0474 Comparison of digestion and particle-associated bacteria after in situ incubation of different barley varieties in the rumen of cattle

Abstract

Amylopectin (Waxy starch)…………………………………….…………..15 2.4 Role of rumen microbes in grain digestion………………………………………….16 2.4.1 Attachmentbiofilm formation…………………………………………………..16 2.4.2 Role of amylolytic microbes………………………………………………………18 vi 2.4.3 Role of firbrolytic microbes……………………………………………………….19 2.4.4 Role of proteolytic microbes………………………………………………………20 2.5 Barley grain processing………………………………………………………………20 2.6 Impacet on site of barley grain digestion in ruminant digestive tract…………….22 2.7 Rumen digestibility methodology……………………………………………………23 2.7.1 In situ / in vitro techniques for measuring digestibility in the rumen….………….23 2.8 Rumen molecular microbiology…………………………….………………..….…...24 2.8.1 Molecular techniques for ecological characterization in the rumen ………...…....24 2.8.2 Next generation sequencing technology……………………………………...…...25 2.8.3 454 pyrosequencing……………………………………………………...………..27 2.9 Defining microbial ecology using sequencing of 16S rRNA……………………….28 2.10 Bioinformatic analysis………………………………………………………………..31 2.11 Using QIIME to analyze rRNA sequences……………………………….………….31 2.12 Application of NGS in ruminants……………………………………………………35 2.13 Conclusions……………………………………………………………………………37 2.14 Hypotheses…………………………………………………………………………….38 2.15 Objectives……………………………………………………………………….……..38 3.0 COMPARISON OF DIGESTION AND PARTICLE-ASSOCIATED BACTERIA (PAB) AFTER IN SITU INCUBATION OF DIFFERENT BARLEY VARIETIES IN THE RUMEN OF CATTLE………………………………………………………………..………..39 3.1 Introduction…………………………………………………………………...………39 3.2 Materials and Methods……………………………………………………………….41 3.2.1 Animal and diets…………………………………………………………………..41 3.2.2 Incubation of barley varieities and corn in the rumen…………………………….43 3.2.3 DNA extraction of bacterial biofilms……………………………………………..44 3.2.4 Pyrosequecing of the 16S rRNA gene…………………………………………….44 3.2.5 Sequencing analysis by QIIME 1.9.1 …………………………………………….45 3.2.6 Chemical analysis of barley grain varieties and corn……………………………..45 3.2.7 Scanning electron microscopy (SEM)…………………………………………….47 3.2.8 In situ digestibility of four barley varieties and corn……………………………...48 3.2.9 Rumen particle-associated bacteria sequencing analysis………………….………49 3.3 Results…………………………………………………………………………………49 3.3.1 Chemical composition of barley varieties and corn…………………….…………49 3.3.2 Comparison of digestibility after in situ incubation of different barley varieties in the rumen when heifers were fed low and high grain diets…………….…………52 3.3.3 Differences in particle-associated bacteria (PAB) between corn and the four barley varieties when heifers were fed a low grain diet….…………………….…………57 vii 3.3.4 Differences in particle-associated bacteria (PAB) between corn and the four barley varieties when heifers were fed high grain diet…………………………………...68 3.3.5 Bacterial morphologies associated with barley grain……………………………..77 3.4 Discussion……………………………………………………………………………...79 3.4.1 Digestibility after in situ incubation of different barley varieties when heifers were fed low and high grain diets…………………………………………….…………79 3.4.2 Temporal microbial colonizati...