Nutrition, gastrointestinal microorganisms and metabolites in mastitis occurrence and control.

Mastitis affects almost all mammals including humans and dairy cows. In the dairy industry, bovine mastitis is a disease with a persistently high incidence, causing serious losses to the health of cows, the quality of dairy products, and the economy of dairy farms. Although local udder infection caused by the invasion of exogenous pathogens into the mammary gland was considered the main cause of mastitis, evidence has been established and continues to grow, showing that nutrition factors and gastrointestinal microbiome (GM) as well as their metabolites are also involved in the development of mammary inflammatory response. Suboptimal nutrition is recognized as a risk factor for increased susceptibility to mastitis in cattle, in particular the negative energy balance. The majority of data regarding nutrition and bovine mastitis involves micronutrients. In addition, the dysbiotic GM can directly trigger or aggravate mastitis through entero-mammary gland pathway. The decreased beneficial commensal bacteria, lowered bacterial diversity, and increased pathogens as well as proinflammatory metabolites are found in both the milk and gastrointestinal tract of mastitic dairy cows. This review discussed the relationship between the nutrition (energy and micronutrient levels) and mastitis, summarized the role of GM and metabolites in regulating mastitis. Meanwhile, several non-antibiotics strategies were provided for the prevention and alleviation of mastitis, including micronutrients, probiotics, short-chain fatty acids, high-fiber diet, inulin, and aryl hydrocarbon receptor.

Au@SiO2 SERS nanotags based lateral flow immunoassay for simultaneous detection of aflatoxin B1 and ochratoxin A.

Agricultural products are frequently contaminated by mycotoxins. Multiplex, ultrasensitive, and rapid determination of mycotoxins is still a challenging problem, which is of great significance to food safety and public health. Herein, a surface-enhanced Raman scattering (SERS) based lateral flow immunoassay (LFA) for the simultaneous on-site determination of aflatoxin B1 (AFB1) and ochratoxin A (OTA) on the same test line (T line) was developed, in this study. In practice, two kinds of Raman reporters 4-mercaptobenzoic acid (4-MBA), and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) encoded silica-encapsulated gold nanotags (Au4-MBA@SiO2 and AuDNTB@SiO2) were used as detection markers to identify the two different mycotoxins. Through systematic optimization of the experimental conditions, this biosensor has high sensitivity and multiplexing with the limits of detection (LODs) at 0.24 pg mL-1 for AFB1 and 0.37 pg mL-1 for OTA. These are far below the regulatory limits set by the European Commission, in which the minimum LODs for AFB1 and OTA are 2.0 and 3.0 µg kg-1. In the spiked experiment, the food matrix are corn, rice, and wheat, and the mean recoveries of the two mycotoxins ranged from 91.0% ± 6.3%-104.8% ± 5.6% for AFB1 and 87.0% ± 4.2%-112.0% ± 3.3% for OTA. These results demonstrate that the developed immunoassay has good stability, selectivity, and reliability, which can be used for routine monitoring of mycotoxin contamination.

Protein Expression Profiles in Exosomes of Bovine Mammary Epithelial Cell Line MAC-T Infected with Staphylococcus aureus.

