Relative diversity and community structure analysis of rumen protozoa according to T-RFLP and microscopic methods

Protozoa are common inhabitants of the rumen where they play roles in host nutrition and methanogenesis. Knowledge of how changes in the composition of protozoa communities affect these processes is limited in part due to a lack of efficient methods for protozoa community analysis. In this study, a terminal-restriction fragment length polymorphism (T-RFLP) assay targeting the 18S rRNA gene was developed for comparative analysis of rumen protozoa communities. Comparison of diversity and structure of protozoa communities from hay-fed versus silage/grain-fed cattle via T-RFLP analysis yielded similar overall results to microscopy analysis. According to both methods, Entodinium spp. were more abundant in the silage/grain-fed cattle and protozoa diversity (as calculated using the Shannon index) was higher for the hay-fed cattle due to greater species evenness. Type B protozoa were more prevalent in the hay-fed cattle, whereas Type A protozoa were more prevalent in the silage/grain-fed cattle. Analysis of similarity (ANOSIM) indicated that the protozoa communities from hay-fed and silage/grain-fed cattle were different, and multivariate analysis indicated that pen mates (i.e., cattle fed the same diet and housed together) tended to have similar protozoa communities types. In summary, we present a T-RFLP method for analyzing rumen protozoa communities which complements traditional microscopy approaches but has the advantage of being amenable to high-throughput.