Impact and legacy of the highly cited paper by Blaxter and Clapperton (1965) 'Prediction of the amount of methane produced by ruminants [Br J Nutr 19, 511-522]'.

The paper by K. L. Blaxter and J. L. Clapperton (1965) 'Prediction of the amount of methane produced by ruminants. Br J Nutr 19, 511-522' has been cited 656 times according to Web of Science and continues to be cited with increasing frequency to the present day. The analysis described in the paper, or meta-analysis as it would be known now, is of methane production from cattle and sheep based on forty-eight trials using closed-circuit respiration chambers, all carried out at the Hannah Research Institute, Ayr, UK, between 1955 and 1965. Methane emissions per unit of diet fed were shown to vary depending on diet, level of feeding and individual animal. As such, previous notions that methane emissions were essentially proportional to energy intake were set aside. The main reasons for the paper's continuing citation are the set of equations that can be used to predict methane emissions from ruminants when the technically demanding respiration chambers are unavailable, and that it was the first definitive study to describe the complexities of methane emissions with respect to animals and diets. The paper thus provided abundant insights of the relations between ruminant methane emissions and nutritional biology, and rumen microbiology, in particular, that have informed countless research projects in the intervening half-century. Given the importance of methane as a greenhouse gas in the climate change scenario, these insights are at least as relevant today as they were in 1965.

Bovine Host Genetic Variation Influences Rumen Microbial Methane Production with Best Selection Criterion for Low Methane Emitting and Efficiently Feed Converting Hosts Based on Metagenomic Gene Abundance.

Methane produced by methanogenic archaea in ruminants contributes significantly to anthropogenic greenhouse gas emissions. The host genetic link controlling microbial methane production is unknown and appropriate genetic selection strategies are not developed. We used sire progeny group differences to estimate the host genetic influence on rumen microbial methane production in a factorial experiment consisting of crossbred breed types and diets. Rumen metagenomic profiling was undertaken to investigate links between microbial genes and methane emissions or feed conversion efficiency. Sire progeny groups differed significantly in their methane emissions measured in respiration chambers. Ranking of the sire progeny groups based on methane emissions or relative archaeal abundance was consistent overall and within diet, suggesting that archaeal abundance in ruminal digesta is under host genetic control and can be used to genetically select animals without measuring methane directly. In the metagenomic analysis of rumen contents, we identified 3970 microbial genes of which 20 and 49 genes were significantly associated with methane emissions and feed conversion efficiency respectively. These explained 81% and 86% of the respective variation and were clustered in distinct functional gene networks. Methanogenesis genes (e.g. mcrA and fmdB) were associated with methane emissions, whilst host-microbiome cross talk genes (e.g. TSTA3 and FucI) were associated with feed conversion efficiency. These results strengthen the idea that the host animal controls its own microbiota to a significant extent and open up the implementation of effective breeding strategies using rumen microbial gene abundance as a predictor for difficult-to-measure traits on a large number of hosts. Generally, the results provide a proof of principle to use the relative abundance of microbial genes in the gastrointestinal tract of different species to predict their influence on traits e.g. human metabolism, health and behaviour, as well as to understand the genetic link between host and microbiome.

The rumen microbial metaproteome as revealed by SDS-PAGE.

