Progress 09/28/07 to 12/31/10
Outputs
Progress Report Objectives (from AD-416) 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 (from AD-416) 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. A publication examining species diversity in the microbial community of the rumen is in preparation. Data collection for a second paper examining responses of the rumen microbial community to changes in diet has been completed. ADODR monitoring is performed by phone calls and e-mail.
Impacts
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Publications
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) 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 (from AD-416) 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. Completed deep sequencing of rumen micro-flora from cows fed forage. Bioinformatics has been completed using this sequence information to: 1) identify appropriate isolation procedures for use in metagenomics studies and 2) provide the richest ever characterization of rumen microbial diversity. Manuscript detailing these results has been peer reviewed. Further deep sequencing of samples from cattle adapting to various levels of concentrate feeding has also been completed with resulting data shipped to scientists at USDA-ARS Miles City. ADODR monitoring is done via e-mail and phone calls.
Impacts
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Publications
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) 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 (from AD-416) 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. Significant Activities that Support Special Target Populations We conducted high throughput metagenomic sequencing and data analysis on bovine rumen extrusa samples derived from two crossbred cows fed high- quality grass hay at the Fort Keogh Livestock and Range Research Laboratory, Miles City, Montana. Results show a tremendous level of diversity of eukaryotes, prokaryote and viruses and present the potential for investigating these types of samples further for microbial species that have potential for understanding microbial ecology of the rumen, and the species that hold the secrets for improving animal production, creating renewal gases, and reducing pollution in production settings. Additional samples are being collected to assess effects of diet and changes of diet on rumen microbial diversity. ADODR monitoring includes phone calls and e-mails.
Impacts
(N/A)
Publications
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) 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 (from AD-416) 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. Significant Activities that Support Special Target Populations The Principal Investigator was changed, which resulted in some delay relative to our anticipated progress. Samples were collected from one of two animals for the pilot study and sent to the Venter Institute to get initial sequence data for refinement of sample preparation protocols.
Impacts
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Publications
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