Source: Agricultural Research Service submitted to
PHYSIOLOGICAL REGULATION AND FUNCTIONAL GENOMICS OF RUMINANT NUTRIENT METABOLISM
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0405894
Grant No.
(N/A)
Project No.
1265-31000-089-00D
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 26, 2002
Project End Date
Jul 25, 2007
Grant Year
(N/A)
Project Director
BALDWIN R L
Recipient Organization
Agricultural Research Service
BLDG 003 BARC W RM 331
BELTSVILLE,MD 20705-2351
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
25%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3023310101050%
3023410101050%
Goals / Objectives
The objectives are to identify mechanisms regulating visceral growth and metabolism which are influenced by dietary energy, protein intake, and physiological status by determining the effect of growth factors and nutrients on cell proliferation, differentiation, and metabolism and determining differential gene expression and gene product (proteome) response to changes in nutrient inputs, and delineating cellular control mechanisms affected by nutrient use and absorption patterns and/or changes in visceral organ mass.
Project Methods
Approaches used will include: identification of metabolic (enzyme pathways, nutrient transport) or physiological functions (growth factor expression and receptors) that are regulators of nutrient economy within the animal; development and use of animal models (natural species differences, genetic variation, perturbation with exogenous compounds, and/or level of production) to assess the extent to which differential expression of critical genes can result in altered nutrient economy; and the development and characterization of cDNA libraries, and other genomics and proteomics tools, from ruminant intestine and stomach complex to facilitate gene discovery and to delineate critical pathways relating to nutrient use and visceral tissue growth. On a quantitative basis the influence of dietary energy, protein intake, and physiological status on splachnic and whole body energy dynamics will be assessed by determining the impact of dietary composition and site of substrate delivery on splanchnic nutrient output and tissue accretion in cattle and quantifying both at the whole animal and tissue level, the metabolic ramifications of altering nutrient assimilation.

Progress 07/26/02 to 07/25/07

Outputs
Progress Report Objectives (from AD-416) The objectives are to identify mechanisms regulating visceral growth and metabolism which are influenced by dietary energy, protein intake, and physiological status by determining the effect of growth factors and nutrients on cell proliferation, differentiation, and metabolism and determining differential gene expression and gene product (proteome) response to changes in nutrient inputs, and delineating cellular control mechanisms affected by nutrient use and absorption patterns and/or changes in visceral organ mass. Approach (from AD-416) Approaches used will include: identification of metabolic (enzyme pathways, nutrient transport) or physiological functions (growth factor expression and receptors) that are regulators of nutrient economy within the animal; development and use of animal models (natural species differences, genetic variation, perturbation with exogenous compounds, and/or level of production) to assess the extent to which differential expression of critical genes can result in altered nutrient economy; and the development and characterization of cDNA libraries, and other genomics and proteomics tools, from ruminant intestine and stomach complex to facilitate gene discovery and to delineate critical pathways relating to nutrient use and visceral tissue growth. On a quantitative basis the influence of dietary energy, protein intake, and physiological status on splachnic and whole body energy dynamics will be assessed by determining the impact of dietary composition and site of substrate delivery on splanchnic nutrient output and tissue accretion in cattle and quantifying both at the whole animal and tissue level, the metabolic ramifications of altering nutrient assimilation. Significant Activities that Support Special Target Populations The effect of photoperiod on expression of genes involved in gluconeogenesis and fatty acid synthesis in liver of growing steers was evaluated by quantitative realtime RT-PCR to determine whether short-day photoperiod treatment during the dry period may provide protection against fatty liver and metabolic syndrome in dairy cattle in early lactation. Results indicated significant changes in expression of genes related to fatty acid synthesis and gluconeogenesis in response to short- day photoperiod treatment that may be beneficial for reducing the incidence or severity of fatty liver in dairy cows. Differential expression of genes in liver of growing bulls in response to dietary restriction and re-feeding was examined by quantitative realtime RT-PCR based on genes identified by preliminary microarray analysis. Results indicated approximately 62% of genes tested were validated by realtime RT-PCR. Additional animals will be evaluated by microarray to obtain a more robust data set for analysis. Evaluated metabolic flux of substrates through Krebs cycle intermediates in isolated rumen epithelial (REC) and duodenal mucosal (DMC) cells preparations. Partial catabolism of glucose to lactate in these cells may play a role in preserving 3-carbon units from glucose for recycling. Increasing the supply of the amino acid glutamate to REC and DMC increased its flux to Krebs cycle intermediates thereby reducing anaplerosis from other substrates entering at or beyond �-ketoglutarate. Catabolism of glutamine (GLN) by gut cells has been previously demonstrated, but in the current study, GLN entry to the Krebs cycle was limited. Extent and type of energetic substrates used by the ruminant gastrointestinal tract (GIT) varies with energy intake and diet energy density such that GIT of cattle fed high forage-low concentrate rations use more energy and amino acids than those fed low forage-high concentrate rations. This results in a reduction in post-absorptive accretion of amino acids by carcass and decreased efficiency. We demonstrated differential expression of selected regulatory genes involved in substrate catabolism by the GIT in response to altered nutrient supply using complex rations. Accomplishments By optimizing urea recycling in productive ruminants, there is potential to decrease nitrogen (N) excretion into the environment and increase the conversion efficiency of N in feed to milk and meat. Transfer of N in the form of urea from the blood to the gastrointestinal tract (GIT) and back from the GIT into circulation in the form of microbial proteins may serve as a practical tool for producers to enhance nutrient use efficiency. Low ruminally undegradable protein content in diets resulted in greater urea transfer to the GIT and greater return of recycled urea to the blood. Blood flow to the kidney and clearance of blood urea per liter were unchanged indicating that any regulation of recycling is not at the kidney. Moreover, urea transporter (bUT-B2) expression in ruminal epithelium was unchanged despite the wide range in ruminally degraded protein. Rate of transfer of urea across the rumen wall is independent of rumen and blood urea concentrations, thus increasing the proportion of BUN and rumen ammonia N (RAN) transferred with low protein diets results in decreased BUN and RAN concentrations. Altering site of dietary protein digestion has a clear impact on N recycling and the potential to serve as a management tool for the producer to enhance on farm nutrient use efficiency. This research contributes to Component 1: Understanding, Improving, and Effectively Using Animal Genetic and Genomic Resources, specifically, Problem Statement 1B: Identify Functional Genes and Their Interactions; and Component 2: Enhancing Animal Adaptation, Well-Being and Efficiency in Diverse Production Systems, specifically Problem Statement 2C: Improving Efficiency of Nutrient Utilization and Conversion to Animal Products of the current NP101 Action Plan. Technology Transfer Number of Active CRADAS and MTAS: 1 Number of Non-Peer Reviewed Presentations and Proceedings: 5

Impacts
(N/A)

Publications

  • Sunny, N.E., Owens, S.L., Baldwin, R.L., El-Kadi, S.W., Kohn, R.A., Bequette, B.J. 2007. Salvage of blood urea nitrogen in sheep is highly dependent on plasma urea concentration and the efficiency of capture within the diegestive tract. Journal of Animal Science. 85(4):1006-1013.
  • Mccleod, K.E., Baldwin, R.L., Solomon, M.B., Baumann, R.G. 2007. Influence of ruminal and postruminal carbohydrate infusion on visceral organ mass and adipose tissue accretion in growing beef steers. Journal of Animal Science. 85:2256-2270.
  • Baldwin, R.L., Baumann, R.G., Mcleod, K.R. 2007. Influence of abomasal carbohyrdates on subcutaneous, omental, and mesenteric adipose lipogenic and lypolytic rates in growing beef steers. Journal of Animal Science. 85:2271-2282.


