Effects of grain intervention on hypothalamic function and the metabolome of blood and milk in dairy cows.

BACKGROUND: The hypothalamus plays a crucial role in the health and productivity of dairy cows, yet studies on its functionality and its impact on peripheral circulation in these animals are relatively scarce, particularly regarding dietary interventions. Therefore, our study undertook a comprehensive analysis, incorporating both metabolomics and transcriptomics, to explore the effects of a grain-based diet on the functionality of the hypothalamus, as well as on blood and milk in dairy cows. RESULTS: The hypothalamic metabolome analysis revealed a significant reduction in prostaglandin E2 (PGE2) level as a prominent response to the grain-based diet introduction. Furthermore, the hypothalamic transcriptome profiling showed a notable upregulation in amino acid metabolism due to the grain-based diet. Conversely, the grain-based diet led to the downregulation of genes involved in the metabolic pathway from lecithin to PGE2, including phospholipase A2 (PLA2G4E, PLA2G2A, and PLA2G12B), cyclooxygenase-2 (COX2), and prostaglandin E synthase (PTGES). Additionally, the plasma metabolome analysis indicated a substantial decrease in the level of PGE2, along with a decline in adrenal steroid hormones (tetrahydrocortisol and pregnenolone) following the grain-based diet introduction. Analysis of the milk metabolome showed that the grain-based diet significantly increased uric acid level while notably decreasing PGE2 level. Importantly, PGE2 was identified as a critical metabolic marker in the hypothalamus, blood, and milk in response to grain intervention. Correlation analysis demonstrated a significant correlation among metabolic alterations in the hypothalamus, blood, and milk following the grain-based diet. CONCLUSIONS: Our findings suggest a potential link between hypothalamic changes and alterations in peripheral circulation resulting from the introduction of a grain-based diet.

High-grain diet feeding alters ileal microbiota and disrupts bile acid metabolism in lactating dairy cows.

Bile acids (BAs) play an important role in the regulation of lipid metabolic homeostasis, but little is known about their metabolism in dairy cows fed a high-grain (HG) diet. In the present study, we examined the bacterial community, BA profile, and the FXR/FGF19 signaling pathway in the ileum and liver to investigate the gut microbe-BA metabolism interactions response to HG diet and the changes in the subsequent enterohepatic circulation of dairy cows. The results showed that the ileal bacterial community was altered, with an increase of Paraclostridium, Anaerobutyricum, Shuttleworthia, and Stomatobaculum in the relative abundance in the HG group. Moreover, real-time polymerase chain reaction (PCR) showed that the abundance of total bacteria and bacterial bile-salt hydrolase (BSH) genes was increased in the ileal digesta in the HG group. Meanwhile, HG feeding also decreased the total BA content in the digesta of jejunum and ileum and in feces. HG feeding altered the BA profile in the ileal digesta by increasing unconjugated BAs and decreasing conjugated BAs. In addition, the intestinal FXR/FGF19 signaling pathway was activated. The expression of CYP7A1 (cholesterol 7α-hydroxylase) was depressed, which inhibited BAs synthesis in the liver of cows fed HG. Overall, HG feeding altered the ileal bacterial community and BA profile, and activated FXR/FGF19 signaling pathway, resulting in a decrease of BA level in the ileal digesta via the inhibition of hepatic BA synthesis. The findings provided novel insights into understanding the relationship between gut microbiota and the homeostasis of BAs in dairy cows fed a HG diet.

Bile acids plays an important role in regulating lipids metabolism in animals and human. Dairy cows fed high-grain (HG) diet generally suffer abnormal lipids metabolism. However, if there is a relationship between the bile acids metabolism and abnormal lipids metabolism in dairy cows fed HG diet is unclear. This study found that HG diet altered the bacterial community and bile aids composition in the ileum of dairy cows. HG also activated the FXR/FGF19 signaling pathway in the ileum, and inhibited the bile acid synthesis in the liver, which might be the reason for the reduced level of bile acid in the digesta of small intestine. The reduced bile acid level in the small intestine might affect the digestion and absorption of the dietary lipids in dairy cows fed HG diet.

Metagenomics analysis reveals differences in rumen microbiota in cows with low and high milk protein percentage.