BACKGROUND: Ruminal digestion is carried out by large numbers of bacteria, archaea, protozoa and fungi. Understanding the microbiota is important because ruminal fermentation dictates the efficiency of feed utilisation by the animal and is also responsible for major emissions of the greenhouse gas, methane. Recent metagenomic and metatranscriptomic studies have helped to elucidate many features of the composition and activity of the microbiota. The metaproteome provides complementary information to these other -omics technologies. The aim of this study was to explore the metaproteome of bovine and ovine ruminal digesta using 2D SDS-PAGE. RESULTS: Digesta samples were taken via ruminal fistulae and by gastric intubation, or at slaughter, and stored in glycerol at -80 °C. A protein extraction protocol was developed to maximise yield and representativeness of the protein content. The proteome of ruminal digesta taken from dairy cows fed a high concentrate diet was dominated by a few very highly expressed proteins, which were identified by LC-MS/MS to be structural proteins, such as actin and α- and ß-tubulins, derived from ciliate protozoa. Removal of protozoa from digesta before extraction of proteins revealed the prokaryotic metaproteome, which was dominated by enzymes involved in glycolysis, such as glyceraldehyde-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, phosphoglycerate kinase and triosephosphate isomerase. The enzymes were predominantly from the Firmicutes and Bacteroidetes phyla. Enzymes from methanogenic archaea were also abundant, consistent with the importance of methane formation in the rumen. Gels from samples from dairy cows fed a high proportion of grass silage were consistently obscured by co-staining of humic compounds. Samples from beef cattle and fattening lambs receiving a predominantly concentrate diet produced clearer gels, but the pattern of spots was inconsistent between samples, making comparisons difficult. CONCLUSION: This work demonstrated for the first time that 2D-PAGE reveals key structural proteins and enzymes in the rumen microbial community, despite its high complexity, and that taxonomic information can be deduced from the analysis. However, technical issues associated with feed material contamination, which affects the reproducibility of electrophoresis of different samples, limits its value.

Metabolism of α-linolenic acid during incubations with strained bovine rumen contents: products and mechanisms.

Description of α-linolenic acid (cis-9,cis-12,cis-15-18 : 3, ALA) metabolism in the rumen is incomplete. Ruminal digesta samples were incubated with ALA and buffer containing water or deuterium oxide to investigate the products and mechanisms of ALA biohydrogenation. Geometric Δ9,11,15-18 : 3 isomers were the main intermediates formed from ALA. An increase in the n+1 isotopomers of Δ9,11,15-18 : 3 was due to 2H labelling at C-13. Isomers of Δ9,11,13-18 : 3, cis-7,cis-12,cis-15-18 : 3 and cis-8,cis-12,cis-15-18 : 3 were also formed. No increase in n+1 isotopomers of Δ7,12,15-18 : 3 or Δ8,12,15-18 : 3 was detected. Enrichment in n+2 isotopomers of 18 : 2 products indicated that ALA metabolism continued via the reduction of 18 : 3 intermediates. Isomers of Δ9,11,15-18 : 3 were reduced to Δ11,15-18 : 2 labelled at C-9 and C-13. ALA resulted in the formation of Δ11,13-18 : 2 and Δ12,14-18 : 2 containing multiple 2H labels. Enrichment of the n+3 isotopomer of Δ12,15-18 : 2 was also detected. Metabolism of ALA during incubations with rumen contents occurs by one of three distinct pathways. Formation of Δ9,11,15-18 : 3 appears to be initiated by H abstraction on C-13. Octadecatrienoic intermediates containing cis-12 and cis-15 double bonds are formed without an apparent H exchange with water. Labelling of Δ9,11,13-18 : 3 was inconclusive, suggesting formation by an alternative mechanism. These findings explain the appearance of several bioactive fatty acids in muscle and milk that influence the nutritional value of ruminant-derived foods.

Risks associated with endotoxins in feed additives produced by fermentation.

Increasingly, feed additives for livestock, such as amino acids and vitamins, are being produced by Gram-negative bacteria, particularly Escherichia coli. The potential therefore exists for animals, consumers and workers to be exposed to possibly harmful amounts of endotoxin from these products. The aim of this review was to assess the extent of the risk from endotoxins in feed additives and to calculate how such risk can be assessed from the properties of the additive. Livestock are frequently exposed to a relatively high content of endotoxin in the diet: no additional hazard to livestock would be anticipated if the endotoxin concentration of the feed additive falls in the same range as feedstuffs. Consumer exposure will be unaffected by the consumption of food derived from animals receiving endotoxin-containing feed, because the small concentrations of endotoxin absorbed do not accumulate in edible tissues. In contrast, workers processing a dusty additive may be exposed to hazardous amounts of endotoxin even if the endotoxin concentration of the product is low. A calculation method is proposed to compare the potential risk to the worker, based on the dusting potential, the endotoxin concentration and technical guidance of the European Food Safety Authority, with national exposure limits.

