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.

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.

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

BACKGROUND: The emergence and spread of antimicrobial resistance is the most urgent current threat to human and animal health. An improved understanding of the abundance of antimicrobial resistance genes and genes associated with microbial colonisation and pathogenicity in the animal gut will have a major role in reducing the contribution of animal production to this problem. Here, the influence of diet on the ruminal resistome and abundance of pathogenicity genes was assessed in ruminal digesta samples taken from 50 antibiotic-free beef cattle, comprising four cattle breeds receiving two diets containing different proportions of concentrate. RESULTS: Two hundred and four genes associated with antimicrobial resistance (AMR), colonisation, communication or pathogenicity functions were identified from 4966 metagenomic genes using KEGG identification. Both the diversity and abundance of these genes were higher in concentrate-fed animals. Chloramphenicol and microcin resistance genes were dominant in samples from forage-fed animals (P < 0.001), while aminoglycoside and streptomycin resistances were enriched in concentrate-fed animals. The concentrate-based diet also increased the relative abundance of Proteobacteria, which includes many animal and zoonotic pathogens. A high ratio of Proteobacteria to (Firmicutes + Bacteroidetes) was confirmed as a good indicator for rumen dysbiosis, with eight cases all from concentrate-fed animals. Finally, network analysis demonstrated that the resistance/pathogenicity genes are potentially useful as biomarkers for health risk assessment of the ruminal microbiome. CONCLUSIONS: Diet has important effects on the complement of AMR genes in the rumen microbial community, with potential implications for human and animal health.

Identification, Comparison, and Validation of Robust Rumen Microbial Biomarkers for Methane Emissions Using Diverse Bos Taurus Breeds and Basal Diets.

Previous shotgun metagenomic analyses of ruminal digesta identified some microbial information that might be useful as biomarkers to select cattle that emit less methane (CH4), which is a potent greenhouse gas. It is known that methane production (g/kgDMI) and to an extent the microbial community is heritable and therefore biomarkers can offer a method of selecting cattle for low methane emitting phenotypes. In this study a wider range of Bos Taurus cattle, varying in breed and diet, was investigated to determine microbial communities and genetic markers associated with high/low CH4 emissions. Digesta samples were taken from 50 beef cattle, comprising four cattle breeds, receiving two basal diets containing different proportions of concentrate and also including feed additives (nitrate or lipid), that may influence methane emissions. A combination of partial least square analysis and network analysis enabled the identification of the most significant and robust biomarkers of CH4 emissions (VIP > 0.8) across diets and breeds when comparing all potential biomarkers together. Genes associated with the hydrogenotrophic methanogenesis pathway converting carbon dioxide to methane, provided the dominant biomarkers of CH4 emissions and methanogens were the microbial populations most closely correlated with CH4 emissions and identified by metagenomics. Moreover, these genes grouped together as confirmed by network analysis for each independent experiment and when combined. Finally, the genes involved in the methane synthesis pathway explained a higher proportion of variation in CH4 emissions by PLS analysis compared to phylogenetic parameters or functional genes. These results confirmed the reproducibility of the analysis and the advantage to use these genes as robust biomarkers of CH4 emissions. Volatile fatty acid concentrations and ratios were significantly correlated with CH4, but these factors were not identified as robust enough for predictive purposes. Moreover, the methanotrophic Methylomonas genus was found to be negatively correlated with CH4. Finally, this study confirmed the importance of using robust and applicable biomarkers from the microbiome as a proxy of CH4 emissions across diverse production systems and environments.

Association of Temperament and Acute Stress Responsiveness with Productivity, Feed Efficiency, and Methane Emissions in Beef Cattle: An Observational Study.

The aim of this study was to assess individual differences in temperament and stress response and quantify their impact on feed efficiency, performance, and methane (CH4) emissions in beef cattle. Eighty-four steers (castrated males) (Charolais or Luing) were used. Temperament was assessed using two standardized tests: restlessness when restrained [crush score (CS)] and flight speed (FS) on release from restraint. Over a 56-day period individual animal dry matter intake (DMI) and weekly body weight was measured. Ultrasound fat depth was measured at the end of 56 days. Average daily gain (ADG), feed conversion ratio (FCR), and residual feed intake (RFI) were calculated. After the 56-day test period, animals were transported in groups of six/week to respiration chamber facilities. Blood samples were taken before and 0, 3, 6, and 9 h after transport. Plasma cortisol, creatine kinase (CK), glucose, and free fatty acids (FFA) were determined to assess physiological stress response. Subsequently, CH4 emissions were measured over a 3-day period in individual respiration chambers. CS (1.7 ± 0.09) and FS (1.6 ± 0.60 m/s) were repeatable (0.63 and 0.51, respectively) and correlated (r = 0.36, P < 0.001). Plasma cortisol, CK, and FFA concentrations increased after transport (P = 0.038, P = 0.006, and P < 0.001, respectively). Temperament (CS) and CK concentration were correlated (r = 0.29; P = 0.015). The extreme group analysis reveals that excitable animals (FS; P = 0.032) and higher stress response (cortisol, P = 0.007; FFA, P = 0.007; and CK, P = 0.003) were associated with lower DMI. ADG was lower in more temperamental animals (CS, P = 0.097, and FS, P = 0.030). Fat depth was greater in steers showing calmer CS (P = 0.026) and lower plasma CK (P = 0.058). Temperament did not show any relationship with RFI or CH4 emissions. However, steers with higher cortisol showed improved feed efficiency (lower FCR and RFI) (P < 0.05) and greater CH4 emissions (P = 0.017). In conclusion, agitated temperament and higher stress responsiveness is detrimental to productivity. A greater stress response is associated with a reduction in feed intake that may both increase the efficiency of consumed feed and the ratio of CH4 emissions/unit of feed. Therefore, temperament and stress response should be considered when designing strategies to improve efficiency and mitigate CH4 emissions in beef cattle.