Variation exists in milk protein concentration of dairy cows of the same breed that are fed and managed in the same environment, and little information was available on this variation which might be attributed to differences in rumen microbial composition as well as their fermentation metabolites. This study is aimed at investigating the difference in the composition and functions of rumen microbiota as well as fermentation metabolites in Holstein cows with high and low milk protein concentrations. In this study, 20 lactating Holstein cows on the same diet were divided into two groups (10 cows each), high degree of milk protein group (HD), and low degree of milk protein (LD) concentrations based on previous milk composition history. Rumen content samples were obtained to explore the rumen fermentation parameters and rumen microbial composition. Shotgun metagenomics sequencing was employed to investigate the rumen microbial composition and sequences were assembled via the metagenomics binning technique. Metagenomics revealed that 6 Archaea genera, 5 Bacteria genera, 7 Eukaryota genera, and 7 virus genera differed significantly between the HD and LD group. The analysis of metagenome-assembled genomes (MAGs) showed that 2 genera (g__Eubacterium_H and g__Dialister) were significantly enriched (P < 0.05, linear discriminant analysis (LDA) > 2) in the HD group. However, the LD group recorded an increased abundance (P < 0.05, LDA > 2) of 8 genera (g__CAG-603, g__UBA2922, g__Ga6A1, g__RUG13091, g__Bradyrhizobium, g__Sediminibacterium, g__UBA6382, and g__Succinivibrio) when compared to the HD group. Furthermore, investigation of the KEGG genes revealed an upregulation in a higher number of genes associated with nitrogen metabolism and lysine biosynthesis pathways in the HD group as compared to the LD group. Therefore, the high milk protein concentration in the HD group could be explained by an increased ammonia synthesis by ruminal microbes which were converted to microbial amino acids and microbial protein (MCP) in presence of an increased energy source made possible by higher activities of carbohydrate-active enzymes (CAZymes). This MCP gets absorbed in the small intestine as amino acids and might be utilized for the synthesis of milk protein. KEY POINTS: • Rumen microbiota and their functions differed between cows with high milk protein % and those with low milk protein %. • The rumen microbiome of cows with high milk protein recorded a higher number of enriched genes linked to the nitrogen metabolism pathway and lysine biosynthesis pathway. • The activities of carbohydrate-active enzymes were found to be higher in the rumen of cows with high milk protein %.

Coordinated response of milk bacterial and metabolic profiles to subacute ruminal acidosis in lactating dairy cows.

BACKGROUND: Bovine milk is an important source of nutrition for human consumption, and its quality is closely associated with the microbiota and metabolites in it. But there is limited knowledge about the milk microbiome and metabolome in cows with subacute ruminal acidosis. METHODS: Eight ruminally cannulated Holstein cows in mid lactation were selected for a 3-week experiment. The cows were randomly allocated into 2 groups, fed either a conventional diet (CON; 40% concentrate; dry matter basis) or a high-concentrate diet (HC; 60% concentrate; dry matter basis). RESULTS: The results showed that there was a decreased milk fat percentage in the HC group compared to the CON group. The amplicon sequencing results indicated that the alpha diversity indices were not affected by the HC feeding. At the phylum level, the milk bacteria were dominated by Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes both in the CON and HC groups. At the genus level, the HC cows displayed an improved proportion of Labrys (P = 0.015) compared with the CON cows. Results of both the principal components analysis and partial least squares of discriminant analysis of milk metabolome revealed that samples of the CON and HC groups clustered separately. A total of 31 differential metabolites were identified between the two groups. Of these, the levels of 11 metabolites decreased (α-linolenic acid, prostaglandin E2, L-lactic acid, L-malic acid, 3-hydroxysebacic acid, succinyladenosine, guanosine, pyridoxal, L-glutamic acid, hippuric acid, and trigonelline), whereas the levels of the other 20 metabolites increased in the HC group with respect to the CON group (P < 0.05). CONCLUSION: These results suggested that subacute ruminal acidosis less impacted the diversity and composition of milk microbiota, but altered the milk metabolic profiles, which led to the decline of the milk quality.

Variety of rumen microbial populations involved in biohydrogenation related to individual milk fat percentage of dairy cows.