The rumen microbial metagenome associated with high methane production in cattle.

BACKGROUND: Methane represents 16 % of total anthropogenic greenhouse gas emissions. It has been estimated that ruminant livestock produce ca. 29 % of this methane. As individual animals produce consistently different quantities of methane, understanding the basis for these differences may lead to new opportunities for mitigating ruminal methane emissions. Metagenomics is a powerful new tool for understanding the composition and function of complex microbial communities. Here we have applied metagenomics to the rumen microbial community to identify differences in the microbiota and metagenome that lead to high- and low-methane-emitting cattle phenotypes. METHODS: Four pairs of beef cattle were selected for extreme high and low methane emissions from 72 animals, matched for breed (Aberdeen-Angus or Limousin cross) and diet (high or medium concentrate). Community analysis was carried out by qPCR of 16S and 18S rRNA genes and by alignment of Illumina HiSeq reads to the GREENGENES database. Total genomic reads were aligned to the KEGG genes databasefor functional analysis. RESULTS: Deep sequencing produced on average 11.3 Gb per sample. 16S rRNA gene abundances indicated that archaea, predominantly Methanobrevibacter, were 2.5× more numerous (P = 0.026) in high emitters, whereas among bacteria Proteobacteria, predominantly Succinivibrionaceae, were 4-fold less abundant (2.7 vs. 11.2 %; P = 0.002). KEGG analysis revealed that archaeal genes leading directly or indirectly to methane production were 2.7-fold more abundant in high emitters. Genes less abundant in high emitters included acetate kinase, electron transport complex proteins RnfC and RnfD and glucose-6-phosphate isomerase. Sequence data were assembled de novo and over 1.5 million proteins were annotated on the subsequent metagenome scaffolds. Less than half of the predicted genes matched matched a domain within Pfam. Amongst 2774 identified proteins of the 20 KEGG orthologues that correlated with methane emissions, only 16 showed 100 % identity with a publicly available protein sequence. CONCLUSIONS: The abundance of archaeal genes in ruminal digesta correlated strongly with differing methane emissions from individual animals, a finding useful for genetic screening purposes. Lower emissions were accompanied by higher Succinovibrionaceae abundance and changes in acetate and hydrogen production leading to less methanogenesis, as similarly postulated for Australian macropods. Large numbers of predicted protein sequences differed between high- and low-methane-emitting cattle. Ninety-nine percent were unknown, indicating a fertile area for future exploitation.

Essential oils have different effects on human pathogenic and commensal bacteria in mixed faecal fermentations compared with pure cultures.

A static batch culture system inoculated with human faeces was used to determine the influence of essential oil compounds (EOCs) on mixed faecal microbiota. Bacteria were quantified using quantitative PCR of 16S rRNA genes. Incubation for 24 h of diluted faeces from six individuals caused enrichment of Bifidobacterium spp., but proportions of other major groups were unaffected. Thymol and geraniol at 500 p.p.m. suppressed total bacteria, resulting in minimal fermentation. Thymol at 100 p.p.m. had no effect, nor did eugenol or nerolidol at 100 or 500 p.p.m. except for a slight suppression of Eubacterium hallii. Methyl isoeugenol at 100 or 500 p.p.m. suppressed the growth of total bacteria, accompanied by a large fall in the molar proportion of propionate formed. The relative abundance of Faecalibacterium prausnitzii was unaffected except with thymol at 500 p.p.m. The ability of EOCs to control numbers of the pathogen Clostridium difficile was investigated in a separate experiment, in which the faecal suspensions were amended by the addition of pure culture of C. difficile. Numbers of C. difficile were suppressed by thymol and methyl isoeugenol at 500 p.p.m. and to a lesser extent at 100 p.p.m. Eugenol and geraniol gave rather similar suppression of C. difficile numbers at both 100 and 500 p.p.m. Nerolidol had no significant effect. It was concluded from these and previous pure-culture experiments that thymol and geraniol at around 100 p.p.m. could be effective in suppressing pathogens in the small intestine, with no concern for beneficial commensal colonic bacteria in the distal gut.