Nitrate and Inhibition of Ruminal Methanogenesis: Microbial Ecology, Obstacles, and Opportunities for Lowering Methane Emissions from Ruminant Livestock.

Ruminal methane production is among the main targets for greenhouse gas (GHG) mitigation for the animal agriculture industry. Many compounds have been evaluated for their efficacy to suppress enteric methane production by ruminal microorganisms. Of these, nitrate as an alternative hydrogen sink has been among the most promising, but it suffers from variability in efficacy for reasons that are not understood. The accumulation of nitrite, which is poisonous when absorbed into the animal's circulation, is also variable and poorly understood. This review identifies large gaps in our knowledge of rumen microbial ecology that handicap the further development and safety of nitrate as a dietary additive. Three main bacterial species have been associated historically with ruminal nitrate reduction, namely Wolinella succinogenes, Veillonella parvula, and Selenomonas ruminantium, but others almost certainly exist in the largely uncultivated ruminal microbiota. Indications are strong that ciliate protozoa can reduce nitrate, but the significance of their role relative to bacteria is not known. The metabolic fate of the reduced nitrate has not been studied in detail. It is important to be sure that nitrate metabolism and efforts to enhance rates of nitrite reduction do not lead to the evolution of the much more potent GHG, nitrous oxide. The relative importance of direct inhibition of archaeal methanogenic enzymes by nitrite or the efficiency of capture of hydrogen by nitrate reduction in lowering methane production is also not known, nor are nitrite effects on other members of the microbiota. How effective would combining mitigation methods be, based on our understanding of the effects of nitrate and nitrite on the microbiome? Answering these fundamental microbiological questions is essential in assessing the potential of dietary nitrate to limit methane emissions from ruminant livestock.

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 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.

Archaeal abundance in post-mortem ruminal digesta may help predict methane emissions from beef cattle.

Methane produced from 35 Aberdeen-Angus and 33 Limousin cross steers was measured in respiration chambers. Each group was split to receive either a medium- or high-concentrate diet. Ruminal digesta samples were subsequently removed to investigate correlations between methane emissions and the rumen microbial community, as measured by qPCR of 16S or 18S rRNA genes. Diet had the greatest influence on methane emissions. The high-concentrate diet resulted in lower methane emissions (P < 0.001) than the medium-concentrate diet. Methane was correlated, irrespective of breed, with the abundance of archaea (R = 0.39), bacteria (-0.47), protozoa (0.45), Bacteroidetes (-0.37) and Clostridium Cluster XIVa (-0.35). The archaea:bacteria ratio provided a stronger correlation (0.49). A similar correlation was found with digesta samples taken 2-3 weeks later at slaughter. This finding could help enable greenhouse gas emissions of large animal cohorts to be predicted from samples taken conveniently in the abattoir.

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.

Heat shock induces interferon-TAU gene expression by in vitro-produced bovine blastocysts.

PROBLEM: The type I interferon (IFN), IFN-tau (τ), is the primary embryonic signal for pregnancy maintenance in ruminants. This study determined the effects of heat shock upon IFN-τ (IFNT) gene expression by bovine blastocysts in vitro. METHOD OF STUDY: In vitro-produced blastocyst-stage embryos were exposed to 42°C for 4 hr, and mRNA for heat-shock protein 70 (HSP70) and IFNT quantified. RESULTS: Heat shock increased both HSP70 and IFNT expression. There was a significant correlation between HSP70 and IFNT transcript levels irrespective of whether a blastocyst had been exposed to heat shock or not. CONCLUSION: The increase in IFNT as a result of heat shock suggests that a proportion of the variation in IFNT expression observed in blastocyst-stage embryos is a response to stress.

Effects of cobalt/vitamin B12 status in ewes on ovum development and lamb viability at birth.

