University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Theses and Dissertations in Animal Science Animal Science Department 11-2014 The ffecE ts of HMTBa (2-hydroxy-4-methylthio- butanoic acid) Supplementation on Ruminal Microbial Crude Protein Synthesis and Community Structure in Dairy Cattle Chad J. R. Jenkins University of Nebraska-Lincoln, [email protected] Follow this and additional works at: http://digitalcommons.unl.edu/animalscidiss Part of the Bioinformatics Commons, and the Dairy Science Commons Jenkins, Chad J. R., "The Effects of HMTBa (2-hydroxy-4-methylthio-butanoic acid) Supplementation on Ruminal Microbial Crude Protein Synthesis and Community Structure in Dairy Cattle" (2014). Theses and Dissertations in Animal Science. 99. http://digitalcommons.unl.edu/animalscidiss/99 This Article is brought to you for free and open access by the Animal Science Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Theses and Dissertations in Animal Science by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. THE EFFECTS OF HMTBA (2-HYDROXY-4-METHYLTHIO-BUTANOIC ACID) SUPPLEMENTATION ON RUMINAL MICROBIAL CRUDE PROTEIN SYNTHESIS AND COMMUNITY STRUCTURE IN DAIRY CATTLE by Chad James Robert Jenkins A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science Major: Animal Science Under the Supervision of Professors Paul J. Kononoff and Samodha C. Fernando Lincoln, Nebraska November, 2014 THE EFFECTS OF HMTBA (2-HYDROXY-4-METHYLTHIO-BUTANOIC ACID) SUPPLEMENTATION ON RUMINAL MICROBIAL CRUDE PROTEIN SYNTHESIS AND COMMUNITY STRUCTURE IN DAIRY CATTLE Chad James Robert Jenkins, M.S. University of Nebraska, 2014 Co-advisors: Paul J. Kononoff and Samodha C. Fernando Metabolizable protein (MP) is protein that reaches the small intestine and is available for absorption and utilization by the cow. Dairy rations may be limited in the supply of MP essential to meeting the demands of milk synthesis, however as much as half of the MP flowing to the small intestine may be attributed to microbial origins and is referred to as microbial CP (MCP). Experiment 1 utilized a technique in which DNA was used as a microbial marker to estimate the concentration of bacterial CP (BCP) in the solid and liquid portions of rumen digesta. Rumen digesta was sampled and separated into solid and liquid fractions and microbes were isolated from whole ruminal digesta. Targeting bacterial DNA in samples using real-time PCR, in addition to N analysis, allowed for estimates of the concentration of BCP in the solid and liquid fractions to be attained. The concentration of BCP tended to be higher in the solid portion, highlighting the need to consider both particle and liquid associated bacteria when conducting experiments involving the microbial community. Experiment 2 focused on the ruminal effects of a commercial feed additive when fed with diets low or high in MP. The feed additive, 2-hydroxy-4-methylthio-butanoic acid (HMTBa) molecule (Alimet, Novus Internation, St. Charles, MO), a methionine analog, is believed to result in several positive effects on rumen fermentation, including increased MCP yield. Rumen pH was decreased in response to the additive, while rumen VFA and ammonia were increased. The MCP yield was unaffected across treatments. Nutrient digestibility was increased in cows fed the diet low in MP. Rumen bacterial DNA was sequenced and analyzed bioinformatically; the proportion of Fibrobacteres were increased in cows receiving the additive, and a number of associations of the relative abundance of microorganisms with ruminal observations and treatments were observed. iv ACKNOWLEDGEMENTS I would like to give a special thank you to everyone who helped and encouraged me throughout this project. Your kind words, direction, and support were invaluable to me as I worked my way through this study. First of all, I want to thank my loving wife Leah for standing with me through the ups and downs of my program. You listened, offered encouragement, and put up with a stinky husband who came home late at night from sampling. Additionally, to my parents, without whose advocacy this would not be possible. I can’t imagine having completed this project without you all on my side. Second, thank you to my major advisor, Dr. Paul Kononoff, for your steadfast support and guidance. Your genuine interest in your students, not only academically, but also as people, is truly admirable. Thank you for all of your advice along the way regarding this study and otherwise. Third, thank you to my co-advisor, Dr. Fernando, and the rest of my advisory committee, Dr. Burkey and Dr. Miller. Your classes and instruction were essential in helping me to reach a level of understanding to complete this project, as well as in sparking my interest to new ideas and inspiring me to keep on learning. Fourth, thank you to former graduate students Hugo Ramirez-Ramirez, Ezequias Castillo-Lopez, and Henry Paz for taking me under your wings upon my arrival into this program. You all have gracefully “shown me the ropes” and have become my friends. Thank you to my office mate, Alison Foth—your laughter made every day brighter. It was awesome to have someone with your positivity and enthusiasm to work through this experience alongside. v Finally, thank you to research staffers Erin Marotz and Darren Strizek, and to Kiley Algya and the rest of the undergraduate employees for all of your hard work in the dairy and lab. Your exceptional care for the animals made this work possible. vi “How many are your works, LORD! In wisdom you made them all; the earth is full of your creatures.” Psalm 104:24 “For every animal of the forest is mine, and the cattle on a thousand hills.” Psalm 50:10 vii TABLE OF CONTENTS Chapter Page CHAPTER I INTRODUCTION..............................................................................................................1 REVIEW OF LITERATURE ...........................................................................................3 Protein Digestion in Ruminants .......................................................................................3 Fractionation of Protein Entering the Rumen ...............................................................3 Fractionation of Protein Leaving the Rumen ...............................................................5 Microbes in the Rumen ....................................................................................................5 Bacteria .........................................................................................................................6 Protozoa ........................................................................................................................7 Fractionation and Chemical Composition of Rumen Microorganisms ........................9 Effects of HMTBa Supplementation on Ruminal Microorganisms ...............................10 Microbial Protein Yield ..............................................................................................11 Protozoa Number ........................................................................................................12 Fiber Digestibility .......................................................................................................13 Volatile Fatty Acids ....................................................................................................14 Estimation of Rumen Microbial Protein Production and Utilization .............................15 Digesta Flow Markers ................................................................................................16 Microbial Markers ......................................................................................................17 Purines ........................................................................................................................17 DNA for Microbial Markers .......................................................................................19 DNA Sequencing ............................................................................................................21 Pyrosequencing...........................................................................................................22 viii Semiconductor-based Sequencing. .............................................................................22 SUMMARY ......................................................................................................................24 General Objectives .........................................................................................................24 REFERENCES .................................................................................................................26 TABLES AND FIGURES ...............................................................................................32 CHAPTER II Short Communication: Estimation of bacterial protein in rumen digesta using DNA markers INTERPRETIVE SUMMARY .......................................................................................40 ABSTRACT ......................................................................................................................41 INTRODUCTION............................................................................................................42 MATERIALS AND METHODS ....................................................................................42