EFFECTS OF COMMERCIAL FEED ADDITIVES ON THE GUT MICROBIOTA OF FOOD ANIMALS BY PRAKASH POUDEL A dissertation submitted in partial fulfillment of the requirements for the Doctor of Philosophy Major in Animal Science South Dakota State University 2019 DocuSign Envelope ID: 33CCE236-EF21-4AF6-8A80-6DC42FC018CE ii DISSERTATION ACCEPTANCE PAGE Prakash Poudel This dissertation is approved as a creditable and independent investigation by a candidate for the Doctor of Philosophy degree and is acceptable for meeting the dissertation requirements for this degree. Acceptance of this does not imply that the conclusions reached by the candidate are necessarily the conclusions of the major department. Benoit St-Pierre Advisor Date Joseph P Cassady Department Head Date Dean, Graduate School Date iii ACKNOWLEDGEMENT First of all, I would like to express my thanks to my advisor, Dr. Benoit St-Pierre, for accepting me as a PhD student in your team and providing me the valuable guidance, encouragement, support and knowledge throughout the study of my PhD at South Dakota State University. I extremely felt fortunate to have a professor like you who cared so much about my research activities, always responded my queries, and supported to execute laboratory works in no time. The journey of my study was possible due to your patience and encouragement. You taught me to be an expert on microbiota and a true researcher. Many thanks again for your guidance towards my future career. I am also indebted to receive a great support from my supervisory committee members, Dr. Crystal Levesque, Dr. Joy Scaria, and Dr. Kunsoon Park. Thank you all for your time, directions, support, advices, and inputs towards my research projects. Special thank goes to Dr. Samuel Ryan for supporting on animal trial on Swine facility at SDSU. At this moment, I would also like to thank my previous advisor Dr. David Casper and fellow graduate students Kelly Froehlich and Subash Acharya for helping me to collect rumen samples from the calves on EO experiment. I really appreciate the support and assistance I got from my past and present lab colleagues Vinay, Lee, Angie and other graduate student for sampling and data analysis. I would also like to thank swine facility at SDSU for allowing me to carry out the nursery pig research. I would thankful to Vitech Bio-Chem Corporation, CA for providing the funding of two Peptiva experiments carried out on piglets. iv Finally, I would like to express my heartful gratitude to my beloved wife Kalpana, daughter Anweshi, and son Ayansh, and other family members for their endless support, patience, understanding, and love throughout my study period without which this great accomplishment would not have been possible. Thank you for all those and love you so much. v TABLE OF CONTENTS LIST OF ABBREVIATIONS…………………………………………..……………......xi LIST OF FIGURES……………………………………………….……..……………...xiii LIST OF TABLES……………………………………………………..…………...…...xiv ABSTRACT…………………………………………………………………………….xvi Chapter 1: Literature Review ……………………………………………………………..1 1. Meeting the Food Demand of a Growing World Population .....................................2 1.1 Current Situation and Future Outlook .................................................................2 1.2 Role of the livestock sector .................................................................................3 2. Challenges for livestock production: health and welfare of young animals ...............5 2.1 Overview of challenges.......................................................................................5 2.2 Dairy production .................................................................................................6 2.3 Swine production .............................................................................................. 10 3. The gut microbiome ............................................................................................... 12 3.1 Importance........................................................................................................ 12 3.2 Rumen Microbial ecology ................................................................................. 14 3.3 Microbial succession in the gastrointestinal tract ............................................... 15 3.4 Eubiosis vs dysbiosis ........................................................................................ 17 4. Manipulating the gut microbiome ........................................................................... 18 4.1 Antibiotics ........................................................................................................ 19 vi 4.2 Essential oils ..................................................................................................... 21 4.3 Peptides ............................................................................................................ 26 4.4 Enzymes as supplements in animal feed ............................................................ 29 4.5 Prebiotics in animal diets .................................................................................. 32 5. Hypothesis and Research Objectives ...................................................................... 33 References ................................................................................................................. 34 Chapter 2: Feeding essential oils to neonatal Holstein dairy calves results in increased ruminal Prevotellaceae abundance and propionate concentrations…………...57 Abstract ..................................................................................................................... 58 2. Materials and Methods ........................................................................................ 61 2.1 Sample collection ............................................................................................. 61 2.2 SCFA analysis .................................................................................................. 62 2.3 Microbial DNA isolation and PCR amplification .............................................. 63 2.4 Computational analysis of PCR generated 16S rRNA amplicon sequences ....... 64 2.5 Computational analysis for alpha and beta diversity .......................................... 65 2.6 Statistical Analyses ........................................................................................... 66 3. Results ................................................................................................................ 66 3.1 Comparative analysis of ruminal SCFA between EO supplemented and non-supplemented diets .......................................................................................... 66 vii 3.2 Effects of EO on the taxonomic composition of ruminal bacteria in growing calves ..................................................................................................................... 67 3.3 Effects of EO on the ruminal bacterial community structure in growing calves . 68 3.4 Identification of potential associations between main OTUs and ruminant performance parameters .......................................................................................... 69 4. Discussion .......................................................................................................... 71 5. Conclusion .......................................................................................................... 76 References ................................................................................................................. 85 Chapter 3: Dietary inclusion of a peptide-based feed additive can accelerate the maturation of the fecal bacterial microbiome in weaned pigs……………..…………….93 Abstract ..................................................................................................................... 94 1. Introduction ........................................................................................................ 96 2. Materials and Methods ........................................................................................ 99 2.1 Animal performance trial and sample collection................................................ 99 2.2 Microbial DNA isolation and PCR amplification of the 16S rRNA gene ......... 101 2.3 Computational analysis of PCR generated 16S rRNA amplicon sequences ..... 101 2.4 Statistical analyses .......................................................................................... 103 3. Results .............................................................................................................. 104 3.1 Effect of low inclusion of Peptiva on swine performance during the nursery phase .................................................................................................................... 104 viii 3.2 Effect of diet composition and Peptiva supplementation on the fecal bacterial profile of weaned pigs .......................................................................................... 104 3.3 Comparative analysis of fecal bacterial composition by alpha and beta diversity ............................................................................................................................. 105 3.4 Identification of weaned pig OTUs responding to distinct dietary treatments .. 106 3.5 Associations between main OTUs and dietary treatments................................ 107 4. Discussion ........................................................................................................ 107 4.1 Bacterial succession from Lactobacillaceae