Scottish Blackface ewes from cobalt-deficient farmland were fed a diet containing 0.06 mg cobalt per kg dry matter from approximately 30 days before embryo recovery/transfer until lambing. Ewes remained untreated (-Co; n = 82) or were given an intraruminal cobalt-containing bolus to compensate for the dietary deficit (+Co; n = 82). Ewes used as embryo donors (-Co, n = 17; +Co, n = 16) were artificially inseminated with semen from a single Suffolk sire. Day 6 embryos obtained from -Co and +Co donors were transferred in singleton to -Co and +Co recipients in a 2 x 2 factorial-designed experiment to determine the effects of cobalt/vitamin B12 status during the periconception period (factor 1) and pregnancy (factor 2) on lamb viability at birth. Mean (+/- s.e.m.) circulating concentrations of vitamin B12 in -Co and +Co donors at ovum recovery were 182 +/- 10 and 1288 +/- 64 pmol L(-1), respectively (P < 0.001), and the number of corpora lutea per ewe ovulating was 9.9 +/- 1.6 and 14.4 +/- 1.3, respectively (P < 0.05). Treatment did not affect the proportion of recovered ova that contained >32 cells (viable) or the median stage of development (late morula), but viable ova recovered from -Co v. +Co ewes had a better morphological grade (2.0 +/- 0.1 v. 2.20 +/- 0.04, respectively; P < 0.01). There was no effect of treatment on the proportion of recipient ewes that became pregnant. Circulating concentrations of vitamin B12 were lower in -Co than +Co ewes during pregnancy (P < 0.001) and at birth in lambs born to -Co ewes compared with those born to +Co ewes (P < 0.001). There was no effect of donor or recipient cobalt/vitamin B12 status on lamb birthweight, neonatal vigour or neonatal rectal temperatures, but lambs derived from +Co v. -Co embryo donors were more active in the first 3 days after birth (P < 0.05). Results show that sub-clinical cobalt/vitamin B12 deficiency reduces ovulatory response in superovulated ewes and that periconception nutrition can affect neonatal lamb behaviour.

Granulocyte-macrophage colony-stimulating-factor increases interferon-tau protein secretion in bovine trophectoderm cells.

PROBLEM: Uterine-derived factors are required for optimal conceptus development and secretion of the maternal recognition of pregnancy factor, interferon-tau (IFN-tau). Identifying these factors may lead to the development of schemes for increasing pregnancy success in cattle. METHOD OF STUDY: The objectives were to examine the effects of granulocyte-macrophage colony-stimulating-factor (GM-CSF) on trophectoderm proliferation rates and IFN-tau production, and verify the appropriateness of using an in vitro model of bovine trophectoderm (CT-1 cell). RESULTS: Rate of [(3)H]-thymidine incorporation into DNA was increased by supplementation of CT-1 medium with 10 or 100 ng/mL porcine (po) GM-CSF. GM-CSF supplementation to CT-1 medium also increased IFN-tau secretion. When results were normalized to account for number of CT-1 cells, 10 and 100 ng/mL poGM-CSF increased antiviral activity and IFN-tau concentrations (using an IFN-tau-specific enzyme-linked immunosorbent assay) in CT-1 conditioned medium compared with controls. CONCLUSIONS: These findings indicate that GM-CSF increases proliferation and IFN-tau production in bovine trophectoderm.

Characterisation of peptides in silages made from perennial ryegrass with different silage additives.

The effects of applying either formic acid (5.4 g kg-1 ), a mixture of formic acid (2.7 g kg-1 ) and formaldehyde (1.5 g kg-1 , 81 g kg-1 herbage crude protein) or two concentrations of a cysteine peptidase inhibitor, cystamine (5 or 50 g kg-1 ), to perennial ryegrass (Lolium perenne) on the nitrogen (N) distribution of the resulting silages were investigated, with emphasis on changes in concentration, composition and molecular weight of silage peptides. Herbage (156 g dry matter kg-1 and 141 g water-soluble carbohydrate kg-1 dry matter) was ensiled in triplicate in laboratory silos for 100 days. Formic acid and the formic acid/formaldehyde mixture reduced soluble non-protein N and ammonia N concentrations (P < 0.01); in addition, formic acid increased peptide N concentrations (P < 0.05). Cystamine at 50 g kg-1 reduced ammonia N concentrations (P < 0.01) and increased peptide N concentrations (P < 0.05), but when applied at 5 g kg-1 had little effect. Gel filtration of silage extracts on Sephadex G-25 suggested that a small proportion (0.06-0.11 g kg-1 peptide N) of silage peptides had a chain length of 7-9 amino acids, but remaining peptides were smaller with chain lengths of 2-6 amino acid residues. Amino acid analysis of silage peptides indicated that additive treatment had little effect on peptide amino acid composition but that peptides with a chain length of 7-9 amino acids contained lower proportions of isoleucine and arginine. © 2000 Society of Chemical Industry.