Associating changes in the bacterial community of rumen and faeces and milk fatty acid profiles in dairy cows fed high-starch or starch and oil-supplemented diets.

The experiment reported in this research paper aimed to evaluate the effects of high-starch or starch and oil-supplemented diets on rumen and faecal bacteria, and explore links between the structure of bacterial communities and milk fatty acid (FA) profiles. We used four Holstein dairy cows in a 4 × 4 Latin square design. Cows were fed a diet rich in cereals (high-starch diet with 23% starch content on dry matter (DM) basis), a diet supplemented with saturated FA from Ca salts of palm oil + 18% DM starch, a diet with high content of monounsaturated FA (from extruded rapeseeds) + 18% DM starch or a diet rich in polyunsaturated FA (from extruded sunflower seeds) + 17% DM starch. At the end of each experimental period, cows were sampled for rumen and faecal contents, which were used for DNA extraction and amplicon sequencing. Partial least squares (PLS) regression analysis highlighted diet-related changes in both rumen and faecal bacterial structures. Sparse PLS discriminant analysis was further employed to identify biologically relevant operational taxonomical units (OTUs) driving these differences. Our results show that Butyrivibrio discriminated the high-starch diet and linked positively with higher concentrations of milk odd- and branched-chain FA. YS2-related OTUs were key taxa distinguishing diets supplemented with Ca salts of palm oil or sunflower seeds and correlated positively with linoleic acid in milk. Similarly, diets modulated faecal bacterial composition. However, correlations between changes in faecal and rumen bacteria were poor. With this work, we demonstrated that high-starch or lipid-supplemented diets affect rumen and faecal bacterial community structure, and these changes could have a knock-on effect on milk FA profiles.

Link-HD: a versatile framework to explore and integrate heterogeneous microbial communities.

MOTIVATION: We present Link-HD, an approach to integrate multiple datasets. Link-HD is a generalization of 'Structuration des Tableaux A Trois Indices de la Statistique-Analyse Conjointe de Tableaux', a family of methods designed to integrate information from heterogeneous data. Here, we extend the classical approach to deal with broader datasets (e.g. compositional data), methods for variable selection and taxon-set enrichment analysis. RESULTS: The methodology is demonstrated by integrating rumen microbial communities from cows for which methane yield (CH4y) was individually measured. Our approach reproduces the significant link between rumen microbiota structure and CH4 emission. When analyzing the TARA's ocean data, Link-HD replicates published results, highlighting the relevance of temperature with members of phyla Proteobacteria on the structure and functionality of this ecosystem. AVAILABILITY AND IMPLEMENTATION: The source code, examples and a complete manual are freely available in GitHub https://github.com/lauzingaretti/LinkHD and in Bioconductor https://bioconductor.org/packages/release/bioc/html/LinkHD.html.

Milk metabolome reveals variations on enteric methane emissions from dairy cows fed a specific inhibitor of the methanogenesis pathway.

Metabolome profiling in biological fluids is an interesting approach for exploring markers of methane emissions in ruminants. In this study, a multiplatform metabolomics approach was used for investigating changes in milk metabolic profiles related to methanogenesis in dairy cows. For this purpose, 25 primiparous Holstein cows at similar lactation stage were fed the same diet supplemented with (treated, n = 12) or without (control, n = 13) a specific antimethanogenic additive that reduced enteric methane production by 23% with no changes in intake, milk production, and health status. The study lasted 6 wk, with sampling and measures performed in wk 5 and 6. Milk samples were analyzed using 4 complementary analytical methods, including 2 untargeted (nuclear magnetic resonance and liquid chromatography coupled to a quadrupole-time-of-flight mass spectrometer) and 2 targeted (liquid chromatography-tandem mass spectrometry and gas chromatography coupled to a flame ionization detector) approaches. After filtration, variable selection and normalization data from each analytical platform were then analyzed using multivariate orthogonal partial least square discriminant analysis. All 4 analytical methods were able to differentiate cows from treated and control groups. Overall, 38 discriminant metabolites were identified, which affected 10 metabolic pathways including methane metabolism. Some of these metabolites such as dimethylsulfoxide, dimethylsulfone, and citramalic acid, detected by nuclear magnetic resonance or liquid chromatography-mass spectrometry methods, originated from the rumen microbiota or had a microbial-host animal co-metabolism that could be associated with methanogenesis. Also, discriminant milk fatty acids detected by targeted gas chromatography were mostly of ruminal microbial origin. Other metabolites and metabolic pathways significantly affected were associated with AA metabolism. These findings provide new insight on the potential role of milk metabolites as indicators of enteric methane modifications in dairy cows.

