes' Profile in Dairy Cows Fed High-Concentrate Diets.

As the co-enzyme of pyruvate formate-lyase under ruminal anaerobic condition, thiamine plays a critical role in carbohydrate metabolism in dairy cows. The objective of this study was to investigate the impacts of thiamine supplementation on ruminal carbohydrate-active enzymes. Twelve Holstein dairy cows were randomly assigned into three dietary treatments: control diet (CON; 20% starch, dry matter (DM) basis), high-concentrate diet (HC; 33.2% starch, DM basis) and a high-concentrate diet supplemented with 180 mg thiamine/kg DM (HCT; 33.2% starch, DM basis). Dry matter intake and milk production were recorded for 21 days. Rumen fluid samples were collected, and ruminal pH and volatile fatty acids (VFAs) were measured. The metagenome sequencing technique was used to detect the genes in ruminal microorganisms and identify putative carbohydrate-active enzymes. The total abundances of carbohydrate-active enzymes and fiber-degrading enzymes were both reduced by HC with no effect on starch-degrading enzymes compared with CON. However, the fiber-degrading enzymes and starch-degrading enzymes were both increased after thiamine supplementation. These results indicated that 180 mg thiamine /kg DM might effectively improve rumen carbohydrate metabolism through increasing the abundance of ruminal carbohydrate-active enzymes and consequently balanced the rumen volatile fatty acids and rumen pH, providing a practical strategy in preventing subacute ruminal acidosis in cows offered HC.