Our objective was to investigate the contribution of the rumen microbiome on the individual milk fat percentage (MFP) of Holstein dairy cows under the same nutritional and management conditions. From 92 early lactation dairy cows, the top 10 with the highest MFP (HF; n = 10) and the last 10 with the lowest MFP (LF; n = 10) were selected for the study. As a result, the milk trans-10, cis-12 C18:2 content was significant lower in the HF group than that in the LF group (P < 0.001). The rumen acetate to propionate ratio was significant higher in the HF group than that in the LF group (P = 0.035). According to the results of 16S rRNA gene sequencing, a minor but significant difference existed between the groups (P = 0.040). Three genera of the family Lachnospiraceae and four genera of the order Bacteroidales were identified to be the biomarkers for the LF group and HF group in the LEfSe analysis, respectively. Three microbial modules enriched by the family Lachnospiraceae were positively related to the milk trans-10, cis-12 C18:2 content (r s > 0.60, P < 0.05). According to the results of shotgun metagenome sequencing, three kinds of linoleic acid (LA) isomerase genes were present in the gene pools of the rumen microbiome. Among them, the relative abundance of Bifidobacterium LA isomerase (BBI) was higher in the HF group than that in the LF group (P = 0.007). Three metagenome-assembled genomes (MAGs) with LA isomerase genes were positively correlated to the milk trans-10, cis-12 C18:2 content (r s > 0.40, P < 0.05). Furthermore, all of these three MAGs were found to be able to produce lactate. Taken together, these results indicate that the increased relative abundance of microbial population with the trans-10 biohydrogenation pathway within the rumen microbiome contributes to the decrease of MFP via the increase of rumen trans-10, cis-12 C18:2 production. This study provides a new perspective for the development of measures for improving the milking performance of dairy cows.

The gastrointestinal microbiome in dairy cattle is constrained by the deterministic driver of the region and the modified effect of diet.

BACKGROUND: Dairy cattle (Bos taurus), especially Holstein cows, which are the highest-producing dairy animals and are widely bred to provide milk products to humans, rely critically on their associated gastrointestinal tract (GIT) microbiota to digest plant feed. However, the region-specific taxonomic composition and function of the GIT microbiome in dairy cattle and the mechanistic basis for the diet-induced effects remain to be elucidated.  RESULTS: We collected 120 digesta samples from 10 GIT regions of 12 Holstein cows fed forage- and grain-based diets and characterized their GIT microbiome via functional shotgun metagenomics and the resolution of metagenome-assembled genomes. Our results demonstrated that the GIT microbiome was mainly partitioned into three distinct clusters, four-chambered stomach, small intestine, and large intestine. Moreover, we found that the four-chambered stomach microbiome with the highest diversity had a strong ability to degrade recalcitrant polysaccharide substrates, underpinned by the prevalence of potential cellulosome--producing and plant-derived polysaccharide utilization loci-encoding consortia. In contrast, the post-gastric intestinal microbiome orchestrated alternative fermentation pathways to adapt to nutrient availability and energy acquisition. Diet shifts selectively modified the metabolic cascades of the microbiome in specific GIT regions, evidenced by the loss of fiber-degrading taxa and increased hydrogen sinks in propionate after grain introduction. CONCLUSIONS: Our findings provide new insights into GIT microbial organization and function in dairy cattle by GIT regions and diet regimes, which offers clues for improving animal production and health in the future. Video Abstract.

Live yeast supplementation altered the bacterial community's composition and function in rumen and hindgut and alleviated the detrimental effects of heat stress on dairy cows.

The objective of this study was to investigate the effects of live yeast (LY, Saccharomyces cerevisiae) on the lactation performance, bacterial community, and functions in the rumen and hindgut of dairy cows under heat stress. Thirty-three multiparous (parity 3.9 ± 0.8) Holstein dairy cows (189.1 ± 6.6 d in milk at the beginning of the experiment) were randomly assigned to three groups (11 cows per treatment). Cows in the three groups were fed a diet without yeast (CON), with 10 g yeast/d/head (LY-10), and with 20 g yeast/d/head (LY-20). The yeast product contained 2.0 × 1010 CFU/g. Supplementing LY decreased the rectal temperature and respiratory rate of cows, and increased dry matter intake, milk yield, milk fat yield, milk protein yield, and milk lactose yield (P < 0.001), yet decreased milk urea nitrogen concentration (P = 0.035). Interaction effects of treatment × week were observed for rectal temperature (P < 0.05), respiratory rate (P < 0.05), milk yield (P = 0.015), milk urea nitrogen (P = 0.001), milk protein yield (P = 0.008), and milk lactose yield (P = 0.030). In rumen, LY increased the concentrations of acetate, isobutyrate, isovaterate, valerate, total volatile fatty acids (VFAs), and NH3-N (P < 0.05). Miseq sequencing of the 16S rRNA genes showed that LY increased the relative abundance of Prevotella and Prevotellaceae UCG-003 at the genus level with a series of enriched pathways in the metabolism of carbohydrates and protein. In fecal samples, LY did not affect the profile of VFAs (P > 0.05). Clostridium sensu stricto 1 (P = 0.013) and Actinobacillus (P = 0.011) increased in the relative abundance by LY, whereas Bacteroides (P = 0.016) and Oscillospirales UCG-010 (P = 0.005) decreased with a series of enriched pathways in carbohydrate metabolism, secondary bile acid biosynthesis. In summary, LY supplementation altered the bacterial community's composition and function in rumen and hindgut, and simultaneously alleviated the detrimental effects of heat stress on dairy cows. These findings provide extended insight into the effects of LY in the rumen and hindgut of dairy cows exposed to heat stress.