Identification of rumen microbial biomarkers linked to methane emission in Holstein dairy cows.

Mitigation of greenhouse gas emissions is relevant for reducing the environmental impact of ruminant production. In this study, the rumen microbiome from Holstein cows was characterized through a combination of 16S rRNA gene and shotgun metagenomic sequencing. Methane production (CH4 ) and dry matter intake (DMI) were individually measured over 4-6 weeks to calculate the CH4 yield (CH4 y = CH4 /DMI) per cow. We implemented a combination of clustering, multivariate and mixed model analyses to identify a set of operational taxonomic unit (OTU) jointly associated with CH4 y and the structure of ruminal microbial communities. Three ruminotype clusters (R1, R2 and R3) were identified, and R2 was associated with higher CH4 y. The taxonomic composition on R2 had lower abundance of Succinivibrionaceae and Methanosphaera, and higher abundance of Ruminococcaceae, Christensenellaceae and Lachnospiraceae. Metagenomic data confirmed the lower abundance of Succinivibrionaceae and Methanosphaera in R2 and identified genera (Fibrobacter and unclassified Bacteroidales) not highlighted by metataxonomic analysis. In addition, the functional metagenomic analysis revealed that samples classified in cluster R2 were overrepresented by genes coding for KEGG modules associated with methanogenesis, including a significant relative abundance of the methyl-coenzyme M reductase enzyme. Based on the cluster assignment, we applied a sparse partial least-squares discriminant analysis at the taxonomic and functional levels. In addition, we implemented a sPLS regression model using the phenotypic variation of CH4 y. By combining these two approaches, we identified 86 discriminant bacterial OTUs, notably including families linked to CH4 emission such as Succinivibrionaceae, Ruminococcaceae, Christensenellaceae, Lachnospiraceae and Rikenellaceae. These selected OTUs explained 24% of the CH4 y phenotypic variance, whereas the host genome contribution was ~14%. In summary, we identified rumen microbial biomarkers associated with the methane production of dairy cows; these biomarkers could be used for targeted methane-reduction selection programmes in the dairy cattle industry provided they are heritable.

Indirect measures of methane emissions of Sahelian zebu cattle in West Africa, role of environment and management.

In Northern Senegal, traditional cattle management systems (TRAD) which depend on natural forages coexist with intensified systems (INT) which rely on periodic supplementation with crop residues and local concentrates. This study aims to estimate the effects of seasons and management systems on the methane emissions of Gobra zebu, in relation to the diet's chemical composition and feed intake. Six Gobra zebu cows per management system were individually monitored over 10 months, diet and faeces were sampled each season and their chemical composition and dry matter intake (DMI) were predicted by near infrared spectroscopy. Each diet was fermented in vitro to assess methane production and volatile fatty acid concentration. The DMI and digestible organic matter intake (DOMI) decreased (P < 0.0001) during the dry seasons for both systems in the same range, but INT improved the crude protein of the diets (P < 0.0001). Enteric methane production (mmol.g-1 dry matter) was lower for TRAD than INT, except during the rainy season when TRAD cows experienced a higher increase (P = 0.002). The methanogenic potential (methane production in vitro × DMI) varied with the seasons and the system with more accentuated variations for TRAD (P < 0.0001). Methanogenic potential shows true reflection of the effects of the seasons and management systems. The results highlight that enteric methane emissions varied with seasonal changes and that intensifying the diet induced no mitigating effect.

Changes in the Rumen Microbiota of Cows in Response to Dietary Supplementation with Nitrate, Linseed, and Saponin Alone or in Combination.

