A metagenome expression library of bulk DNA extracted from the rumen content of a dairy cow was established in a phage lambda vector and activity-based screening employed to explore the functional diversity of the microbial flora. Twenty-two clones specifying distinct hydrolytic activities (12 esterases, nine endo-beta-1,4-glucanases and one cyclodextrinase) were identified in the library and characterized. Sequence analysis of the retrieved enzymes revealed that eight (36%) were entirely new and formed deep-branched phylogenetic lineages with no close relatives among known ester- and glycosyl-hydrolases. Bioinformatic analyses of the hydrolase gene sequences, and the sequences and contexts of neighbouring genes, suggested tentative phylogenetic assignments of the rumen organisms producing the retrieved enzymes. The phylogenetic novelty of the hydrolases suggests that some of them may have potential for new applications in biocatalysis.

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We conducted a lactation trial with a fresh forage diet in order to evaluate 1) the effects of monensin on nitrogen metabolism, and 2) the Cornell Net Carbohydrate and Protein System (CNCPS). Thirty Holstein cows in midlactation (eight fitted with ruminal fistulas) were gradually introduced to a fresh forage diet. A concentrate mix based on corn meal was fed before the a.m. and p.m. milking times 0730 and 1730 h, then the fresh forage was fed at 0830 and 1830 h. Fifteen cows each were allocated to a control (no monensin) and a treatment group receiving 350 mg/cow per day of monensin in the p.m. concentrate feeding. A 7-d fecal and urine collection period and a 3-d rumen sampling period were conducted with the fistulated cows. After the lactation study was concluded, the fistulated cows were fed forage regrowth and a 3-d rumen sampling period was repeated. Monensin increased milk production by 1.85 kg. Milk fat and protein concentrations decreased and milk fat and protein yields increased, but the effects were nonsignificant. Monensin did not significantly affect DMI. Ruminal ammonia and the acetate-to-propionate ratio decreased with the addition of monensin in both fed forages. Monensin decreased fecal N output, and increased apparent N digestibility by 5.4%. Because of the decrease in ruminal ammonia and increase in apparent N digestibility, we concluded monensin was sparing amino acids from wasteful rumen degradation with a fresh forage diet. The precision of the CNCPS in predicting performance was high (r2 = 0.76), and the bias was low (overprediction of 3.6%). These results indicate that the CNCPS can be used for dairy cows consuming fresh forage and gives realistic predictions of performance.

The internal transcribed spacer (ITS) is a popular barcode marker for fungi and in particular the ITS1 has been widely used for the anaerobic fungi (phylum Neocallimastigomycota). A good number of validated reference sequences of isolates as well as a large number of environmental sequences are available in public databases. Its highly variable nature predisposes the ITS1 for low level phylogenetics; however, it complicates the establishment of reproducible alignments and the reconstruction of stable phylogenetic trees at higher taxonomic levels (genus and above). Here, we overcame these problems by proposing a common core secondary structure of the ITS1 of the anaerobic fungi employing a Hidden Markov Model-based ITS1 sequence annotation and a helix-wise folding approach. We integrated the additional structural information into phylogenetic analyses and present for the first time an automated sequence-structure-based taxonomy of the ITS1 of the anaerobic fungi. The methodology developed is transferable to the ITS1 of other fungal groups, and the robust taxonomy will facilitate and improve high-throughput anaerobic fungal community structure analysis of samples from various environments.

To obtain information on the diversity of ruminal methanogens in grazing animals, three ruminal methanogens from grazing cattle were characterized and identified. Two of the isolates were rod-shaped, with one staining Gram-positive and being non-motile (BRM9), and the other (BRM16) staining Gram-negative and being motile. These isolates grew only on H(2)/CO(2) and formate, and optimally at 38 degrees C and pH 6.5-7.0. The third isolate (CM1) was non-motile, pseudosarcina-shaped, and grew on H(2)/CO(2), acetate, and methyl-containing compounds, with optimal growth at 40 degrees C and pH 6.5. DNA was prepared from the three isolates, and their 16S rRNA genes were sequenced. Phenotypic data and comparisons of nearly complete 16S rDNA sequences showed that BRM9, BRM16, and CM1 are strains of Methanobacterium formicicum, Methanomicrobium mobile, and Methanosarcina barkeri respectively. To the best of our knowledge, this is the first information on ruminal methanogens in cattle maintained under grazing management.