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Impact of Starch Source on Equine Hindgut Microbial Ecology

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Impact of Starch Source on Equine Hindgut Microbial Ecology University of Kentucky UKnowledge Theses and Dissertations--Animal and Food Sciences Animal and Food Sciences 2015 IMPACT OF STARCH SOURCE ON EQUINE HINDGUT MICROBIAL ECOLOGY Brittany Elizabeth Davis Harlow University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Harlow, Brittany Elizabeth Davis, "IMPACT OF STARCH SOURCE ON EQUINE HINDGUT MICROBIAL ECOLOGY" (2015). Theses and Dissertations--Animal and Food Sciences. 55. https://uknowledge.uky.edu/animalsci_etds/55 This Doctoral Dissertation is brought to you for free and open access by the Animal and Food Sciences at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Animal and Food Sciences by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Brittany Elizabeth Davis Harlow, Student Dr. Laurie M. Lawrence, Major Professor Dr. David L. Harmon, Director of Graduate Studies IMPACT OF STARCH SOURCE ON EQUINE HINDGUT MICROBIAL ECOLOGY DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Animal and Food Sciences in the College of Agriculture, Food and Environment at the University of Kentucky By Brittany Elizabeth Davis Harlow Lexington, Kentucky Directors: Dr. Michael D. Flythe, Research Microbiologist, USDA, ARS, MWA, FAPRU, Lexington, Kentucky & Dr. Laurie M. Lawrence, Professor of Animal Science, Lexington, Kentucky 2015 Copyright © Brittany Elizabeth Davis Harlow 2015 ABSTRACT OF DISSERTATION IMPACT OF STARCH SOURCE ON EQUINE HINDGUT MICROBIAL ECOLOGY Grain inclusion in equine diets can allow starch to reach the hindgut where bacteria compete for the substrate. The hypothesis was that starch introduction would cause a source-dependent press disturbance in equine fecal microflora. Fecal cell suspensions were prepared by differential centrifugation and re-suspension in media with ground corn, oats or wheat. At 24 h, corn had more amylolytics and Group D Gram-positive cocci (GPC), and fewer lactate-utilizing (LU) bacteria and lactobacilli than oats, with wheat being intermediate. Predominant amylolytics were identified by their 16S RNA gene sequence as Enterococcus faecalis (corn, wheat) and Streptococcus bovis (oats). In an in vivo experiment, 30 horses were assigned to 1 of 6 treatments: CO (hay only), HC (high corn), HO (high oats), LC (low corn), LO (low oats), and LW (low wheat middlings). The study consisted of a 2 wk adaptation (forage only diet) followed by a 2 wk treatment period, during which horses were adapted to their final starch intake (high, 2 g kg BW-1; low, 1 g kg BW-1). Both HC and LC had fewer lactobacilli and LU and more GPC than CO. In contrast, LO and HO had more lactobacilli and LU, and fewer GPC. LW had higher lactobacilli and GPC than CO. The highest number of amylolytics was observed in HC, followed by LC and LW. The predominant amylolytic isolates from corn and wheat horses were E. faecalis. Both experiments identified a negative correlation between lactobacilli and amylolytics, indicating a potential competitive relationship (r = -0.89, in vitro; r = -0.95, in vivo). The next experiment was conducted to determine if a Lactobacillus addition would mitigate amylolytic proliferation, specifically GPC, with corn fermentation. This experiment was conducted as described above with ground corn ± live or dead (autoclaved) L. reuteri. The addition of L. reuteri, regardless of viability, decreased amylolytics and GPC. To identify the mechanism of action, an E. faecalis isolate was co-incubated with dead L. reuteri cells or supernatant. The supernatant depleted the intracellular K+ of E. faecalis. This result demonstrates that one aspect of competition between lactobacilli and enterococci could be a membrane active antimicrobial. KEYWORDS: starch, amylolytic, enterococcus, competition, hindgut acidosis, equine Brittany E. D. Harlow December 9, 2015 IMPACT OF STARCH SOURCE ON EQUINE HINDGUT