Comparative effects of nisin and monensin supplementation on growth performance, rumen fermentation, nutrient digestion, and plasma metabolites of fattening Hu sheep.

Introduction: This study was conducted to compare the effects of nisin (NIS) and ionophore antibiotic monensin (MON) on the growth performance, rumen fermentation, nutrient digestion and plasma metabolites of fattening Hu sheep. Methods: Thirty-six male Hu sheep (23.5 ± 1.0 kg) were divided into two blocks based on BW (low BW and high BW). Sheep within each block were then allotted to 9 pens respectively (two sheep/pen). Pens within each block were randomly assigned to one of three dietary treatments: (1) basal diet (CON); (2) basal diet + 40 mg/kg DM of MON; (3) basal diet + 274.5 mg/kg DM of NIS. The study lasted 9 weeks, with the initial 2 weeks for adaptation and the subsequent 7 weeks for treatment. Results: The results showed that both NIS and MON addition had no impacts on average daily gain (ADG), dry matter intake (DMI), and feed conservation rate (G:F) of sheep (p > 0.05). The digestibility of ether extract (EE) was lower in the MON-fed and NIS-fed sheep (p < 0.01) than in the CON group, whereas crude protein (CP) digestibility was higher in the MON-fed sheep compared to those fed NIS (p < 0.05). Both NIS and MON supplementation decreased acetate levels and acetate/propionate ratio in the rumen of Hu sheep (p < 0.05). Sheep fed MON exhibited higher total cholesterol concentrations (p < 0.05) compared to the CON and NIS groups. However, there were no significant differences in other plasma metabolites, including blood urea nitrogen (BUN), total bile acid, triglyceride, total protein, albumin, globulin, glucose, etc., among the three groups (p > 0.05). Discussion: In conclusion, dietary addition of NIS and MON altered the rumen fermentation mode by reducing acetate levels, with no discernible effects on the growth performance of the fattening Hu sheep.

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.

Infant age inversely correlates with gut carriage of resistance genes, reflecting modifications in microbial carbohydrate metabolism during early life.

The infant gut microbiome is increasingly recognized as a reservoir of antibiotic resistance genes, yet the assembly of gut resistome in infants and its influencing factors remain largely unknown. We characterized resistome in 4132 metagenomes from 963 infants in six countries and 4285 resistance genes were observed. The inherent resistome pattern of healthy infants (N = 272) could be distinguished by two stages: a multicompound resistance phase (Months 0-7) and a tetracycline-mupirocin-ß-lactam-dominant phase (Months 8-14). Microbial taxonomy explained 40.7% of the gut resistome of healthy infants, with Escherichia (25.5%) harboring the most resistance genes. In a further analysis with all available infants (N = 963), we found age was the strongest influencer on the resistome and was negatively correlated with the overall resistance during the first 3 years (p < 0.001). Using a random-forest approach, a set of 34 resistance genes could be used to predict age (R 2 = 68.0%). Leveraging microbial host inference analyses, we inferred the age-dependent assembly of infant resistome was a result of shifts in the gut microbiome, primarily driven by changes in taxa that disproportionately harbor resistance genes across taxa (e.g., Escherichia coli more frequently harbored resistance genes than other taxa). We performed metagenomic functional profiling and metagenomic assembled genome analyses whose results indicate that the development of gut resistome was driven by changes in microbial carbohydrate metabolism, with an increasing need for carbohydrate-active enzymes from Bacteroidota and a decreasing need for Pseudomonadota during infancy. Importantly, we observed increased acquired resistance genes over time, which was related to increased horizontal gene transfer in the developing infant gut microbiome. In summary, infant age was negatively correlated with antimicrobial resistance gene levels, reflecting a composition shift in the gut microbiome, likely driven by the changing need for microbial carbohydrate metabolism during early life.

Nutrigenetic and Epigenetic Mechanisms of Maternal Nutrition-Induced Glucolipid Metabolism Changes in the Offspring.

