Comparative survey of rumen microbial communities and metabolites across one caprine and three bovine groups, using bar-coded pyrosequencing and ¹H nuclear magnetic resonance spectroscopy

Abstract

Pyrosequencing of 16S rRNA genes (targeting Bacteria and Archaea) and (1)H nuclear magnetic resonance were applied to investigate the rumen microbiota and metabolites of Hanwoo steers in the growth stage (HGS), Hanwoo steers in the late fattening stage (HFS), Holstein-Friesian dairy cattle (HDC), and Korean native goats (KNG) in the late fattening stage. This was a two-part investigation. We began by comparing metabolites and microbiota of Hanwoo steers at two stages of husbandry. Statistical comparisons of metabolites and microbial communities showed no significant differences between HFS and HGS (differing by a dietary shift at 24 months and age [67 months versus 12 months]). We then augmented the study by extending the investigation to HDC and KNG. Overall, pyrosequencing of 16S rRNA genes showed that the rumens had highly diverse microbial communities containing many previously undescribed microorganisms. Bioinformatic analysis revealed that the bacterial sequences were predominantly affiliated with four phyla-Bacteroidetes, Firmicutes, Fibrobacteres, and Proteobacteria-in all ruminants. However, interestingly, the bacterial reads belonging to Fibrobacteres were present at a very low abundance (<0.1%) in KNG. Archaeal community analysis showed that almost all of these reads fell into a clade related to, but distinct from, known cultivated methanogens. Statistical analyses showed that the microbial communities and metabolites of KNG were clearly distinct from those of other ruminants. In addition, bacterial communities and metabolite profiles of HGS and HDC, fed similar diets, were distinctive. Our data indicate that bovine host breeds override diet as the key factor that determines bacterial community and metabolite profiles in the rumen.

Fig 1

Rarefaction curves of bacterial (A) and archaeal (B) 16S rRNA gene sequences from the rumens of HGS, HFS, HDC, and KNG. The operational taxonomic units (OTUs) were calculated by the RDP pipeline with a 3% dissimilarity.

Fig 2

Bacterial (A and B) and archaeal (C and D) taxonomic compositions of the rumens of Hanwoo steers (two growth stages), Holstein cows, and Korean native goats. The data portray phylum (A and C) and genus (B and D) level 16S rRNA pyrotagged gene sequences found in HGS, HFS, HDC, and KNG (see Table 1 for the treatment key). Sequences were classified using the RDP naive Bayesian rRNA Classifier with an 80% confidence threshold. The minor group in panel C is composed of the genera, each showing a percentage of reads <0.4% of the total reads in all of the samples.

Fig 3

Phylogenetic dendrogram based on 16S rRNA gene sequences showing the phylogenetic relationships of archaeal sequencing reads and selected reference sequences found in Hanwoo steers (two growth stages: HFS and HGS), Holstein cows (HDC), and Korean native goats (KNG). The tree was constructed using 430 representative sequences derived by CD-HIT (97% similarity level) from the total archaeal pyrosequencing reads (29,843 reads). For simplicity, a clade of representative reads is shown as a shaded triangle. The numbers in parentheses indicate the numbers of the representative sequences and their corresponding total sequences, respectively. Bootstrap values are shown as percentages of 1,000 replicates, when greater than 50% at the branch points. Aquifex pyrophilus Kol5a^T (M83548) was used as an outgroup. The scale bar indicates 0.05 inferred nucleotide substitution per position.

Fig 4

Score plot of principal-component analysis (PCA) of rumen metabolites by PC1 and PC2. The treatment animals were Hanwoo steers (two growth stages: HGS and HFS), Holstein cows (HDC), and Korean native goats (KNG). The score plot was constructed using the mean values of the ¹H NMR peaks from triplicate analyses. The ellipse represents the 95% Hotelling confidence region.

Fig 5

Hierarchical clustering of bacterial (A) and archaeal (B) communities in the rumens of Hanwoo steers (two growth stages), Holstein cows, and Korean native goats using the unweighted UniFrac method in HGS, HFS, HDC, and KNG. The scale bars represent the unweighted UniFrac distances.

Fig 6

PCoA results showing the relationships of bacterial (A) and archaeal (B) communities of the rumens in HGS, HFS, HDC, and KNG. The PCoA plots were constructed using the unweighted UniFrac method.