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.

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.

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.

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.

Inoculation with rumen fluid in early life accelerates the rumen microbial development and favours the weaning process in goats.

BACKGROUND: Newborn ruminants possess an underdeveloped rumen which is colonized by microorganisms acquired from adult animals and the surrounding environment. This microbial transfer can be limited in dairy systems in which newborns are separated from their dams at birth. This study explores whether the direct inoculation of fresh or autoclaved rumen fluid from adult goats to newborn kids has a beneficial effect on rumen microbial development and function. RESULTS: Repetitive inoculation of young kids with fresh rumen fluid from adult goats adapted to forage (RFF) or concentrate diets (RFC) accelerated microbial colonization of the rumen during the pre-weaning period leading to high protozoal numbers, a greater diversity of bacterial (+ 234 OTUs), methanogens (+ 6 OTUs) and protozoal communities (+ 25 OTUs) than observed in control kids (CTL) without inoculation. This inoculation also increased the size of the core bacterial and methanogens community and the abundance of key rumen bacteria (Ruminococcaceae, Fibrobacteres, Veillonellaceae, Rikenellaceae, Tenericutes), methanogens (Methanobrevibacter ruminantium, Methanomicrobium mobile and Group 9), anaerobic fungi (Piromyces and Orpinomyces) and protozoal taxa (Enoploplastron, Diplodinium, Polyplastron, Ophryoscolex, Isotricha and Dasytricha) before weaning whereas CTL kids remained protozoa-free through the study. Most of these taxa were positively correlated with indicators of the rumen microbiological and physiological development (higher forage and concentrate intakes and animal growth during the post-weaning period) favoring the weaning process in RFF and RFC kids in comparison to CTL kids. Some of these microbiological differences tended to decrease during the post-weaning period, although RFF and RFC kids retained a more complex and matured rumen microbial ecosystem than CTL kids. Inoculation with autoclaved rumen fluid promoted lower development of the bacterial and protozoal communities during the pre-weaning period than using fresh inocula, but it favored a more rapid microbial development during the post-weaning than observed for CTL kids. CONCLUSIONS: This study demonstrated that inoculation of young ruminants with fresh rumen fluid from adult animals accelerated the rumen microbial colonization which was associated with an earlier rumen functional development. This strategy facilitated a smoother transition from milk to solid feed favoring the animal performance during post-weaning and minimizing stress.

Fresh Rumen Liquid Inoculant Enhances the Rumen Microbial Community Establishment in Pre-weaned Dairy Calves.

The development of the functional rumen in calves involves a complex interplay between the host and host-related microbiome. Attempts to modulate rumen microbial community establishment may therefore have an impact on weaning success, calf health, and animal performance later in life. In this experiment, we aimed to elucidate how rumen liquid inoculum from an adult cow, provided to calves during the pre-weaning period, influences the establishment of rumen bacterial, archaeal, fungal, and ciliate protozoan communities in monozygotic twin calves (n = 6 pairs). The calves were divided into treatment (T-group) and control (C-group) groups, where the T-group received fresh rumen liquid as an oral inoculum during a 2-8-week period. The C-group was not inoculated. The rumen microbial community composition was determined using bacterial and archaeal 16S ribosomal RNA (rRNA) gene, protozoal 18S rRNA gene, and fungal ITS1 region amplicon sequencing. Animal weight gain and feed intake were monitored throughout the experiment. The T-group tended to have a higher concentrate intake (Treatment: p < 0.08) and had a significantly higher weekly weight gain (Treatment: p < 0.05), but no significant difference in volatile fatty acid concentrations between the groups was observed. In the T-group, the inoculum stimulated the earlier establishment of mature rumen-related bacterial taxa, affecting significant differences between the groups until 6 weeks of age. The inoculum also increased the archaeal operational taxonomic unit (OTU) diversity (Treatment: p < 0.05) but did not affect the archaeal quantity. Archaeal communities differed significantly between groups until week 4 (p = 0.02). Due to the inoculum, ciliate protozoa were detected in the T-group in week 2, while the C-group remained defaunated until 6 weeks of age. In week 8, Eremoplastron dilobum was the dominant ciliate protozoa in the C-group and Isotricha sp. in the T-group, respectively. The Shannon diversity of rumen anaerobic fungi reduced with age (Week: p < 0.01), and community establishment was influenced by a change of diet and potential interaction with other rumen microorganisms. Our results indicate that an adult cow rumen liquid inoculum enhanced the maturation of bacterial and archaeal communities in pre-weaning calves' rumen, whereas its effect on eukaryotic communities was less clear and requires further investigation.