Maternal nutrition during pregnancy regulates the offspring's metabolic homeostasis, including insulin sensitivity and the metabolism of glucose and lipids. The fetus undergoes a crucial period of plasticity in the uterus; metabolic changes in the fetus during pregnancy caused by maternal nutrition not only influence fetal growth and development but also have a long-term or even life-long impact for the offspring. Epigenetic modifications, such as DNA methylation, histone modification, and non-coding RNAs, play important roles in intergenerational and transgenerational effects. In this context, this narrative review comprehensively summarizes and analyzes the molecular mechanisms underlying how maternal nutrition, including a high-fat diet, polyunsaturated fatty acid diet, methyl donor nutrient supplementation, feed restriction, and protein restriction during pregnancy, impacts the genes involved in glucolipid metabolism in the liver, adipose tissue, hypothalamus, muscle, and oocytes of the offspring in terms of the epigenetic modifications. This will provide a foundation for the further exploration of nutrigenetic and epigenetic mechanisms for integrative mother-child nutrition and promotion of the offspring's health through the regulation of maternal nutrition during pregnancy. Note: This paper is part of the Nutrition Reviews Special Collection on Precision Nutrition.

Acid-base properties of non-protein nitrogen affect nutrients intake, rumen fermentation and antioxidant capacity of fattening Hu sheep.

This study conducted a comparison of the effects of non-protein nitrogen with different acid-base properties on feed intake, rumen fermentation, nutrient digestion and antioxidant capacity in fattening Hu sheep. Sixteen fattening male sheep (31.43 ± 2.41 kg) with permanent rumen cannulas were randomly assigned to two dietary treatments: 1% urea and 1.78% ammonium chloride (NH4Cl, AC). A 42 days experimental period was conducted, with 14 days for adaptation and 28 days for treatment. Daily feed intake was recorded and various samples including feed, feces, rumen fluid, and blood were collected at different time points during the final week. The results indicated that the urea group had significantly higher dry matter intake, average daily gain, and gain efficiency in comparison to the AC group (p < 0.01). There was no difference in rumen pH and concentration of ammonia nitrogen between different groups (p > 0.05), but the rumen pH of urea group was higher than that of the AC group at 1 and 3 h after feeding (p < 0.05). The urea group exhibited higher concentrations of total volatile fatty acids (VFA) and individual VFAs compared to the AC group at all-time points (p < 0.01). Compared to the urea group, the intake of all nutrients decreased in the AC group (p < 0.01), but the digestibility of dry matter and organic matter increased significantly (p < 0.01), and the digestibility of CP had an increasing trend (p = 0.06) in the AC group. Additionally, the urea group had lower levels of serum glucagon-like peptide-1, peptide YY, Cl, total protein and globulin than the AC group (p < 0.05). The overall levels of HCO3-, superoxide dismutase, glutathione peroxidase, catalase, albumin/globulin, blood urea nitrogen and total cholesterol in the urea group increased significantly compared to the AC group (p < 0.05). It was concluded that adding urea to the high-concentrate diet resulted in increased rumen pH and improved rumen fermentation and growth performance in fattening sheep compared to NH4Cl addition. Furthermore, urea addition improved sheep's antioxidant capacity and maintained their acid-base balance more effectively as compared to NH4Cl.

Fructo-oligosaccharide supplementation enhances the growth of nursing dairy calves while stimulating the persistence of Bifidobacterium and hindgut microbiome's maturation.

The colonization and development of the gut microbiome in dairy calves play a crucial role in their overall health and future productivity. Despite the widely proposed benefits of inulin-related products on the host, there is insufficient information about how supplementing fructo-oligosaccharides (FOS) affects the colonization and development of the gut microbiome in calves. In a randomized intervention trial involving newborn male Holstein dairy calves, we investigated the effect of FOS on the calf hindgut microbiome, short-chain fatty acids (SCFA), growth performance, and the incidence of diarrhea. The daily administration of FOS exhibited a time-dependent increase in the ADG and the concentration of SCFA. Concurrently, FOS delayed the natural decline of Bifidobacterium, promoting the maturation and stabilization of the hindgut microbiome. These findings not only contribute to a theoretical understanding of the judicious application of prebiotics but also hold significant practical implications for the design of early life dietary interventions in the rearing of dairy calves.

AMPK Suppression Due to Obesity Drives Oocyte mtDNA Heteroplasmy via ATF5-POLG Axis.

