Active Rumen Bacterial and Protozoal Communities Revealed by RNA-Based Amplicon Sequencing on Dairy Cows Fed Different Diets at Three Physiological Stages

Abstract

Seven Italian Simmental cows were monitored during three different physiological stages, namely late lactation (LL), dry period (DP), and postpartum (PP), to evaluate modifications in their metabolically-active rumen bacterial and protozoal communities using the RNA-based amplicon sequencing method. The bacterial community was dominated by seven phyla: Proteobacteria, Bacteroidetes, Firmicutes, Spirochaetes, Fibrobacteres, Verrucomicrobia, and Tenericutes. The relative abundance of the phylum Proteobacteria decreased from 47.60 to 28.15% from LL to DP and then increased to 33.24% in PP. An opposite pattern in LL, DP, and PP stages was observed for phyla Verrucomicrobia (from 0.96 to 4.30 to 1.69%), Elusimicrobia (from 0.32 to 2.84 to 0.25%), and SR1 (from 0.50 to 2.08 to 0.79%). The relative abundance of families Succinivibrionaceae and Prevotellaceae decreased in the DP, while Ruminococcaceae increased. Bacterial genera Prevotella and Treponema were least abundant in the DP as compared to LL and PP, while Ruminobacter and Succinimonas were most abundant in the DP. The rumen eukaryotic community was dominated by protozoal phylum Ciliophora, which showed a significant decrease in relative abundance from 97.6 to 93.9 to 92.6 in LL, DP, and PP, respectively. In conclusion, the physiological stage-dependent dietary changes resulted in a clear shift in metabolically-active rumen microbial communities.

Keywords: RNA; active rumen microbiota; cDNA; physiological stages; protozoa.

Figure 1

Principal-coordinate analysis (PCO) of the active rumen bacterial (A) and eukaryotic (B) communities during different physiological stages. The bacterial OTU abundance data were normalized by total sum normalization, while eukaryotic OTU abundance data were transformed by Hellinger transformation (total sum normalization combined with square root transformation) prior to the calculation of Bray–Curtis dissimilarity matrices. The three physiological stages are indicated by different colored triangles: red triangles, LL; blue triangles, DP; grey triangles, PP. The LL and PP is a major cluster with 25% similarity in the bacterial dataset, and with 60% similarity in the eukaryotic dataset.

Figure 2

Distribution of bacterial-OTUs and active rumen bacterial taxa in different groups. (A) Venn diagram of OTUs specific to the V3–V4 region of bacterial 16S rRNA. The bacterial OTUs identified during different physiological stages were grouped as either unique or shared OTUs between LL, DP, and PP microbiome. (B) Relative abundance (percent reads out of total reads) of nine bacterial phyla and 36 bacterial families are shown. Phyla with relative abundance <1% were grouped as “Other”. Usually, family (f) was indicated; when family was not identified, lower identified taxa were indicated. The significantly different bacterial taxa between physiological stages are indicated by p values (ANOVA). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 3

Heatmap at genus-level of active rumen bacterial communities of different physiological stages. The twenty most abundant bacterial genera (excluding the unclassified bacteria) during LL, DP, and PP were included in the heatmap.

Figure 4

Distribution of eukaryotic-OTUs and active rumen eukaryotic taxa in different groups. (A) A total of 256 OTUs specific to the V9 region of eukaryotic 18S rRNA, identified during different physiological stages, were included in the Venn diagram and grouped as either unique or shared OTUs between the LL, DPs and PP microbiome. (B) Relative abundances of five eukaryotic phyla and 20 eukaryotic families are shown. Phyla with relative abundance <1% were grouped as “Other”. The significantly different bacterial taxa between physiological stages are indicated by p values (ANOVA). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 5

Heatmap at genus-level of active rumen eukaryotic communities of different physiological stages. The twenty most abundant genera (excluding the unclassified taxa) during LL, DP, and PP were included in the heatmap.