Mastitis is a common and widespread infectious disease in dairy farms around the world, resulting in reduced milk production and quality. Staphylococcus aureus is one of the main pathogenic bacteria causing subclinical mastitis in dairy cows. S. aureus can activate inflammatory signaling pathways in bovine mammary epithelial cells. Exosomes produced by cells can directly transfer pathogen-related molecules from cell to cell, thus affecting the process of infection. Protein is the material basis of the immune defense function in the body; therefore, a comprehensive comparison of proteins in exosomes derived from S. aureus-infected (SA group) and normal (control group [C group]) bovine mammary epithelial MAC-T cells was performed using shotgun proteomics by a DIA approach. A total of 7,070 proteins were identified and quantified. Compared with the C group, there were 802 differentially expressed proteins (DEPs) identified in the SA group (absolute log2 fold change [|log2FC|] of ≥0.58; false discovery rate [FDR] of <0.05), among which 325 proteins were upregulated and 477 were downregulated. The upregulated proteins, including complement 3 (C3), integrin alpha-6 (ITGA6), apolipoprotein A1 (APOA1), annexin A2 (ANXA2), tripeptidyl peptidase II (TPP2), keratin 8 (KRT8), and recombinant desmoyokin (AHNAK), are involved mostly in host defense against pathogens, inflammation, and cell structure maintenance. KEGG enrichment analysis indicated that DEPs in S. aureus infection were involved in the complement and coagulation cascade, phagosome, extracellular matrix (ECM)-receptor interaction, and focal adhesion pathways. The results of this study provide novel information about proteins in the exosomes of MAC-T cells infected with S. aureus and could contribute to an understanding of the infectious mechanism of bovine mastitis. IMPORTANCE Mastitis is a widespread infectious disease in dairy farms, resulting in reduced milk production and quality. Staphylococcus aureus is one of the main pathogenic bacteria causing subclinical mastitis. Exosomes contain proteins, lipids, and nucleic acids, which are involved in many physiological and pathological functions. The expression of proteins in exosomes derived from bovine mammary epithelial cells infected by S. aureus is still barely understood. These results provide novel information about MAC-T-derived exosomal proteins, reveal insights into their functions, and lay a foundation for further studying the biological function of exosomes during the inflammatory response.

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.

Rumen-protected glucose supplementation alters fecal microbiota and its metabolic profiles in early lactation dairy cows.

Introduction: Negative energy balance (NEB) is the pathological basis of metabolic disorders in early lactation dairy cows. Rumen-protected glucose (RPG) is a feed additive to relieve NEB of cows in early lactation. The aims of the current study were to evaluate the impact of different doses of RPG supply on fecal microbiota and metabolome in early lactation dairy cows, and their correlation with each other. Methods: A total of 24 multiparous Holstein dairy cows in early lactation were randomly assigned to one of four treatments for the first 35 days of the early lactation period, as follows: control group, a basal diet without RPG (CON); low RPG, a basal diet plus 200 g/d RPG (LRPG); medium RPG, a basal diet plus 350 g/d RPG (MRPG); or HRPG, high RPG, a basal diet plus 500 g/d RPG (HRPG). After 35 days, fecal samples were obtained from cows in all groups individually and using 16S rRNA gene sequencing to evaluate their microbiotas, while their metabolites were evaluated through metabolomics. Results: As expected, Firmicutes and Bacteroidetes were the core bacteria phyla. After RPG supplementation, there were an increase in Firmicutes and a decrease in Bacteroidetes. MRPG increased the relative abundance of cellulolytic bacteria, including Ruminococcaceae_UCG-005, Lachnospiraceae_UCG-008, Lachnospiraceae_FCS020_group, and Ruminiclostridium_9, while it decreased the relative abundance of Alistipes, Prevotellaceae_UCG-003, and Dorea. RPG supplementation could regulate the carbohydrate metabolism and amino acid metabolism pathway significantly and relieve lipolysis in dairy cows. Correlation analysis of fecal microbiome and metabolome showed that some major differential bacteria were the crucial contributors to differential metabolites. Conclusion: In conclusion, RPG supplementation can affect the fecal microbial components and microbial metabolism, and 350 g RPG might be the ideal dose as a daily supplement.

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.

Discrepancies among healthy, subclinical mastitic, and clinical mastitic cows in fecal microbiome and metabolome and serum metabolome.