Hydrogen and methane emissions from beef cattle and their rumen microbial community vary with diet, time after feeding and genotype.

The aims of the present study were to quantify hydrogen (H2) and methane (CH4) emissions from beef cattle under different dietary conditions and to assess how cattle genotype and rumen microbial community affected these emissions. A total of thirty-six Aberdeen Angus-sired (AAx) and thirty-six Limousin-sired (LIMx) steers were fed two diets with forage:concentrate ratios (DM basis) of either 8:92 (concentrate) or 52:48 (mixed). Each diet was fed to eighteen animals of each genotype. Methane (CH4) and H2 emissions were measured individually in indirect respiration chambers. H2 emissions (mmol/min) varied greatly throughout the day, being highest after feed consumption, and averaged about 0·10 mol H2/mol CH4. Higher H2 emissions (mol/kg DM intake) were recorded in steers fed the mixed diet. Higher CH4 emissions (mol/d and mol/kg DM intake) were recorded in steers fed the mixed diet (P< 0·001); the AAx steers produced more CH4 on a daily basis (mol/d, P< 0·05) but not on a DM intake basis (mol/kg DM intake). Archaea (P= 0·002) and protozoa (P< 0·001) were found to be more abundant and total bacteria (P< 0·001) less abundant (P< 0·001) on feeding the mixed diet. The relative abundance of Clostridium cluster IV was found to be greater (P< 0·001) and that of cluster XIVa (P= 0·025) lower on feeding the mixed diet. The relative abundance of Bacteroides plus Prevotella was greater (P= 0·018) and that of Clostridium cluster IV lower (P= 0·031) in the LIMx steers. There were no significant relationships between H2 emissions and microbial abundance. In conclusion, the rate of H2 production immediately after feeding may lead to transient overloading of methanogenic archaea capacity to use H2, resulting in peaks in H2 emissions from beef cattle.

Effect of DNA extraction and sample preservation method on rumen bacterial population.

The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P < 0.01) in eDNA samples. The qPCR indicated significantly higher amount of Firmicutes in iDNA sample frozen with glycerol (P < 0.01). Deep sequencing analysis of iDNA samples revealed the prevalence of Bacteroidetes and similarity of samples frozen with and without cryoprotectants, which differed from sample stored with ethanol at room temperature. Centrifugation and consequent filtration of rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile.

Ammonia production by human faecal bacteria, and the enumeration, isolation and characterization of bacteria capable of growth on peptides and amino acids.

BACKGROUND: The products of protein breakdown in the human colon are considered to be detrimental to gut health. Amino acid catabolism leads to the formation of sulfides, phenolic compounds and amines, which are inflammatory and/or precursors to the formation of carcinogens, including N-nitroso compounds. The aim of this study was to investigate the kinetics of protein breakdown and the bacterial species involved. RESULTS: Casein, pancreatic casein hydrolysate (mainly short-chain peptides) or amino acids were incubated in vitro with suspensions of faecal bacteria from 3 omnivorous and 3 vegetarian human donors. Results from the two donor groups were similar. Ammonia production was highest from peptides, followed by casein and amino acids, which were similar. The amino acids metabolized most extensively were Asp, Ser, Lys and Glu. Monensin inhibited the rate of ammonia production from amino acids by 60% (P = 0.001), indicating the involvement of Gram-positive bacteria. Enrichment cultures were carried out to investigate if, by analogy with the rumen, there was a significant population of asaccharolytic, obligately amino acid-fermenting bacteria ('hyper-ammonia-producing' bacteria; HAP) in the colon. Numbers of bacteria capable of growth on peptides or amino acids alone averaged 3.5% of the total viable count, somewhat higher than the rumen. None of these were HAP, however. The species enriched included Clostridium spp., one of which was C. perfringens, Enterococcus, Shigella and Escherichia coli. CONCLUSIONS: Protein fermentation by human faecal bacteria in the absence of sugars not only leads to the formation of hazardous metabolic products, but also to the possible proliferation of harmful bacteria. The kinetics of protein metabolism were similar to the rumen, but HAP bacteria were not found.