Due to the exclusive maternal transmission, oocyte mitochondrial dysfunction reduces fertility rates, affects embryonic development, and programs offspring to metabolic diseases. However, mitochondrial DNA (mtDNA) are vulnerable to mutations during oocyte maturation, leading to mitochondrial nucleotide variations (mtSNVs) within a single oocyte, referring to mtDNA heteroplasmy. Obesity (OB) accounts for more than 40% of women at the reproductive age in the USA, but little is known about impacts of OB on mtSNVs in mature oocytes. It is found that OB reduces mtDNA content and increases mtSNVs in mature oocytes, which impairs mitochondrial energetic functions and oocyte quality. In mature oocytes, OB suppresses AMPK activity, aligned with an increased binding affinity of the ATF5-POLG protein complex to mutated mtDNA D-loop and protein-coding regions. Similarly, AMPK knockout increases the binding affinity of ATF5-POLG proteins to mutated mtDNA, leading to the replication of heteroplasmic mtDNA and impairing oocyte quality. Consistently, AMPK activation blocks the detrimental impacts of OB by preventing ATF5-POLG protein recruitment, improving oocyte maturation and mitochondrial energetics. Overall, the data uncover key features of AMPK activation in suppressing mtSNVs, and improving mitochondrial biogenesis and oocyte maturation in obese females.

Early-life ruminal microbiome-derived indole-3-carboxaldehyde and prostaglandin D2 are effective promoters of rumen development.

BACKGROUND: The function of diverse ruminal microbes is tightly linked to rumen development and host physiology. The system of ruminal microbes is an excellent model to clarify the fundamental ecological relationships among complex nutrient-microbiome-host interactions. Here, neonatal lambs are introduced to different dietary regimes to investigate the influences of early-life crosstalk between nutrients and microbiome on rumen development. RESULTS: We find starchy corn-soybean starter-fed lambs exhibit the thickest ruminal epithelia and fiber-rich alfalfa hay-fed lambs have the thickest rumen muscle. Metabolome and metagenome data reveal that indole-3-carboxaldehyde (3-IAld) and prostaglandin D2 (PGD2) are the top characteristic ruminal metabolites associated with ruminal epithelial and muscular development, which depend on the enhanced ruminal microbial synthesis potential of 3-IAld and PGD2. Moreover, microbial culture experiment first demonstrates that Bifidobacterium pseudolongum is able to convert tryptophan into 3-IAld and Candida albicans is a key producer for PGD2. Transcriptome sequencing of the ruminal epithelia and smooth muscle shows that ruminal epithelial and muscular development is accompanied by Wnt and Ca2+ signaling pathway activation. Primary cell cultures further confirm that 3-IAld promotes ruminal epithelial cell proliferation depending on AhR-wnt/ß-catenin signaling pathway and PGD2 accelerates ruminal smooth muscle cell proliferation via Ca2+ signaling pathway. Furthermore, we find that 3-IAld and PGD2 infusion promote ruminal epithelial and musculature development in lambs. CONCLUSIONS: This study demonstrates that early-life ruminal microbiome-derived 3-IAld and PGD2 are effective promoters of rumen development, which enhances our understanding of nutrient-microbiome-host interactions in early life.

Unraveling the phylogenomic diversity of Methanomassiliicoccales and implications for mitigating ruminant methane emissions.

BACKGROUND: Methanomassiliicoccales are a recently identified order of methanogens that are diverse across global environments particularly the gastrointestinal tracts of animals; however, their metabolic capacities are defined via a limited number of cultured strains. RESULTS: Here, we profile and analyze 243 Methanomassiliicoccales genomes assembled from cultured representatives and uncultured metagenomes recovered from various biomes, including the gastrointestinal tracts of different animal species. Our analyses reveal the presence of numerous undefined genera and genetic variability in metabolic capabilities within Methanomassiliicoccales lineages, which is essential for adaptation to their ecological niches. In particular, gastrointestinal tract Methanomassiliicoccales demonstrate the presence of co-diversified members with their hosts over evolutionary timescales and likely originated in the natural environment. We highlight the presence of diverse clades of vitamin transporter BtuC proteins that distinguish Methanomassiliicoccales from other archaeal orders and likely provide a competitive advantage in efficiently handling B12. Furthermore, genome-centric metatranscriptomic analysis of ruminants with varying methane yields reveal elevated expression of select Methanomassiliicoccales genera in low methane animals and suggest that B12 exchanges could enable them to occupy ecological niches that possibly alter the direction of H2 utilization. CONCLUSIONS: We provide a comprehensive and updated account of divergent Methanomassiliicoccales lineages, drawing from numerous uncultured genomes obtained from various habitats. We also highlight their unique metabolic capabilities involving B12, which could serve as promising targets for mitigating ruminant methane emissions by altering H2 flow.