Progress 10/01/05 to 09/30/06

Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? A comprehensive understanding of the physiological limitations to nutrient assimilation and the regulatory mechanisms dictating performance are fundamental to the successful installation of new feeding paradigms to optimize nutrient delivery in the ruminant. Limitations can be alleviated by targeted use of nutritional regimes, exogenous compounds and feed additives, and ultimately, will be used as genetic selection criteria. The goals are: to determine critical regulatory steps and identify genetic controls, to determine metabolic limits, and to characterize nutrient use within the animal in order to aid in the development of tools (feeding models, pharmacological treatment, and selection criteria) to alleviate metabolic and genetic limitations restricting animal performance. Our approach is to (1) identify metabolic (enzyme pathways, nutrient transport) and physiological functions (growth factor expression and receptors) that are regulators of nutrient economy within the animal; (2) develop and use of animal models (natural species differences, genetic variation, perturbation with exogenous compounds, and/or level of production) to assess the extent to which differential expression of critical genes can result in altered nutrient economy; and; (3) develop and characterize cDNA libraries from ruminant intestine and stomach complex to facilitate gene discovery and to delineate critical pathways relating to nutrient use and visceral tissue growth. The gut and liver tissues of ruminants account for 30 to 50% of whole body energy use and 30 to 40% of total body protein turnover, while representing only 8 to 10% of the total body mass. Thus, without an understanding of the dynamics of visceral nutrient use, improvements in the current models for animal feeding can not be made. The potential improvements include, but are not limited to, increasing the nutrients available to the animal's productive tissues, decreasing the excretion of excess nutrients due to improperly balanced diets, and ultimately decreasing the cost and environmental impact of animal production. Feeding is the primary cost of both dairy and beef production and thus, savings in this area are crucial to successful animal production. Potential impact of incorporating these research findings into production and research models is phenomenal considering that a nominal 2% increase in energetic efficiency is worth $500 million to the dairy and beef industries annually in feed costs alone. Given both the expense of feed protein sources and new legislation related to nitrogen loss from the farm, the fiscal impact of use of improved feeding programs is even greater, and implementation of strategies to improve animal nitrogen use will lead to a decrease in nitrogen excretion. This cannot be accomplished without the development of better and more complete feeding paradigms. 2. List by year the currently approved milestones (indicators of research progress) 18 months (2002 and 2003) 1. Determine the impact of individual growth factors and specific nutrients on rumen epithelial cell and enterocyte proliferation, metabolic activity (amino acid metabolism, oxidative energy expenditures, and specific nutrient transport). (Experiment 1.4) 2. Develop incubation conditions to facilitate investigation of amino acid (primarily non-essential) use by isolated rumen epithelial cells in short-term cultures (Experiment 1.1). Specific objectives are to establish parameter estimates (rate and extent of oxidation, use for protein synthesis, or other metabolic outcomes) for pathways of amino acid use by the absorptive tissues of the ruminant and to quantify the capacity for other nutritional and physiological factors to alter these metabolic characteristics of the tissue. 3. Determine impact of site of delivery and form of carbohydrate infusion on lipogenesis and lipolysis in primary adipose depots of ruminants. Complete sample collection phase of Experiment 2.1 including all intestinal and adipose tissues for subsequent analysis. Specific objectives are to prepare tissues of the gastrointestinal tract and adipose depots for analysis of transport and metabolically important gene expression in response to alterations in nutrient delivery. 36 months (2004 and 2005) 4. Develop and characterize a cDNA library resource (Experiment 1.5) to facilitate the discovery of unknown pathways and mechanisms. Library will represent bovine intestinal tissues harvested at different physiological stages including: 1) neonatal and mature, 2) lactating and dry, 3) growing animals fed differing energy densities. 5. Establish specific parameter estimates (rate and extent of oxidation, use for protein synthesis, or other metabolic outcomes) for pathways of amino acid use by isolated small intestinal enterocytes and to quantifiably assess the relative ability of other nutritional and physiological factors to alter these metabolic characteristics (Experiment 1.2). 6. Quantify amino acid metabolism in isolated rumen epithelial cells and small intestinal enterocytes harvested from dairy cows fed diets varying in energy density (i.e., forage vs. concentrate) at various physiological states (e.g. stage of lactation, dry) in order to establish the contribution of this important regulatory pathway for nitrogen use by ruminants. (Experiment 1.3) 60 months (2006 and 2007) 7. Determine if the increased mesenteric adiposity observed with post- ruminal delivery of carbohydrate is the result of a physiological bias or is a result of exceeding the genetic capacity for product formation (milk or meat) (Experiment 2.2). 4a List the single most significant research accomplishment during FY 2006. Primary substrate flux and contribution to Krebs cycle metabolism of isolated rumen epithelial cells (REC) and small intestinal mucosal tissues (DMC) were evaluated to determine the impact of luminal environment on intermediary metabolism in ruminants. Rates of glucose conversion to lactate represented 25% of lactate flux in REC from bulls fed a high concentrate diet and in DMC from bulls fed both high concentrate and high forage diets. In contrast, in REC from bulls fed a high forage diet, glucose contributed only 12% of lactate flux. Partial catabolism of glucose to lactate, and possibly alanine, may play a role in preserving 3-carbon units for gluconeogenesis in the liver, and the extent of the salvage is affected by diet type. Flux of amino acids leucine and valine to ketoisocaproic and ketoisovaleric acid increased with leucine (17-63%) and valine (19-82%) supply, however neither entered Krebs cycle intermediates. Glutamate was the largest contributor to a- ketoglutarate flux and increased (9-41%) with supply. While glutamine catabolism by gastrointestinal cells has been previously demonstrated, a- ketoglutarate flux from glutamine did not exceed 3%. Increasing supply of glutamate to REC and DMC increases the flux of Krebs cycle intermediates from glutamate, thereby reducing the entry of other substrates entering at or beyond a-ketoglutarate. Thus, metabolic adaptations to diet are detectable and determination of the roles of substrate, enzyme, transporter, gene, and post-transcriptional regulation are needed to enhance nutrient delivery to the productive tissues of the ruminant. 4b List other significant research accomplishment(s), if any. Sample collection has been completed and transcript profiling of genes of interest has been initiated in targeted tissues to evaluate gene expression in intestinal tissues exhibiting observed differences in nutrient use efficiency induced by dietary alterations (concentrate vs. forage), physiological status (i.e., stage of lactation), and by direct infusion of specific nutrients. 5. Describe the major accomplishments to date and their predicted or actual impact. A cDNA library resource was created from normal gut tissues representing different physiological stages to facilitate the search for specific metabolic pathways, transporters, growth factor receptors, and growth factors that have profound effects on ruminant visceral energy and protein metabolism. A novel normalized cDNA library was synthesized from lactating dairy cow and neonatal calf intestine to facilitate gene discovery and delineate critical pathways relating to nutrient use and visceral tissue growth. This library (8BOV) was synthesized, sequenced and characterized. This library will be an indispensable resource in the study of genetic regulation of ruminant nutrient metabolism and growth. This resource is having direct impacts as evidenced by the 19,110 additional EST sequences deposited in GenBank, by ongoing external interest by cooperators in this sequence information, and by requests for materials by colleagues. A total of 1,123 sequence elements from these EST represent genes encoding proteins in other animal systems that are now represented in bovine. This resource has facilitated the development of second generation tools (bmet microarray), an in-house oligoucleotide- based NimbleGen array, as well as, commercially available arrays via database accession. Urea recycling is an important mechanism that improves the efficiency of nitrogen use. Urea synthesis by ruminant gut tissues may be a strategic target to reduce ammonia absorption and improve nitrogen use. Using an in vitro isolated cell system and innovative stable isotopic approaches, we demonstrated that ruminant gut tissues have the capability to synthesize urea from substrate intermediates in the ornithine-urea cycle, in particular from arginine degradation by ruminal epithelial cells. Using these basic findings, University of MD scientists have successfully submitted a patent application for the use of stimulatory compounds for up-regulation of the argino-succinate synthetase gene. Essential amino acids (AA) and glucose metabolism by the visceral organs was assessed in sheep when the protein supply to the small intestines was increased from marginal to adequate for maintenance and growth requirements. Efficiency of absorption of the essential AA remained fixed regardless of protein supply. Specifically, branched chain AA and lysine were metabolized in increasing amounts, thus maintaining net absorption. Because lysine is most often a limiting AA for growth and milk production in ruminants fed corn or corn-silage based diets, the basis for gastrointestinal tract metabolism of lysine and the branched chain AA will need to be understood more completely and represented in feeding models. While variable, visceral organs are the greatest users of nutrients in the productive ruminant on a weight basis, impacting availability of nutrients for productive function. Balancing nutrient delivery with tissue requirements may prove to be a valuable tool for producers to improve the efficiency and effectiveness of feeding regimes. To determine the capability of ruminant gut tissues to detoxify ammonia nitrogen via ornithine-urea cycle or alanine synthesis, we assessed metabolism in vitro. Utilization of ammonia nitrogen for net alanine synthesis increased with ammonia concentration for both ruminal epithelial and duodenal mucosal cells. Alanine synthesis may be a significant metabolic pathway for ruminant gut tissues to detoxify ammonia nitrogen when it is presented luminally at high concentrations, and a key regulatory target to increase efficiency of nitrogen utilization in ruminant animals. Studies of interactions of amino acid metabolism with alternative energy substrates were completed. Manipulation of ammonia N use by the gut tissues is a potential physiological approach to enhance efficiency of N use. Using a carbohydrate infusion model, we have characterized alterations in adipose metabolism across three primary adipose depots in steers. Our findings support the concept that alterations in tissue nutrient specificity and enzyme expression result from changes in diet composition and in the site of delivery of starch from the rumen to the small intestine. Increases in fat synthesis and breakdown with increased energy and altered site of glucose delivery are consistent with the observed changes in adiposity of these steers. Increased efficiency of nutrient use observed by steers fed diets with higher energy densities can be partially explained by alterations in gut tissue metabolism of energy substrates and the subsequent alterations in adipose depot lipogenic rates. Quantitative RT-PCR assays were developed and employed to measure expression of genes in adipose tissue known to be involved in fatty acid metabolism including FAS, ACC, Spot-14, SERBP, SERBP-1, SERBP- 2, and ChREBP genes. We demonstrated up-regulation of lipogenic metabolism genes as a result of postruminal carbohydrate delivery and identified Spot-14 as a putative critical controlling element in the response. There is conflicting data concerning the extent of up-regulation of sodium-dependent glucose co-transporter 1 (SGLT1) in response to carbohydrate in the small intestinal lumen. A carbohydrate infusion into either the rumen or abomasum was used to determine the effects of glucose and starch hydrolysate on activity and abundance of SGLT1 in the small intestine of steers. While activity and abundance exhibited a differential pattern along the small intestine, dietary effects were not observed. Moreover, an inverse relationship between glucose uptake and SGLT1 abundance suggests that regulation of glucose transport capacity is complex, involving factors other than SGLT1 abundance. Nutrient uptake by the gut tissues affects hepatic metabolism and subsequently, nutrient supply to peripheral tissues, yet the metabolic basis for oxidative metabolism of energy substrates by the gut tissues is not well understood. Intestinal enterocyte primary cultures have been used successfully to develop unique kinetic parameter estimates for hexose oxidation. These results add to previous findings by this unit describing the kinetic parameters for substrate oxidation by ruminant intestinal epithelial tissue. Together these add important inputs into the refinement of mechanistic models. Once fully developed, these models will be used to balance accurately nutrient delivery to productive tissues. Diets will then be formulated to ensure excessive losses to the environment are minimized and animal health is not compromised. To identify genetic mechanisms regulating visceral growth and metabolism of cattle, a study was completed using growing beef steers to evaluate changes in gene expression in the liver in response to feed restriction and re-feeding. The goal of the study was to identify gene pathways controlling compensatory growth and improved feed efficiency. As expected, improved feed:gain ratios were observed in animals experiencing a period of dietary restriction. A microarray containing oligonucleotides representing over 53,000 potential gene transcripts produced within the laboratory in collaboration with NimbleGen as part of project 1265-31000- 086-00D, Functional Genomics of Dairy Production, was used to characterize these changes in hepatic gene expression over time. Analyses to identify potential genes regulating enhanced feed efficiency are ongoing and results from this work should identify gene targets for selection to improve feed efficiency. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A rumen epithelial cell isolation procedure has been refined and the procedure has been transferred to other scientists either through visits to Beltsville or via presentations at national meetings. Moreover, the coupling of stable isotope capabilities with short-term cell culture has been used to provide a greater understanding of rumen tissue metabolism and its impact on substrate detoxification and supply to the ruminant. This unit has used the same isolated cell system to develop novel parameter estimates for ruminal energy substrate use that will be applied to development of robust feeding models. Partial efficiency of metabolizable energy use for tissue gain for ruminal and intestinal digested starch has been determined. This unique work was highlighted in an invited presentation at the national meeting for the National Society of Animal Scientists. The developed partial efficiency estimates will be used to adjust the NRC net energy values of cereal grains and will impact nutritional feeding strategies and ration formulations in the beef cattle industry. Impact of cereal grain processing on Net Energy for Lactation (NEL) was used in the current edition of Nutrient Requirements of Dairy Cattle to adjust the energy values of cereal grains. Genetic sequences (19,110) were deposited in GenBank, making them available to the global research community free of charge. Finally, the sequences have been provided to cooperators to facilitate the development of comprehensive microarrays using oligonucleotides based on 8BOV sequences.

