IMPACT OF DIET COMPOSITION ON RUMEN BACTERIAL PHYLOGENETICS A Thesis Submitted to the College of Graduate Studies and Research In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In the Department of Animal and Poultry Science University of Saskatchewan Saskatoon By RENÉE MAXINE PETRI Copyright Renee M. Petri, March, 2013. All rights reserved. Permission to Use In presenting this thesis in partial fulfilment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to make other use of material in this thesis in whole or part should be addressed to: Dr. Andrew Van Kessel Head of the Department of Animal and Poultry Science University of Saskatchewan Saskatoon, Saskatchewan S7N 5A8 i ABSTRACT Two experiments were conducted to determine the effects of various forage to concentrate ratios on the rumen microbial ecosystem and rumen fermentation parameters using culture- independent methods. In the first experiment, cattle were fed either a high concentrate (HC) or a high concentrate without forage (HCNF) diet. Comparison of rumen fermentation parameters between these two diets showed that duration of time spent below pH 5.2 and rumen osmolality were higher for HCNF. Calculations using Simpson’s index showed a greater diversity of dominant species for HCNF than in HC based on 16S rRNA PCR-DGGE. Real-time real-time PCR showed populations of Fibrobacter succinogenes (P=0.01) were lower in HCNF than HC diets. Ruminococcus spp., F. succinogenes and Selenomonas ruminantium were present at higher (P≤0.05) concentrations in solid than in liquid digesta in both diets. The second experiment compared cattle as they adapted from a strictly forage to a concentrate diet, after which they were subject to an acidotic challenge and a recovery period (Forage, Mixed Forage, High Grain, Acidosis and Recovery). A total of 153,621 high-quality bacterial sequences were obtained from biopsied rumen epithelium, and 407,373 sequences from the solid and liquid phases of rumen contents. Only 14 epithelial genera representing >1.0% of the epimural population differed (P ≤ 0.05) among dietary treatments. However, clustering showed a closer relation in bacterial profiles for the Forage and Mixed Forage diets as compared to the High Grain, Acidosis and Recovery diets. Several epithelial identified genera including Atopobium, Desulfocurvus, Fervidicola, Lactobacillus and Olsenella increased as a result of acidosis. However, any changes in bacterial populations during the acidosis challenge were not sustained during the recovery period. This indicates a high level of stability within the rumen epimural community. An epithelial core microbiome was determined which explained 21% of the enumerable rumen population across all treatment samples. Cluster analysis of the solid and liquid phase rumen ii bacterial showed that these populations differed (P ≤ 0.10) between forage and grain-based diets. Rumen core microbiome analysis found 32 OTU’s representing 10 distinct bacterial taxa in whole rumen contents for all dietary treatments. Heifers that developed clinical acidosis vs the subclinical acidosis showed increases in the genera Acetitomaculum, Lactobacillus, Prevotella, and Streptococcus. Variation in microbial taxa as an effect of both treatment and animal was evident in the solid and liquid fractions of the rumen digesta. However, impacts of a dietary treatment were transient and despite an acidotic challenge, rumen microbiota were able to recover within a week of perturbation. The bacterial populations in the rumen are highly diverse as indicated by DGGE analysis and showed clear distinction between not only dietary treatments, individual animals, but also between epithelial, liquid and solid associated populations on the same diet. Molecular techniques provide an increased understanding of the impact of dietary change on the nature of rumen bacterial populations and conclusions derived using these techniques may not match those previously derived using traditional laboratory culturing techniques. iii ACKNOWLEDGMENTS I would like to express my deep appreciation and gratitude to my advisors, Dr. Tim McAllister and Dr. John McKinnon for the support, guidance and mentorship provided to me, all the way from when I was first considering applying to a PhD program, through to completion of this degree. John, thank you for encouraging me to continue on past my Master’s program, I am truly fortunate to have had the opportunity to work you throughout my graduate programs. Tim, your keen intellect and drive for knowledge is surpassed only by your skills in support and motivation of graduate students, I am honored to have had the chance to work with you. I would also like to thank my committee members, Drs. Tim Mutsvangwa, Karen Beauchemin, Andrew Van Kessel and Steve Hendrick for the friendly guidance, thought provoking suggestions, and the general collegiality that each of them offered to me over the years. In a similar vein, I’d like to recognize Drs. Robert Forster, Ron Teather, Trevor Alexander, Greg Penner and the late Denis Krause for the contributions that each of them made to my intellectual growth during my program. I must also express my appreciation to the staff of the Lethbridge Research Centre for their assistance and collaborative work throughout my program. Primarily, Shaun Cook, Wendy Smart, Lyn Paterson, Pam Lussier, and Krysty Munns. Finally, I’d be remiss if I didn’t acknowledge the innumerable sacrifices made by my husband, Daniel, in shouldering daily burdens, diaper changes and long distances all while also pursuing his PhD program. iv DEDICATION To the most amazing little boy I know. Finn, you are my biggest accomplishment. v TABLE OF CONTENTS Page ABSTRACT .................................................................................................................................... ii ACKNOWLEDGMENTS ............................................................................................................. iv LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES ....................................................................................................................... xi ABBREVIATIONS ..................................................................................................................... xiii 1.0 GENERAL INTRODUCTION ..................................................................................................1 2.0 LITERATURE REVIEW ..........................................................................................................4 2.1 Microbial Ecology in the Rumen .......................................................................................... 4 2.2 Role of Microbes in Digestion .............................................................................................. 7 2.2.1 Forage Diets .................................................................................................................8 2.2.2 Mixed Forage-Concentrate Diets ...............................................................................11 2.3 Impact of Digestive Disturbances on Ruminal Microbiome .............................................. 16 2.4 Rumen Microbial Component of Acidosis ......................................................................... 19 2.5 Individual Animal Variation ............................................................................................... 20 2.6 Rumen Molecular Microbiology......................................................................................... 21 2.6.1 Current Molecular Techniques for Ecological Characterization in the Rumen .........23 2.6.2 Fingerprinting Techniques .........................................................................................27 2.6.3 Quantification Techniques .........................................................................................30 2.6.4 Next-generation sequencing technology ....................................................................31 2.7 Summary ............................................................................................................................. 34 2.8 Hypothesis........................................................................................................................... 35 3.0 CHARACTERIZATION OF RUMEN BACTERIAL DIVERSITY IN FEEDLOT CATTLE ……………………………………………………………………………………………………38 3.1 Introduction ......................................................................................................................... 38 3.2Materials and Methods ........................................................................................................