Mastitis is generally considered a local inflammatory disease caused by the invasion of exogenous pathogens and resulting in the dysbiosis of microbiota and metabolites in milk. However, the entero-mammary pathway theory may establish a possible link between some endogenous gut bacteria and the occurrence and development of mastitis. In the current study, we attempted to investigate differences in the gut microbiota profile and metabolite composition in gut and serum from healthy cows and those with subclinical mastitis and clinical mastitis. Compared with those of healthy cows, the microbial community diversities in the feces of cows with subclinical mastitis (SM) and clinical mastitis (CM) were lower. Lower abundance of Bifidobacterium, Romboutsia, Lachnospiraceae_NK3A20_group, Coprococcus, Prevotellaceae_UCG-003, Ruminococcus, and Alistipes, and higher abundance of the phylum Proteobacteria and the genera Escherichia-Shigella and Streptococcus were observed in CM cows. Klebsiella and Paeniclostridium were significantly enriched in the feces of SM cows. Several similarities were observed in feces and serum metabolites in mastitic cows. Higher levels of proinflammatory lipid products (20-trihydroxy-leukotriene-B4, 13,14-dihydro-15-keto-PGE2, and 9,10-dihydroxylinoleic acids) and lower levels of metabolites involved in secondary bile acids (deoxycholic acid, 12-ketolithocholic acid), energy (citric acid and 3-hydroxyisovalerylcarnitine), and purine metabolism (uric acid and inosine) were identified in both SM and CM cows. In addition, elevated concentrations of IL-1ß, IL-6, tumor necrosis factor-α and decreased concentrations of glutathione peroxidase and superoxide dismutase were detected in the serum of SM and CM cows. Higher serum concentrations of triglyceride and total cholesterol and lower concentrations of high-density lipoproteins in mastitic cows might be related to changes in the gut microbiota and metabolites. These findings suggested a significant difference in the profile of feces microbiota and metabolites in cows with different udder health status, which might increase our understanding of bovine mastitis.

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.

Effects of Milk-Derived Extracellular Vesicles on the Colonic Transcriptome and Proteome in Murine Model.

Evidence shows that effective nutritional intervention can prevent or mitigate the risk and morbidity of inflammatory bowel disease (IBD). Bovine milk extracellular vesicles (mEVs), a major bioactive constituent of milk, play an important role in maintaining intestinal health. The aims of this study were to assess the effects of mEV pre-supplementation on the colonic transcriptome and proteome in dextran sulphate sodium (DSS)-induced acute colitis, in order to understand the underlying molecular mechanisms of mEV protection against acute colitis. Our results revealed that dietary mEV supplementation alleviated the severity of acute colitis, as evidenced by the reduced disease activity index scores, histological damage, and infiltration of inflammatory cells. In addition, transcriptome profiling analysis found that oral mEVs significantly reduced the expression of pro-inflammatory cytokines (IL-1ß, IL-6, IL-17A and IL-33), chemokine ligands (CXCL1, CXCL2, CXCL3, CXCL5, CCL3 and CCL11) and chemokine receptors (CXCR2 and CCR3). Moreover, oral mEVs up-regulated 109 proteins and down-regulated 150 proteins in the DSS-induced murine model, which were involved in modulating amino acid metabolism and lipid metabolism. Collectively, this study might provide new insights for identifying potential targets for the therapeutic effects of mEVs on colitis.

Electrochemical Biosensor Using Nitrogen-Doped Graphene/Au Nanoparticles/DNAzyme for Ca2+ Determination.

An electrochemical biosensor for detecting Ca2+ concentration was proposed using glass carbon electrodes (GCEs) modified with nitrogen-doped graphene (NGR), gold nanoparticles (AuNPs) and DNAzyme. The resistance signal was amplified through two methods: electrochemical reduction of AuNPs on the NGR surface to increase the specific surface area of the electrode and strengthen the adsorption of DNAzyme; and increasement of the DNAzyme base sequence. The process of electrode modification was characterized by scanning electron microscopy, Raman spectroscopy, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). Experimental parameters' influence, such as the deposition time of gold nanoparticles and the detection time, were assessed by electrochemical methods. The linear ranges of the electrochemical biosensor were in the range from 5 × 10-6 to 5 × 10-5 and 5 × 10-5 to 4 × 10-4 M, with a detection limit of 3.8 × 10-6 M. The concentration of Ca2+ in the serum of dairy cows was determined by the biosensor with satisfactory results, which could be potentially used to diagnose subclinical hypocalcemia.

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.

