Investigation and subsequent manipulation of the intestinal microbiota of pigs, with a view to optimising feed efficiency By Ursula Mary McCormack (B.Ag.Sc) A thesis submitted for the degree of Doctor of Philosophy Waterford Institute of Technology Research Supervisors Dr. Gillian E. Gardiner Dr. Peadar G. Lawlor Submitted to Waterford Institute of Technology September 2017 Declaration No element of the work described in this thesis has been previously submitted for a degree at this or any other institution. The work in this thesis has been performed entirely by the author. Signature: ____________________________ Date: ___________________________ i Table of Contents Declaration ..................................................................................................................... i Table of Tables ............................................................................................................. xi Table of Figures .......................................................................................................... xiii List of abbreviations ................................................................................................... xvi Abstract ............................................................................................................................. 1 1. Literature Review .......................................................................................................... 2 1.1 Introduction ............................................................................................................. 3 1.2. Feed efficiency measures ....................................................................................... 4 1.3. Factors affecting feed efficiency in pigs ................................................................ 6 1.3.1 Breed/genetics .................................................................................................. 6 1.3.2. Diet .................................................................................................................. 7 1.3.3. Feeding to match requirements ....................................................................... 8 1.3.4. Management and microenvironment............................................................... 9 1.3.5. Health status .................................................................................................. 11 1.4. Benefits of improving feed efficiency in pigs ...................................................... 12 1.4.1. Lower production costs ................................................................................. 12 1.4.2. Improved carcass quality .............................................................................. 12 1.4.3. Reduced environmental impact ..................................................................... 13 1.5. Potential biomarkers for feed efficiency .............................................................. 13 1.5.1. Immune markers for feed efficiency ............................................................. 13 1.5.2. Serum metabolites ......................................................................................... 16 ii 1.5.3. Salivary cortisol ............................................................................................ 17 1.5.4. Gut morphology ............................................................................................ 17 1.6. The intestinal microbiota of pigs .......................................................................... 19 1.6.1. Establishment, composition and diversity of the porcine intestinal microbiota ................................................................................................................................. 19 1.6.2. Role of the intestinal microbiota in pigs ....................................................... 24 1.6.3. Potential impact of the intestinal microbiota on feed efficiency in pigs ....... 25 1.6.4. Analysing the intestinal microbiota .............................................................. 27 1.7. Manipulating the intestinal microbiota, with a view to optimising feed efficiency in pigs .......................................................................................................................... 31 1.7.1. Feed additives, with a focus on prebiotics .................................................... 31 1.7.2 Inoculation of live bacteria............................................................................. 36 1.8. Conclusions .......................................................................................................... 42 1.9. Overall objectives of the research ........................................................................ 43 2. Exploring a possible link between the intestinal microbiota and feed efficiency in pigs .................................................................................................................................. 44 2.1. Abstract ................................................................................................................ 45 2.2. Importance ............................................................................................................ 46 2.3. Introduction .......................................................................................................... 47 2.4. Materials and Methods ......................................................................................... 50 2.4.1. Ethical approval ............................................................................................ 50 2.4.2. Animal management and sample collection ................................................. 50 2.4.3. Salivary cortisol analysis .............................................................................. 53 iii 2.4.4. Histological analysis of ileal tissue ............................................................... 53 2.4.5. 16S rRNA gene amplicon sequencing of faecal and intestinal microbiota ... 53 2.4.6. Prediction of microbial function ................................................................... 54 2.4.7. Total bacterial quantification using quantitative PCR .................................. 54 2.4.8. Volatile fatty acid analyses of faecal and intestinal digesta samples ............ 55 2.4.9. Statistical analysis ......................................................................................... 55 2.5. Results .................................................................................................................. 58 2.5.1. Growth performance of pigs ranked by residual feed intake ........................ 58 2.5.2. Salivary cortisol and ileal histology in pigs ranked by residual feed intake . 58 2.5.3. Microbial load and diversity in pigs ranked by residual feed intake ............ 59 2.5.4. Intestinal microbiota composition in pigs ranked by residual feed intake .... 59 2.5.5. Intestinal microbiota correlations with RFI value ......................................... 62 2.5.6. Predictive functional analysis of the intestinal microbiota of pigs ranked by residual feed intake ................................................................................................. 63 2.5.7. Volatile fatty acid concentrations in the faeces and digesta of pigs ranked by residual feed intake ................................................................................................. 64 2.5.8. Correlations between microbial composition and physiological traits in pigs ranked by residual feed intake ................................................................................ 64 2.6. Discussion ............................................................................................................ 65 2.7. Conclusion ............................................................................................................ 71 2.8. Tables and Figures ................................................................................................ 72 2.9. Supplementary Information .................................................................................. 80 iv 3. Porcine feed efficiency (FE)-associated intestinal microbiota and physiological traits: finding consistent cross-locational biomarkers for residual feed intake (RFI) ............... 93 3.1. Abstract ................................................................................................................ 94 3.2. Introduction .......................................................................................................... 96 3.3. Materials and methods .......................................................................................... 98 3.3.1. Ethical approval ............................................................................................ 98 3.3.2. Animal management, performance records and sampling ............................ 98 3.3.3. Salivary cortisol analysis ............................................................................ 100 3.3.5. Immunological analyses .............................................................................. 101 3.3.8. Microbiota profiling .................................................................................... 104 3.3.9. Microbial function prediction ..................................................................... 104 3.3.10. Volatile fatty acid concentrations in faeces and digesta ........................... 105 3.3.11. Statistical analyses ...................................................................................