Impacts
(N/A)

Publications

  • Kalscheur, K.F., Baldwin, R.L., Glenn, B.P., Kohn, R.A. 2006. Milk Producton of Dairy Cows Fed Differing cConcentrations of Rumen Degraded Protein. Journal of Dairy Sci. 89:249-259.
  • El-Kadi, S.W., Baldwin, R.L., Sunny, N.E., Owens, S.L., Bequette, B.J. 2006. Metabolism of amino acids and glucose by the sheep gatrointestinal tract in response to post-ruminal protein supplies. Journal of Nutrition. 136(5):1261-1269.
  • Baldwin, R.L., Mcleod, K., Baumann, R.G., Connor, E.E. 2006. Influence of carbohydrate infusion on lipogenic enzyme and regulatory protein gene expression in growing beef steer [abstract]. Experimental Biology Meeting. FASEB Journal 20, Abstract #LB380.
  • Peterson, A.B., Baldwin, R.L., Kohn, R. 2006. Effect of particle size on passage rates from the rumen to the duodenum of lactating dairy cows [abstract]. American Dairy Science and Society of Animal Science. 89:242.
  • Peterson, A.B., Baldwin, R.L., Bequette, B.J., Kohn, R.A. 2006. Effect of ruminally degraded protein source on nitrogen metabolism in Holstein cows [abstract]. Journal of Dairy Science. 89(Suppl. 1)180.