Dietary supplementation with linseed, saponins, and nitrate is a promising methane mitigation strategy in ruminant production. Here, we aimed to assess the effects of these additives on the rumen microbiota in order to understand underlying microbial mechanisms of methane abatement. Two 2-by-2 factorial design studies were conducted simultaneously, which also allowed us to make a broad-based assessment of microbial responses. Eight nonlactating cows were fed diets supplemented with linseed or saponin in order to decrease hydrogen production and nitrate to affect hydrogen consumption; also, combinations of linseed plus nitrate or saponin plus nitrate were used to explore the interaction between dietary treatments. Previous work assessed effects on methane and fermentation patterns. Rumen microbes were studied by sequencing 18S and 16S rRNA genes and ITS1 amplicons. Methanogen activity was monitored by following changes in mcrA transcript abundance. Nitrate fed alone or in combination in both studies dramatically affected the composition and structure of rumen microbiota, although impacts were more evident in one of the studies. Linseed moderately modified only bacterial community structure. Indicator operational taxonomic unit (OTU) analysis revealed that both linseed and nitrate reduced the relative abundance of hydrogen-producing Ruminococcaceae Linseed increased the proportion of bacteria known to reduce succinate to propionate, whereas nitrate supplementation increased nitrate-reducing bacteria and decreased the metabolic activity of rumen methanogens. Saponins had no effect on the microbiota. Inconsistency found between the two studies with nitrate supplementation could be explained by changes in microbial ecosystem functioning rather than changes in microbial community structure.IMPORTANCE This study aimed at identifying the microbial mechanisms of enteric methane mitigation when linseed, nitrate, and saponins were fed to nonlactating cows alone or in a combination. Hydrogen is a limiting factor in rumen methanogenesis. We hypothesized that linseed and saponins would affect hydrogen producers and nitrate would affect hydrogen consumption, leading to reduced methane production in the rumen. Contrary to what was predicted, both linseed and nitrate had a deleterious effect on hydrogen producers; linseed also redirected hydrogen consumption toward propionate production, whereas nitrate stimulated the growth of nitrate-reducing and, hence, hydrogen-consuming bacterial taxa. This novel knowledge of microbial mechanisms involved in rumen methanogenesis provides insights for the development and optimization of methane mitigation strategies.

Recent Advances in Enteric Methane Mitigation and the Long Road to Sustainable Ruminant Production.

Mitigation of methane emission, a potent greenhouse gas, is a worldwide priority to limit global warming. A substantial part of anthropogenic methane is emitted by the livestock sector, as methane is a normal product of ruminant digestion. We present the latest developments and challenges ahead of the main efficient mitigation strategies of enteric methane production in ruminants. Numerous mitigation strategies have been developed in the last decades, from dietary manipulation and breeding to targeting of methanogens, the microbes that produce methane. The most recent advances focus on specific inhibition of key enzymes involved in methanogenesis. But these inhibitors, although efficient, are not affordable and not adapted to the extensive farming systems prevalent in low- and middle-income countries. Effective global mitigation of methane emissions from livestock should be based not only on scientific progress but also on the feasibility and accessibility of mitigation strategies.

Microbiota members from body sites of dairy cows are largely shared within individual hosts throughout lactation but sharing is limited in the herd.

BACKGROUND: Host-associated microbes are major determinants of the host phenotypes. In the present study, we used dairy cows with different scores of susceptibility to mastitis with the aim to explore the relationships between microbiota composition and different factors in various body sites throughout lactation as well as the intra- and inter-animal microbial sharing. RESULTS: Microbiotas from the mouth, nose, vagina and milk of 45 lactating dairy cows were characterized by metataxonomics at four time points during the first lactation, from 1-week pre-partum to 7 months post-partum. Each site harbored a specific community that changed with time, likely reflecting physiological changes in the transition period and changes in diet and housing. Importantly, we found a significant number of microbes shared among different anatomical sites within each animal. This was between nearby anatomic sites, with up to 32% of the total number of Amplicon Sequence Variants (ASVs) of the oral microbiota shared with the nasal microbiota but also between distant ones (e.g. milk with nasal and vaginal microbiotas). In contrast, the share of microbes between animals was limited (< 7% of ASVs shared by more than 50% of the herd for a given site and time point). The latter widely shared ASVs were mainly found in the oral and nasal microbiotas. These results thus indicate that despite a common environment and diet, each animal hosted a specific set of bacteria, supporting a tight interplay between each animal and its microbiota. The score of susceptibility to mastitis was slightly but significantly related to the microbiota associated to milk suggesting a link between host genetics and microbiota. CONCLUSIONS: This work highlights an important sharing of microbes between relevant microbiotas involved in health and production at the animal level, whereas the presence of common microbes was limited between animals of the herd. This suggests a host regulation of body-associated microbiotas that seems to be differently expressed depending on the body site, as suggested by changes in the milk microbiota that were associated to genotypes of susceptibility to mastitis.