Sensitivity of pathogenic and commensal bacteria from the human colon to essential oils.

The microbiota of the intestinal tract plays an important role in colonic health, mediating many effects of dietary components on colonic health and during enteric infections. In the context of the increasing incidence of antibiotic resistance in gut bacteria, complementary therapies are required for the prevention and treatment of enteric infections. Here we report the potential application of essential oils (EO) and pure EO compounds to improve human gut health. Nerolidol, thymol, eugenol and geraniol inhibited growth of the pathogens Escherichia coli O157 : H7(VT(-)), Clostridium difficile DSM1296, Clostridium perfringens DSM11780, Salmonella typhimurium 3530 and Salmonella enteritidis S1400 at a half-maximal inhibitory concentration (IC(50)) varying from 50 to 500 p.p.m. Most EO showed greater toxicity to pathogens than to commensals. However, the beneficial commensal Faecalibacterium prausnitzii was sensitive to EO at similar or even lower concentrations than the pathogens. The EO showed dose-dependent effects on cell integrity, as measured using propidium iodide, of Gram-positive bacteria. These effects were not strongly correlated with growth inhibition, however, suggesting that cell membrane damage occurred but was not the primary cause of growth inhibition. Growth inhibition of Gram-negative bacteria, in contrast, occurred mostly without cell integrity loss. Principal component analysis showed clustering of responses according to bacterial species rather than to the identity of the EO, with the exception that responses to thymol and nerolidol clustered away from the other EO. In conclusion, the selective effects of some EO might have beneficial effects on gut health if chosen carefully for effectiveness against different species.

Dietary fish oil supplements modify ruminal biohydrogenation, alter the flow of fatty acids at the omasum, and induce changes in the ruminal Butyrivibrio population in lactating cows.

Four lactating cows fitted with ruminal cannulae and fed a grass silage-based diet were used in a 4 × 4 Latin square with 28-d periods to investigate the effects of incremental dietary fish oil (FO) supplementation (0, 75, 150, or 300 g/d) on the flow of fatty acids at the omasum and populations of rumen bacteria capable of biohydrogenation. FO decreased silage intake and ruminal volatile fatty acid concentrations and promoted an increase in molar butyrate and propionate proportions at the expense of acetate. Extensive ruminal biohydrogenation of 20:5(n-3) and 22:6(n-3) resulted in corresponding increases in numerous 20- and 22-carbon unsaturated fatty acids at the omasum. Omasal flow of several 20-, 21-, and 22-carbon all-cis (n-3) PUFA exceeded the intake from FO. Supplements of FO also induced a dose-dependent decrease in 18:0 and increased trans 18:1 and trans 18:2 flow at the omasum. Trans-11 was the major 18:1 intermediate in digesta, while FO induced quadratic increases in trans-10 18:1 flow, reaching a maximum of 300 g/d. FO had no substantial influence on omasal flow of CLA. Results suggest that one or more fatty acids in FO inhibit the reduction of trans-18:1 and trans-18:2 intermediates by ruminal microorganisms. qPCR based on 16S rRNA genes in omasal digesta indicated that key Butyrivibrio spp. declined linearly in response to FO. Dose-dependent increases in ruminal outflow of biohydrogenation intermediates containing one or more trans double bonds in response to FO has major implications for host metabolism and the nutritional quality of ruminant foods.

As yet uncultured bacteria phylogenetically classified as Prevotella, Lachnospiraceae incertae sedis and unclassified Bacteroidales, Clostridiales and Ruminococcaceae may play a predominant role in ruminal biohydrogenation.

