BackgroundMethane 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.MethodsFour 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.ResultsDeep 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2032-0) contains supplementary material, which is available to authorized users.

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.

The aims of the present study were to quantify hydrogen (H 2 ) and methane (CH 4 ) 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 (CH 4 ) and H 2 emissions were measured individually in indirect respiration chambers. H 2 emissions (mmol/min) varied greatly throughout the day, being highest after feed consumption, and averaged about 0·10 mol H 2 /mol CH 4 . Higher H 2 emissions (mol/kg DM intake) were recorded in steers fed the mixed diet. Higher CH 4 emissions (mol/d and mol/kg DM intake) were recorded in steers fed the mixed diet (P, 0·001); the AAx steers produced more CH 4 on a daily basis (mol/d, P, 0·05) but not on a DM intake basis (mol/kg DM intake). Archaea (P¼ 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 (P¼ 0·025) lower on feeding the mixed diet. The relative abundance of Bacteroides plus Prevotella was greater (P¼ 0·018) and that of Clostridium cluster IV lower (P¼ 0·031) in the LIMx steers. There were no significant relationships between H 2 emissions and microbial abundance. In conclusion, the rate of H 2 production immediately after feeding may lead to transient overloading of methanogenic archaea capacity to use H 2 , resulting in peaks in H 2 emissions from beef cattle.

Adding nitrate to or increasing the concentration of lipid in the diet are established strategies for reducing enteric methane (CH4) emissions, but their effectiveness when used in combination has been largely unexplored. This study investigated the effect of dietary nitrate and increased lipid included alone or together on CH4 emissions and performance traits of finishing beef cattle. The experiment was a 2×4 factorial design comprising two breeds (cross-bred Aberdeen Angus (AAx) and cross-bred Limousin (LIMx) steers) and four dietary treatments (each based on 550 g forage : 450 g concentrate/kg dry matter (DM)). The four dietary treatments were assigned according to a 2×2 factorial design where the control treatment contained rapeseed meal as the main protein source, which was replaced either with nitrate (21.5 g nitrate/kg DM); maize distillers dark grains (MDDG, which increased diet ether extract from 24 to 37 g/kg DM) or both nitrate and MDDG. Steers (n=20/dietary treatment) were allocated to each of the four treatments in equal numbers of each breed with feed offered ad libitum. After 28 days adaptation to dietary treatments, individual animal intake, performance and feed efficiency were recorded for 56 days. Thereafter, CH4 emissions were measured over 13 weeks (six steers/week). Increasing dietary lipid did not adversely affect animal performance and showed no interactions with dietary nitrate. In contrast, addition of nitrate to diets resulted in poorer live-weight gain (P<0.01) and increased feed conversion ratio (P<0.05) compared with diets not containing nitrate. Daily CH4 output was lower (P<0.001) on nitrate-containing diets but increasing dietary lipid resulted in only a non-significant reduction in CH4. There were no interactions associated with CH4 emissions between dietary nitrate and lipid. Cross-bred Aberdeen Angus steers achieved greater live-weight gains (P<0.01), but had greater DM intakes (P<0.001), greater fat depth (P<0.01) and poorer residual feed intakes (P<0.01) than LIMx steers. Cross-bred Aberdeen Angus steers had higher daily CH4 outputs (P<0.001) but emitted less CH4 per kilogram DM intake than LIMx steers (P<0.05). In conclusion, inclusion of nitrate reduced CH4 emissions in growing beef cattle although the efficacy of nitrate was less than in previous work. When increased dietary lipid and nitrate inclusion were combined there was no evidence of an interaction between treatments and therefore combining different nutritional treatments to mitigate CH4 emissions could be a useful means of achieving reductions in CH4 while minimising any adverse effects.

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