Analysis of methanogens in the bovine rumen by polymerase chain reaction single-strand conformation polymorphism

Abstract

The polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method reported by Schwieger and Tebbe (1998) was used to analyze the diversity of methanogens inhabiting the rumen. Partial 16S rRNA gene fragments were amplified from DNA extracted from rumen contents by PCR with archaea-specific primers, Ar1000F and Ar1500R, or methanogen-specific primers, M301F and M915R, with one primer phosphorylated at the 5′ end. The amplified DNA fragments were analyzed by SSCP gel electrophoresis after the phosphorylated strands of the PCR products were digested with λ exonuclease. When we analyzed samples collected from the six Holstein cows used in a previous study, in which cows were given feed with or without α-cyclodextrin-horseradish oil complex (CD-HR), nine and six bands were identified in the profiles generated by PCR products amplified with archaea-specific and methanogen-specific primers, respectively. While dendrogram analysis based on SSCP gel profiles found that the methanogens from each rumen showed a particular composition of methanogens, the profiles of the methanogens isolated from two of three cows fed with CD-HR fell into the same branch in the dendrogram constructed from the profiles. Therefore, this study demonstrates the potential of the PCR-SSCP method in the methanogenic community analysis of the rumen and in investigating changes in the methanogenic community due to the addition of CD-HR to the rumen.