Fecal microbial gene transfer contributes to the high-grain diet-induced augmentation of aminoglycoside resistance in dairy cattle.

A high-grain (HG) diet can rapidly lower the rumen pH and thus modify the gastrointestinal microbiome in dairy cattle. Although the prevalence of antibiotic resistance is strongly linked with the gut microbiome, the influences of HG diet on animals' gut resistome remain largely unexplored. Here, we examined the impact and mechanism of an HG diet on the fecal resistome in dairy cattle by metagenomically characterizing the gut microbiome. Eight lactating Holstein cattle were randomly allocated into two groups and fed either a conventional (CON) or HG diet for 3 weeks. The fecal microbiome and resistome were significantly altered in dairy cattle from HG, demonstrating an adaptive response that peaks at day 14 after the dietary transition. Importantly, we determined that feeding an HG diet specifically elevated the prevalence of resistance to aminoglycosides (0.11 vs 0.24 RPKG, P < 0.05). This diet-induced resistance increase is interrelated with the disproportional propagation of microbes in Lachnospiraceae, indicating a potential reservoir of aminoglycosides resistance. We further showed that the prevalence of acquired resistance genes was also modified by introducing a different diet, likely due to the augmented frequency of lateral gene transfer (LGT) in microbes (CON vs HG: 254 vs 287 taxa) such as Lachnospiraceae. Consequently, we present that diet transition is associated with fecal resistome modification in dairy cattle and an HG diet specifically enriched aminoglycosides resistance that is likely by stimulating microbial LGT.IMPORTANCEThe increasing prevalence of antimicrobial resistance is one of the most severe threats to public health, and developing novel mitigation strategies deserves our top priority. High-grain (HG) diet is commonly applied in dairy cattle to enhance animals' performance to produce more high-quality milk. We present that despite such benefits, the application of an HG diet is correlated with an elevated prevalence of resistance to aminoglycosides, and this is a combined effect of the expansion of antibiotic-resistant bacteria and increased frequency of lateral gene transfer in the fecal microbiome of dairy cattle. Our results provided new knowledge in a typically ignored area by showing an unexpected enrichment of antibiotic resistance under an HG diet. Importantly, our findings laid the foundation for designing potential dietary intervention strategies to lower the prevalence of antibiotic resistance in dairy production.

Early concentrate starter introduction induces rumen epithelial parakeratosis by blocking keratinocyte differentiation with excessive ruminal butyrate accumulation.

INTRODUCTION: Rumen epithelial parakeratosis, a common disease in ruminants caused by abnormalities in the ruminal stratified squamous epithelial keratinization process, negatively impacts ruminant health and performance. However, we still lack a comprehensive perception of the underlying mechanisms and the predisposing factors for this disorder. OBJECTIVES: Here, we investigated rumen epithelial cell heterogeneity, differentiation trajectories, and cornification to clarify the rumen epithelial keratinization process and discern the key ruminal metabolites contributing to rumen epithelial parakeratosis. METHODS: Twenty-four 14-day-old lambs were divided into three groups, including only milk feeding, milk plus alfalfa hay feeding, and milk plus corn-soybean concentrate starter feeding. At 42 days of age, the lambs were slaughtered, and rumen tissues were collected for single-cell RNA-sequencing (scRNA-seq), immunofluorescence, and quantitative real-time PCR (qRT-PCR) analyses. Ruminal fluid samples were collected for metabolomic analyses. Rumen epithelial organoid was used to verify the key ruminal metabolites contributing to parakeratosis. RESULTS: As expected, we observed that concentrate starter introduction resulted in rumen epithelial parakeratosis. Moreover, scRNA-seq analysis revealed a developmental impediment in the transition from differentiated keratinocytes to terminally differentiated keratinocytes (TDK) in lambs with concentrate starter introduction. Immunofluorescence and qRT-PCR analyses further verified the location and expression of marker genes of TDK. Metabolomic analysis showed a robust positive correlation between ruminal butyrate levels and rumen epithelial keratinization. More importantly, we successfully established a rumen organoid model capable of facilitating the study of the keratinization process in the rumen epithelia and further confirmed that high dose butyrate indeed contributed to rumen epithelial parakeratosis. CONCLUSION: Collectively, concentrate starter introduction induces ruminal epithelial parakeratosis by blocking keratinocyte differentiation with excessive ruminal butyrate accumulation in a neonatal lamb model. These findings enhance our understanding of rumen epithelial keratinization and provide valuable insights for addressing rumen epithelial parakeratosis using early nutritional intervention strategies.

