Assessment of the microbial ecology of ruminal methanogens in cattle with different feed efficiencies

Abstract

Cattle with high feed efficiencies (designated "efficient") produce less methane gas than those with low feed efficiencies (designated "inefficient"); however, the role of the methane producers in such difference is unknown. This study investigated whether the structures and populations of methanogens in the rumen were associated with differences in cattle feed efficiencies by using culture-independent methods. Two 16S rRNA libraries were constructed using approximately 800-bp amplicons generated from pooled total DNA isolated from efficient (n = 29) and inefficient (n = 29) animals. Sequence analysis of up to 490 randomly selected clones from each library showed that the methanogenic composition was variable: less species variation (22 operational taxonomic units [OTUs]) was detected in the rumens of efficient animals, compared to 27 OTUs in inefficient animals. The methanogenic communities in inefficient animals were more diverse than those in efficient ones, as revealed by the diversity indices of 0.84 and 0.42, respectively. Differences at the strain and genotype levels were also observed and found to be associated with feed efficiency in the host. No difference was detected in the total population of methanogens, but the prevalences of Methanosphaera stadtmanae and Methanobrevibacter sp. strain AbM4 were 1.92 (P < 0.05) and 2.26 (P < 0.05) times higher in inefficient animals, while Methanobrevibacter sp. strain AbM4 was reported for the first time to occur in the bovine rumen. Our data indicate that the methanogenic ecology at the species, strain, and/or genotype level in the rumen may play important roles in contributing to the difference in methane gas production between cattle with different feed efficiencies.

FIG. 1.

Diagram of OTUs identified by the Mothur program at the 97% similarity level within and between libraries 1 (L-RFI animals) and 2 (H-RFI animals). Representative OTUs are presented by the clone identification numbers, with GenBank accession numbers in parentheses.

FIG. 2.

Distribution of methanogenic species on the basis of their sequences, classified as methanogens from library 1 (L-RFI animals) and library 2 (H-RFI animals). NT7, M. ruminantium NT7; 30Y, Methanobrevibacter sp. strain 30Y; AbM4, Methanobrevibacter sp. strain AbM4; SM9, M. smithii SM9; PS, M. smithii PS; CW, M. thaueri CW; FM1, Methanobrevibacter sp. strain FM1; CSIRO1.33, Methanobacteriales archaeon CSIRO1.33 clone. The y axis shows that the percentages of >70% for more than 70% of the sequences were Methanobrevibacter ruminantium NT7 sequences in both libraries.

FIG. 3.

(A) Genotype analysis of all sequences with 99% identity with the Methanobrevibacter ruminantium NT7 strain. The bars indicate the number of sequences of each genotype in the 16S rRNA library generated from L-RFI and H-RFI animals. The arrows point out the genotypes that existed in both L-RFI and H-RFI animals. (B) Example of SNPs shown in the sequences belonging to this category. The position with an asterisk represents the nucleotide position with SNPs. The base with a square indicates the particular SNPs of each sequence.

FIG. 4.

Phylogenetic analysis of methanogen partial 16S rRNA sequences obtained in this study. Representative sequences were generated by the Mothur program at a 3% difference level. GenBank sequences are identified by accession number. Bootstrap values (>50%) from 1,000 replications are indicated on the tree. 1, Methanococcales; 2, Methanosarcinales; 3, Methanomicrobiales; 4, Methanobacteriales; ▴, representative OTUs appearing in both libraries; ⋄, representative OTUs appearing only in library 1 (L-RFI animals); ○, representative OTUs appearing only in library 2 (H-RFI animals).