RNA-SEQ REVEALS NOVEL GENES AND PATHWAYS INVOLVED IN BOVINE MAMMARY INVOLUTION DURING THE DRY PERIOD AND UNDER ENVIRONMENTAL HEAT STRESS By BETHANY M. DADO SENN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2018 © 2018 Bethany M. Dado Senn To my family, the true dairy enthusiasts ACKNOWLEDGMENTS To my advisor, mentor, and friend Dr. Jimena Laporta, I am humbled and grateful to have served as your graduate student as you provided invaluable advice and kindness throughout my projects. Your open-door policy has facilitated my growth both personally and professionally. Thank you for the opportunity to research lactation physiology, volunteer and teach, and pursue a degree at the University of Florida. I extend my appreciation to my committee members Dr. Geoffrey Dahl and Dr. Pete Hansen for utilizing their many years of experience to provide useful critiques and additional insight into my analysis and interpretations. Thank you to Dr. Hansen for the use of Ingenuity Pathway Analysis® and to Dr. Dahl for his heat stress expertise. I thank the faculty and staff in the Department of Animal Sciences at the University of Florida, especially Dr. Francisco Peñagaricano for his vital RNA- sequencing and statistical contribution to my thesis project. Further thanks to Dr. Corwin Nelson, Dr. Stephanie Wohlgemuth, and Dr. John Bromfield for use of lab space and research support. Special appreciation goes to Joyce Hayen, Pam Krueger, and Renee Parks-James and the UF Dairy Unit staff. I also express appreciation to the Animal Molecular and Cellular Biology program, the Brélan E. Moritz family, and the National Dairy Shrine for funding a portion of my education. I am grateful for my supportive laboratory community for their assistance with projects and papers, not to mention the memories and laughter accumulated from long nights in the lab. Special thanks to Dr. Amy Skibiel for being an incredible role model and mentoring me through assays, presentations, and paper writing, Catalina Mejia Bonilla for being my first UF friend and research confidante, Marcela Marérro-Perez and Sena Field for bringing joy into research, Thiago Fabris for his guidance on-farm, and 4 Debora da Silva, Carolina Collazos, Fabiana Corra, and Therus Brown for their assistance with various aspects of my research projects including sample collection, analysis, and presentation practice. Thanks to my undergraduate role models Dr. Laura Hernandez, Dr. Hasan Khatib, Dr. Marina Danes, Dr. Michel Wattiaux, Ryan Pralle, Nicole Gross, and Patti Hurtgen for helping me find academic direction and pointing me to UF. Thank you to my friends near and far—Jessi and Cody Getschel, Saager Paliwal, Eleanor Miller, Katey Scholz, Mykayla Getschel, Alexus and Josh Berndt, Mackenzie Dickson, and the Flores, Tyler, Sy, and Guernsey families—for listening to my crazy lab stories, offering solutions to my dilemmas, and being truly genuine friends throughout the journey. I would like to give special thanks to my loving family. Thank you to my parents, Rick and Gwen Dado, for serving as excellent examples of academics and dairy producers. To my siblings Ethan, Trent, and Meikah Dado, thank you for praying for me and setting the bar high for success. I thank my extended families, specially my Grandma Thelma Betzold, Grandpa Gary Dado, and Grandma Arlene Dado, and my in- laws Jim, Deb, and Ted Senn and Jeremy and Tracy Keifenheim for the many phone calls inquiring about my research. And to my husband, Travis Senn: thank you for moving across the country for me, challenging me academically and spiritually, and providing for our beautiful future. I look forward to all our adventures to come. Finally, I give thanks to my Heavenly Father who has granted me strength and patience for the journey and the talents and resources to serve others through this degree. To God be the Glory. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF OBJECTS ....................................................................................................... 10 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 13 CHAPTER 1 LITERATURE REVIEW .......................................................................................... 15 The Bovine Mammary Gland Dry Period ................................................................ 15 Physiology of the Dry Period ............................................................................ 16 Molecular Regulators of Mammary Involution and Redevelopment ................. 18 Heat Stress in Dairy Cattle ...................................................................................... 21 Heat Stress During the Dry Period ................................................................... 25 Mammary Gene Expression under Heat Stress ............................................... 27 RNA-Sequencing Technology ................................................................................. 30 Transcriptome Analysis Technology Comparisons ........................................... 32 RNA-Sequencing Application in Bovine Research ........................................... 34 Summary ................................................................................................................ 35 2 RNA-SEQ REVEALS NOVEL GENES AND PATHWAYS INVOLVED IN BOVINE MAMMARY INVOLUTION DURING THE DRY PERIOD AND UNDER ENVIRONMENTAL HEAT STRESS ....................................................................... 37 Abstract ................................................................................................................... 37 Introduction ............................................................................................................. 38 Materials and Methods............................................................................................ 40 Animals, Treatments, and Experimental Design ............................................... 40 Mammary Tissue Collection and RNA Extraction ............................................. 40 Library Generation and RNA Sequencing ........................................................ 41 Identification of Differentially Expressed Genes, Pathways, and Regulators ... 42 Results .................................................................................................................... 44 Physiological Parameters and Milk Yield .......................................................... 44 Ingenuity® Pathways Analysis (IPA®) Regulator and Network Analysis............ 47 Differentially Expressed Genes and Regulators Impacted by Heat Stress ....... 48 Discussion .............................................................................................................. 49 Conclusions ............................................................................................................ 59 6 3 GENERAL DISCUSSION AND SUMMARY ............................................................ 87 APPENDIX: TABLES IN LINKS .................................................................................... 92 LIST OF REFERENCES ............................................................................................... 93 BIOGRAPHICAL SKETCH .......................................................................................... 109 7 LIST OF TABLES Table page 2-1 Primer sequences for genes utilized for quantitative real-time PCR (qRT- PCR) validation of RNA-Seq results in bovine mammary tissue......................... 60 2-2 Top KEGG pathways and MeSH terms along with their corresponding DEGs in bovine mammary tissue during transition between lactation to involution. ...... 61 2-3 Top KEGG pathways and MeSH terms along with their corresponding DEGs inbovine mammary tissue during early involution. .............................................. 69 2-4 Differentially expressed genes (DEGs) in bovine mammary tissue during steady-state involution and redevelopment. ....................................................... 71 2-5 Differentially expressed genes (DEGs) in bovine mammary tissue between heat-stressed and cooled cows during the dry period. ....................................... 73 8 LIST OF FIGURES Figure page 2-1 Pictorial representation of experimental design. ................................................ 79 2-2 Volcano plot of DEGs in bovine mammary tissue during early involution (D3 vs. D-3 and D7 vs. D3). ...................................................................................... 80 2-3 Significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Medical Subject Headings (MeSH) terms in bovine mammary tissue during early involution (D3 vs. D-3 and D7 vs. D3). ................................. 81 2-4 Ingenuity® Pathway Analysis (IPA®) upstream regulators and summary network in bovine mammary tissue comparing D3 vs. D-3 relative to dry-off. .... 82 2-5 Ingenuity® Pathway Analysis (IPA®) upstream regulators