Progress 10/01/04 to 09/30/05

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? A comprehensive understanding of the physiological limitations to nutrient assimilation and the regulatory mechanisms dictating performance are fundamental to the successful installation of new feeding paradigms to optimize nutrient delivery in the ruminant. Limitations can be alleviated by targeted use of nutritional regimes, exogenous compounds and feed additives, and ultimately, will be used as genetic selection criteria. The goals are: to determine critical regulatory steps and identify genetic controls, to determine metabolic limits, and to characterize nutrient use within the animal in order to aid in the development of tools (feeding models, pharmacological treatment, and selection criteria) to alleviate metabolic and genetic limitations restricting animal performance. Our approach is to (1) identify metabolic (enzyme pathways, nutrient transport) and physiological functions (growth factor expression and receptors) that are regulators of nutrient economy within the animal; (2) develop and use of animal models (natural species differences, genetic variation, perturbation with exogenous compounds, and/or level of production) to assess the extent to which differential expression of critical genes can result in altered nutrient economy; and; (3) develop and characterize cDNA libraries from ruminant intestine and stomach complex to facilitate gene discovery and to delineate critical pathways relating to nutrient use and visceral tissue growth. The gut and liver tissues of ruminants account for 30 to 50% of whole body energy use and 30 to 40% of total body protein turnover, while representing only 8 to 10% of the total body mass. Thus, without an understanding of the dynamics of visceral nutrient use, improvements in the current models for animal feeding can not be made. The potential improvements include, but are not limited to, increasing the nutrients available to the animal's productive tissues, decreasing the excretion of excess nutrients due to improperly balanced diets, and ultimately decreasing the cost and environmental impact of animal production. Feeding is the primary cost of both dairy and beef production and thus, savings in this area are crucial to successful animal production. Potential impact of incorporating these research findings into production and research models is phenomenal considering that a nominal 2% increase in energetic efficiency is worth $500 million to the dairy and beef industries annually in feed costs alone. Given both the expense of feed protein sources and new legislation related to nitrogen loss from the farm, the fiscal impact of use of improved feeding programs is even greater, and implementation of strategies to improve animal nitrogen use will lead to a decrease in nitrogen excretion. This cannot be accomplished without the development of an improved understanding of the interactions between nutrients and genes and their inclusion in the development of more complete feeding paradigms. 2. List the milestones (indicators of progress) from your Project Plan. 1. Determine the impact of individual growth factors and specific nutrients on rumen epithelial cell and enterocyte proliferation, metabolic activity (amino acid metabolism, oxidative energy expenditures, and specific nutrient transport). (Experiment 1.4) 18 months (2002 to 2003). 2. Develop incubation conditions to facilitate investigation of amino acid (primarily non-essential) use by isolated rumen epithelial cells in short-term cultures (Experiment 1.1). Specific objectives are to establish parameter estimates (rate and extent of oxidation, use for protein synthesis, or other metabolic outcomes) for pathways of amino acid use by the absorptive tissues of the ruminant and to quantify the capacity for other nutritional and physiological factors to alter these metabolic characteristics of the tissue. 3. Determine impact of site of delivery and form of carbohydrate infusion on lipogenesis and lipolysis in primary adipose depots of ruminants. Complete sample collection phase of Experiment 2.1 including all intestinal and adipose tissues for subsequent analysis. Specific objectives are to prepare tissues of the gastrointestinal tract and adipose depots for analysis of transport and metabolically important gene expression in response to alterations in nutrient delivery. 36 months (2004 and 2005). 4. Develop and characterize a cDNA library resource (Experiment 1.5) to facilitate the discovery of unknown pathways and mechanisms. Library will represent bovine intestinal tissues harvested at different physiological stages including: 1) neonatal and mature, 2) lactating and dry, 3) growing animals fed differing energy densities. 5. Establish specific parameter estimates (rate and extent of oxidation, use for protein synthesis, or other metabolic outcomes) for pathways of amino acid use by isolated small intestinal enterocytes and to quantifiably assess the relative ability of other nutritional and physiological factors to alter these metabolic characteristics. (Experiment 1.2) 6. Quantify amino acid metabolism in isolated rumen epithelial cells and small intestinal enterocytes harvested from dairy cows fed diets varying in energy density (i.e., forage vs. concentrate) at various physiological states (e.g. stage of lactation, dry) in order to establish the contribution of this important regulatory pathway for nitrogen use by ruminants. (Experiment 1.3) 60 months (2006 to 2007) 7. Determine if the increased mesenteric adiposity observed with postruminal delivery of carbohydrate is the result of a physiological bias or is a result of exceeding the genetic capacity for product formation (milk or meat). (Experiment 2.2) 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 3. Develop a cDNA library and second generation genomics tools. Milestone Fully Met 4. Establish specific parameter estimates for pathways of amino acid use by isolated small intestinal enterocytes. Milestone Substantially Met 5. Quantify amino acid metabolism in isolated rumen epithelial cells and small intestinal enterocytes. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Year 4, 2006 Milestone 3 - This work represents an extension of an FY 2005 milestone. Complete microarray analysis of changes in hepatic gene expression in response to feed restriction and re-feeding and initiate realtime PCR studies to validate microarray results. Begin microarray analysis of intestinal epithelial changes associated with forage vs. concentrate feeding. Collect samples, assess microarray results, and initiate realtime PCR validation experiments. Milestone 5 - Continued experiments will further define metabolic pathways of interest for understanding inefficient metabolism in the ruminant gut using stable isotopic approaches in conjunction with in vivo dietary manipulation of intestinal mass and metabolism. Milestone 6 - Initiate experiment using a starch infusion model to manipulate adipose tissue metabolism and collect samples for use on the microarray applications available to the laboratory. Milestone: 7 - Nitrogen use and microbial efficiency will be assessed using in vivo approaches and whole animal N efficincy will be addressed. Year 5, 2007 Milestone 3 - This work represents an extension of an FY 2005 milestone. Initiate follow-up studies on specific genes of interest identified from the microarray study to further characterize their role in modulation of feed efficiency in growing cattle. Initiate follow-up experiments to address specific mechanisms affecting intestinal growth and metabolism using the forage vs. concentrate feeding model. Milestone 6 - Further develop and use adipose metabolism models in lactating and developing ruminants in vivo (Stage of lactation and development) to facilitate enhanced provision of nutrients to support requirements for lactation. Milestone: 7 - Conduct new experiments to assess the mechanistic control of specifically identified genes of interest affecting metabolism of the intestine and gut tissues in the lactating dairy cow and critical tissues. Year 6, 2008. This will represent the first year of a new project if approved and will be based largely on findings from the existing project. Metabolic challenges facing lactating dairy cattle in early lactation or transition periods are great and often result in inefficient use of nutrients for production and ultimately affect animal health. Models will be used to assess genomic level alterations that are causative to economically important metabolic diseases. Initiate studies to manipulate use of nutrients by dairy cattle in the transition period and evaluate the impact on cow metabolic health and subsequent lactation. 