High-throughput sequencing techniques to analyze microbial communities in the gastrointestinal tract of broiler chickens Daniel Enrique, Borda Molina FACULTY OF AGRICULTURAL SCIENCES Institute of Animal Science – University of Hohenheim Livestock Microbial Ecology Jun.-Prof. Dr. rer. nat. Amélia Camarinha Silva HIGH-THROUGHPUT SEQUENCING TECHNIQUES TO ANALYZE MICROBIAL COMMUNITIES IN THE GASTROINTESTINAL TRACT OF BROILER CHICKENS DISSERTATION submitted in fulfilment of the requirements for the degree “Doktor der Agrarwissenschaften” (Dr. sc. agr. / PhD in Agricultural Sciences) to the Faculty of Agricultural Sciences presented by Daniel Enrique, Borda Molina (M.Sc. – Microbiologist) born on 17.07.1987 in Bogotá, Colombia Stuttgart-Hohenheim, 2018 The presented studies were realized at the Institute of Animal Science and at the Agricultural Research Station Lindenhöfe (72800, Eningen), of the University of Hohenheim in Stuttgart, Germany. The animal experiments were performed in February 2015 and April 2016 under the supervision of Jun.-Prof. Dr. Amélia Camarinha Silva (University of Hohenheim, Head of the “Livestock Microbial Ecology”) and the co- supervision of Prof. Dr. Markus Rodehutscord (University of Hohenheim, Head of the “Animal Nutrition”). University of Hohenheim, Faculty of Agricultural Science, December 2018 Dean of the Faculty of Agricultural Sciences: Prof. Dr. Ralf T. Vögele Date of examination: 10th December 2018 Chair of the committee: Prof. Dr. J. Bennewitz First Reviewer, First Examiner: Jun.-Prof. Dr. rer. Nat. A. Camarinha Silva Second Reviewer: Prof. Dr. C. Hölzel Second Examiner: Prof. Dr. J. Seifert Third Examiner: Prof. Dr. M. Rodehutscord MY FAMILY TABLE OF CONTENTS TABLE OF CONTENTS Table of contents ....................................................................................................... i - iii List of tables ................................................................................................................... iv List of figures .......................................................................................................... v - viii List of abbreviations .................................................................................................. ix - x 1. Introduction ...................................................................................................... 1 – 33 1.1 General Introduction ................................................................................................ 3 1.2 Recent Techniques to Investigate Broiler Chicken GIT Microbiota .......................... 5 1.3 Microbial Colonization and the Influence of Digestive Activities in the GIT ........... 7 1.4 Effect of the Age in the GIT of Broiler Chickens ................................................... 10 1.5 Influence of the Microbiota in Broiler Chicken Metabolism ................................... 11 1.6 Diet Components in Broiler Chicken Nutrition, and Its Effects on Microbiota ...... 15 1.7 Scope and Work Hypothesis ................................................................................... 20 1.8 References .............................................................................................................. 21 2. Current perspectives of the chicken gastrointestinal tract and its microbiome …………………………………………………………………………………….. 35 – 63 2.1 Abstract .................................................................................................................. 38 2.2 Introduction ............................................................................................................ 38 2.3 Exploring the Composition and Function of the Chicken Gut Microbiome ........... 40 2.3.1 Targeted Amplicon Sequencing of the 16S rRNA Gene ..................................... 40 2.3.2 Metagenomic Shotgun Sequencing ....................................................................... 43 2.3.3 Metaproteomics .................................................................................................... 46 2.4 Chicken Feeding and Its Influence on the Microbiota ............................................ 48 2.5 Future Perspectives ................................................................................................. 52 2.6 Acknowledgments .................................................................................................. 54 2.7 References .............................................................................................................. 55 3. Insights into broilers’ gut microbiota fed with phosphorus, calcium and phytase supplemented diets ……………………………………………………………..… 65 – 94 3.1 Abstract .................................................................................................................. 68 3.2 Introduction ............................................................................................................ 69 3.3 Materials and Methods ........................................................................................... 70 3.3.1 Animal Sampling .................................................................................................. 70 3.3.2 Broiler Performance Analysis .............................................................................. 71 3.3.3 DNA Extraction and Illumina Amplicon Sequencing .......................................... 71 ____ i TABLE OF CONTENTS 3.3.4 Multivariate Analysis ........................................................................................... 72 3.4 Results and Discussion ............................................................................................ 73 3.4.1 Global Overview of Broiler Performance and the Microbial Community in Crop, Ileum and Caeca ............................................................................................................. 73 3.4.2 Diet Effect in the Crop Microbial Community ...................................................... 77 3.4.3 Diet Effect on the Microbial Community in the Ileum ......................................... 79 3.4.4 Diet Effect on the Microbial Community in the Caeca ........................................ 82 3.5 Conclusions ............................................................................................................. 85 3.6 Authors Contributions ............................................................................................. 85 3.7 Acknowledgments ................................................................................................... 86 3.8 Supplementary Material .......................................................................................... 86 3.9 References ............................................................................................................... 88 4. Effects of protease and phytase supplements on small intestinal microbiota and amino acid digestibility in broiler chickens …………………………………... 95 – 124 4.1 Abstract ................................................................................................................... 98 4.2 Introduction ............................................................................................................. 99 4.3 Material and Methods ........................................................................................... 101 4.3.1 Experimental Diets .............................................................................................. 101 4.3.2 Birds and Experimental Procedures ................................................................... 104 4.3.3 Chemical Analyses ............................................................................................. 105 4.3.4 DNA Extraction, Illumina Amplicon Sequencing and Data Analysis ................ 105 4.3.5 Calculations and Statistical Analysis .................................................................. 106 4.4 Results ................................................................................................................... 107 4.4.1 Growth Performance and Prececal Amino Acid Digestibility ............................. 107 4.4.2 Microbial Communities in the Terminal Small Intestine ..................................... 109 4.5 Discussion ............................................................................................................. 113 4.5.1 Prececal Amino Acid Digestibility ...................................................................... 113 4.5.2 Microbial Communities in the Terminal Small Intestine ................................... 114 4.6 Supplementary Material ......................................................................................... 117 4.7 Acknowledgements .............................................................................................. 117 4.8 Conflicts of interest ............................................................................................... 117 4.9 Notes ..................................................................................................................... 117 4.10 References ............................................................................................................. 118 5. General discussion …………………………………………………………..125 – 173 5.1 Methods Standardization ....................................................................................... 127 5.2 The Chicken GIT Microbiota from culturing to high-throughput sequencing ........ 130 5.3 Sample Variability ...............................................................................................