Dairy cows are exposed to severe heat stress under hot and humid climates in summer in south China, resulting in a decline in feed intake and milk yield. Therefore, we investigated the effect of live yeast (LY, Saccharomyces cerevisiae) supplementation on the milk performance, bacterial community, and functions in the rumen and hindgut of dairy cows under heat stress. Thirty-three dairy cows were randomly assigned to control (CON, without yeast addition), treatment 1 (LY-10, with 10 g yeast/d/head) and treatment 2 (LY-20, with 20 g yeast/d/head). Supplementing LY decreased the rectal temperature and respiratory rate of the dairy cows and increased feed intake and milk performance. Live yeast enhanced fermentation in the rumen but did not affect it in the hindgut. Live yeast altered the microbiota in the rumen and hindgut, with an enrichment of bacteria in the pathways of the metabolism of carbohydrates, protein, and other substances. In all, LY supplementation had beneficial effects on dairy cows under heat stress by affecting the microbiota and fermentation in the rumen and hindgut.

Genome-centric investigation of bile acid metabolizing microbiota of dairy cows and associated diet-induced functional implications.

Although the importance of bile acid (BA)-related microbial strains and enzymes is increasingly recognized for monogastric animals, a lack of knowledge about BA metabolism in dairy cows limits functional applications aimed at the targeted modulation of microbe-host interactions for animal production and health. In the present study, 108 content samples from six intestinal regions of dairy cows were used for shotgun metagenomic sequencing. Overall, 372 high-quality metagenome-assembled genomes (MAGs) were involved in BA deconjugation, oxidation, and dehydroxylation pathways. Furthermore, the BA-metabolizing microbiome predominately occurred in the large intestine, resulting in the accumulation of secondary unconjugated BAs. Comparative genomic analysis revealed that the bile salt hydrolase (BSH)-carrying microbial populations managed with the selective environment of the dairy cow intestine by adopting numerous host mucin glycan-degrading abilities. A sequence similarity network analysis classified 439 BSH homologs into 12 clusters and identified different clusters with diverse evolution, taxonomy, signal peptides, and ecological niches. Our omics data further revealed that the strains of Firmicutes bacterium CAG-110 processed the increased abundance of BSHs from Cluster 1, coinciding with the changes in the colon cholic acid concentration after grain introduction, and were intricately related to intestinal inflammation. This study is the first to use a genome-centric approach and whole intestine-targeted metabolomics to reveal microbial BA metabolism and its diet-induced functional implications in dairy cows. These findings provide insight into the manipulation of intestinal microorganisms for improving host health.

Fermented soybean meal modified the rumen microbiome to enhance the yield of milk components in Holstein cows.

