Taxon abundance, diversity, co-occurrence and network analysis of the ruminal microbiota in response to dietary changes in dairy cows

Abstract

The ruminal microbiome, comprising large numbers of bacteria, ciliate protozoa, archaea and fungi, responds to diet and dietary additives in a complex way. The aim of this study was to investigate the benefits of increasing the depth of the community analysis in describing and explaining responses to dietary changes. Quantitative PCR, ssu rRNA amplicon based taxa composition, diversity and co-occurrence network analyses were applied to ruminal digesta samples obtained from four multiparous Nordic Red dairy cows fitted with rumen cannulae. The cows received diets with forage:concentrate ratio either 35:65 (diet H) or 65:35 (L), supplemented or not with sunflower oil (SO) (0 or 50 g/kg diet dry matter), supplied in a 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments and four 35-day periods. Digesta samples were collected on days 22 and 24 and combined. QPCR provided a broad picture in which a large fall in the abundance of fungi was seen with SO in the H but not the L diet. Amplicon sequencing showed higher community diversity indices in L as compared to H diets and revealed diet specific taxa abundance changes, highlighting large differences in protozoal and fungal composition. Methanobrevibacter ruminantium and Mbb. gottschalkii dominated archaeal communities, and their abundance correlated negatively with each other. Co-occurrence network analysis provided evidence that no microbial domain played a more central role in network formation, that some minor-abundance taxa were at nodes of highest centrality, and that microbial interactions were diet specific. Networks added new dimensions to our understanding of the diet effect on rumen microbial community interactions.

Fig 1. qPCR of bacterial 16S rRNA, archaeal mcrA, protozoal 18S rRNA genes and fungal region between 18S rRNA and ITS1 of rumen digesta samples.

Samples collected from lactating cows fed four diets: high (H) or low (L) proportion of concentrates without oil, or supplemented with SO (HSO and LSO, respectively). Results are expressed as copy number per ng of extracted DNA. Error bars represent standard deviations (SD), n = 4 per diet.

Fig 2. Relative abundance of bacteria, archaea, ciliate protozoa and fungi among diets.

Bubble-charts represent taxonomy at the phylum level for bacteria and genus level for the rest. Diets are as follows: high (H) or low (L) proportion of concentrates without oil, or supplemented with SO (HSO and LSO, respectively).

Fig 3. Bacterial, archaeal, protozoal and fungal diversity (Simpson’s index) as measured from amplicon sequence data in four diets.

Diets are as follows: high (H) or low (L) proportion of concentrates without oil, or supplemented with SO (HSO and LSO, respectively).

Fig 4. NMDS plots of bacteria, archaea, ciliate protozoa and fungi, based on amplicon sequence data.

Individual animals are represented by dots, colored based on the diet: high (H—black) or low (L—green) proportion of concentrates without oil, or supplemented with SO (HSO–red, and LSO—blue).

Fig 5. Graphical networks representing the interactions between rumen microorganisms within three diet comparisons.

a) L-H, b) L-LSO, and c) H-HSO diet comparisons. Nodes correspond to microbial taxa while green and red edges represent positive and negative partial correlations above 0.25, respectively. Microbial taxa are colored by taxonomy: archaea—light gray, ciliate protozoa—white, fungi—dark grey, bacteria: Bacteroidetes—green, Firmicutes—moss green, Proteobacteria—brown, others—blue.