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.

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.

Hydrogenotrophic methanogens of the mammalian gut: Functionally similar, thermodynamically different-A modelling approach.

Methanogenic archaea occupy a functionally important niche in the gut microbial ecosystem of mammals. Our purpose was to quantitatively characterize the dynamics of methanogenesis by integrating microbiology, thermodynamics and mathematical modelling. For that, in vitro growth experiments were performed with pure cultures of key methanogens from the human and ruminant gut, namely Methanobrevibacter smithii, Methanobrevibacter ruminantium and Methanobacterium formicium. Microcalorimetric experiments were performed to quantify the methanogenesis heat flux. We constructed an energetic-based mathematical model of methanogenesis. Our model captured efficiently the dynamics of methanogenesis with average concordance correlation coefficients of 0.95 for CO2, 0.98 for H2 and 0.97 for CH4. Together, experimental data and model enabled us to quantify metabolism kinetics and energetic patterns that were specific and distinct for each species despite their use of analogous methane-producing pathways. Then, we tested in silico the interactions between these methanogens under an in vivo simulation scenario using a theoretical modelling exercise. In silico simulations suggest that the classical competitive exclusion principle is inapplicable to gut ecosystems and that kinetic information alone cannot explain gut ecological aspects such as microbial coexistence. We suggest that ecological models of gut ecosystems require the integration of microbial kinetics with nonlinear behaviours related to spatial and temporal variations taking place in mammalian guts. Our work provides novel information on the thermodynamics and dynamics of methanogens. This understanding will be useful to construct new gut models with enhanced prediction capabilities and could have practical applications for promoting gut health in mammals and mitigating ruminant methane emissions.

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.

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.

Addressing Global Ruminant Agricultural Challenges Through Understanding the Rumen Microbiome: Past, Present, and Future.

The rumen is a complex ecosystem composed of anaerobic bacteria, protozoa, fungi, methanogenic archaea and phages. These microbes interact closely to breakdown plant material that cannot be digested by humans, whilst providing metabolic energy to the host and, in the case of archaea, producing methane. Consequently, ruminants produce meat and milk, which are rich in high-quality protein, vitamins and minerals, and therefore contribute to food security. As the world population is predicted to reach approximately 9.7 billion by 2050, an increase in ruminant production to satisfy global protein demand is necessary, despite limited land availability, and whilst ensuring environmental impact is minimized. Although challenging, these goals can be met, but depend on our understanding of the rumen microbiome. Attempts to manipulate the rumen microbiome to benefit global agricultural challenges have been ongoing for decades with limited success, mostly due to the lack of a detailed understanding of this microbiome and our limited ability to culture most of these microbes outside the rumen. The potential to manipulate the rumen microbiome and meet global livestock challenges through animal breeding and introduction of dietary interventions during early life have recently emerged as promising new technologies. Our inability to phenotype ruminants in a high-throughput manner has also hampered progress, although the recent increase in "omic" data may allow further development of mathematical models and rumen microbial gene biomarkers as proxies. Advances in computational tools, high-throughput sequencing technologies and cultivation-independent "omics" approaches continue to revolutionize our understanding of the rumen microbiome. This will ultimately provide the knowledge framework needed to solve current and future ruminant livestock challenges.

Effectiveness of Interventions to Modulate the Rumen Microbiota Composition and Function in Pre-ruminant and Ruminant Lambs.