The study was conducted to evaluate the rumen microbiota as well as the milk composition and milk component yields of Holstein cows supplemented with fermented soybean meal (FSBM). Eighteen Holstein cows in their 2nd parity with 54.38 ± 11.12 SD days in milking (DIM) were divided into two dietary groups (CON and TRT) of nine cows per group. The cows in the TRT group received 300 g of FSBM per cow per day in addition to the conventional diet, while each cow in the CON group was supplemented with 350 g of soybean meal (SBM) in their diet daily throughout the 28-day feeding trial. Rumen bacterial composition was detected via 16S rRNA sequencing, and the functional profiles of bacterial communities were predicted. Milk composition, milk yield, as well as rumen fermentation parameters, and serum biochemistry were also recorded. The inclusion of FSBM into the diets of Holstein cows increased the milk urea nitrogen (MUN), milk protein yield, fat corrected milk (FCM), and milk fat yield while the milk somatic cell count (SCC) was decreased. In the rumen, the relative abundances of Fibrobacterota, and Spirochaetota phyla were increased in the TRT group, while the percentage of Proteobacteria was lower. In addition, the supplementation of FSBM to Holstein cows increased the acetate percentage, rumen pH, and acetate to propionate ratio, while the proportion of propionate and propionate % was observed to decrease in the TRT group. The KEGG pathway and functional prediction revealed an upregulation in the functional genes associated with the biosynthesis of amino acids in the TRT group. This enrichment in functional genes resulted in an improved synthesis of several essential amino acids including lysine, methionine, and branch chain amino acids (BCAA) which might be responsible for the increased milk protein yield. Future studies should employ shotgun metagenomics, transcriptomics, and metabolomics technology to investigate the effects of FSBM on other rumen microbiomes and milk protein synthesis in the mammary gland in Holstein cows. KEY POINTS: • The supplementation of fermented soybean meal (FSBM) to Holstein cows modified the proportion of rumen bacteria. • Predicted metabolic pathways and functional genes of rumen bacteria revealed an enrichment in pathway and genes associated with biosynthesis of amino acids in the group fed FSBM. • The cows supplemented with FSBM record an improved rumen fermentation. • Cows supplemented with FSBM recorded an increased yield of milk protein and milk fat.

Metagenomic and Metabolomic Insights Into the Mechanism Underlying the Disparity in Milk Yield of Holstein Cows.

This study was conducted to investigate the metabolic mechanism underlying the disparity in the milk yield of Holstein cows. Eighteen lactating Holstein cows in their second parity and 56 (±14.81 SD) days in milking (DIM) were selected from 94 cows. Based on the milk yield of the cows, they were divided into two groups of nine cows each, the high milk yield group (HP) (44.57 ± 2.11 kg/day) and the low milk yield group (LP) (26.71 ± 0.70 kg/day). The experimental cows were fed the same diet and kept under the same management system for more than 60 days. Rumen metagenomics revealed that two Archaea genera, one Bacteria genus, eight Eukaryota genera, and two Virus genera differ between the HP and LP groups. The analysis of metabolites in the rumen fluid, milk, and serum showed that several metabolites differed between the HP and LP groups. Correlation analysis between the predominant microbiota and milk yield-associated metabolites (MP-metabolites) revealed that four Bacteria and two Eukaryota genera have a positive relationship with MP-metabolites. Pathway enrichment analysis of the differential metabolites revealed that five pathways were enriched in all the samples (two pathways in the milk, two pathways in the serum, and one pathway in the rumen fluid). Further investigation revealed that the low milk yield observed in the LP group might be due to an upregulation in dopamine levels in the rumen fluid and milk, which could inhibit the release of prolactin or suppress the action of oxytocin in the udder resulting in reduced milk yield. On the other hand, the high milk yield in the HP group is attributed to an upregulation in citrulline, and N-acetylornithine, which could be used as substrates for energy metabolism in the citric acid cycle and ultimately gluconeogenesis.

Response of Milk Performance, Rumen and Hindgut Microbiome to Dietary Supplementation with Aspergillus oryzae Fermentation Extracts in Dairy Cows.

Fungal additives had beneficial effects on milk performance in dairy cows. Previous studies investigating the effects of fungal additives mainly focused on the rumen, such influences on the hindgut remain limited. This study aimed to investigate the effects of Aspergillus oryzae fermentation extracts (AOE) on the milk performance and microbiome in the rumen and hindgut using 16S rRNA gene sequencing. Twenty lactating multiparous Holstein cows were randomly assigned to control and treatment (5 g AOE per cow per day). The results showed that AOE increased the milk yield, milk protein and lactose concentration, but did not affect the milk fat concentration. Feeding AOE did not affect the ruminal VFA pattern, pH, NH3-N, and microbial cell protein production, but decreased lipopolysaccharide concentration and tended to decrease lactate concentration. The addition of AOE increased the fecal pH and the proportions of propionate, isovalerate and valerate, and decreased the acetate to propionate ratio. PCoA analysis showed that AOE did not affect the overall ruminal bacterial population composition. Only three genera changed slightly in relative abundance. In the feces, PCoA analysis showed that AOE changed the bacterial population composition. Feeding AOE increased the relative abundances of Ruminococcaceae UCG-010 and unclassified Clostridiales vadinBB60 group, and decreased Christensenellaceae R-7 group, unclassified Muribaculaceae, Prevotellaceae UCG-001 and Romboutsia. Spearman correlation showed unclassified Clostridiales vadinBB60 group was positively correlated with propionate proportion. Overall, we present that AOE not only functioned in rumen, but also in hindgut. The hindgut microbiome changes might play an important role in the milk performance improvement of dairy cows.