Changes in the Profile of Fecal Microbiota and Metabolites as Well as Serum Metabolites and Proteome After Dietary Inulin Supplementation in Dairy Cows With Subclinical Mastitis.

The occurrence and development of mastitis is linked to dysbiostic gastrointestinal microbiota. Inulin is a dietary prebiotic that improves the profile of intestinal flora. Our previous study showed that inulin supplementation could improve the ruminal microbes of subclinical mastitis (SCM) cows. The current study attempted to further investigate the response of hindgut (fecal) microbiome and metabolites, serum metabolism, and protein expression to inulin in the in SCM cows. Different levels of inulin (0, 100, 200, 300, and 400 g/day per cow) were supplemented in SCM cows. Compared with control group, Bacteroides and Bifidobacteria were increased, and Paeniclostridium, Ruminococcaceae, Coprococcus, and Clostridia were decreased in the feces of inulin groups, and accompanied with elevated propionate and butyrate concentrations, while secondary bile acid (SBA) metabolites were increased and proinflammatory lipid oxidation products were dropped in both feces and serum. In serum, inulin intake suppressed the levels of triglyceride (TG) and low-density lipoprotein (LDL). Serum proteome analysis found that CD44 antigen, phosphatidylinositol-glycan-specific phospholipase D, apolipoprotein A-II, and superoxide dismutase [Cu-Zn] were upregulated, while cathelicidin-1, haptoglobin, serpin A3, inter-alpha-trypsin inhibitor heavy chain H4 were downregulated in inulin groups. These findings suggested further evidence for inulin supplementation in amelioration of inflammatory symptoms in SCM cows, which might provide alternative treatment for mastitis.

Quantitative Detection of Mastitis Factor IL-6 in Dairy Cow Using the SERS Improved Immunofiltration Assay.

Interleukin-6 (IL-6) is generally used as a biomarker for the evaluation of inflammatory infection in humans and animals. However, there is no approach for the on-site and rapid detection of IL-6 for the monitoring of mastitis in dairy farm scenarios. A rapid and highly sensitive surface enhanced Raman scattering (SERS) immunofiltration assay (IFA) for IL-6 detection was developed in the present study. In this assay, a high sensitivity gold core silver shell SERS nanotag with Raman molecule 4-mercaptobenzoic acid (4-MBA) embedded into the gap was fabricated for labelling. Through the immuno-specific combination of the antigen and antibody, antibody conjugated SERS nanotags were captured on the test zone, which facilitated the SERS measurement. The quantitation of IL-6 was performed by the readout Raman signal in the test region. The results showed that the detection limit (LOD) of IL-6 in milk was 0.35 pg mL-1, which was far below the threshold value of 254.32 pg mL-1. The recovery of the spiking experiment was 87.0-102.7%, with coefficients of variation below 9.0% demonstrating high assay accuracy and precision. We believe the immunosensor developed in the current study could be a promising tool for the rapid assessment of mastitis by detecting milk IL-6 in dairy cows. Moreover, this versatile immunosensor could also be applied for the detection of a wide range of analytes in dairy cow healthy monitoring.

Synergistic Effects of 3-Nitrooxypropanol with Fumarate in the Regulation of Propionate Formation and Methanogenesis in Dairy Cows In Vitro.