4a What was the single most significant accomplishment this past year? Depot specific lipogenic gene expression in response to altered energy supply in ruminants. Increased efficiency of nutrient use observed by ruminants fed diets with higher energy densities can be partially explained by alterations in gut tissue metabolism of energy substrates and the subsequent alterations in adipose depot lipogenic rates. Methods to quantify the expression of lipogenic genes present in adipose tissue depots by RT-PCR have been developed and used to assess expression of Fatty Acid Synthetase, Acetyl- CoA Carboxylase, Spot-14, SERBP, SERBP-1, SERBP-2, and ChREBP. We demonstrated upregulation of lipogenic metabolism genes as a result postruminal carbohydrate delivery and have identified Spot-14 as a putative critical controlling element in the response. Moreover, these changes correlate well with observed changes in depot adiposity and lipogenic rates in vitro. Deposition and mobilization of adipose depots in the lactating dairy cow and synthesis of adipose tissue in growing steers are critical metabolic processes where efficiency of nutrients use can be manipulated to enhance animal performance and reduce losses to the environment. 4b List other significant accomplishments, if any. Metabolic fates of ammonia nitrogen in ruminant gut cells. To determine the capability of ruminant gut tissues to detoxify ammonia nitrogen via ornithine-urea cycle or alanine synthesis we assessed metabolism in vitro. Utilization of ammonia nitrogen for net alanine synthesis increased with ammonia concentration for both ruminal epithelial and duodenal mucosal cells. Alanine synthesis may be a significant metabolic pathway for ruminant gut tissues to detoxify ammonia nitrogen when it is presented luminally at high concentrations, and a key regulatory target to increase efficiency of nitrogen utilization in ruminant animals (Experiments 1.2 and 1.3). In line with milestones laid out in Experiment 1.4, interactions of amino acid metabolism with alternative energy substrates were completed and manipulation of ammonia N use by the gut tissues is possibly a physiological approach for enhancing efficiency of N use. Hepatic gene expression changes occurring in response to restriction and re-feeding in steers. To identify genetic mechanisms regulating visceral growth and metabolism of cattle, a study was completed using growing beef steers to evaluate changes in gene expression in the liver in response to feed restriction and re-feeding. The goal of the study is to identify gene pathways controlling compensatory growth and improved feed efficiency. As expected, improved feed:gain ratios were observed in animals experiencing a period of dietary restriction. Currently, a microarray containing oligonucleotides representing over 53,000 potential gene transcripts produced within the laboratory in collaboration with Nimblgen as part of project 1265-31000-086-00D, Functional Genomics of Dairy Production, is being used to characterize these changes in hepatic gene expression over time. Analyses to identify potential genes regulating enhanced feed efficiency are ongoing and results from this work should identify gene targets for selection to improve feed efficiency. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The development of a cDNA library resource to facilitate the search for specific metabolic pathways, transporters, growth factor receptors, and growth factors which have profound effects on ruminant visceral energy and protein metabolism. A novel normalized cDNA library has been synthesized from lactating dairy cow and neonatal calf intestine to facilitate gene discovery and delineate critical pathways relating to nutrient use and visceral tissue growth. This library (8BOV) has been synthesized, sequenced and characterized. This library will be an indispensable resource in the study of genetic regulation of ruminant nutrient metabolism and growth. This resource is already having a direct impact as evidenced by the 19,110 NEW EST sequences deposited in GenBank and by ongoing external interest by cooperators in this sequence information and by requests for materials by colleagues. A total of 1, 123 sequence elements from these EST represt genes encoding proteins in other animal systems that are now represented in bovine. This resource has facilitated the development of second generation tools (bmet microarray), our inhouse Nimblegen array , as well as, commercially available arrays via database accession. Urea recycling is an important mechanism that improves the efficiency of nitrogen utilization. Using a unique coupling of isolated epithelial cells and stable isotopes, we demonstrated that ruminant gut tissues are capable of synthesizing urea, particularly by ruminal epithelial cells degrading Arginine. Urea synthesis by ruminant gut tissues may be a strategic target to reduce ammonia absorption and improve nitrogen utilization. In fact, University of MD scientists, using these basic findings, have successfully submitted a patent application for the use of stimulatory compounds for up-regulation of the argino-succinate synthetase gene. Using a carbohydrate infusion model we have defined alterations in adipose metabolism across three primary adipose depots. These results support the concept that alterations in tissue nutrient specificity and enzyme expression result from changes in diet composition that result in a shift in the site of delivery of starch from the rumen to the small intestine. Increases in fat synthesis and breakdown with increased energy and altered site of glucose delivery are consistent with the observed changes in adiposity of these steers. Increased efficiency of nutrient use observed by steers fed diets with higher energy densities can be partially explained by alterations in gut tissue metabolism of energy substrates and the subsequent alterations in adipose depot lipogenic rates. There is conflicting data concerning the extent of up-regulation of sodium-dependent glucose co-transporter 1 (SGLT1) in response to carbohydrate in the small intestinal lumen. A carbohydrate infusion into either the rumen or abomasum was used to determine the effect of glucose and starch hydrolysate on activity and abundance of SGLT1 in the small intestine of steers. While activity and abundance exhibited a differential pattern along the small intestine, dietary effects were not observed. Moreover, an inverse relationship between glucose uptake and SGLT1 abundance suggests that regulation of glucose transport capacity is complex, involving factors other than SGLT1 abundance. Current dairy and feedlot finishing rations are formulated to deliver large amounts of starch post-ruminally based on the premise that increased energetic efficiency will be realized. While feed energy efficiency is enhanced by these dietary alterations it is sometimes not the extent expected. Thus, to understand losses in efficiency, these data are helpful in assessing the costs of absorption under varying conditions. Nutrient uptake by the gut tissues affect hepatic metabolism and subsequently, nutrient supply to peripheral tissues, yet the metabolic basis for oxidative metabolism of energy substrates by the gut tissues is not well understood. Intestinal enterocyte primary cultures have been used successfully to develop unique kinetic parameter estimates for hexose oxidation. These results add to previous findings by this unit describing the kinetic parameters for substrate oxidation by ruminant intestinal epithelial tissue. Together these add important inputs into the refinement of mechanistic models. Once fully developed, these models will be used to balance accurately nutrient delivery to productive tissues, diets will then be formulated to ensure excessive losses to the environment are minimized and animal health is not compromised. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A rumen epithelial cell isolation procedure has been refined and the procedure has been transferred to other scientists either through visits to Beltsville or via presentations at national meetings. Moreover, the coupling of stable isotope capabilities with short-term cell culture has been used to provide a greater understanding of rumen tissue metabolism and its impact on substrate detoxification and supply to the ruminant. This unit has used the same isolated cell system in the development of novel parameter estimates for ruminal energy substrate use which will be used in the development of robust feeding models. Partial efficiency of metabolizable energy use for tissue gain for ruminal and intestinal digested starch has been determined. This unique work was highlighted in an invited presentation at the national meeting for the National Society of Animal Scientists. The developed partial efficiency estimates will be used to adjust the NRC net energy values of cereal grains and will impact nutritional feeding strategies and ration formulations in the beef cattle industry. Impact of cereal grain processing on NEl was used in the current edition of Nutrient Requirements of Dairy Cattle to adjust the energy values of cereal grains. Genetic sequences (19,110) were deposited in GenBank, making them available to the global research community free of charge. Additionally, individual clones are being requested and provided to researchers interested in novel EST not present, or not present with the same quality, as in 8BOV. Finally, the sequences have been provided to cooperators to facilitate the development of comprehensive microarrays using oligonucleotides based on 8BOV sequences.

