NUTRIENT DIGESTION AND NITROGEN METABOLISM OF DRIED FERMENTATION BIOMASS AND VARIOUS FRACTIONS OF RUMEN MICROBES A THESIS SUBMITTED TO THE FACULTY OF UNIVERSITY OF MINNESOTA BY Abigail Joy Carpenter IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE Marshall D. Stern, advisor August, 2012 © Abigail Carpenter 2012 ACKNOWLEDGEMENTS I would not have made it to the end of my Master’s degree without the help and support of many people. First, I am forever grateful to my advisor, Dr. Marshall Stern, for the opportunities that he has given me at the University of Minnesota. Thanks also to my committee members, Dr. Marcia Endres and Dr. Douglas Mashek, for their knowledge, support, and especially for their flexibility! Other faculty members deserve thanks also, particularly Dr. Brian Crooker for technical assistance and Drs. Alfredo DiCostanzo and Grant Crawford for adopting me as an honorary member of the Beef Team. Thank you to the graduate students who came before me, Martin Ruiz-Moreno and Elizabeth (Liz) Binversie, for your experience and teaching in the lab. Special thanks to my fellow Master’s student, Sam Fessenden, for your help and lab expertise and (seemingly) infinite patience over the past year. My family has been a source of strength and encouragement to me for as long as I can remember, and has continued to be so over the past two years. Thank you to my parents, Dan and Sandy Carpenter, for nurturing my excitement for science and learning from a young age, and to my “little” brothers, Mitch and Enoch, for being geeky enough that I don’t feel like I’m the weird one. Thanks also to my “sisters,” Katie Timmerman and Megan Sprague, for letting me unload about frustrations in the lab, even though I’m sure you often had no idea what I was talking about! i TABLE OF CONTENTS Acknowledgements ......................................................................................... i Table of Contents .......................................................................................... ii List of Tables ............................................................................................... iv List of Figures ............................................................................................... v LITERATURE REVIEW .................................................................................... 1 Introduction .................................................................................................. 1 Overview of N metabolism in ruminants .......................................................... 1 Microbial protein ........................................................................................... 3 Mechanisms of proteolysis and MPS ............................................................. 3 Factors affecting MPS ................................................................................. 6 Predicting MPS ......................................................................................... 16 Chemical composition of rumen microbes ................................................... 17 Amino acid digestion and metabolism ........................................................... 20 Factors affecting AA composition of rumen microbes ................................... 21 Amino acid flow from the rumen ................................................................ 22 Manipulating AA flow from the rumen ........................................................ 23 Utilization of absorbed AA in animal tissues ................................................ 24 Intestinal digestion of crude protein in ruminants ........................................... 24 Nitrogen passage to the small intestine ...................................................... 24 Intestinal digestibility of protein flowing from the rumen ............................. 33 Dried Fermentation Biomass ......................................................................... 35 Summary .................................................................................................... 36 Objectives .................................................................................................. 36 Experiment 1 EFFECT OF DRIED FERMENTATION BIOMASS ON MICROBIAL FERMENTATION IN CONTINUOUS CULTURE AND IN VITRO INTESTINAL DIGESTIBILITY…37 Overview ...................................................................................... 37 Introduction .................................................................................. 38 Materials and Methods ................................................................... 39 ii Results and Discussion ................................................................... 42 Implications .................................................................................. 48 Experiment 2 CHEMICAL COMPOSITION AND IN VITRO INTESTINAL DIGESTIBILITY OF ISOLATED RUMEN MICROBIAL FRACTIONS ........................................ 61 Overview ...................................................................................... 61 Introduction .................................................................................. 62 Materials and Methods ................................................................... 62 Results and Discussion ................................................................... 64 Implications .................................................................................. 70 LITERATURE CITED ..................................................................................... 76 iii LIST OF TABLES EXPERIMENT 1 1 Ingredient and chemical composition of experimental diets. ..................... 62 2 Amino acid composition of experimental diets. ........................................ 63 3 Effect of dietary N source on in situ CP degradation. ............................... 64 4 Effect of dietary N source on digestion. .................................................. 65 5 Effect of dietary N source on pH in continuous culture fermenters. ........... 66 6 Effect of dietary N source on total VFA concentration and VFA molar proportions. ......................................................................................... 67 7 Effect of dietary N source on N metabolism in continuous culture fermenters........................................................................................... 68 8 Effect of dietary N source on amino acid flow. ........................................ 69 9 Effect of dietary N source on percent change of input of AA. .................... 70 10 Effect of dietary N source on in vitro intestinal protein digestion. ............ 71 EXPERIMENT 2 1 Ingredient and chemical composition of diets fed to ruminally-cannulated steers .................................................................................................. 88 2 Characteristics of isolated rumen microbial fractions. ............................... 89 3 Amino acid composition of isolated rumen microbial fractions (wt/wt). ...... 90 4 Amino acid composition of isolated rumen microbial fractions (g/100 g of total AA). ........................................................................... 91 iv LIST OF FIGURES EXPERIMENT 1 1 Effect of dietary treatment on average, maximum, and minimum hourly pH in continuous culture fermenters over time. ............................................... 72 EXPERIMENT 2 1 Procedure used to isolate LAB, SAB, and LAP from whole rumen contents. ............................................................................................. 92 v LITERATURE REVIEW INTRODUCTION Nitrogen metabolism in the rumen consists of dietary crude protein degradation and protein synthesis by microorganisms. Nitrogen flowing from the rumen is made up of dietary, endogenous, and microbial N. Some dietary N will be degraded in the rumen (RDP, rumen degradable protein) and the rest will escape as rumen undegradable protein (RUP). Sources of RDP are dietary non-protein N (NPN) and dietary protein. Non-protein N includes DNA, RNA, ammonia (NH3), amino acids (AA), and peptides. Protein that has been deaminated to form NH3 can be incorporated into microbial N, and some species of bacteria can also incorporate peptides and AA for microbial protein synthesis (MPS). Microbial protein that enters the small intestine is the major form of N utilized by the ruminant, and is a high-quality AA source that is generally balanced for maintenance and moderate production. Therefore, maximizing MPS in a way that is efficient to the animal has the potential to increase milk production. Researchers have investigated how to manipulate flows of differing N fractions such as microbial N from the rumen with intent to influence growth, milk output, and overall productive efficiencies. Overview of N metabolism in ruminants Actual protein and AA requirements in ruminants have not been determined. Because of MPS, N recycling, and interactions between them, it is difficult to determine requirements of ruminants for individual AA, RDP, RUP, and even CP. Therefore, ruminants are commonly fed on a crude protein (CP) basis. From a practical standpoint, a meta-analysis showed maximum milk production was achieved when 22.8% of the diet is CP (Ipharraguerre and Clark, 2005). However, there is considerable variation between studies that may be explained by source of CP. Quality, amount, and source of CP in the diet all have an effect on whether a milk response is observed. Relative amounts of RDP and RUP can affect MPS and N recycling as well as what AA are