Metabolic influence of core ciliates within the rumen microbiome.

Protozoa comprise a major fraction of the microbial biomass in the rumen microbiome, of which the entodiniomorphs (order: Entodiniomorphida) and holotrichs (order: Vestibuliferida) are consistently observed to be dominant across a diverse genetic and geographical range of ruminant hosts. Despite the apparent core role that protozoal species exert, their major biological and metabolic contributions to rumen function remain largely undescribed in vivo. Here, we have leveraged (meta)genome-centric metaproteomes from rumen fluid samples originating from both cattle and goats fed diets with varying inclusion levels of lipids and starch, to detail the specific metabolic niches that protozoa occupy in the context of their microbial co-habitants. Initial proteome estimations via total protein counts and label-free quantification highlight that entodiniomorph species Entodinium and Epidinium as well as the holotrichs Dasytricha and Isotricha comprise an extensive fraction of the total rumen metaproteome. Proteomic detection of protozoal metabolism such as hydrogenases (Dasytricha, Isotricha, Epidinium, Enoploplastron), carbohydrate-active enzymes (Epidinium, Diplodinium, Enoploplastron, Polyplastron), microbial predation (Entodinium) and volatile fatty acid production (Entodinium and Epidinium) was observed at increased levels in high methane-emitting animals. Despite certain protozoal species having well-established reputations for digesting starch, they were unexpectedly less detectable in low methane emitting-animals fed high starch diets, which were instead dominated by propionate/succinate-producing bacterial populations suspected of being resistant to predation irrespective of host. Finally, we reaffirmed our abovementioned observations in geographically independent datasets, thus illuminating the substantial metabolic influence that under-explored eukaryotic populations have in the rumen, with greater implications for both digestion and methane metabolism.

Rumen microbial and fermentation characteristics are affected differently by bacterial probiotic supplementation during induced lactic and subacute acidosis in sheep.

BACKGROUND: Ruminal disbiosis induced by feeding is the cause of ruminal acidosis, a digestive disorder prevalent in high-producing ruminants. Because probiotic microorganisms can modulate the gastrointestinal microbiota, propionibacteria- and lactobacilli-based probiotics were tested for their effectiveness in preventing different forms of acidosis. RESULTS: Lactic acidosis, butyric and propionic subacute ruminal acidosis (SARA) were induced by feed chalenges in three groups of four wethers intraruminally dosed with wheat, corn or beet pulp. In each group, wethers were either not supplemented (C) or supplemented with Propionibacterium P63 alone (P) or combined with L. plantarum (Lp + P) or L. rhamnosus (Lr + P). Compared with C, all the probiotics stimulated lactobacilli proliferation, which reached up to 25% of total bacteria during wheat-induced lactic acidosis. This induced a large increase in lactate concentration, which decreased ruminal pH. During the corn-induced butyric SARA, Lp + P decreased Prevotella spp. proportion with a concomitant decrease in microbial amylase activity and total volatile fatty acids concentration, and an increase in xylanase activity and pH. Relative to the beet pulp-induced propionic SARA, P and Lr + P improved ruminal pH without affecting the microbial or fermentation characteristics. Regardless of acidosis type, denaturing gradient gel electrophoresis revealed that probiotic supplementations modified the bacterial community structure. CONCLUSION: This work showed that the effectiveness of the bacterial probiotics tested depended on the acidosis type. Although these probiotics were ineffective in lactic acidosis because of a deeply disturbed rumen microbiota, some of the probiotics tested may be useful to minimize the occurrence of butyric and propionic SARA in sheep. However, their modes of action need to be further investigated.

