Table 1.
Protozoa concentration (log (numbers +1) per mL of rumen fluid) from sheep either protozoa-free (P1), faunated with holotrich protozoa (P2) or with a complete protozoal population (P3).
Table 2.
Fermentation parameters of rumen samples from sheep either protozoa-free (P1), faunated with holotrich protozoa (P2) or with a complete protozoal population (P3).
Table 3.
Richness (Ri) and Shannon-Wiener (Sh) indexes of NGS (OTU matrix from Ion Torrent data) or T-RFLP (peaks in the amplicons of 16S rRNA gene digested using HhaI, HaeIII, MspI and RsaI.
Figure 1.
Correlation between T-RFLP and NGS.
Correlation plot between NGS (OTU matrix from Ion Torrent data) and T-RFLP (peaks in the amplicons of 16S rRNA gene digested using HhaI, HaeIII, MspI and RsaI (dendrogram shows amalgamation of data from all four enzymes) on both Richness (Figure 2a) and Shannon-Wienner index (Figure 2b). Genomic DNA was obtained from rumen samples from 8 animals (A1 to A8) either protozoa-free (P1, crosses), faunated with holotrich protozoa (P2, black triangles) o with a complete protozoal population (P3, black squares).
Figure 2.
Dendrogram of the rumen bacterial population analysed by T-RFLP(a) or NGS(b).
Dendrogram using the combination of Manhattan distances and UPGMA (unweighted pair group method with arithmetic mean) of T-RFLP (peaks in the amplicons of 16S rRNA gene digested using HhaI, HaeIII, MspI and RsaI (dendrogram shows amalgamation of data from all four enzymes) or NGS (OTU matrix from Ion Torrent data). Genomic DNA was obtained from rumen samples from 8 animals (A1 to A8) either protozoa-free (P1, crosses), faunated with holotrich protozoa (P2, black triangles) o with a complete protozoal population (P3, black squares).
Figure 3.
PCA plot of the rumen bacterial structure analysed by T-RFLP(a) or NGS(b).
Principal Component Analysis of T-RFLP (Figure 4a, peaks in the amplicons of 16S rRNA gene digested using HhaI, HaeIII, MspI and RsaI (dendrogram shows amalgamation of data from all four enzymes) or NGS (Figure 4b, OTU matrix from Ion Torrent data). Genomic DNA was obtained from rumen samples from 8 animals either protozoa-free (P1, black crosses), faunated with holotrich protozoa (P2, black triangles) or with a complete protozoal population (P3, black squares).
Table 4.
Spearson's correlation (rho value) between Richness (Ri) and Shannon-Wiener (Sh) indexes of NGS (OTU matrix from Ion Torrent data) or T-RFLP (peaks in the amplicons of 16S rRNA gene digested using HhaI, HaeIII, MspI and RsaI) and fermentation parameters of rumen samples.
Figure 4.
CCA plot of rumen bacterial communities analysed by T-RFLP(a) or NGS(b) and considering fermentation parameters.
Canonical correspondence analysis of NGS (OTU matrix from Ion Torrent data) or T-RFLP (peaks in the amplicons of 16S rRNA gene digested using HhaI, HaeIII, MspI and RsaI. The formula used in the analysis was the following y (either NGS or T-RFLP data) = pH+Ammonia (NH3)+Acetate (C2)+Propionate (C3)+Butyrate (C4). Genomic DNA was obtained from rumen samples from 8 animals either protozoa-free (P1, black crosses), faunated with holotrich protozoa (P2, black triangles) o with a complete protozoal population (P3, black squares). Blue vectors indicate the effect of the constraining variables (pH, Ammonia (NH3), Acetate (C2), Propionate (C3) and Butyrate (C4).
Figure 5.
Phyla-level classification of OTUs from NGS at 97%.
Phyla-level classification of OTUs from NGS at 97%. Genomic DNA was obtained from rumen samples from 8 animals (A1 to A8) either protozoa-free (P1), faunated with holotrich protozoa (P2) o with a complete protozoal population (P3).
Table 5.
Relative abundance (%) of the main phyla identified in rumen fluid from protozoa-free (P1), faunated with holotrich protozoa (P2) o with a complete protozoal population (P3) animals.
Figure 6.
Bacterial genera significantly impacted by rumen protozoa.
Boxplots of bacterial genera found to shift in their relative abundance (as percentage) when progressive colonization of rumen protozoa is applied. Samples come from rumen fluid from protozoa-free (P1), faunated with holotrich protozoa (P2) o with a complete protozoal population (P3) animals.