Responsive changes of rumen microbiome and metabolome in dairy cows with different susceptibility to subacute ruminal acidosis.

Subacute ruminal acidosis (SARA) represents one of the most important digestive disorders in intensive dairy farms, and dairy cows are individually different in the severity of SARA risk. The objectives of the current study were to investigate differences in the ruminal bacterial community and metabolome in dairy cattle with different susceptibility to SARA. In the present study, 12 cows were initially enrolled in the experiment. Based on average ruminal pH, 4 cows with the lowest ruminal pH were assigned to the susceptible group (SUS, pH = 5.76, n = 4) and 4 cows with the highest ruminal pH assigned to the tolerant group (TOL, pH = 6.10, n = 4). Rumen contents from susceptible (SUS, n = 4) and tolerant (TOL, n = 4) dairy cows were collected through rumen fistula to systematically reveal the rumen microbial and metabolic alterations of dairy cows with different susceptibility to SARA using multi-omics approaches (16S and 18S rRNA gene sequencing and metabolome). The results showed that despite being fed the same diet, SUS cows had lower ruminal pH and higher concentrations of total volatile fatty acids (VFA) and propionate than TOL cows (P < 0.05). No significant differences were observed in dry matter intake, milk yield, and other milk compositions between the SUS and TOL groups (P > 0.05). The principal coordinates analysis based on the analysis of molecular variance indicated a significant difference in bacterial composition between the two groups (P = 0.01). More specifically, the relative abundance of starch-degrading bacteria (Prevotella spp.) was greater (P < 0.05), while the proportion of fiber-degrading bacteria (unclassified Ruminococcaceae spp., Ruminococcus spp., Papillibacter, and unclassified Family_XIII) was lower in the rumen of SUS cows compared with TOL cows (P < 0.05). Community analysis of protozoa showed that there were no significant differences in the diversity, richness, and community structure (P > 0.05). Metabolomics analysis revealed that the concentrations of organic acids (such as lactic acid), biogenic amines (such as histamine), and bacterial degradation products (such as hypoxanthine) were significantly higher in the SUS group compared to the TOL group (P < 0.05). These findings revealed that the higher proportion of starch-degrading bacteria/lower fiber-degrading bacteria in the rumen of SUS cows resulted in higher VFA-producing capacity, in particular propionate. This caused a disruption in metabolic homeostasis in the rumen which might be the reason for the higher susceptibility to SARA. Overall, these findings enhanced our understanding of the ruminal microbiome and metabolic changes in cows susceptible to SARA.

Multi-omics Analysis Revealed Coordinated Responses of Rumen Microbiome and Epithelium to High-Grain-Induced Subacute Rumen Acidosis in Lactating Dairy Cows.

Subacute ruminal acidosis (SARA) is a major metabolic disease in lactating dairy cows caused by the excessive intake of high-concentrate diets. Here, we investigated the synergistic responses of rumen bacteria and epithelium to high-grain (HG)-induced SARA. Eight ruminally cannulated lactating Holstein cows were randomly assigned to 2 groups for a 3-week experiment and fed either a conventional (CON) diet or an HG diet. The results showed that the HG-feeding cows had a thickened rumen epithelial papilla with edge injury and a decreased plasma ß-hydroxybutyrate concentration. The 16S rRNA gene sequencing results demonstrated that HG feeding caused changes in rumen bacterial structure and composition, which further altered rumen fermentation and metabolism. Cooccurrence network analysis revealed that the distribution of the diet-sensitive bacteria responded to the treatment (CON or HG) and that all diet-sensitive amplicon sequence variants showed low to medium degrees of cooccurrence. Metabolomics analysis indicated that the endothelial permeability-increasing factor prostaglandin E1 and the polyamine synthesis by-product 5'-methylthioadenosine were enriched under HG feeding. Transcriptome analysis suggested that cholesterol biosynthesis genes were upregulated in the rumen epithelium of HG cows. The gene expression changes, coupled with more substrate being available (total volatile fatty acids), may have caused an enrichment of intracellular cholesterol and its metabolites. All of these variations could coordinately stimulate cell proliferation, increase membrane permeability, and trigger epithelial inflammation, which eventually disrupts rumen homeostasis and negatively affects cow health. IMPORTANCE Dairy cows are economically important livestock animals that supply milk for humans. The cow's rumen is a complex and symbiotic ecosystem composed of diverse microorganisms, which has evolved to digest high-fiber diets. In modern dairy production, SARA is a common health problem due to overfeeding of high-concentrate diets for an ever-increasing milk yield. Although extensive studies have been conducted on SARA, it remains unclear how HG feeding affects rumen cross talk homeostasis. Here, we identified structural and taxonomic fluctuation for the rumen bacterial community, an enrichment of certain detrimental metabolites in rumen fluid, and a general upregulation of cholesterol biosynthesis genes in the rumen epithelium of HG-feeding cows by multi-omics analysis. Based on these results, we propose a speculation to explain cellular events of coordinated rumen bacterial and epithelial adaptation to HG diets. Our work provides new insights into the exploitation of molecular regulation strategies to treat and prevent SARA.