Rumen protozoa and methanogenesis: not a simple cause-effect relationship.

Understanding the interactions between hydrogen producers and consumers in the rumen ecosystem is important for ruminant production and methane mitigation. The present study explored the relationships between rumen protozoa, methanogens and fermentation characteristics. A total of six donor sheep harbouring (F, faunated) or not (D, defaunated) protozoa in their rumens (D animals were kept without protozoa for a period of a few months (D - ) or for more than 2 years (D+)) were used in in vitro and in vivo experiments. In vitro the absence of protozoa decreased NH3 and butyrate production and had no effect on methane. In contrast, the liquid-associated bacterial and methanogens fraction of D+ inocula produced more methane than D -  and F inoculum (P < 0·05). In vivo fermentation parameters of donor animals showed the same trend on NH3 and butyrate and showed that D+ animals were high methane emitters, while D -  were the lowest ( - 35 %). The concentration of dissolved dihydrogen measured after feeding followed the opposite trend. Methane emissions did not correlate with the relative abundance of methanogens in the rumen measured by quantitative PCR, but there was a trend for higher methanogens concentration in the solid-associated population of D+ animals compared with D -  animals. In contrast, PCR-denaturing gradient gel electrophoresis profiles of methanogens' methyl coenzyme-M reductase A gene showed a clear clustering in liquid-associated fractions for all three groups of donors but fewer differences in solid-associated fractions. These results show that the absence of protozoa may affect differently the methanogen community and methane emissions in wethers.

Impact of Tannin Supplementation on Proteolysis during Post-Ruminal Digestion in Wethers Using a Dynamic In Vitro System: A Plant (Medicago sativa) Digestomic Approach.

The aim of this study was to characterize the effects of tannins on plant protein during sheep digestion using a digestomic approach combining in vivo (rumen) conditions and an in vitro digestive system (abomasum and small intestine). Ruminal fluid from wethers infused with a tannin solution or water (control) was introduced into the digester, and protein degradation was followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Tannin infusion in the rumen led to a clear decrease in protein degradation-related fermentation end-products, whereas ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) protein was more abundant than in control wethers. In the simulated abomasum, peptidomic analysis showed more degradation products of RuBisCo in the presence of tannins. The effect of RuBisCo protection by tannins continued to impact Rubisco digestion into early-stage intestinal digestion but was no longer detectable in late-stage intestinal digestion. The peptidomics approach proved a potent tool for identifying and quantifying the type of protein hydrolyzed throughout the gastrointestinal tract.

Seven steps to enhance Open Science practices in animal science.

The Open Science movement aims at ensuring accessibility, reproducibility, and transparency of research. The adoption of Open Science practices in animal science, however, is still at an early stage. To move ahead as a field, we here provide seven practical steps to embrace Open Science in animal science. We hope that this paper contributes to the shift in research practices of animal scientists towards open, reproducible, and transparent science, enabling the field to gain additional public trust and deal with future challenges to guarantee reliable research. Although the paper targets primarily animal science researchers, the steps discussed here are also applicable to other research domains.

A Meta-analysis Describing the Effects of the Essential oils Blend Agolin Ruminant on Performance, Rumen Fermentation and Methane Emissions in Dairy Cows.

There is an increasing pressure to identify feed additives which increase productivity or decrease methane emissions. This paper aims to elucidate the effects of supplementing a specific essential oils blend Agolin® Ruminant on the productivity of dairy cows in comparison to non-treated animals. A total of 23 in vivo studies were identified in which Agolin was supplemented at 1 g/d per cow; then a meta-analysis was performed to determine the response ratio on milk yield, rumen fermentation, methane emissions and health. Results indicated that an adaptation period of at least 4 weeks of treatment is required. Whereas short-term studies showed minor and inconsistent effects of Agolin, long-term studies (>4 weeks of treatment) revealed that Agolin supplementation increases milk yield (+3.6%), fat and protein corrected milk (+4.1%) and feed efficiency (+4.4%) without further changes in milk composition and feed intake. Long-term treatment also decreased methane production per day (-8.8%), per dry matter intake (-12.9%) and per fat and protein corrected milk yield (-9.9%) without changes in rumen fermentation pattern. In conclusion, despite the mode of action is still unclear and the small number of studies considered, these findings show that Agolin represents an encouraging alternative to improve productivity in dairy cows.