Microbial biohydrogenation of dietary poly-unsaturated fatty acids (PUFA) to saturated fatty acids (SFA) in the rumen results in the high ratio of SFA/PUFA in ruminant products, such as meat and milk. In vitro, Butyrivibrio proteoclasticus-related bacteria extensively biohydrogenate PUFA to SFA, yet their contribution in the rumen has not been confirmed. The aim of this study was to evaluate the role of Butyrivibrio proteoclasticus group bacteria in ruminal biohydrogenation and to assess the possible role of other bacteria. Fish oil at 0%, 1.5% and 3% dry matter intake was fed to eight Holstein × Friesian steers, in order to elicit changes in the extent of PUFA biohydrogenation. Fatty acid and B. proteoclasticus group 16S rRNA concentrations in rumen digesta were determined. Correlation between digesta 18:0 concentration and B. proteoclasticus group 16S rRNA concentration was low. Terminal restriction fragment length polymorphism and denaturing gradient gel electrophoresis (DGGE) coupled with multivariate statistics revealed that many terminal restriction fragments (T-RFs) and DGGE bands were linked to cis-9, trans-11 conjugated linoleic acid (CLA), 18:1 trans-11 and 18:0 ruminal concentrations. MiCA T-RF predictive identification software showed that these linked T-RFs were likely to originate from as yet uncultured bacteria classified as Prevotella, Lachnospiraceae incertae sedis, and unclassified Bacteroidales, Clostridiales and Ruminococcaceae. Sequencing of linked DGGE bands also revealed that as yet uncultured bacteria classified as Prevotella, Anaerovoax (member of the Lachnospiraceae incertae sedis family), and unclassified Clostridiales and Ruminococcaceae may play a role in biohydrogenation.

Olsenella umbonata sp. nov., a microaerotolerant anaerobic lactic acid bacterium from the sheep rumen and pig jejunum, and emended descriptions of Olsenella, Olsenella uli and Olsenella profusa.

Strain A2 is an anaerobic, variably Gram-stain-positive, non-spore-forming, small and irregularly rod-shaped bacterium from the ruminal fluid of a sheep that has been described informally as a representative of 'Olsenella (basonym Atopobium) oviles'. Three phenotypically similar bacterial strains (lac15, lac16 and lac31(T)) were isolated in concert with Veillonella magna lac18(T) from the mucosal jejunum of a pig. A phylogenetic analysis based on 16S rRNA gene sequences revealed that strains A2, lac15, lac16 and lac31(T) formed a genetically coherent group (100 % interstrain sequence similarity) within the bigeneric Olsenella-Atopobium branch of the family Coriobacteriaceae, class Actinobacteria. This group was most closely related to the type strains of the two recognized Olsenella species, namely Olsenella uli (sequence similarity of 96.85 %) and Olsenella profusa (sequence similarity of 97.20 %). The sequence similarity to the type strain of Atopobium minutum, the type species of the genus Atopobium, was 92.33 %. Unlike those of O. uli and O. profusa, outgrown colonies of strains A2, lac15, lac16 and lac31(T) were opaque and greyish-white with an umbonate elevation on solid culture media. The four novel strains were characterized as being well-adapted and presumably indigenous to the gastrointestinal tract of homoeothermic vertebrates: they were mesophilic, microaerotolerant, neutrophilic and acidotolerant, bile-resistant, mucin-utilizing and markedly peptidolytic lactic acid bacteria. The results of DNA-DNA hybridizations, cellular fatty acid analysis and other differential phenotypic (physiological and biochemical) tests confirmed that strains A2, lac15, lac16 and lac31(T) represent a novel species of the genus Olsenella. On the basis of the genotypic and phenotypic results, we therefore describe Olsenella umbonata sp. nov., with lac31(T) ( = CCUG 58604(T)  = DSM 22620(T)  = JCM 16156(T)) as the type strain and A2 ( = CCUG 58212  = DSM 22619  = JCM 16157) as an additionally available reference strain. Also, based on our data, we propose emended descriptions of the genus Olsenella and the species Olsenella uli and Olsenella profusa.

