2010 Annual Report
1a.Objectives (from AD-416)
The overall objectives of the research project are to apply modern genomic tools to the identification and characterization of genetic pathways, physiological mechanisms, and microbial-host interactions that modulate nutrient uptake, partitioning, and loss in cattle. The specific objectives include development of resources for identification of selectable markers of nutrient efficiency, identification and characterization of genetic pathways and/or genomic regions influencing critical regulatory pathways of nutrient efficiency and transport, and development of intervention strategies to enhance nutrient uptake and partitioning in cattle.
1b.Approach (from AD-416)
To identify and characterize factors affecting nutrient use efficiency in cattle, resources will be developed including a phenotypic database of dairy efficiency and corresponding DNA and tissue collections, methods for live animal intestinal tissue collection suitable for gene expression studies, and sub-populations of dairy cattle to investigate inflammation and nutrient use interactions. Novel DNA sequencing technology will also be evaluated for its utility in characterizing changes in rumen microbial populations during rumen development. Transcript profiling techniques including microarray and quantitative real-time PCR will be used to establish molecular markers of proliferation, development and differentiation of the bovine gastrointestinal tract, and to identify metabolic and hormone pathways controlling nutrient metabolism in the ruminant gastrointestinal tract. Finally, two dietary strategies that potentially affect the rumen microbial population and production efficiency will be studied for their effects on nutrient use efficiency in cattle.
Progress was made in several studies addressing the project objectives and FY 2010 National Program Components on development of genome-enabling resources, identifying critical genes and their interactions, reducing animal stress related to nutrient use efficiency, and improving overall efficiency of nutrient use by dairy cattle. For instance, gene expression analysis was completed for 2 animal studies including one aimed at validating molecular markers for rumen development and growth in adult cattle during transition from a forage- versus a concentrate-dominated diet in collaboration with the Fort Keogh Livestock and Range Research Laboratory, Miles City, MT; and one examining the effects of a gut peptide on intestinal development and function in calves in collaboration with the University of Kentucky. Evaluation of residual feed intake, a measure of feed conversion efficiency to milk production, was continued within the Beltsville Dairy Herd and genetic marker testing was initiated in the herd to map regions of the cattle genome controlling feed efficiency. Genetic testing of dairy heifers from the Beltsville herd for a specific variant in a gene involved in immune function and inflammation was also completed. An experiment was initiated using these heifers to evaluate the association between variation in this gene and response to an immune challenge, and subsequent health and milk production traits. Progress was made in the validation and use of specific protein nitration biomarkers for characterizing dairy cattle that are "hyper-sensitive" to immune challenges.
Characterization of microbial populations of the rumen was also completed in pre-weaned calves using newly developed molecular biology and DNA sequencing tools established in the laboratory. Further, an animal experiment was completed to determine the effects of dietary amino acid supply on ruminal microbial community structure and dynamics using metagenomic sequencing of microbial DNA present in the gut contents. This information will assist in the development of specific dietary interventions to enhance nutrient uptake and partitioning in cattle. To permit biopsy of gut tissues in live animals repeatedly over time during various experimental treatments, cannulae were surgically placed in 2 intestinal compartments (rumen and small intestine) of 10 dairy cows. These animals are now being used to examine changes in gene expression in the cow gut in response to dietary manipulation. Study of metabolic flux within 2 cell types from the cattle gut was continued to improve knowledge of how nutrient supply to the gut influences amino acid use and to develop feeding strategies that minimize inefficiencies in the digestive process. Data analysis was completed from an animal study examining forage versus concentrate feeding on amino acid use by the cattle gut and specific genes were identified that may be involved in the observed increase in nutrient use for energy production in response to a forage-based ration. Combined, these investigations will lead to better animal management practices to improve production efficiency of dairy cattle and reduce nutrient losses to the environment.
Applied the latest molecular biology tools to study nutritional regulation of the transcriptome of cattle. Butyrate is a normal byproduct of carbohydrate fermentation produced by microbes present in the rumen and is a critical energy source for the cow. Two molecular technologies (next-generation sequencing and chromatin immunoprecipitation) were combined to map key protein targets encoded in the bovine genome that are responsive to butyrate. In addition, microarray technology was used to identify a specific type of genetic material, called microRNAs, as targets of butyrate action. The microRNAs function in the regulation of gene expression. These basic research projects provide systematic and novel insights into relationships between nutrition and regulation of gene expression in cattle by showing microRNA involvement in butyrate response.
Validated a cell culture model to study heat stress. Controlling production losses enable cattle producers to increase production efficiency of their herds by ensuring that the metabolic costs for maintenance of health are minimized. Heat stress is common in dairy cattle during summer months and can exacerbate the detrimental effects of infectious and metabolic diseases. An immortalized cell line from a specific cell type present in the mammary gland of cattle was developed and used to demonstrate significant changes in cell form and structure in response to a temperature increase as small as 2.5°C and as brief as 1 hour. This development will allow the study of cellular and molecular changes during heat stress without the use of live animals and expensive environmentally-controlled chambers.
