Effect of DNA Extraction Methods and Sampling Techniques on the Apparent Structure of Cow and Sheep Rumen Microbial Communities

Gemma Henderson,Samantha J Noel,Sandra Kittelmann,Michael Zethof,Garry C Waghorn,Faith Cox,Peter H Janssen,Vahideh Heidarian Miri,Stefan Bertilsson

doi:10.1371/journal.pone.0074787

Abstract

Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial community are crucial. Fifteen different methods were used to extract DNA from cow and sheep rumen samples. The DNA yield and quality, and its suitability for downstream PCR amplifications varied considerably, depending on the DNA extraction method used. DNA extracts from nine extraction methods that passed these first quality criteria were evaluated further by quantitative PCR enumeration of microbial marker loci. Absolute microbial numbers, determined on the same rumen samples, differed by more than 100-fold, depending on the DNA extraction method used. The apparent compositions of the archaeal, bacterial, ciliate protozoal, and fungal communities in identical rumen samples were assessed using 454 Titanium pyrosequencing. Significant differences in microbial community composition were observed between extraction methods, for example in the relative abundances of members of the phyla Bacteroidetes and Firmicutes. Microbial communities in parallel samples collected from cows by oral stomach-tubing or through a rumen fistula, and in liquid and solid rumen digesta fractions, were compared using one of the DNA extraction methods. Community representations were generally similar, regardless of the rumen sampling technique used, but significant differences in the abundances of some microbial taxa such as the Clostridiales and the Methanobrevibacter ruminantium clade were observed. The apparent microbial community composition differed between rumen sample fractions, and Prevotellaceae were most abundant in the liquid fraction. DNA extraction methods that involved phenol-chloroform extraction and mechanical lysis steps tended to be more comparable. However, comparison of data from studies in which different sampling techniques, different rumen sample fractions or different DNA extraction methods were used should be avoided.

Highlights

Ruminants such as cattle, sheep, deer, yak, buffalo and goats are of great importance for the production of meat and dairy products, wool, and leather
The aim of this study was to determine the effects that 15 different DNA extraction, two rumen sampling methods, and sample fractionation had on parameters such as DNA quality and quantity, as well as on absolute microbial numbers and relative microbial community composition
A similar observation was made by Ariefdjohan and colleagues [9], who found that specific yields of DNA increased when less faecal material was used for DNA extraction

Summary

Introduction

Sheep, deer, yak, buffalo and goats are of great importance for the production of meat and dairy products, wool, and leather. Ruminants have a complex digestive system, and digestion of feed takes place initially in the rumen. There, microbes play a key role in the breakdown of feed components such as fibre, producing short chain fatty acids that provide energy for the host. Rumen microbes are essential providers of animal energy and nutrition, and play a key role in the productivity and health of ruminants. Rumen archaea produce the greenhouse gas methane as a metabolic end product. This methane gas is eructated by ruminants and represents 2 to 12% dietary gross energy lost to the animal [1]. Understanding the functions and compositions of rumen microbial communities is required to improve animal productivity and to reduce the amount of energy lost as methane

Objectives

Methods

Results

Conclusion