3-Nitrooxypropanol (3-NOP) is effective at reducing ruminal methane emissions in ruminants. But it also causes a drastic increase in hydrogen accumulation, resulting in feed energy waste. Fumarate is a key precursor for propionate formation and plays an important role in rumen hydrogen metabolism. Therefore, this study examined the effects of 3-NOP combined with fumarate on volatile fatty acids, methanogenesis, and microbial community structures in dairy cows in vitro. The in vitro culture experiment was performed using a 2-by-2 factorial design, two 3-NOP levels (0 or 2 mg/g dry matter [DM]) and two fumarate levels (0 or 100 mg/g DM), including 3 runs with 4 treatments, 4 replicates, and 4 blanks containing only the inoculum. Rumen fluid was collected from three lactating Holstein cows with permanent ruminal fistulas. The combination of 3-NOP and fumarate reduced methane emissions by 11.48% without affecting dry matter degradability. The propionate concentration increased and the acetate/propionate ratio decreased significantly. In terms of bacteria, the combination of 3-NOP and fumarate reduced the abundances of Ruminococcus and Lachnospiraceae_NK3A20_group and increased the abundances of Prevotella and Succiniclasticum. For archaea, the combination of 3-NOP and fumarate significantly increased the abundances of Methanobrevibacter_sp._AbM4, while the abundance of operational taxonomic unit 581 (OTU581) (belonging to an uncultured_rumen_methanogen_g__Methanobrevibacter strain) was significantly decreased. These results indicated that the combination of 3-NOP and fumarate could alleviate the accumulation of hydrogen and enhance the inhibition of methanogenesis compared with 3-NOP only in dairy cows. IMPORTANCE The global problem of climate change and the greenhouse effect has become increasingly severe, and the abatement of greenhouse gases has received great attention from the international community. Methane produced by ruminants during digestion not only aggravates the greenhouse effect but also causes a waste of feed energy. As a methane inhibitor, 3-nitrooxypropanol can effectively reduce methane emissions from ruminants. However, when it inhibits methane emissions, the emission of hydrogen increases sharply, resulting in the waste of feed resources. Fumarate is a propionic acid precursor that can promote the metabolism of hydrogen to propionic acid in animals. Therefore, we studied the effects of the combined addition of 3-nitrooxypropanol and fumarate on methanogenesis, rumen fermentation, and rumen flora. It is of great significance to inhibit methane emission from ruminants and slow down the greenhouse effect.

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.

Glucogenic and lipogenic diets affect in vitro ruminal microbiota and metabolites differently.

This study was conducted to evaluate the effects of two glucogenic diets (C: ground corn and corn silage; S: steam-flaked corn and corn silage) and a lipogenic diet (L: sugar beet pulp and alfalfa silage) on the ruminal bacterial and archaeal structures, the metabolomic products, and gas production after 48 h in vitro fermentation with rumen fluid of dairy cows. Compared to the C and S diets, the L dietary treatment leaded to a lower dry matter digestibility (DMD), lower propionate production and ammonia-nitrogen concentration. The two glucogenic diets performed worse in controlling methane and lactic acid production compared to the L diet. The S diet produced the greatest cumulative gas volume at any time points during incubation compared to the C and L diet. The metabolomics analysis revealed that the lipid digestion especially the fatty acid metabolism was improved, but the amino acid digestion was weakened in the L treatment than in other treatments. Differences in rumen fermentation characteristics were associated with (or resulting from) changes in the relative abundance of bacterial and archaeal genera. The rumen fluid fermented with L diet had a significantly higher number of cellulolytic bacteria, including the genera of Ruminococcus, Butyrivibrio, Eubacterium, Lachnospira, unclassified Lachnospiraceae, and unclassified Ruminococcaceae. The relative abundances of amylolytic bacteria genera including Selenomonas_1, Ruminobacter, and Succinivibrionaceae_UCG-002 were higher in samples for diets C and S. The results indicated that the two glucogenic diets leaded to a higher relative abundance of bacteria which functions in succinate pathway resulting in a higher propionate production. The steam-flaked corn diet had a higher gas production and lower level of metabolites in fatty acids and amino acids. Most highly abundant bacteria were observed to be not sensitive to dietary alterations of starch and fiber, except for several amylolytic bacteria and cellulolytic bacteria. These finding offered new insights on the digesting preference of ruminal bacteria, which can assist to improve the rumen functioning.

Dietary supplementation with inulin improves lactation performance and serum lipids by regulating the rumen microbiome and metabolome in dairy cows.