Impacts
(N/A)

Publications

  • Moallem, U., Dahl, G.E., Duffey, E.K., Capuco, A.V., Mcleod, K.R., Baldwin, R.L., Erdman, R.A. 2004. Bovine somatotropin and rumen undergradable protein effects in prepubertal heifers: Effects on body composition and organ tissue weights. Journal of Dairy Science. 87(11):3869-3880.
  • Baldwin, R.L., Rodriquez, S.M., Guimaraes, K.C., Matthews, J.C., Mcleod, K. M., Harmon, D.L. 2004. Influence of abomasal carbohydrates on small intestinal sodium-dependent glucose co-transporter activity and abundance in steers. Journal of Animal Science. 82:(10):3015-3023.
  • Baumann, R.G., Baldwin, R.L., Van Tassell, C.P., Matukumalli, L.K., Sonstegard, T.S. 2005. Characterization of a normalized cDNA library from bovine intestinal muscle and epithelial tissues. Animal Biotechnology. 16(1):17-30.
  • Peterson, A.B., Baldwin, R.L., Kohn, R. 2005. Effect of ruminally degraded protein source on production performance in Holstein cows [abstract]. Journal of Animal Science. 88(1)99.


Progress 10/01/03 to 09/30/04

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? The gut and liver tissues of ruminants account for 30 to 50% of whole body energy use and 30 to 40% of total body protein turnover, while representing only 8 to 10% of the total body mass. Thus, without an understanding of the dynamics of visceral nutrient use, improvements in the current models for animal feeding can not be made. The potential improvements include, but are not limited to, increasing the nutrients available to the animal's productive tissues, decreasing the excretion of excess nutrients due to improperly balanced diets, and ultimately decreasing the cost and environmental impact of animal production. Feeding is the primary cost of both dairy and beef production and thus, savings in this area are crucial to successful animal production. Potential impact of incorporating these research findings into production and research models is phenomenal considering that a nominal 2% increase in energetic efficiency is worth $500 million to the dairy and beef industries annually in feed costs alone. Given both the expense of feed protein sources and new legislation related to nitrogen loss from the farm, the fiscal impact of use of improved feeding programs is even greater, and implementation of strategies to improve animal nitrogen use will lead to a decrease in nitrogen excretion. This cannot be accomplished without the development of better and more complete feeding paradigms. A comprehensive understanding of the physiological limitations to nutrient assimilation and the regulatory mechanisms dictating performance are fundamental to the successful installation of new feeding paradigms to optimize nutrient delivery in the ruminant. Limitations can be alleviated by targeted use of nutritional regimes, exogenous compounds and feed additives, and ultimately, will be used as genetic selection criteria. The goals are: to determine critical regulatory steps and identify genetic controls, to determine metabolic limits, and to characterize nutrient use within the animal in order to aid in the development of tools (feeding models, pharmacological treatment, and selection criteria) to alleviate metabolic and genetic limitations restricting animal performance. Our approach is to (1) identify metabolic (enzyme pathways, nutrient transport) and physiological functions (growth factor expression and receptors) that are regulators of nutrient economy within the animal; (2) develop and use of animal models (natural species differences, genetic variation, perturbation with exogenous compounds, and/or level of production) to assess the extent to which differential expression of critical genes can result in altered nutrient economy; and; (3) develop and characterize cDNA libraries from ruminant intestine and stomach complex to facilitate gene discovery and to delineate critical pathways relating to nutrient use and visceral tissue growth. 2. List the milestones (indicators of progress) from your Project Plan. Month Outcome 18 - Complete experiment 1.1 and experiment 2.1. 36 - The cDNA library resource (Expt.1.5) will be developed and characterized, microarray applications will be developed based on related experimental needs. Complete experiment 1.2 and 1.3. Initiate experiment 2.2. 60 - Experiments 2.2 and initial experiments for 1.4 completed. 18 month Milestones: Completion of Experiment 1.1 - Objectives are to establish parameter estimates (rate and extent of oxidation, use for protein synthesis, or other metabolic outcomes) for pathways of amino acid use by isolated ruminal epithelial cells and to quantifiably assess the ability of other nutritional and physiological factors to alter these metabolic characteristics. Development of in vitro incubation conditions for the investigation of amino acid (primarily non-essential) use and activity by isolated rumen epithelial cells in short-term cultures. Completion of sample collection phase of Experiment 2.1 with the Objective to determine impact of site and form of carbohydrate infusion on lipogenesis and lipolysis in growing steers. 36 month Milestones: Develop and characterize a cDNA resource to facilitate the discovery of unknown pathways and mechanisms from pooled bovine tissues harvested at different physiological stages including: 1) neonatal and mature, 2) lactating and dry, 3) growing animals fed differing energy densities. Completion of Experiment 1.2 - Objectives of this series of experiments will be to establish specific parameter estimates (rate and extent of oxidation, use for protein synthesis, or other metabolic outcomes) for pathways of amino acid use by isolated small intestinal enterocytes and to quantifiably assess the relative ability of other nutritional and physiological factors to alter these metabolic characteristics. Completion of Experiment 1.3 - Objective of these experiments will be to quantify ornithine cycle activity in isolated rumen epithelial cells and small intestinal enterocytes harvested from dairy cows fed diets varying in energy density (i.e., forage vs. concentrate) at various physiological states (e.g. stage of lactation, dry) in order to establish the contribution of this important regulatory pathway for nitrogen use by ruminants. Initiate Experiment 2.2 - Objective of this experiment is to determine if the increased mesenteric adiposity observed with postruminal delivery of carbohydrate is the result of a physiological bias or is a result of exceeding the genetic capacity for lean tissue growth. 60 month Milestones: Completion of Experiment 2.2 and initiate Experiments outlined in 1.4 designed to determine the impact of individual growth factors and specific nutrients on rumen epithelial cell and enterocyte proliferation, metabolic activity (amino acid metabolism, oxidative energy expenditures, and specific nutrient transport). 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. A cDNA library resource has been developed and characterized and a manuscript is being developed (36 month milestone). This resource has already been shared extensively through submission of gene sequence information to GenBank. Rumen epithelial and duodenal epitheial cells isolated from sheep intestinal tissues have been used successfully to develop non-radioactive methods for the assessment of Nitrogen metabolism (ammonia detoxification, urea synthesis and amino acid metabolism) through the use of stable isotopic techniques (Experiments 1.2 and 1.3). In line with milestones laid out in Experiment 1.4, interactions of amino acid metabolism with alternative energy substrates were initiated. The large experiment 2.2 was not initiated, however complementary (Whole animal N flux experiments) and cooperative experiments with other research scientists, has resulted in the development of the necessary resource population of surgically prepared cows (10 duodenally and ruminally cannulated cows) to facilitate the conduct of a modified experiment 2.2 in the upcoming FY2005 and FY2006. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Year 3, 2005 - Experiments are planned for the upcoming year, FY2005 with small adaptations to the project plan. One major change is the inclusion of a lactating cow model as opposed to the initially proposed growing beef steer model outlined. While meaningful data would be collected in either case, the lactating dairy cow model provides greater diversity of adipose tissue gene expression which is a global objective of the CRIS. - Continued research is underway relating to the expression of putative regulatory elements affecting physiological control of adipose synthesis and degradation. - Development of a Bovine metabolism microarray will be completed in cooperation and printed for implementation of microarray research with inclusion of 8BOV unique elements. - Initial assessments the impact of starch infusion on expression of metabolic gene expression through the use of a Bovine metabolism specific microarray will be made. - Nitrogen use and microbial efficiency will be assessed using in vivo approaches. Modelling of the data will continue with cooperators. Enterocyte metabolism of nitrogen will be further assessed using isolated ruminant enterocytes and stable isotopic approaches to further understand inefficiencies in ruminants fed for production agriculture. - Continue development of the adipose deposition research model resulting from the Chlortetracycline and starch infusion models for use as a potential assay for developing efficient nutrient use animals. Year 4, 2006 - Continue to develop and use long-term (2 weeks) cultures of primary cell isolates will be used to assess the impact of growth factors on expression of metabolic and growth related genes. - Selected genes and their controls, putative regulators of intestinal growth and metabolism, affecting animal performance will be identified and assessed as potential tools for the Dairy and Beef industries. - Continued experiments will further define metabolic pathways of interest for understanding inefficient metabolism in the ruminant gut. - Control of nitrogen use by the enterocyte in the ruminant will be challenged with in vivo and in vitro approaches to assess the extent to which basal amino acid use is required to support normal cellular function. - Further develop and use the adipose development model in experiments in vivo. Year 5, 2007 - Follow up experiments to assess the mechanistic control of specifically identified genes of interest affecting metabolism of the intestine and gut tissues in the lactating dairy cow and critical tissues. 4. What were the most significant accomplishments this past year? The development of a cDNA library resource to facilitate the search for specific metabolic pathways, transporters, growth factor receptors, and growth factors which have profound effects on ruminant visceral energy and protein metabolism. A novel normalized cDNA library has been synthesized from lactating dairy cow and neonatal calf intestine to facilitate gene discovery and delineate critical pathways relating to nutrient use and visceral tissue growth. This library (8BOV) has been synthesized, sequenced and characterized and this library will be an indispensable resource in the study of genetic regulation of ruminant nutrient metabolism and growth. This resource is already having a direct impact as evidenced by the 19,110 new EST sequences deposited in GenBank and by ongoing external interest by cooperators in this sequence information and by requests for materials by colleagues. A total of 1, 123 sequence elements from these EST represent genes encoding proteins in other animal systems that are now represented in bovine. B. Other Significant Accomplishment(s), if any. In ruminants, the poor efficiency of converting dietary protein into milk or muscle protein results partly from the extensive degradation of protein in the rumen resulting in ammonia absorption and ultimately, excretion of nitrogen in urine. It was not known if a mechanism to synthesize urea from absorbed ammonia existed in ruminant gut tissues, in particular the rumen epithelia. Such a mechanism would be an attractive target pathway for reducing the toxic side-effects of ammonia absorption in the animal and for promoting the local recycling of urea to the rumen for microbial protein synthesis. Using an in vitro isolated cell system and innovative stable isotopic approaches we demonstrated, that ruminant gut tissues have the capability to synthesize urea from substrate intermediates in the ornithine-urea cycle, in particular from arginine degradation by ruminal epithelial cells. This urea recycling is an important mechanism that improves the efficiency of nitrogen utilization. Urea synthesis by ruminant gut tissues may be a strategic target to reduce ammonia absorption and improve nitrogen utilization. C. Significant activities that support special target populations. None D. Progress Report opportunity to submit additional programmatic information to your Area Office and NPS (optional for all in-house (""D"") projects and the projects listed in Appendix A; mandatory for all other subordinate projects). None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The impact of site of carbohydrate digestion on visceral tissue growth and adipose accretion have been determined. Substrate and substrate supply impact rate and composition of tissue accretion. We have demonstrated that the observed increase in adiposity with carbohydrate delivery post-ruminally is associated with increases in overall lipogenesis from both glucose and acetate carbon (as assessed in short- term incubations in vitro) and have initiated RT-PCR analysis of fatty acid synthetase and AcetylCoA-Carboxylase expression to determine if they are causative in the increase in observed adiposity with post-ruminal delivery of carbohydrates. There is conflicting data concerning the extent of up-regulation of sodium-dependent glucose co-transporter 1 (SGLT1) in response to carbohydrate in the small intestinal lumen. A carbohydrate infusion into either the rumen or abomasum was used to determine the effect of glucose and starch hydrolysate on activity and abundance of SGLT1 in the small intestine of steers. While activity and abundance exhibited a differential pattern along the small intestine, dietary effects were not observed. Moreover, an inverse relationship between glucose uptake and SGLT1 abundance suggests that regulation of glucose transport capacity is complex, involving factors other than SGLT1 abundance. Nutrient uptake by the gut tissues affect hepatic metabolism and subsequently, nutrient supply to peripheral tissues, yet the metabolic basis for oxidative metabolism of energy substrates by the gut tissues is not well understood. Intestinal enterocyte primary cultures have been used successfully to develop unique kinetic parameter estimates for hexose oxidation. These results add to previous findings by this unit describing the kinetic parameters for substrate oxidation by ruminant intestinal epithelial tissue. Together these add important inputs into the refinement of mechanistic models. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A rumen epithelial cell isolation procedure has been refined and the procedure has been transferred to other scientists either through visits to Beltsville or via presentations at national meetings. Moreover, the coupling of stable isotope capabilities with short-term cell culture has been used to provide a greater understanding of rumen tissue metabolism and its impact on substrate detoxification and supply to the ruminant. This unit has used the same isolated cell system in the development of novel parameter estimates for ruminal energy substrate use which will be used in the development of robust feeding models. Partial efficiency of metabolizable energy use for tissue gain for ruminal and intestinal digested starch has been determined. This unique work was highlighted in an invited presentation at the national meeting for the National Society of Animal Scientists. The developed partial efficiency estimates will be used to adjust the NRC net energy values of cereal grains and will impact nutritional feeding strategies and ration formulations in the beef cattle industry. Impact of cereal grain processing on NEl was used in the current edition of Nutrient Requirements of Dairy Cattle to adjust the energy values of cereal grains. Genetic sequences (19,110) were deposited in GenBank, making them available to the global research community free of charge. Additionally, individual clones are being requested and provided to researchers interested in novel EST not present, or not present with the same quality, as in 8BOV. Finally, the sequences have been provided to cooperators to facilitate the development of comprehensive microarrays using oligonucleotides based on 8BOV sequences.

