Diversity and fluctuation in ciliate protozoan population in the rumen of cattle

Abstract

The purpose of this study was to investigate the diversity and fluctuation in the ciliate protozoan population in the rumen of cattle. DNA was extracted from the rumen of three ruminally cannulated, crossbred cattle and a polymerase chain reaction (PCR)-derived clone library was constructed, using a specific primer set targeting 18S ribosomal RNA genes of ciliate protozoa. DNA fragments of seven selected clones were validated for standard DNA of the protozoa-specific real-time PCR assay. Furthermore, population fluctuation of ciliate protozoa and methanogens in the cattle rumen was determined by real-time PCR. A total of 60 clones were sequenced, phylogenetically analyzed, and classified into 24 operational taxonomic units (OTUs) based on a 99% similarity criterion. More than 80% sequences were phylogenetically placed in the genus Entodinium. The rest of the sequences were placed in the genus Diploplastron (5%), Dasytricha (8.3%) and Isotricha (3.3%). The results suggest that Entodinium was the dominant group in the rumen of cattle used in this study. The ciliate protozoan population showed no significant change in numbers during the monitoring period and reached a peak at 3 h after feeding. Changes in the protozoa population were lower than those of the methanogens.