This study investigated the effects of inulin on rumen fermentation parameters, ruminal microbiome and metabolites, as well as lactation performance and serum indexes in dairy cows. Sixteen Holstein dairy cows with similar body conditions were randomly divided into 2 groups (n = 8 per group), with inulin addition at 0 and 200 g/d per cow. The experiment lasted for 6 weeks, including a 1-week adaptation period and a 5-week treatment period. At the end of the experimental period, the milk, serum and rumen fluid were sampled and analyzed. The microbiome and metabolome in the rumen fluid were analyzed via 16S rRNA sequencing and untargeted metabolomics, respectively. The results showed that supplementation with inulin (200 g/d per cow) increased the milk yield (P = 0.001), milk protein (P = 0.032), lactose rate (P = 0.004) and proportion of saturated fatty acids (SFA) in milk (P < 0.001), but decreased the proportion of unsaturated fatty acids (USFA) (P = 0.041). Rumen pH (P = 0.040) and the concentration of NH3-N (P = 0.024) were decreased; however, acetate (P < 0.001), propionate (P = 0.003), butyrate (P < 0.001) and lactic acid (LA) (P = 0.043) were increased. The total cholesterol (TC) (P = 0.008) and triglycerides (TG) (P = 0.01) in serum were also reduced. Additionally, inulin addition elevated the relative abundance of several beneficial symbiotic and short-chain fatty acid (SCFA)-producing bacteria, such as Muribaculaceae (false discovery rate [FDR]-adjusted P < 0.01), Acetitomaculum (FDR-adjusted P = 0.043), and Butyrivibrio (FDR-adjusted P = 0.036), while elevating the levels of L-lysine (FDR-adjusted P = 4.24 × 10-3), L-proline (FDR-adjusted P = 0.0158), and L-phenylalanine (FDR-adjusted P = 0.027). In contrast, several pathogens and ruminal bacteria abundant in high-fat diets, such as Escherichia-Shigella (FDR-adjusted P = 0.022), Erysipelotrichaceae __UCG-004 (FDR-adjusted P < 0.01) and RF39 (FDR-adjusted P = 0.042) were decreased along with the reduction of lysophosphatidylcholine (LysoPC) (18:1 (9Z)) (FDR-adjusted P = 1.03 × 10-3), LysoPC (16:0) (FDR-adjusted P = 0.0108), LysoPC (18:2 (9Z, 12Z)) (FDR-adjusted P = 1.65 × 10-3) and 8-methylnonenoate. In conclusion, dietary inulin supplementation could increase the relative abundance of commensal microbiota and SCFA-producing bacteria, upregulate amino acidmetabolism and downregulate lipid metabolism in the rumen of dairy cows, which might further improve lactation performance and the level of serum lipids.

Effects of oral milk extracellular vesicles on the gut microbiome and serum metabolome in mice.

Milk extracellular vesicles (EVs) are rich in abundant bioactive macromolecules, such as glycoconjugates, proteins, lipids and nucleic acids, and these vesicles might transmit signals to human consumers. However, it remains to be determined whether milk EVs import new pathogens to humans or are beneficial for human health. Here, C57BL/6 female and male mice were randomly divided into 4 EV dose levels (0, 1.5 × 109 p g-1, 1.0 × 1010 p g-1 and 1.5 × 1010 p g-1). Based on the alterations in body weight, the control group (0 p g-1, PBS) and the middle treatment group (1.0 × 1010 p g-1) were chosen for further analysis of the effects of EVs on the gut microbiota and blood metabolites in mice, by 16S rRNA gene sequencing and untargeted metabolomics, respectively. We found that milk EVs increased the abundance of "beneficial" microbes such as Akkermansia, Muribaculum and Turicibacter, while decreased the level of "harmful" bacteria Desulfovibrio. Serum metabolites showed that EVs mainly changed the lipid and amino acid metabolism, and especially increased several serum anti-inflammatory factors, which might be beneficial for inflammation and other metabolic diseases. The results of KEGG analysis suggested that the enriched pathways were the intestinal immune network for IgA production, retinol metabolism, and D-glutamine and D-glutamate metabolism. Taken together, the positive effect of milk EVs on serum nutrient metabolism without promoting "harmful" bacterial colonization in female and male mice may indicate that they are safe bioactive molecules, and some of the changes they induce may provide protection against certain diseases.

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.