Impacts
(N/A)

Publications

  • Oba, M., Baldwin, R.L., Owens, S.L., Bequette, B.J. 2004. Urea synthesis by ruminal epithelial and duodenal mucosal cells from growing sheep. Journal of Dairy Science. 87(6):1803-1805.
  • Oba, M., Baldwin, R.L., Owens, S.L., Bequette, B.J. 2004. Utilization of ammonia-n by ruminal epithelial and duodenal mucosal cells isolated from growing sheep [abstract]. BARC Poster Day.
  • Oba, M., Baldwin, R.L., Owens, S.L., Bequette, B.J. 2004. Use of ammonia-n by ruminal epithelial and duodenal mucosal cells isolated from growing sheep. [abstract] ADSA/ASAS/PAS Joint Meeting, St. Louis, MO. vol. 83(suppl. 1, p.219
  • Baumann, R.G., Baldwin, R.L., Sonstegard, T.S., Van Tassell, C.P., Matukumalli, L. 2004. Construction of a normalized cDNA library of the bovine intestine: 8BOV - Gene identification and metabolic pathway discovery [abstract]. BARC Poster Day. p. 32.
  • McLeod, K.R., Baldwin, R.L., Rumsey, T.S., Elsasser, T.H., Kahl, S., Streeter, M.N. 2003. Influence of subtherapeutic chlortetracycline and dietary protein on circulating concentration of insulin-like growth factor- 1 in growing beef steers. Journal of Animal and Veterinary Advances. 2:531- 35.
  • Oba, M., Baldwin, R.L., Bequette, B.J. 2004. Oxidation of glucose, glutamate, and glutamine by isolated ovine enterocytes in vitro is decreased by presence of other metabolic fuels. Journal of Dairy Science. 82(2):479-486.
  • Baldwin, R.L., Mcleod, K.R., Klotz, J.L., Heitmann, R.N. 2004. Rumen development, intestinal growth and hepatic metabolism in the pre-and post- weaning ruminant. Journal of Dairy Science. 87:E. Suppl.:E55-E65.