The rumen microbiome: balancing food security and environmental impacts.

Ruminants produce edible products and contribute to food security. They house a complex rumen microbial community that enables the host to digest their plant feed through microbial-mediated fermentation. However, the rumen microbiome is also responsible for the production of one of the most potent greenhouse gases, methane, and contributes about 18% of its total anthropogenic emissions. Conventional methods to lower methane production by ruminants have proved successful, but to a limited and often temporary extent. An increased understanding of the host-microbiome interactions has led to the development of new mitigation strategies. In this Review we describe the composition, ecology and metabolism of the rumen microbiome, and the impact on host physiology and the environment. We also discuss the most pertinent methane mitigation strategies that emerged to balance food security and environmental impacts.

Metagenomic analysis of the cow, sheep, reindeer and red deer rumen.

The rumen microbiota comprises a community of microorganisms which specialise in the degradation of complex carbohydrates from plant-based feed. These microbes play a highly important role in ruminant nutrition and could also act as sources of industrially useful enzymes. In this study, we performed a metagenomic analysis of samples taken from the ruminal contents of cow (Bos Taurus), sheep (Ovis aries), reindeer (Rangifer tarandus) and red deer (Cervus elaphus). We constructed 391 metagenome-assembled genomes originating from 16 microbial phyla. We compared our genomes to other publically available microbial genomes and found that they contained 279 novel species. We also found significant differences between the microbiota of different ruminant species in terms of the abundance of microbial taxonomies, carbohydrate-active enzyme genes and KEGG orthologs. We present a dataset of rumen-derived genomes which in combination with other publicly-available rumen genomes can be used as a reference dataset in future metagenomic studies.

Metabolism of conjugated linoleic acids and 18 : 1 fatty acids by ruminal bacteria: products and mechanisms.

Cultures of ruminal bacteria known to metabolize unsaturated fatty acids were grown in medium containing 50 microg ml(-1) of geometric and positional isomers of conjugated linoleic acid (CLA) or 18 : 1 fatty acids and 37.4 % deuterium oxide to investigate the mechanisms responsible for fatty acid metabolism. Butyrivibrio fibrisolvens JW11 converted cis-9,trans-11-18 : 2 and trans-9,trans-11-18 : 2 to trans-11-18 : 1 as the main product, labelled at C-9, and metabolized trans-10,cis-12-18 : 2 to trans-10-18 : 1, labelled at C-13, and smaller amounts of trans-12-18 : 1 and cis-12-18 : 1. Butyrivibrio proteoclasticus P-18 did not grow in the presence of cis-9,trans-11-18 : 2 or trans-10,cis-12-18 : 2, but grew in medium containing trans-9,trans-11-18 : 2, forming 18 : 0. Propionibacterium acnes, a ruminal species that isomerizes linoleic acid to trans-10,cis-12-18 : 2, did not metabolize CLA isomers further. B. fibrisolvens metabolized small amounts of trans-10-18 : 1, trans-11-18 : 1 and cis-9-18 : 1, but the products formed were not detected. B. proteoclasticus, on the other hand, carried out substantial conversion of 18 : 1 substrates to 18 : 0. P. acnes hydrated cis-9-18 : 1 and trans-11-18 : 1 to 10-OH-18 : 0, which was further oxidized to yield 10-O-18 : 0. The deuterium enrichment in the intermediates formed during incubations with 9,11 geometric isomers of CLA was about half that of the products from trans-10,cis-12 CLA and 18 : 1 isomers, suggesting that the reduction of 9,11 geometric isomers CLA by ruminal bacteria occurs via different mechanisms compared with the metabolism of other unsaturated fatty acids.

Identification of Complex Rumen Microbiome Interaction Within Diverse Functional Niches as Mechanisms Affecting the Variation of Methane Emissions in Bovine.

