Project Number: 5434-31000-016-02
Project Type: Grant

Start Date: Sep 28, 2007
End Date: Dec 31, 2009

Objective:
Efficient use of feed drives profitability and sustainability of beef production. Ruminants typically consume diets high in fiber. Microbes (archaea, bacteria, fungi, protozoa, and viruses) digest fiber within a complex rumen ecosystem. Describing rumen microbial populations is difficult due to the anaerobic environment and inability to culture a many of the species. Metagenomics has provided unique insights into other microbial ecosystems. Bacteria represent a very large proportion of ruminal organisms and are dominant in fermenting feed. Therefore, primary objectives are to generate and sequence 16s rDNA libraries for eubacteria and archaea from rumen contents to determine: 1) number of prokaryotic species in the rumen and 2) how number and type of species vary in response to diet. Secondarily, we will attempt to determine diversity of fungi, protozoa, and viruses.

Approach:
Like-age crossbred (Black Angus x Hereford) heifers will be ruminally fistulated. Initial discovery will use two animals grazing late summer forage. Rumen contents will be collected. DNA will be extracted from the liquid and solid (food particles) fractions at Fort Keogh. Some additional technology development may be required to separate the microbes from food and to isolate the viruses. Extracted DNA will be sent to the Venter Institute for rDNA library construction and sequencing in support of the first primary objective. Separate libraries will be generated for the eubacterial and archaeal sequences from both liquor and solid rumen contents. As the number of eubacterial and archaeal species is not currently known, it is necessary to perform pilot sequencing of 16s rDNA clones to determine how many sequences will be representative of the number of different species. Therefore, these samples will be analyzed by first constructing four libraries for each animal (eubacteria and archaea from both liquor and solid contents) then harvesting 6000 shotgun sequences (~850 base pair reads, 3000 each direction) from each bacterial library, 2000 sequences from each archaeal library and conducting bioinformatic analyses to estimate the number and diversity of species present. Upon receipt of these results, we will assess whether an alternative sampling strategy would be more informative in analyzing diversity under other dietary conditions. In the event that we are successful in separating fungi and protozoa from food particles and(or) isolating viruses from the rumen contents, these samples will also be sent to the Venter Institute for library construction and sequencing. Subsequently, differences in eubacterial and archaeal populations resulting from dietary effects will be assessed. Ruminally fistulated heifers (n=4/diet) will be fed alfalfa, corn silage, and high concentrate (emulating a feed lot ration) diets. Again, rumen contents will be collected and DNA will be extracted from the liquid and solid fractions. The extracted DNA will be sent to the Venter Institute for 16s rDNA library construction and sequencing. Separate libraries will be generated for the eubacterial and archaeal species with 6,000 shotgun sequences harvested for each bacterial library and 2,000 sequences harvested for each archaeal library. Bioinformatic analyses will again to estimate the number and diversity of species present. Manuscripts resulting from this work will be developed jointly and submitted to refereed journals.