Monolithic molecular imprinted polymer fiber for recognition and solid phase microextraction of ephedrine and pseudoephedrine in biological samples prior to capillary electrophoresis analysis.

A novel capillary electrophoresis (CE) method coupled with monolithic molecular imprinted polymer (MIP) fiber based solid phase microextraction (SPME) was developed for selective and sensitive determination of ephedrine (E) and pseudoephedrine (PE). With in situ polymerization in a silica capillary mold and E as template, the MIP fibers could be produced in batch reproducibly and each fiber was available for 50 extraction cycles without significant decrease in extraction ability. Using the MIP fiber under optimized extraction conditions, CE detection limits of E and PE were greatly lowered from 0.20 to 0.00096 µg/mL and 0.12 to 0.0011 µg/mL, respectively. Analysis of urine and serum samples by the MIP-SPME-CE method was also performed, with results indicating that E and PE could be selectively extracted. The recoveries and relative standard deviations (RSDs) for sample analysis were found in the range of 91-104% and 3.8-9.1%, respectively.

Simultaneous stacking of cationic and anionic compounds in single run capillary zone electrophoresis by two-end field amplified sample injection.

In order to extend the application of field amplified sample injection (FASI) in high throughput analysis, a convenient and simple procedure, namely two-end field amplified sample injection (TE-FASI), was developed for the simultaneous stacking of cationic and anionic compounds in a single run capillary zone electrophoresis (CZE). Following the capillary-filling with a buffer of high conductivity, water plug was loaded into each end of the capillary; and two high-field strength zones were generated at both heads of the column when high voltage was applied. Therefore, under suppressed EOF cations and anions can be selectively FASI stacked at anode and cathode head, respectively. After separation, the stacked anions and cations are detected by a common detector placed in the center of the capillary. Under the optimized conditions, the limits of detection for the model cationic (matrine and oxymatrine) and anionic (5-sulfosalicylic acid) compounds were determined as 0.2, 0.2 and 0.06 ng/mL, respectively. Compared with non-stacking conditions, the sensitivities of these compounds were enhanced 1003-, 1330- and 1380-fold, respectively. The results of reproducibility, linearity and real sample analysis show that the proposed procedure is promising to be applied for the simultaneous quantification detection of trace cationic and anionic analytes.

Light-emitting-diode-induced chemiluminescence detection for capillary electrophoresis.

In this work, an LED-induced-chemiluminescence (LED-CL) system was developed to extend the application of CL detection in CE. In the LED-CL, the analyte photooxidizes luminol under the irradiation of LEDs and generates CL. Taking the advantage of the small size nature of LEDs, the constructed photoreactor is greatly miniaturized, and especially suitable as a CE detector. The feasibility of the proposed detector was evaluated by detection of riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) after CE separation. Under the optimized conditions, the LODs for RF, FMN and FAD were 0.007, 0.02 and 0.1 microg/mL, respectively, better than those by UV detection. The RSDs were 3.4, 3.6 and 4.1% for 0.5 microg/mL RF, 2 microg/mL FMN and 5 microg/mL FAD, respectively. The LED-CL detector features low cost, miniaturization, fast response, high sensitivity and good reproducibility.

Integration of isoelectric focusing with multi-channel gel electrophoresis by using microfluidic pseudo-valves.

