USING RNA-SEQ TO CHARACTERIZE THE BIOLOGICAL BASIS OF VARIATION IN FEED EFFICIENCY IN BROILER CHICKENS by Nan Zhou A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Master of Science in Animal Science Spring 2015 © 2015 Nan Zhou All Rights Reserved ProQuest Number: 1596914 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. ProQuest 1596914 Published by ProQuest LLC (2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106 - 1346 USING RNA-SEQ TO CHARACTERIZE THE BIOLOGICAL BASIS OF VARIATION IN FEED EFFICIENCY IN BROILER CHICKENS by Nan Zhou Approved: __________________________________________________________ Behnam Abasht, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee Approved: __________________________________________________________ Limin Kung, Jr., Ph.D. Chair of the Department of Animal and Food Sciences Approved: __________________________________________________________ Mark W. Rieger, Ph.D. Dean of the College of Agriculture and Natural Resources Approved: __________________________________________________________ James G. Richards, Ph.D. Vice Provost for Graduate and Professional Education ACKNOWLEDGMENTS The work was partly funded by Delaware Bioscience Center for Advanced Technology and Heritage Breeders, LLC. Thank to my collaborators, Behnam Abasht and William R Lee, for their great contributions on this work. The work can’t be completed without you. I would like to thank my committee members for their support. First, I want to thank Behnam Abasht for providing me many good opportunities and teaching me so many things. You brought me to the U.S. and gave me a very warm “home” for the last two and a half years. You made me to be a better person. Thank you to Larry Cogburn, Walter Bottje and William R Lee for serving on my thesis committee and helping me throughout my degree. Your support has been greatly appreciated over the last two years. Thank you to my lab mates, Zhu Zhuo, Marie Mutryn and Weixuan Fu, for helping me with my study and live. Thank you all my friends for helping me and making me not alone during the last two and a half years. I want to give a special thank to my parents for their support and unconditional love. You are always my greatest treasure. iii TABLE OF CONTENTS LIST OF TABLES ....................................................................................................... vii LIST OF FIGURES ..................................................................................................... viii ABSTRACT .................................................................................................................. ix Chapter 1 BACKGROUND ................................................................................................ 1 1.1 The Definition of Feed Efficiency ............................................................ 1 1.2 The Significance of Improving Feed Efficiency ....................................... 2 1.3 Factors Affecting Feed Efficiency in Chickens ........................................ 3 1.3.1 External Factors ............................................................................ 3 1.3.2 Internal Factors .............................................................................. 4 1.4 Previous Studies in Chicken Feed Efficiency ........................................... 5 1.4.1 Mitochondrial Function and Feed Efficiency ............................... 5 1.4.2 Global Gene Expression Study of Feed Efficiency ....................... 7 1.5 Introduction to RNA-seq Technique ......................................................... 8 1.6 Introduction to Skeletal Muscle .............................................................. 10 1.6.1 Skeletal Muscle Metabolism ....................................................... 10 1.6.2 Skeletal Muscle Fiber Types ....................................................... 11 1.7 Adverse Effects of Intense Genetic Selection ......................................... 12 1.8 Project Introduction ................................................................................. 13 REFERENCES ................................................................................................. 15 2 MESSENGER RNA SEQUENCING AND PATHWAY ANALYSIS PROVIDE NOVEL INSIGHTS INTO THE BIOLOGICAL BASIS OF CHICKENS’ FEED EFFICIENCY ................................................................. 20 2.1 Background ............................................................................................. 20 2.2 Methods ................................................................................................... 22 iv 2.2.1 Animals and Sample Collection .................................................. 22 2.2.2 RNA Isolation ............................................................................. 23 2.2.3 RNA-seq Library Preparation and Sequencing ........................... 24 2.2.4 Mapping Reads to the Chicken Reference Genome ................... 24 2.2.5 Differential Expression Analysis ................................................ 25 2.2.6 Nanostring nCounter® Gene Expression Assay ......................... 25 2.2.7 Ingenuity Pathway Analysis ........................................................ 26 2.3 Results and Discussion ............................................................................ 26 2.3.1 Phenotype Measurements ............................................................ 26 2.3.2 Transcriptional Profile of Chicken Breast Muscle ...................... 27 2.3.3 Gene Differential Expression Analysis ....................................... 29 2.3.4 Confirmation of RNA-seq Data .................................................. 31 2.3.5 Overview of IPA Analysis .......................................................... 33 2.3.6 Increased Muscle Growth and Remodeling in High-FE Chickens. ..................................................................................... 37 2.3.7 Growth Hormone (GH) and IGFs/PI3K/AKT Signaling Pathway Over-represented in the Differentially Expressed Genes….. ..................................................................................... 43 2.3.8 Inflammatory Response in the Breast Muscle of High-FE Broilers… .................................................................................... 48 2.3.9 Free Radical Scavenging Enriched in the Differentially Expressed Genes between High- and Low-FE Broilers .............. 52 2.3.10 Transcriptional Regulation of Hypoxia-inducible Factor-1α (HIF1α).. ...................................................................................... 58 2.4 Conclusions ............................................................................................. 61 REFERENCES ................................................................................................. 64 3 CHARACTERIZATION OF METABOLIC DIFFERENCES IN THE BREAST MUSCLE OF HIGH AND LOW FEED EFFICIENCY CHICKENS USING RNA-SEQ ...................................................................... 79 3.1 Introduction ............................................................................................. 79 3.2 Methods ................................................................................................... 81 3.2.1 Animals and RNA-seq Analysis ................................................. 81 3.2.2 Ingenuity Pathway Analysis ........................................................ 82 3.2.3 Ultimate pH (pHu) Measurement and Analysis .......................... 82 3.2.4 Determination of Breast Muscle Glycogen Content ................... 83 3.2.5 Quantitative Reverse Transcription-PCR (qRT-PCR) ................ 83 v 3.3 Results and Discussion ............................................................................ 85 3.3.1 Carbohydrate Metabolism ........................................................... 85 3.3.1.1 Glycerol-3-Phosphate Shuttle ...................................... 89 3.3.1.2 Transcriptional Control of Carbohydrate Metabolism . 92 3.3.2 Glycogen Depletion in Breast Muscle of the HFE Chickens ...... 93 3.3.3 Transcriptional Regulation of Lipid Metabolism ........................ 96 3.3.3.1 Cholesterol Biosynthesis and Reverse Cholesterol Transport… .................................................................. 97 3.3.3.2 Lipid Catabolism .......................................................... 99 3.3.3.3 Lipogenesis ................................................................ 101 3.3.4 Amino Acid Metabolism ........................................................... 102 3.3.4.1 The Regulation of Amino Acid Metabolism .............. 103 3.3.4.2 Glutamine Metabolism ............................................... 106 3.3.5 Metabolism and Inflammatory Response .................................. 107 3.3.5.1 Effects of Inflammatory Response on Metabolic Regulation.. ................................................................ 107 3.3.5.2 Lipid-derived Inflammatory Mediators ...................... 108 3.4 Conclusions 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