Modulating the assembly of the ruminal microbiota might have practical implications in production. We tested how an early-life dietary intervention in lambs influences the diversity and function of the ruminal microbiota during and after the intervention. Microbiota resilience during a repeated dietary intervention was also tested. The treatment, aiming to mitigate enteric methane emissions, combined garlic essential oil and linseed oil. Fifty-six lambs and their dams were allocated to two groups and treatment (T1) or placebo (C1) was drenched from birth until 10 weeks of life. Lambs were weaned at 8 weeks. From 16 to 20 weeks, lambs in each group were divided in two subgroups that received (T1-T2 and C1-T2) or not (T1-C2 and C1-C2) the same treatment. Measurements were done at 8, 14, and 20 weeks. Average daily gain was similar between groups. Methane production was reduced by treatment at 8 and 20 weeks but at 14 weeks it was similar between C1 and T1. Interestingly, early-life treated lambs displayed a numerical increase (P = 0.12) in methane emissions at 20 weeks compared with non-treated lambs. Concentration of VFA was not affected by the intervention at 8 or 14 weeks but a lower concentration was observed in T2 lambs compared with C2 at week 20. Metataxonomics (rRNA gene) revealed differences in archaeal communities between groups of lambs when treatment was applied (weeks 8 and 20); whereas, in accord with methane emissions, these differences disappeared when treatment was discontinued (week 14). Protozoal community structure was not affected by treatment. In contrast, bacterial community structure differed between treated and non-treated lambs during and after the intervention. Rumen and urine LC-MS and NMR metabolomics at week 20 separated C2 from T2 lambs and correlation analysis highlighted interactions between microbes and metabolites, notably that of methylated compounds and Methanomassiliicocceae methanogens. This study demonstrates that a long-term early-life intervention induced modifications in the composition of the rumen bacterial community that persisted after the intervention ceased with little or no effect on archaeal and protozoal communities. However, there was no persistency of the early-life intervention on methanogenesis indicating resilience for this function.

Plane of nutrition affects the phylogenetic diversity and relative abundance of transcriptionally active methanogens in the bovine rumen.

Methane generated during enteric fermentation in ruminant livestock species is a major contributor to global anthropogenic greenhouse gas emissions. A period of moderate feed restriction followed by ad libitum access to feed is widely applied in cattle management to exploit the animal's compensatory growth potential and reduce feed costs. In the present study, we utilised microbial RNA from rumen digesta samples to assess the phylogenetic diversity of transcriptionally active methanogens from feed-restricted and non-restricted animals. To determine the contribution of different rumen methanogens to methanogenesis during dietary restriction of cattle, we conducted high-throughput mcrA cDNA amplicon sequencing on an Illumina MiSeq and analysed both the abundance and phylogenetic origin of different mcrA cDNA sequences. When compared to their unrestricted contemporaries, in feed-restricted animals, the methanogenic activity, based on mcrA transcript abundance, of Methanobrevibacter gottschalkii clade increased while the methanogenic activity of the Methanobrevibacter ruminantium clade and members of the Methanomassiliicoccaceae family decreased. This study shows that the quantity of feed consumed can evoke large effects on the composition of methanogenically active species in the rumen of cattle. These data potentially have major implications for targeted CH4 mitigation approaches such as anti-methanogen vaccines and/or tailored dietary management.

The Structural and Functional Capacity of Ruminal and Cecal Microbiota in Growing Cattle Was Unaffected by Dietary Supplementation of Linseed Oil and Nitrate.

Microorganisms in the digestive tract of ruminants differ in their functionality and ability to use feed constituents. While cecal microbiota play an important role in post-rumen fermentation of residual substrates undigested in the rumen, limited knowledge exists regarding its structure and function. In this trial we investigated the effect of dietary supplementation with linseed oil and nitrate on methane emissions and on the structure of ruminal and cecal microbiota of growing bulls. Animals were allocated to either a CTL (control) or LINNIT (CTL supplemented with 1.9% linseed and 1.0% nitrates) diet. Methane emissions were measured using the GreenFeed system. Microbial diversity was assessed using amplicon sequencing of microbial genomic DNA. Additionally, total RNA was extracted from ruminal contents and functional mcrA and mtt genes were targeted in amplicon sequencing approach to explore the diversity of functional gene expression in methanogens. LINNIT had no effect on methane yield (g/kg DMI) even though it decreased methane production by 9% (g/day; P < 0.05). Methanobrevibacter- and Methanomassiliicoccaceae-related OTUs were more abundant in cecum (72 and 24%) compared to rumen (60 and 11%) irrespective of the diet (P < 0.05). Feeding LINNIT reduced the relative abundance of Methanomassiliicoccaceae mcrA cDNA reads in the rumen. Principal component analysis revealed significant differences in taxonomic composition and abundance of bacterial communities between rumen and cecum. Treatment decreased the relative abundance of a few Ruminococcaceae genera, without affecting global bacterial community structure. Our research confirms a high level of heterogeneity in species composition of microbial consortia in the main gastrointestinal compartments where feed is fermented in ruminants. There was a parallel between the lack of effect of LINNIT on ruminal and cecal microbial community structure and functions on one side and methane emission changes on the other. These results suggest that the sequencing strategy used here to study microbial diversity and function accurately reflected the absence of effect on methane phenotypes in bulls treated with linseed plus nitrate.