Two-dimensional (2D) protein separation is achieved in a plastic microfluidic device by integrating isoelectric focusing (IEF) with multi-channel polyacrylamide gel electrophoresis (PAGE). IEF (the first dimension) is carried out in a 15 mm-long channel while PAGE (the second dimension) is in 29 parallel channels of 65 mm length that are orthogonal to the IEF channel. An array of microfluidic pseudo-valves is created for introducing different separation media, without cross-contamination, in both dimensions; it also allows transfer of proteins from the first to the second dimension. Fabrication of pseudo-valves is achieved by photo-initiated, in situ gel polymerization; acrylamide and methylenebisacrylamide monomers are polymerized only in the PAGE channels whereas polymerization does not take place in the IEF channel where a mask is placed to block the UV light. IEF separation medium, carrier ampholytes, can then be introduced into the IEF channel. The presence of gel pseudo-valves does not affect the performance of IEF or PAGE when they are investigated separately. Detection in the device is achieved by using a laser induced fluorescence imaging system. Four fluorescently-labeled proteins with either similar pI values or close molecular weight are well separated, demonstrating the potential of the 2D electrophoresis device. The total separation time is less than 10 minutes for IEF and PAGE, an improvement of 2 orders of magnitude over the conventional 2D slab gel electrophoresis.

Fluorescent detection of peptides and amino acids for capillary electrophoresis via on-line derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole.

An in-capillary derivatization of amino acids and peptides with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed for their subsequent capillary electrophoretic analysis with laser-induced fluorescence detection (lambda (ex)=488 nm). The in-capillary derivatization was achieved in zone-passing mode by introducing successive plugs of sample and NBD-F into a fused silica capillary previously equilibrated with an alkaline borate buffer. To prevent NBD-F hydrolysis and to achieve a reliable derivatization, NBD-F was prepared daily in absolute ethanol and a plug of absolute ethanol was introduced between the sample and NBD-F reagent plugs. Various parameters influencing the derivatization efficiency were investigated and the optimum conditions were as follows: background electrolyte (BGE), 20 mM borate buffer (pH 8.8); introduction time, 4 s for sample and 2 s for NBD-F; molar ratio of NBD-F/sample, above 215; temperature, 45 degrees C for amino acids and 35 degrees C for peptides; applied voltage, +15 kV. The validation of the in-capillary derivatization method under optimal conditions showed a good linearity between the heights of the derivative peaks and the concentrations of the amino acids. The intra-day relative standard deviations of the migration times and the peak heights were less than 1.3% and 4.6%, respectively. The efficient derivatization and separation of a mixture of valine, alanine, glutamic acid and aspartic acid were achieved using this technique. Peptides such as buccaline and beta-protein fragment 1-42 could also be derivatized using the developed in-capillary derivatization procedure.

Poly(N,N-dimethylacrylamide)-grafted polyacrylamide: a self-coating copolymer for sieving separation of native proteins by CE.

The potential of a series of newly synthesized poly(N,N-dimethylacrylamide) (PDMA) grafted polyacrylamide (PAM) copolymers (P(AM-PDMA)) as a replaceable separation medium for protein analysis was studied. A comparative study with and without copolymers was performed; the separation efficiency, analysis reproducibility and protein recovery proved that the P(AM-PDMA) copolymers were efficient in suppressing the adsorption of basic proteins onto the silica capillary wall. Furthermore, the size-dependent retardation of native proteins in a representative P(AM-PDMA) copolymer was demonstrated by Ferguson analysis. The results showed that the P(AM-PDMA) copolymers combine the good coating property of PDMA and the sieving property of PAM and could be applied as a sieving matrix for the analysis of native proteins.

Non-aqueous capillary electrophoresis for simultaneous separation and determination of three major active components in traditional medicinal preparations.

A simple and sensitive non-aqueous capillary electrophoresis method has been developed for simultaneous assay of three bioactive components (puerarin, daidzein and wogonin) in three traditional medicinal preparations for the first time. Optimum separation of the analytes was obtained on a 47 cm x 75 microm i.d. capillary using a non-aqueous buffer system of 20% acetonitrile, 25 mm ammonium acetate and apparent pH 9.00, with applied voltage and capillary temperature of 20 kV and 16 degrees C, respectively. The relative standard deviations (RSDs) of the migration times and the peak areas of the three analytes were in the ranges 2.5--4.0% and 3.2--3.9%, respectively. Detection limits of puerarin, daidzein and wogonin were 0.090, 0.145 and 0.090 microg mL(-1), respectively. In the tested concentration range, good linear relationships (correlation coef fi cients: 0.9998 for puerarin, 0.9998 for daidzein and 0.9978 for wogonin) between peak areas and concentrations of the analytes were observed. This method has been successfully applied to simultaneous determination of the three bioactive components with recoveries from 91.0 to 114.0%.