Progress 10/01/02 to 09/30/03

Outputs
1. What major problem or issue is being resolved and how are you resolving it? A comprehensive understanding of the physiological limitations to nutrient assimilation and the regulatory mechanisms dictating performance are fundamental to the successful installation of new feeding paradigms to optimize nutrient delivery in the ruminant. Limitations can be alleviated by targeted use of nutritional regimes, exogenous compounds and feed additives, and ultimately, will be used as genetic selection criteria. The goals of this project are to determine critical regulatory steps and identify genetic controls, determine metabolic limits, and to characterize nutrient use within the animal in order to aid in the development of tools (feeding models, pharmacological treatment, and selection criteria) to alleviate metabolic and genetic limitations restricting animal performance. Our approach is to (1) identify metabolic (enzyme pathways, nutrient transport) and physiological functions (growth factor expression and receptors) that are regulators of nutrient economy within the animal; (2) develop and use of animal models (natural species differences, genetic variation, perturbation with exogenous compounds, and/or level of production) to assess the extent to which differential expression of critical genes can result in altered nutrient economy; (3) develop and characterize cDNA libraries from ruminant intestine and stomach complex to facilitate gene discovery and to delineate critical pathways relating to nutrient use and visceral tissue growth. 2. How serious is the problem? Why does it matter? The gut and liver tissues of ruminants account for 30 to 50% of whole body energy use and 30 to 40% of total body protein turnover, while representing only 8 to 10% of the total body mass. Thus, without an understanding of the dynamics of visceral nutrient use, improvements in the current models for animal feeding can not be made. The potential improvements include, but are not limited to, increasing the nutrients available to the animal's productive tissues, decreasing the excretion of excess nutrients due to improperly balanced diets, and ultimately decreasing the cost and environmental impact of animal production. Feeding is the primary cost of both dairy and beef production and thus, savings in this area are crucial to successful animal production. Potential impact of incorporating these research findings into production and research models is phenomenal considering that a nominal 2% increase in energetic efficiency is worth $500 million to the dairy and beef industries annually in feed costs alone. Given both the expense of feed protein sources and new legislation related to nitrogen loss from the farm, the fiscal impact of use of improved feeding programs is even greater and implementation of strategies to improve animal nitrogen use will lead to a decrease in nitrogen excretion. This cannot be accomplished without the development of better and more complete feeding paradigms. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? Research coincides with target areas in the Food Animal Production National Program (NP101) action plan including: Components 5.1, 5.4 Nutrient Intake and Use - Regulating gene function, Minimizing production losses; Component 4.3 Genomic - Tools and reagents; Component 6.2 - Growth and Development - Tissue growth and development. 4. What were the most significant accomplishments this past year? A. Development of a cDNA library resource facilitates the search for specific metabolic pathways, transporters, growth factor receptors, and growth factors which have profound effects on ruminant visceral energy and protein metabolism. A novel normalized cDNA library has been synthesized from lactating dairy cow and neonatal calf intestine to facilitate gene discovery and delineate critical pathways relating to nutrient use and visceral tissue growth by the Bovine Functional Genomics Laboratory. This library (8BOV) has been synthesized and is currently being characterized. This library will be an indispensable resource in the study of genetic regulation of ruminant nutrient metabolism. B. In ruminants, the poor efficiency of converting dietary protein into milk or muscle protein results partly from the extensive degradation of protein in the rumen resulting in ammonia absorption and ultimately, excretion of nitrogen in urine. It was not known if a mechanism to synthesize urea from absorbed ammonia existed in ruminant gut tissues, in particular the rumen epithelia. This tissue which would be an attractive target pathway for reducing the toxic side-effects of ammonia absorption in the animal and for promoting the local recycling of urea to the rumen for microbial protein synthesis. Using an in vitro isolated cell system and innovative stable isotopic approaches we demonstrated, that ruminant gut tissues have the capability to synthesize urea from substrate intermediates in the ornithine-urea cycle, in particular from arginine degradation by ruminal epithelial cells. This urea recycling is an important mechanism that improves the efficiency of nitrogen utilization, and urea synthesis by ruminant gut tissues may be a strategic target to reduce ammonia absorption and improve nitrogen utilization. The impact of site of carbohydrate digestion on visceral tissue growth and adipose accretion have been determined. These results indicate that substrate and substrate supply impact rate and composition of tissue accretion. We have demonstrated that the observed increase in adiposity with carbohydrate delivery post-ruminally is associated with increases in overall lipogenesis from both glucose and acetate carbon (as assessed in short-term incubations in vitro) and have initiated RT-PCR analysis of fatty acid synthetase and AcetylCoA-Carboxylase expression to determine if they are causative in the increase in observed adiposity with post- ruminal delivery of carbohydrates. There is conflicting data concerning the extent of up-regulation of sodium-dependent glucose co-transporter 1 (SGLT1) in response to carbohydrate in the small intestinal lumen. A carbohydrate infusion into either the rumen or abomasum was used to determine the effect of glucose and starch hydrolysate on activity and abundance of SGLT1 in the small intestine of steers. While activity and abundance exhibited a differential pattern along the small intestine, dietary effects were not observed. Moreover, an inverse relationship between glucose uptake and SGLT1 abundance suggests that regulation of glucose transport capacity is complex, involving factors other than SGLT1 abundance. C. None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Nutrient uptake by the gut tissues affects hepatic metabolism and subsequently, nutrient supply to peripheral tissues, yet the metabolic basis for oxidative metabolism of energy substrates by the gut tissues is not well understood. Intestinal enterocyte primary cultures have been used successfully to develop unique kinetic parameter estimates for hexose oxidation. These results add to previous findings by this unit describing the kinetic parameters for substrate oxidation by ruminant intestinal epithelial tissue. Together these add important inputs into the refinement of mechanistic models. The impact of site of carbohydrate digestion on visceral tissue growth and adipose accretion have been determined. These results indicate that substrate and substrate supply impact rate and composition of tissue accretion. Followup studies are being conducted to test this hypothesis and data are being used to challenge and refine mechanistic models. Established rumen epithelial cell isolation procedures were refined for use with growing dairy cattle and used to establish uniform reporting criteria to facilitate cross laboratory comparative research. This work was completed in conjunction with the University of Tennessee and has resulted in the completion of two Masters of Animal Science Degrees. 6. What do you expect to accomplish, year by year, over the next 3 years? Continued development of the cDNA library resource will be a primary focus for the next year. Whole animal infusion experiments directed and developing differential gene expression of the adipose depot in lactating Dairy Cattle will be conducted over the next two years. Additionally, the continuation of enterocyte metabolism analysis in vitro will be conducted over the next two years. In the third year, specific microarrays will be developed and used to define specific metabolic pathways that are influential in the mechanisms defined in the ongoing research. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A rumen epithelial cell isolation procedure has been refined and the procedure has been transferred to other scientists either through visits to Beltsville or via presentations at national meetings. This method is being used to provide a greater understanding of rumen tissue metabolism and its impact on substrate detoxification and supply to the ruminant. This unit has used the same isolated cell system in the development of novel parameter estimates for ruminal energy substrate use which will be used in the development of robust feeding models. Partial efficiency of metabolizable energy use for tissue gain for ruminal and intestinal digested starch have been determined. This unique work was highlighted in an invited presentation at the national meeting for the National Society of Animal Scientists. The developed partial efficiency estimates will be used to adjust the NRC net energy values of cereal grains and will impact nutritional feeding strategies and ration formulations in the beef cattle industry. Impact of cereal grain processing on NEl were used in the current edition of Nutrient Requirements of Dairy Cattle to adjust the energy values of cereal grains.

Impacts
(N/A)

Publications

  • Oba, M., R. L. Baldwin, VI, S. L. Owens, B. J. Bequette. Urea Synthesis by ruminal epithelial cells and duodenal mucosal cells isolated from growing sheep. Journal of Dairy Science. 2003. v. 86(Suppl. 1). p. 59.
  • Oba, M., R. L. Baldwin, VI, S. L. Owens, B. J. Bequette. Effect of VFA on [15N]ammonia utilization for amino acid and urea synthesis by ruminal epithelial and duodenal mucosal cells isolated from growing sheep. Journal of Dairy Science. 2003. v. 86(Suppl. 1). p. 280.
  • Oba, M., R. L. Baldwin, VI, B. J. Bequette. Oxidation of glucose, glutamate, and glutamine by isolated ovine enterocytes in vitro is decreased by presence of other metabolic fuels. Journal of Dairy Science. 2003. v. 86(Suppl. 1). p. 226.
  • Rodriquez, S. M., K. C. Guimaraes, J. C. Mathews, K. R. McLeod, R. L. Baldwin, VI, D. L. Harmon. 2003. Influence of abomasal carbohydrates on small intestinal sodium-dependent glucose co-transporter activity and abundance in steers. Journal of Dairy Science. 2003. v. 86(Suppl. 1). p. 266.
  • Baldwin, R. L., VI, J. Klotz, R. N. Heitmann, K. R. McLeod. Effects of intestinal development on calf growth. Journal of Dairy Science. 2003. v. 86(Suppl. 1). p. 136.
  • Baldwin, R. L., VI, K. R. McLeod, A. V. Capuco. Effect of stage of lactation on visceral tissue mass and intestinal proliferation. Journal of Dairy Science. 2002. v. 85(Suppl.1). p. 321.
  • McLeod, K. R., R.L. Baldwin, M.B. Solomon, A. V. Capuco, D. L. Harmon. Influence of ruminal and postruminal carbohydrate infusion on visceral organ mass and adipose tissue accretion in growing beef steers. Journal of Animal Science. 2002. v. 80(Suppl. 1). p. 238.