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International audience; Four rumen and duodenallycannulated dairy cows were fed diets with two levels of degradable protein (110 vs. 80% of requirements) and two types of carbohydrates (NDF/starch of 2.8 and 1.3) according to a 4x4 Latin square. Rumen contents were sampled at 0, 2.5 and 5h after feeding and lyophilized for DNA extraction. Ruminal abundance of bacteria, protozoa, anaerobic fungi, methanogens and ten bacterial species were determined by quantitative PCR. Ruminal levels of microorganisms with a long generation time decreased significantly after feeding. Low protein diets decreased the rumen concentration of total bacteria (-13%) and the relative abundance of P. bryantii (-37%), methanogens (-21%), anaerobic fungi (-27%) and cellulolytic bacteria such as R. albus (-39%), F. succinogenes (-21%) and B. fibrisolvens (- 28%). High starch diets led to a decrease of ruminal amount of protozoa (-28%), fungi (- 42%), methanogens (-25%), R. albus (-20%) and S. bovis (-51%) but promoted an increase of R. flavefaciens (+78%), S. ruminantium (+43%) and M. elsdenii (P = 0.05) with respect to those levels observed in high fibrous diets. A negative relationship was observed between the efficiency of N utilization by the cow and the ruminal abundance of cellulolytic bacteria, fungi, protozoa and methanogens.

International audience; Four rumen-cannulated dairy cows were fed with two levels of degradable protein (110 vs. 80% of requirements) and two type of carbohydrates (NDF/starch of 2.8 and 1.3) according to a 4x4 Latin square. Rumen contents were sampled at 0, 2.5 and 5h after feeding for DNA extraction. Bacterial and anaerobic fungal populations were studied by using TRFLP and ARISA respectively. Sampling time modified neither the microbial population structure nor the biodiversity indexes. Bacterial population composition was affected by the type of carbohydrate used but did not modify the bacterial diversity, suggesting a shift in the bacterial species profile but the maintenance of population structure. Bacterial clustering was also affected by the protein level, particularly in animals upplemented with starch. Anaerobic fungal population was also affected by the type of carbohydrate, and fibrous diet increased the number of amplicons (P

International audience; A rapid method has been developed for molecular identification of rumen ciliates without the need for cultivation. Total DNA was isolated from single protozoal cells by the Chelex method and nearly complete protozoal 18S rRNA genes were amplified and subjected to restriction fragment length polymorphism analysis. On the basis of restriction patterns generated a molecular key was elaborated allowing identification of protozoa solely by a molecular technique without prior knowledge of morphology. No differences were observed between identical species originating from different animals or geographic locations, or between morphological variants of the same species. The ARDREA analysis described here provides a rapid and convenient way for identification and diversity studies of rumen protozoa.

International audience; The rumen, a specialized foregut in herbivorous animals (Ruminantia), hosts microbiota of archaea, eubacteria, anaerobic fungi, and ciliated protozoa. Microscopic observations re-veal that the endosymbiotic ciliates account for about 50% of the total microbial mass in the rumen: up to 100 billions of ciliates may populate the rumen of a single cow. These ciliates play an important role in fibre digestion and the modulation of the fermentation pro-files. Classical (morphological) studies have identified more than 250 species of ciliates living in the various ruminants1,2. The number of species in an individual host is known to be highly variable, but the average number of species has been reported about 20 in cat-tle3, but less in sheep3 and goats4. However, as in all protists, the limited number of distinct morphological traits hampers an assessment of the ciliate diversity in the rumen. Molecular studies on rumen ciliates are rare, mainly due to difficulties in culturing these ciliates in vi-tro. Here we describe a molecular approach to study the phylogenetic diversity of rumen protozoa. The diversity of rumen protozoa was analysed by sequencing of 18S rDNA li-braries that have been created from PCR-amplified DNA of total rumen contents of cow, sheep, and a goat. These sequences were compared with the sequences of rumen ciliates obtained from GenBank and ERCULE. The phylogenetic analysis suggests that the ciliate population of the ruminal ecosystem can be much more diverse than previously assumed.

International audience; Here we describe the first molecular approach to study the diversity of rumen protozoa of wild animals. Total DNA was extracted from the rumen content of the red deer (Cervus elaphus) and the 18S rRNA genes were amplified by PCR. A library of ciliate 18S rRNA genes was created and a sample of 35 clones were sequenced partially. With the aid of the sequences of validated rumen ciliate representatives from the culture collection obtained in the frame of the ERCULE project (www.ercule.com) and those deposited in the EMBL gene databank, we were able to identify the phylogenetic position of the ciliates found in the rumen of a single red deer. Notably, none was identical to a previously analysed 18S rDNA sequence and many of the sequences were quite different from those known from the 364 sequences recovered from an analysis of the ciliate communities in cow sheep and goat. Interestingly, several of the clones recovered from the red deer clustered with Polyplastron, a genus that hasn't been reported for these animals until now (1).