MICROBIAL ECOLOGY By Brittany Elizabeth Davis Harlow Michael D. Flythe. Ph.D. Laurie M. Lawrence. Ph.D. Directors of Dissertation Dr. David L. Harmon, Ph.D. Director of Graduate Studies December 9, 2015 This work is dedicated to my Appaloosa Sweette ACKNOWLEDGMENTS I would like to extend a thank you to the following persons for their contributions: Dr. Michael Flythe for his guidenance, advice and his endless hours of teaching and mentoring. He is responsible for shaping me as a scientist. Dr. Laurie Lawrence for taking a chance on me, challenging me to think critically and training me to be independent. Dr. Robert Harmon, Dr. David Harmon, and Dr. Isabelle Kagan for serving on my PhD committee. I appreciate all of the advice and guidenance you have provided. Dr. Geza Bruckner for agreeing to serve as my outside examiner. Dr. Glen Aiken for his assistance with the statistical analyses and continued support. USDA scientists, technicians and staff for allowing me to use their laboratories and for taking me in as one of their own. Research technicians Gloria Gellin, Susan Hayes and Andrea Crum for their assistance and contributions to my research projects. A very long list of students that assisted in day-to-day execution of research studies and have supplied me with much needed support and friendship. My husband Aaron for always standing by me, loving me, supporting me, and funding my horse habit. My parents Jill and Tom, and my siblings for all of the moral support and understanding. My grandparents Virginia and Ken Freutel for talking to me on the phone every day, keeping me update on the news and proof reading all of my manuscripts and this dissertation. My boxers Moose and Murdoch, bearded dragon Oscar and horses Mojo and Sweette for keeping me sane. All of the wonderful horses that participated in my research studies: Nola, Star, Super T, Wildfire, Alyssa, Diggins, Maestro, Moses, Leroy, Eggs, Paddy, Easy, Dino, Oliver, Nip, Kilo, Tad, Rancho, Tonosi, Matty, Smart Balance, Emerlaude, Emi, Good Solution, Quick Flite, WSIF, Hosannah, Distinctive View, Rima, Silver and even George. Funding Sources: Prairie Oat Grower’s Association, USDA-ARS and Waltham/Buckeye iii TABLE OF CONTENTS Acknowledgments………………….…………………………………………….............iii List of Tables…………………………………………………………………………..…ix List of Figures…………………………...………………………………………….……..x Chapter 1: Introduction…………………………………………………………….……...1 Chapter 2: Literature Review………………………………………………………….......3 Gastrointestinal Anatomy…………………………………………………………3 The Norml Intestinal Microflora…………………………………………………..5 Animal-Microflora Symbiotic Relationship……………………………………....7 Effect of Diet on Hindgut Microflora……………………………………………..8 Starch Digestion…………………………………………………………………...9 Foregut Enzymatic Digestion…………………………………………....10 Hindgut Fermentative Digestion………………………………………....12 Factors Affecting Starch Digestion……………………………………………....16 Cereal Grain Components………………………………………………..16 Starch Granule: Surface Area to Volume Ratio………………………….18 Starch Granule: Amylose Content……………………………………….19 Starch Granule: Microbial Attachment and Penetration…….….………..20 Grain Processing………………………………………………….……...20 Gastrointestinal Acidosis Pathogenesis…………………………………….……21 Effect of Acidosis on Animal Physiology……………………………….………24 Intestinal Lining……………………………………….…………………24 Metabolic Acidosis………………………………………………………25 Normal Flora and Microbial Toxic Products…………………………….26 Treatment/Prevention of Acidosis……………………………………………….28 Antibiotics………………………………………………………………..28 Buffers……………………………………………………………………28 Probiotics………………………………………………………………...29 PhD Dissertation Research Justification…………………………………………30 Dissertation Objectives…………………………………………………………..31 Chapter 3: Effect of Dietary Starch Source on Equine Fecal Microbial Communities ……………………………………………………………………………………………32 Introduction………………………………………………………………………32 Materials and Methods…………………………………………………………...32 Animals…………………………………………………………………..33 Experimental design and diets………………………………………...…33
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