Identified associations between variation in an immune-related gene and milk production traits. Understanding differences among individual animals in their susceptibility to stresses to the immune system is important for identifying superior (or inferior) animals for milk production. Two length polymorphisms in an immune-related gene called tumor necrosis factor alpha (TNF-alpha) were identified in cattle and shown to be related to an increase in TNF-alpha protein expression in the blood, and various measures of milk production. These results may reflect an increased efficiency of the mammary gland tissue to recover after injury, or improved aspects of immune function in certain individuals. The knowledge gained from this work can be applied to identifying mechanisms underlying differences in milk production efficiency of dairy cattle, and used in the development of selection criteria for improved mammary gland function and health.
Developed bioinformatics pipelines to analyze metagenomic datasets. The rumen microbial community has profound effects on the health and ability of the cow to digest and extract nutrients from its feed. Our laboratory has initiated several studies to examine changes in the rumen microbial population in response to dietary changes, developmental changes, and disease states (e.g., parasitic infection) using next-generation sequencing technology. Limitations of this approach include the extensive data sets that are generated from this technology, statistical analysis of the data, and biological interpretation of the results. To facilitate these studies, we have successfully developed bioinformatics pipelines to improve our efficiency and ability to analyze large, metagenomic datasets.
Examined molecular mechanisms of improved milk production and immune function in dairy cows receiving heat stress abatement prior to calving. Heat stress contributes to nutrient loss of dairy cattle through energy being spent on non-productive processes. Controlling these losses enables dairy producers to increase production efficiency of their herds by reducing metabolic costs for maintenance and health. In collaboration with the University of Florida, dairy cows were either provided cooling (sprinklers and fans) for the ~45-day period prior to calving known as the dry period, or no cooling during the dry period. The cows that were not cooled experienced heat stress. Relative to heat-stressed cows, cooled cows showed improvements in their transition into milking and exhibited gene expression changes in the signaling pathway for the hormone prolactin, which could support greater milk yield. In addition, improvements in the function of the immune system were observed in cooled cows. Cooling exclusively during the dry period is a promising management strategy to improve the cow's transition into milking, although additional study on its effects on the incidence of diseases related to calving are needed to confirm the practical application of these findings.
Characterized a gut hormone pathway in cattle. Glucagon-like peptide 2 (GLP-2) is a hormone released from specialized cells in the gut. Previous research in rodents and pigs showed that GLP-2 treatment promotes growth and reduces loss of cells lining the intestinal tract. In addition, it enhances nutrient absorption and barrier function of the intestinal lining. The functions of GLP-2 in the gut of cattle and other ruminants are not well studied. Therefore, the gene and protein expression of members of the GLP-2 pathway were evaluated in 9 regions of the bovine gut at different stages of development and lactation. In addition, the correlation of expression of the hormone and its receptor with markers for cell growth, cell death, nutrient transporters, enzyme activity, and blood flow were evaluated. Our findings supported a functional role of GLP-2 in cattle and suggest that GLP-2 therapy may be useful to improve intestinal function and nutrient absorption in ruminant species. This research contributes to basic knowledge of the ruminant intestinal tract and suggests that GLP-2 treatment is a possible means to improve gut function and healing after injury in cattle.
Demonstrated metabolic and gene expression-related changes in response to Vitamin E treatment in bovine cells. Our recent findings indicate that the form of Vitamin E called alpha-tocopherol is more than just a simple fat-soluble antioxidant. Through integrated global gene expression information and knowledge of cell regulation, it was determined that the action of alpha-tocopherol is mediated by specific receptors in the cell nucleus, such as estrogen receptor and androgen receptor. This work provides evidence that alpha-tocopherol may interact with receptors or gene transcription factors that control a myriad of cell functions and ultimately affect production efficiency in dairy cattle. Knowledge gained from this research may be used to develop effective interventions that enhance nutrient uptake and use by livestock.
Connor, E.E., Kahl, S., Elsasser, T.H., Parker, J., Li, R.W., Van Tassell, C.P., Baldwin, R.L., Barao, S.M. 2009. Enhanced mitochondrial complex gene function and reduced liver size may mediate improved feed efficiency of beef cattle during compensatory growth. Functional and Integrative Genomics. 10(1):39-51.
Amaral, B.C., Connor, E.E., Tao, S., Hayen, J., Bubolz, J., Dahl, G.E. 2010. Heat stress abatement during the dry period influences prolactin signaling in lymphocytes. Domestic Animal Endocrinology. 38(1):38-45.
Connor, E.E., Li, R.W., Baldwin, R.L., Li, C. 2009. Gene expression in the digestive tissues of ruminants and their relationships with feeding and digestive processes. Animal. 4(7):993-1007.