AimsTo investigate the effect of donor animal species and their feeding on the composition of the active fraction of rumen microbiota established during invitro experiments with different forages. Methods and ResultsRumen simulation experiments were conducted with maize silage (MS) and grass silage (GS) as substrates. Four experimental runs were performed with ruminal contents of sheep and cows fed either with hay and concentrate or with MS or GS, respectively. Liquid- and solid-associated microbes (LAM and SAM) were fixed for fluorescence insitu hybridization after 14days of incubation. The LAM were characterized by higher proportions of Lachnospiraceae, Bacteroidetes, Deltaproteobacteria and Methanobacteriaceae at hay-concentrate-based feeding of the donor animals whereas counts of Gammaproteobacteria were lower. Similar results were found for SAM. Hay-concentrate-based feeding caused a higher ratio of Bacteria:Archaea in SAM. Incubations with GS seemed to enhance Bacteroidetes, Deltaproteobacteria and Archaea whereas numbers of Gammaproteobacteria were enhanced in MS experiments. The effect of the donor animal species was limited to the number of Archaea establishing invitro being higher for sheep than for cows. ConclusionsThe feeding of donor animals, more than the animal species itself, affects the composition of the ruminal microbial community that establishes invitro. Significance and Impact of the StudyData suggest the need of a standardized approach for studying the rumen microbiota in a rumen simulation. Moreover, this study provides fundamental data on the composition of the ruminal microbial community when different diets are fed to donor animals.