Methods in Gut Microbial Ecology for Ruminants

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Ciliate protozoa, Burk A. Dehority; Part 3. Forster; 3. Denman and Christopher S. McSweeney; Part 4.

The symbiotic rumen microbiome and cattle performance: a brief review

Denaturing gradient gel electrophoresis, Svetlana A. Kocherginskaya, Isaac K. Cann and Roderick I. Mackie; 4. Bacteriophage populations, Athol V. Klieve and Rosalind A. Gilbert; 4. Anaerobic fungal populations, Jayne L.

Canadian Journal of Microbiology

Brookman and Matthew J. Nicholson; 4. Denman, Makoto Mitsumori and Christopher S. McSweeney; Part 5. Wright, Kiyoshi Tajima and Rustam I. Aminov; Part 6. Northern blot analysis to investigate the abundance of micro-organisms, Denis O. Krause; 6. Whole cell probing with fluorescently labelled probes for in situ analysis of microbial populations, Linda L. Blackall; 6. Ginige; Part 7.

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  • Methods in gut microbial ecology for ruminants [2005]?

Genomic Analysis of Microbial Ecosystems; 7. Metagenomic analysis of the microbiomes in ruminants and other herbivores Mark Morrison, Sarah E. According to the manufacturer, RNAprotect bacteria reagent was designed to prevent both degradation of RNA transcripts and induction of genes, and thus provide immediate stabilization of the gene expression profile of bacteria. However, at least in this study, this reagent reduced both RNA yield and quality.

Immediate freezing and grinding of samples in liquid nitrogen followed by mixing in TRIzol reagent yielded RNA of high quality and stability.

Makkar, Harinder P.S.

The addition of RNAprotect to the procedure increased the complexity of the extraction. Therefore, in the present study, we found no value to including RNAprotect in the RNA extraction procedure for either the liquid or solid fraction of rumen contents. A bp length of the celF gene was amplified by using W series primers i. A possible explanation for this may be related to the fact that random hexamer primers were used for the RT.

These reaction conditions are unavoidably biased towards the generation of fragmented, short-length first-strand cDNA products as opposed to whole-length sequences.

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It was possible that compared with the other two genes, a high transcription level of celF existed in the isolated total RNA and resulted in enough whole-length cDNA product to be detectable by PCR amplification. According to these authors, celF had the lowest transcript level of the three highly transcribed genes under their experiment conditions. However, our study analyzed solid ruminal contents from muskoxen, in which the distribution of and relation to known Fibrobacter species is unknown.

CelF previously named EGF was reported to be one of the major cellulose binding proteins identified in F. Considering that the Fibrobacter genus is a major contributor to fibrolytic activity within the rumen Stewart et al. Assembly of the reads revealed many near full-length transcripts, attesting to the quality of the RNA. To date, about transcripts encoding putative cellulases have been identified, including many from rumen fungi, and subsequent analysis has not only confirmed our assumption that the majority of the eukaryotic RNA is of microbial origin, but also revealed a complex eukaryotic community and gene pool Qi et al.

The fact that eukaryotic RNA could be isolated from the rumen solids is encouraging, as at least a portion of this eukaryotic RNA likely arises from either rumen protozoa or fungi as opposed to undegraded dietary RNA or RNA from host cells sloughed from the ruminal epithelium. Fungi possess potent glycoside hydrolase enzyme activities, and rumen solids could prove to be a rich source of these genes.

Metatranscriptomics is a rapidly emerging field and has shown considerable potential as a means of identifying novel biocatalysts Warnecke and Hess ; Sorek and Cossart Our method makes it practical to obtain large quantities of high-quality total RNA, enabling sequence-based approaches to identify genes coding for novel enzymes from environmental samples. The procedure could be easily adapted to other environments, such as compost, leaf cutter ant gardens, or soil, with little difficulty. For the rumen environment, the method enables the investigation of the impact of the host, diet, and other conditions that affect ruminal function and gene expression within this unique ecosystem.

The authors wish to thank Lyn Paterson and Edith Valle for rumen sample handling and technical assistance. Advanced Search. All Journals Journal. Canadian Journal of Microbiology. Corresponding author: Robert J. Forster e-mail: Robert. Forster agr. In this paper Top of page Introduction Methods Results Discussion References Abstract The rumen is one of the most powerful fibrolytic fermentation systems known. Comparison of RNA yield and quality isolated from the rumen fluid RF by using different isolation procedures.

Acknowledgements The authors wish to thank Lyn Paterson and Edith Valle for rumen sample handling and technical assistance. Development of a RT-qPCR method for the quantification of Fibrobacter succinogenes S85 glycoside hydrolase transcripts in the rumen content of gnotobiotic and conventional sheep.

Fiber-degrading systems of different strains of the genus Fibrobacter.

Methods in gut microbial ecology for ruminants

Canadian Council on Animal Care. Guide to the care and use of experimental animals. Google Scholar.

  • Methods in Gut Microbial Ecology for Ruminants by Harinder P.S. Makkar | | Booktopia.
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  • Recent developments in nucleic acid based techniques for use in rumen manipulation;

Cheng, K. Compartmentation in the rumen. In The rumen microbial ecosystem. Edited by P.

Hobson and C. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate—phenol—chloroform extraction. An efficient RNA extraction method for estimating gut microbial diversity by polymerase chain reaction. Composition, spatial distribution, and diversity of the bacterial communities in the rumen of cows fed different forages. FEMS Microbiol. Quantification and chemical composition of mixed bacteria harvested from solid fractions of rumen digesta: effect of detachment procedure.

Microbial Ecology with Jack Gilbert

Feed Sci. Evaluation of procedures for detaching particle-associated microbes from forage and concentrate incubated in Rusitec fermenters: efficiency of recovery and representativeness of microbial isolates. Microbial attachment and feed digestion in the rumen. Isolation and characterization of endoglucanases 1 and 2 from Bacteroides succinogenes S Invited review: adhesion mechanisms of rumen cellulolytic bacteria. Dairy Sci. Mitsumori M, Minato H.