The origin of N isotopic discrimination and its relationship with feed efficiency in fattening yearling bulls is diet-dependent.

Nitrogen (N) isotopic discrimination (i.e. the difference in natural 15N abundance between the animal proteins and the diet; Δ15N) is known to correlate with N use efficiency (NUE) and feed conversion efficiency (FCE) in ruminants. However, results from the literature are not always consistent across studies, likely due to isotopic discrimination pathways that may differ with the nature of diets. The objective of the present study was to assess at which level, from rumen to tissues, Δ15N originates and becomes related to NUE and FCE in fattening yearling bulls when they are fed two contrasted diets. Twenty-four Charolais yearling bulls were randomly divided into two groups and fed during 8 months, from weaning to slaughter, either 1) a high starch diet based on corn silage supplying a balanced N to energy ratio at the rumen level (starch) or 2) a high fiber diet based on grass silage supplying an excess of rumen degradable N (fiber). All animals were slaughtered and samples of different digestive pools (ruminal, duodenal, ileal and fecal contents), animal tissues (duodenum, liver and muscle), blood and urine were collected for each animal. Ruminal content was further used to isolate liquid-associated bacteria (LAB), protozoa and free ammonia, while plasma proteins were obtained from blood. All samples along with feed were analyzed for their N isotopic composition. For both diets, the digestive contribution (i.e. the N isotopic discrimination occurring before absorption) to the Δ15N observed in animal tissues accounted for 65 ± 11%, leaving only one third to the contribution of post-absorptive metabolism. Concerning the Δ15N in digestive pools, the majority of these changes occurred in the rumen (av. Δ15N = 2.12 ± 0.66‰), with only minor 15N enrichments thereafter (av. Δ15N = 2.24 ± 0.41‰), highlighting the key role of the rumen on N isotopic discrimination. A strong, significant overall relationship (n = 24) between Δ15N and FCE or NUE was found when using any post-absorptive metabolic pool (duodenum, liver, or muscle tissues, or plasma proteins; 0.52 < r < 0.73; P ≤ 0.01), probably as these pools reflect both digestive and post-absorptive metabolic phenomena. Fiber diet compared to starch diet had a lower feed efficiency and promoted higher (P ≤ 0.05) Δ15N values across all post-absorptive metabolic pools and some digestive pools (ruminal, duodenal, and ileal contents). The within-diet relationship (n = 12) between Δ15N and feed efficiency was not as strong and consistent as the overall relationship, with contrasted responses between the two diets for specific pools (diet x pool interaction; P ≤ 0.01). Our results highlight the contrasted use of N at the rumen level between the two experimental diets and suggests the need for different equations to predict FCE or NUE from Δ15N according to the type of diet. In conclusion, rumen digestion and associated microbial activity can play an important role on N isotopic discrimination so rumen effect related to diet may interfere with the relationship between Δ15N and feed efficiency in fattening yearling bulls.

Inhibition of enteric methanogenesis in dairy cows induces changes in plasma metabolome highlighting metabolic shifts and potential markers of emission.

There is scarce information on whether inhibition of rumen methanogenesis induces metabolic changes on the host ruminant. Understanding these possible changes is important for the acceptance of methane-reducing practices by producers. In this study we explored the changes in plasma profiles associated with the reduction of methane emissions. Plasma samples were collected from lactating primiparous Holstein cows fed the same diet with (Treated, n = 12) or without (Control, n = 13) an anti-methanogenic feed additive for six weeks. Daily methane emissions (CH4, g/d) were reduced by 23% in the Treated group with no changes in milk production, feed intake, body weight, and biochemical indicators of health status. Plasma metabolome analyses were performed using untargeted [nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS)] and targeted (LC-MS/MS) approaches. We identified 48 discriminant metabolites. Some metabolites mainly of microbial origin such as dimethylsulfone, formic acid and metabolites containing methylated groups like stachydrine, can be related to rumen methanogenesis and can potentially be used as markers. The other discriminant metabolites are produced by the host or have a mixed microbial-host origin. These metabolites, which increased in treated cows, belong to general pathways of amino acids and energy metabolism suggesting a systemic non-negative effect on the animal.