Undernutrition-induced substance metabolism and energy production disorders affected the structure and function of the pituitary gland in a pregnant sheep model.

Introduction: Undernutrition spontaneously occurs in ewes during late gestation and the pituitary is an important hinge in the neurohumoral regulatory system. However, little is known about the effect of undernutrition on pituitary metabolism. Methods: Here, 10 multiparous ewes were restricted to a 30% feeding level during late gestation to establish an undernutrition model while another 10 ewes were fed normally as controls. All the ewes were sacrificed, and pituitary samples were collected to perform transcriptome, metabolome, and quantitative real-time PCR analysis and investigate the metabolic changes. Results: PCA and PLS-DA of total genes showed that undernutrition changed the total transcriptome profile of the pituitary gland, and 581 differentially expressed genes (DEGs) were identified between the two groups. Clusters of orthologous groups for eukaryotic complete genomes demonstrated that substance transport and metabolism, including lipids, carbohydrates, and amino acids, energy production and conversion, ribosomal structure and biogenesis, and the cytoskeleton were enriched by DEGs. Kyoto encyclopedia of genes and genomes pathway enrichment analysis displayed that the phagosome, intestinal immune network, and oxidative phosphorylation were enriched by DEGs. Further analysis found that undernutrition enhanced the lipid degradation and amino acid transport, repressing lipid synthesis and transport and amino acid degradation of the pituitary gland. Moreover, the general metabolic profiles and metabolic pathways were affected by undernutrition, repressing the 60S, 40S, 28S, and 39S subunits of the ribosomal structure for translation and myosin and actin synthesis for cytoskeleton. Undernutrition was found also to be implicated in the suppression of oxidative phosphorylation for energy production and conversion into a downregulation of genes related to T cell function and the immune response and an upregulation of genes involved in inflammatory reactions enriching phagosomes. Discussion: This study comprehensively analyses the effect of undernutrition on the pituitary gland in a pregnant sheep model, which provides a foundation for further research into the mechanisms of undernutrition-caused hormone secretion and metabolic disorders.

Sex differences in paternal arsenic-induced intergenerational metabolic effects are mediated by estrogen.

BACKGROUND: Gene-environment interactions contribute to metabolic disorders such as diabetes and dyslipidemia. In addition to affecting metabolic homeostasis directly, drugs and environmental chemicals can cause persistent alterations in metabolic portfolios across generations in a sex-specific manner. Here, we use inorganic arsenic (iAs) as a prototype drug and chemical to dissect such sex differences. METHODS: After weaning, C57BL/6 WT male mice were treated with 250 ppb iAs in drinking water (iAsF0) or normal water (conF0) for 6 weeks and then bred with 15-week-old, non-exposed females for 3 days in cages with only normal water (without iAs), to generate iAsF1 or conF1 mice, respectively. F0 females and all F1 mice drank normal water without iAs all the time. RESULTS: We find that exposure of male mice to 250 ppb iAs leads to glucose intolerance and insulin resistance in F1 female offspring (iAsF1-F), with almost no change in blood lipid profiles. In contrast, F1 males (iAsF1-M) show lower liver and blood triglyceride levels than non-exposed control, with improved glucose tolerance and insulin sensitivity. The liver of F1 offspring shows sex-specific transcriptomic changes, with hepatocyte-autonomous alternations of metabolic fluxes in line with the sex-specific phenotypes. The iAsF1-F mice show altered levels of circulating estrogen and follicle-stimulating hormone. Ovariectomy or liver-specific knockout of estrogen receptor α/ß made F1 females resemble F1 males in their metabolic responses to paternal iAs exposure. CONCLUSIONS: These results demonstrate that disrupted reproductive hormone secretion in alliance with hepatic estrogen signaling accounts for the sex-specific intergenerational effects of paternal iAs exposure, which shed light on the sex disparities in long-term gene-environment interactions.

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.

Effects of Fumarate and Nitroglycerin on In Vitro Rumen Fermentation, Methane and Hydrogen Production, and on Microbiota.