A network analysis including relative abundances of all ruminal microbial genera (archaea, bacteria, fungi, and protists) and their genes was performed to improve our understanding of how the interactions within the ruminal microbiome affects methane emissions (CH4). Metagenomics and CH4 data were available from 63 bovines of a two-breed rotational cross, offered two basal diets. Co-abundance network analysis revealed 10 clusters of functional niches. The most abundant hydrogenotrophic Methanobacteriales with key microbial genes involved in methanogenesis occupied a different functional niche (i.e., "methanogenesis" cluster) than methylotrophic Methanomassiliicoccales (Candidatus Methanomethylophylus) and acetogens (Blautia). Fungi and protists clustered together and other plant fiber degraders like Fibrobacter occupied a seperate cluster. A Partial Least Squares analysis approach to predict CH4 variation in each cluster showed the methanogenesis cluster had the best prediction ability (57.3%). However, the most important explanatory variables in this cluster were genes involved in complex carbohydrate degradation, metabolism of sugars and amino acids and Candidatus Azobacteroides carrying nitrogen fixation genes, but not methanogenic archaea and their genes. The cluster containing Fibrobacter, isolated from other microorganisms, was positively associated with CH4 and explained 49.8% of its variability, showing fermentative advantages compared to other bacteria and fungi in providing substrates (e.g., formate) for methanogenesis. In other clusters, genes with enhancing effect on CH4 were related to lactate and butyrate (Butyrivibrio and Pseudobutyrivibrio) production and simple amino acids metabolism. In comparison, ruminal genes negatively related to CH4 were involved in carbohydrate degradation via lactate and succinate and synthesis of more complex amino acids by γ-Proteobacteria. When analyzing low- and high-methane emitters data in separate networks, competition between methanogens in the methanogenesis cluster was uncovered by a broader diversity of methanogens involved in the three methanogenesis pathways and larger interactions within and between communities in low compared to high emitters. Generally, our results suggest that differences in CH4 are mainly explained by other microbial communities and their activities rather than being only methanogens-driven. Our study provides insight into the interactions of the rumen microbial communities and their genes by uncovering functional niches affecting CH4, which will benefit the development of efficient CH4 mitigation strategies.

Genoprotective Effects of Essential Oil Compounds Against Oxidative and Methylated DNA Damage in Human Colon Cancer Cells.

Essential oils (EO) are widely used in foods as flavoring and preservative agents. Many of the biological activities of EO have been attributed to major essential oil compounds (EOC) but their direct interaction with colonic epithelial cells and their genotoxic and genoprotective effects are not well established. In this study, the cytotoxicity and genotoxicity of EOC including nerolidol, thymol, geraniol, methylisoeugenol, eugenol, linalool, and a commercial blend (Agolin) were determined. Furthermore, the genoprotective effects of EOC against oxidative and methylating damage were assessed using the comet assay in HT-29 colorectal adenocarcinoma cells. The majority of EOC were cytotoxic to HT-29 cells at or above 250 ppm after 24 hr exposure. At noncytotoxic doses, none of the EOC was genotoxic in the comet assay. Genoprotection against oxidative DNA damage was observed for nerolidol (at 62.5 ppm), thymol (at 12.5 ppm), geraniol, and methylisoeugenol (both at 125 ppm), as well as linalool and Agolin (both at 250 ppm). Thymol was the most protective compound against oxidative DNA damage and geraniol (at 125 ppm) also protected cells against methylating DNA damage. This study highlights the potential of EOC such as thymol to protect the colonic epithelium against oxidative DNA damage and geraniol against methylating DNA damage. Further in vivo studies are needed to confirm these findings for safety and efficacy to exploit their potential pharmaceutical or nutraceutical uses for colonic health.

Correction to: The rumen microbiome as a reservoir of antimicrobial resistance and pathogenicity genes is directly affected by diet in beef cattle.

Following publication of the original article [1], the authors reported an error in the Additional file 1.