Analysis of the diversity of the glycoside hydrolase family 130 in mammal gut microbiomes reveals a novel mannoside-phosphorylase function.

Mannoside phosphorylases are involved in the intracellular metabolization of mannooligosaccharides, and are also useful enzymes for the in vitro synthesis of oligosaccharides. They are found in glycoside hydrolase family GH130. Here we report on an analysis of 6308 GH130 sequences, including 4714 from the human, bovine, porcine and murine microbiomes. Using sequence similarity networks, we divided the diversity of sequences into 15 mostly isofunctional meta-nodes; of these, 9 contained no experimentally characterized member. By examining the multiple sequence alignments in each meta-node, we predicted the determinants of the phosphorolytic mechanism and linkage specificity. We thus hypothesized that eight uncharacterized meta-nodes would be phosphorylases. These sequences are characterized by the absence of signal peptides and of the catalytic base. Those sequences with the conserved E/K, E/R and Y/R pairs of residues involved in substrate binding would target ß-1,2-, ß-1,3- and ß-1,4-linked mannosyl residues, respectively. These predictions were tested by characterizing members of three of the uncharacterized meta-nodes from gut bacteria. We discovered the first known ß-1,4-mannosyl-glucuronic acid phosphorylase, which targets a motif of the Shigella lipopolysaccharide O-antigen. This work uncovers a reliable strategy for the discovery of novel mannoside-phosphorylases, reveals possible interactions between gut bacteria, and identifies a biotechnological tool for the synthesis of antigenic oligosaccharides.

A catalog of microbial genes from the bovine rumen unveils a specialized and diverse biomass-degrading environment.

BACKGROUND: The rumen microbiota provides essential services to its host and, through its role in ruminant production, contributes to human nutrition and food security. A thorough knowledge of the genetic potential of rumen microbes will provide opportunities for improving the sustainability of ruminant production systems. The availability of gene reference catalogs from gut microbiomes has advanced the understanding of the role of the microbiota in health and disease in humans and other mammals. In this work, we established a catalog of reference prokaryote genes from the bovine rumen. RESULTS: Using deep metagenome sequencing we identified 13,825,880 non-redundant prokaryote genes from the bovine rumen. Compared to human, pig, and mouse gut metagenome catalogs, the rumen is larger and richer in functions and microbial species associated with the degradation of plant cell wall material and production of methane. Genes encoding enzymes catalyzing the breakdown of plant polysaccharides showed a particularly high richness that is otherwise impossible to infer from available genomes or shallow metagenomics sequencing. The catalog expands the dataset of carbohydrate-degrading enzymes described in the rumen. Using an independent dataset from a group of 77 cattle fed 4 common dietary regimes, we found that only <0.1% of genes were shared by all animals, which contrast with a large overlap for functions, i.e., 63% for KEGG functions. Different diets induced differences in the relative abundance rather than the presence or absence of genes, which explains the great adaptability of cattle to rapidly adjust to dietary changes. CONCLUSIONS: These data bring new insights into functions, carbohydrate-degrading enzymes, and microbes of the rumen to complement the available information on microbial genomes. The catalog is a significant biological resource enabling deeper understanding of phenotypes and biological processes and will be expanded as new data are made available.

Bacterial direct-fed microbials fail to reduce methane emissions in primiparous lactating dairy cows.