Rumen microbial communities influence metabolic phenotypes in lambs.

The rumen microbiota is an essential part of ruminants shaping their nutrition and health. Despite its importance, it is not fully understood how various groups of rumen microbes affect host-microbe relationships and functions. The aim of the study was to simultaneously explore the rumen microbiota and the metabolic phenotype of lambs for identifying host-microbe associations and potential biomarkers of digestive functions. Twin lambs, separated in two groups after birth were exposed to practices (isolation and gavage with rumen fluid with protozoa or protozoa-depleted) that differentially restricted the acquisition of microbes. Rumen microbiota, fermentation parameters, digestibility and growth were monitored for up to 31 weeks of age. Microbiota assembled in isolation from other ruminants lacked protozoa and had low bacterial and archaeal diversity whereas digestibility was not affected. Exposure to adult sheep microbiota increased bacterial and archaeal diversity independently of protozoa presence. For archaea, Methanomassiliicoccales displaced Methanosphaera. Notwithstanding, protozoa induced differences in functional traits such as digestibility and significantly shaped bacterial community structure, notably Ruminococcaceae and Lachnospiraceae lower up to 6 folds, Prevotellaceae lower by ~40%, and Clostridiaceae and Veillonellaceae higher up to 10 folds compared to microbiota without protozoa. An orthogonal partial least squares-discriminant analysis of urinary metabolome matched differences in microbiota structure. Discriminant metabolites were mainly involved in amino acids and protein metabolic pathways while a negative interaction was observed between methylotrophic methanogens Methanomassiliicoccales and trimethylamine N-oxide. These results stress the influence of gut microbes on animal phenotype and show the potential of metabolomics for monitoring rumen microbial functions.

Abundance, diversity and activity of sulfate-reducing prokaryotes in heavy metal-contaminated sediment from a salt marsh in the Medway Estuary (UK).

We investigated the diversity and activity of sulfate-reducing prokaryotes (SRP) in a 3.5-m sediment core taken from a heavy metal-contaminated site in the Medway Estuary, UK. The abundance of SRPs was quantified by qPCR of the dissimilatory sulfite reductase gene ß-subunit (dsrB) and taking into account DNA extraction efficiency. This showed that SRPs were abundant throughout the core with maximum values in the top 50 cm of the sediment core making up 22.4% of the total bacterial community and were 13.6% at 250 cm deep. Gene libraries for dsrA (dissimilatory sulfite reductase α-subunit) were constructed from the heavily contaminated (heavy metals) surface sediment (top 20 cm) and from the less contaminated and sulfate-depleted, deeper zone (250 cm). Certain cloned sequences were similar to dsrA found in members of the Syntrophobacteraceae, Desulfobacteraceae and Desulfovibrionaceae as well as a large fraction (60%) of novel sequences that formed a deep branching dsrA lineage. Phylogenetic analysis of metabolically active SRPs was performed by reverse transcription PCR and single strand conformational polymorphism analysis (RT-PCR-SSCP) of dsrA genes derived from extracted sediment RNA. Subsequent comparative sequence analysis of excised SSCP bands revealed a high transcriptional activity of dsrA belonging to Desulfovibrio species in the surface sediment. These results may suggest that members of the Desulfovibrionaceae are more active than other SRP groups in heavy metal-contaminated surface sediments.