This study aimed to investigate the effects of fumarate and nitroglycerin on rumen fermentation, methane and hydrogen production, and microbiota. In vitro rumen fermentation was used in this study with four treatment groups: control (CON), fumarate (FA), nitroglycerin (NG) and fumarate plus nitroglycerin (FN). Real-time PCR and 16S rRNA gene sequencing were used to analyze microbiota. The results showed that nitroglycerin completely inhibited methane production and that this resulted in hydrogen accumulation. Fumarate decreased the hydrogen accumulation and improved the rumen fermentation parameters. Fumarate increased the concentration of propionate and microbial crude protein, and decreased the ratio of acetate to propionate in FN. Fumarate, nitroglycerin and their combination did not affect the abundance of bacteria, protozoa and anaerobic fungi, but altered archaea. The PCoA showed that the bacterial (Anosim, R = 0.747, p = 0.001) and archaeal communities (Anosim, R = 0.410, p = 0.005) were different among the four treatments. Compared with CON, fumarate restored Bacteroidetes, Firmicutes, Spirochaetae, Actinobacteria, Unclassified Ruminococcaceae, Streptococcus, Treponema and Bifidobacterium in relative abundance in FN, but did not affect Succinivibrio, Ruminobacter and archaeal taxa. The results indicated that fumarate alleviated the depressed rumen fermentation caused by the inhibition of methanogenesis by nitroglycerin. This may potentially provide an alternative way to use these chemicals to mitigate methane emission in ruminants.

Conjugative transfer of streptococcal prophages harboring antibiotic resistance and virulence genes.

Prophages play important roles in the transduction of various functional traits, including virulence factors, but remain debatable in harboring and transmitting antimicrobial resistance genes (ARGs). Herein we characterize a prevalent family of prophages in Streptococcus, designated SMphages, which harbor twenty-five ARGs that collectively confer resistance to ten antimicrobial classes, including vanG-type vancomycin resistance locus and oxazolidinone resistance gene optrA. SMphages integrate into four chromosome attachment sites by utilizing three types of integration modules and undergo excision in response to phage induction. Moreover, we characterize four subtypes of Alp-related surface proteins within SMphages, the lethal effects of which are extensively validated in cell and animal models. SMphages transfer via high-frequency conjugation that is facilitated by integrative and conjugative elements from either donors or recipients. Our findings explain the widespread of SMphages and the rapid dissemination of ARGs observed in members of the Streptococcus genus.

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.

Effects of high-grain diet feeding on fatty acid profiles in milk, blood, muscle, and adipose tissue, and transcriptional expression of lipid-related genes in muscle and adipose tissue of dairy cows.

BACKGROUND: High-grain (HG) diets affect lipid metabolism in the liver and mammary tissue of dairy cows, but its effects on muscle and adipose tissue have not been wide evaluated. Thus, the aim of this study is to clarify this issue. METHODS: Twelve Holstein cows were randomly divided into two groups: conventional diet group (CON, n = 6) and the HG diet group (n = 6). On day 7 of week 4, rumen fluid was sampled to measure pH, milk was sampled to measure components, and blood was sampled to measure biochemical parameters and fatty acid composition. After the experiment, cows were slaughtered to collect muscle and adipose tissue for fatty acid composition and transcriptome analysis. RESULTS: HG feeding decreased the ruminal pH, milk's fat content and long-chain fatty acid proportion (P < 0.05) and increased the proportion of short- and medium-chain fatty acids in the milk (P < 0.05) as compared with CON diets. The concentrations of blood cholesterol, low-density lipoprotein, and polyunsaturated fatty acids in the HG cows were lower than those in CON cows (P < 0.05). In muscle tissue, HG feeding tended to increase the triacylglycerol (TG) concentration (P < 0.10). Transcriptome analysis revealed changes in the biosynthesis of the unsaturated fatty acids pathway, the regulation of lipolysis in the adipocytes pathway, and the PPAR signalling pathway. In adipose tissue, HG feeding increased the concentration of TG and decreased the concentration of C18:1 cis9 (P < 0.05). At the transcriptome level, the fatty acid biosynthesis pathway, linoleic acid metabolism pathway, and PPAR signalling pathway were activated. CONCLUSION: HG feeding leads to subacute rumen acidosis and a decreased milk fat content. The fatty acid profiles in the milk and plasma of dairy cows were changed by HG feeding. In muscle and adipose tissue, HG feeding increased TG concentration and up-regulated the expression of genes related to adipogenesis, while down-regulated the expression of genes related to lipid transport. These results complement our knowledge of the fatty acid composition of muscle and adipose tissue in dairy cows and expand our understanding of the mechanisms by which HG diets affect lipid metabolism in muscle and adipose tissue.

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.