Bacterial Community Dynamics and Molecular Ecology within the Rumen of Bos indicus Cattle Fed Tropical Diets

Andrew Wilson (2010). Bacterial Community Dynamics and Molecular Ecology within the Rumen of Bos indicus Cattle Fed Tropical Diets PhD Thesis, School of Animal Studies, The University of Queensland.

       
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Author Andrew Wilson
Thesis Title Bacterial Community Dynamics and Molecular Ecology within the Rumen of Bos indicus Cattle Fed Tropical Diets
School, Centre or Institute School of Animal Studies
Institution The University of Queensland
Publication date 2010
Thesis type PhD Thesis
Supervisor Prof. Dennis Poppi
Assoc. Prof. Athol Klieve
Ms. Diane Ouwerkerk
Assoc. Prof. Stuart McLennan
Total pages 213
Total colour pages 13
Total black and white pages 200
Subjects 07 Agricultural and Veterinary Sciences
Abstract/Summary This study investigated community structure and dynamics of bacterial populations within the rumen of Bos indicus cattle fed diets based upon tropical pasture species common in northern Australian beef production systems, with the aim of identifying bacterial communities and member species that might correlate to increased efficiency of microbial growth, increased rate of fibre breakdown and increases in the amount of microbial protein produced in the rumen. The initial experiment (Experiment 1) focussed upon the effects of four hay types (tropical Speargrass (2.6 % CP), Mitchell/Flinders grass (3.0 % CP), Pangola (7.6 % CP) and Ryegrass (20.0 % CP)) upon rumen bacterial speciation and dynamics across liquid- and solid-associated fractions of digesta. The following experiment (Experiment 2) investigated the use of targeted nitrogen supplements (urea, casein and branched-chain amino acids + phenylalanine) in an attempt to enhance the efficiency of microbial protein production and growth of fibrolytic bacteria on two common tropical pasture types (Mitchell/Flinders grass and Pangola). Following these experiments, investigation into the phylogenetic identification of dominant bacteria, the metabolic activity of rumen species and the isolation of dominant novel cellulolytic bacteria were attempted. It was hypothesised that alterations in dietary composition, both in the case of hay type and the addition of nitrogen supplements, would account for clear changes in rumen bacterial community composition and the efficiency of microbial protein production. The analysis of rumen bacterial communities from Experiment 1 examined the distribution of bacterial species in relation to fibrous digesta and their response to dietary change by denaturing gradient gel electrophoresis (DGGE). Bacteria were divided into four fractions: the liquid fraction, the loosely associated fraction, the closely associated fraction and the tightly adherent fraction. No significant differences were observed between fractions associated with solid digesta, however it was demonstrated that distinct microbial populations existed in the liquid- and solid-associated phases of the rumen community. Furthermore, hay type demonstrated limited significant effects upon the diversity of species detectable by DGGE within these communities, with common and dominant bands most likely to be significant contributors to microbial protein remaining stable across all treatments. Parallel rumen function data demonstrated a significant improvement in the efficiency of microbial crude protein production (eMCP) with improving diet quality. Phylogenetic sequence analysis confirmed that the bacteria responsible for the most dominant bands detected were members of the Firmicutes phylum that had not previously been isolated or characterised. Isolation attempts failed to acquire any novel bacteria directly associated with these bands however the cellulolytic bacterium Clostridium chartatabidum was able to be successfully cultured. Attempts to improve the efficiency of microbial protein production in Experiment 2 demonstrated that the provision of supplemental rumen degradable nitrogen (RDN) in different forms produced variable effects within the rumen bacterial community, dependent upon the nutritional value of the basal hay with which they were supplied. Analysis of DGGE patterns demonstrated no significant effect of RDN supplementation upon the rumen bacterial community across both tropical hay types and moreover no subsequent improvement in eMCP values was observed. Once again, a number of dominant bacteria were detected across a number of animals on all dietary treatments, but alterations in the banding patterns of sub-dominant community members were also evident. In an attempt to explain the limited responses observed within the rumen bacterial community, an examination of bacterial metabolic activity was carried out. A reverse-transcriptase protocol for the comparison of RNA-derived DGGE profiles to gDNA-derived profiles was evaluated and trialled with a small culture experiment. The application of this assay to examine bacterial metabolic activity within a steer prior to and up to 8 hours after feeding revealed no apparent changes in dominant banding patterns in either 16S rRNA-derived or 16S rRNA gene-derived profiles. This suggests a community-based proportional response by all bacterial species, where metabolic activity (number of copies of 16S rRNA) increased directly in proportion to presence within the population, with an increase in nutrient availability in the rumen. Overall, the results from this study suggest the presence of a stable functional “core” of dominant bacterial species that appear to be important in the breakdown of digesta within the rumen of Bos indicus cattle, despite considerable variability in the nutritive value of the diets fed in this study. This “core” population is central to the function of the rumen and thus may be heavily involved in influencing the observed community-based response to improved nutritional conditions. Phylogenetic analysis revealed that the most dominant members of this stable core were not related to any previously documented cellulolytic or fibre-associated bacteria and these species remain uncharacterised. Additionally, due to their dominance within rumen bacterial community profiles, they are likely to contribute significantly to the amount of microbial protein received by the animal. A significant shift in microbial populations was not observed despite an improvement in diet quality or supplementation, even though eMCP increased with increasing hay quality. Considerable scope exists for further work to understand the ecology of the rumen and what factors govern its speciation, dynamics and growth to increase the efficiency of microbial protein production and associated animal performance in tropical production systems.
Keyword Bos indicus
Rumen
Tropical
Microbiology
Microbial Ecology
eMCP
DGGE
Additional Notes The following page numbers (by document page number)are to be printed in colour: Title, 88, 128, 140, 141, 142, 165, 166, 167, 168, 177, 178, 179.

 
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