Quantitative fluorescence in situ hybridization of microbial communities in the rumens of cattle fed different diets

Abstract

At present there is little quantitative information on the identity and composition of bacterial populations in the rumen microbial community. Quantitative fluorescence in situ hybridization using newly designed oligonucleotide probes was applied to identify the microbial populations in liquid and solid fractions of rumen digesta from cows fed barley silage or grass hay diets with or without flaxseed. Bacteroidetes, Firmicutes, and Proteobacteria were abundant in both fractions, constituting 31.8 to 87.3% of the total cell numbers. They belong mainly to the order Bacteroidales (0.1 to 19.2%), hybridizing with probe BAC1080; the families Lachnospiraceae (9.3 to 25.5%) and Ruminococcaceae (5.5 to 23.8%), hybridizing with LAC435 and RUM831, respectively; and the classes Deltaproteobacteria (5.8 to 28.3%) and Gammaproteobacteria (1.2 to 8.2%). All were more abundant in the rumen communities of cows fed diets containing silage (75.2 to 87.3%) than in those of cows fed diets containing hay (31.8 to 49.5%). The addition of flaxseed reduced their abundance in the rumens of cows fed silage-based diets (to 45.2 to 58.7%) but did not change markedly their abundance in the rumens of cows fed hay-based diets (31.8 to 49.5%). Fibrolytic species, including Fibrobacter succinogenes and Ruminococcus spp., and archaeal methanogens accounted for only a small proportion (0.4 to 2.1% and 0.2 to 0.6%, respectively) of total cell numbers. Depending on diet, between 37.0 and 91.6% of microbial cells specifically hybridized with the probes used in this study, allowing them to be identified in situ. The identities of other microbial populations (8.4 to 63.0%) remain unknown.

FIG. 1.

Alignments of the probe sequences and their target sites and sequences of corresponding sites in reference bacteria or clones. The probe names in parentheses after the abbreviated names are according to Oligonucleotide Probe Database nomenclature (2). Only the nucleotides that are different from target sequences are shown. E, empty space; R., Ruminococcus; P., Prevotella; F., Fibrobacter.

FIG. 2.

Images of digest samples from the rumens of cows fed hay- or silage-based diets with and without flaxseed after color combination. Images from probes are labeled in red, and those from DAPI staining are in green. The yellow (combination of red and green), including those partly colored cells in panels A to F, hybridized with probes BAC1080, GAM42a, SRBmix, RUM831, LAC435, and ARCH915, respectively. A few cells (arrows) hybridizing with SRBmix (C) were not stained by DAPI. Bars, 10 μm.