Direct-fed microbials (DFM) are considered as a promising technique to improve animal productivity without affecting animal health or harming the environment. The potential of three bacterial DFM to reduce methane (CH4) emissions, modulate ruminal fermentation, milk production and composition of primiparous dairy cows was examined in this study. As previous reports have shown that DFM respond differently to different diets, two contrasting diets were used in this study. Eight lactating primiparous cows were randomly divided into two groups that were fed a corn silage-based, high-starch diet (HSD) or a grass silage-based, high-fiber diet (HFD). Cows in each dietary group were randomly assigned to four treatments in a 4 × 4 Latin square design. The bacterial DFM used were selected for their proven CH4-reducing effect in vitro. Treatments included control (without DFM) and 3 DFM treatments: Propionibacterium freudenreichii 53-W (2.9 × 1010 colony forming units (CFU)/cow per day), Lactobacillus pentosus D31 (3.6 × 1011 CFU/cow per day) and Lactobacillus bulgaricus D1 (4.6 × 1010 CFU/cow per day). Each experimental period included 4 weeks of treatment and 1 week of wash-out, with measures performed in the fourth week of the treatment period. Enteric CH4 emissions were measured during 3 consecutive days using respiration chambers. Rumen samples were collected for ruminal fermentation parameters and quantitative microbial analyses. Milk samples were collected for composition analysis. Body weight of cows were recorded at the end of each treatment period. Irrespective of diet, no mitigating effect of DFM was observed on CH4 emissions in dairy cows. In contrast, Propionibacterium increased CH4 intensity by 27% (g CH4/kg milk) in cows fed HSD. There was no effect of DFM on other fermentation parameters and on bacterial, archaeal and protozoal numbers. Similarly, the effect of DFM on milk fatty acid composition was negligible. Propionibacterium and L. pentosus DFM tended to increase body weight gain with HSD. We conclude that, contrary to the effect previously observed in vitro, bacterial DFM Propionibacterium freudenreichii 53-W, Lactobacillus pentosus D31 and Lactobacillus bulgaricus D1 did not alter ruminal fermentation and failed to reduce CH4 emissions in lactating primiparous cows fed high-starch or high-fiber diets.

Methanogenic potential of tropical feeds rich in hydrolyzable tannins1,2.

The present study was carried out to determine the effect of Acacia nilotica, a tropical plant rich in hydrolyzable tannins (HT), on rumen fermentation and methane (CH4) production in vitro. We used leaves and pods from A. nilotica alone and combined. The combination of HT from A. nilotica leaves and pods and condensed tannins (CT) from Calliandra calothyrsus and Leucaena leucocephala were also evaluated to assess potential differences in biological activity between HT and CT. Four series of 24-h incubations were performed using rumen contents of 4 sheep fed a tropical grass (natural grassland based on Dichanthium spp.). A first experiment tested different levels of replacement of this tropical forage (control [CTL] without tannins) by A. nilotica leaves or pods: 0:100, 25:75, 50:50, 75:25 and 100:0. A second experiment tested the mixture of A. nilotica leaves and pods in different proportions: 100:0, 75:25, 50:50, 25:75, and 0:100. A third experiment tested the 50:50 combination of A. nilotica leaves or pods with C. calothyrsus and L. leucocephala. Acacia nilotica pods and leaves had a high content of HT (350 and 178 g/kg DM, respectively), whereas C. calothyrsus and L. leucocephala had a high content of CT (361 and 180 g/kg DM, respectively). The inclusion of HT from A. nilotica leaves and pods decreased CH4 production dose-dependently (P < 0.01). Total replacement of the CTL by A. nilotica decreased CH4 production by 64 and 55% with leaves and pods, respectively. Pods were richer in HT than leaves, but their antimethanogenic effect did not differ (P > 0.05). Although A. nilotica leaves and pods inhibited fermentation, as indicated by the lower gas production and VFA production (P < 0.01), this effect was less pronounced than for CH4. Volatile fatty acid production decreased by 12% in leaves and by 30% in pods when compared with the CTL alone. Positive associative effect was reported for VFA, when HT-rich sources and CT-rich sources were mixed. Combining the 2 sources of HT did not show associative effects on fermentation or CH4 production (P > 0.05). Hydrolyzable tannin-rich sources were more effective in suppressing methanogenesis than CT-rich sources. Our results show that HT-rich A. nilotica leaves and pods have the potential to reduce ruminal CH4 production.