2008 Annual Report 1a.Objectives (from AD-416) Objective of this research are; 1)Develop a series of trout feeds that replace fish meal and fish oil with novel and traditional plant-derived ingredients. . 2)Determine optimal supplementation levels of vitamins and limiting amino acids for plant-based feeds. . 3)Identify and evaluate genetic, biochemical and physiological responses of trout fed grain-based feeds. . 4)Identify rainbow trout families with improved phenotypes for growth and utilization of an alternative plant-protein fish feed through a genetic selection program. New funds will be used to expand objective one of the project plan: 1) Objective 1 of the project consists of developing trout feeds formulated with traditional plant-derived ingredients in place of fish meal and fish oil. Within this area of research the experimental diets are to be tested to determine the optimal supplementation level of vitamins and limiting amino acids necessary to obtain maximal growth. The financial increase to the project will aid in developing ingredient enhancement through the development of biological and mechanical concentration methods for the formulation of nutrient complete feeds. New funds will be used to expand Objective One of the research project plan: 1) Develop a series of trout feeds that replace fishmeal and fish oil with traditional and enhanced plant-derived ingredients. The focus should be on ingredient enhancement through biological and mechanical concentration of required nutrients for complete feed development. Expedite expanded capacity to increase select cultivars in trout feeding trials. This includes expanding strains being developed at Leetown, WV. Dr. Keshun Liu: Objectives include;. 1)evaluate current methods of dry and wet fractionization for ways to improve performance or reduce cost of production;. 2)evaluate chemical and enzymatic treatments for effectiveness and production costs,. 3)evaluate nutritional quality of protein concentrates, and. 4)develop improved analytical methods for some key grain nutrients, such as beta-glucan, protein, to assist breeding screen for improved grain quality. Dr. Frederic Barrows Objectives;. 1)identify the most effective organism and blend of grains to result in a high protein low carbohydrate ingredient, 2) identify optimal processing and incubating conditions, 3) evaluate digestibility and palatability of each ingredients with in-vivo testing, 4) evaluate effect of long term feeding of experimental ingredient on growth, and product quality. Dr. Kenneth Overturf Objectives: . 1)installation of 10 heath tray incubators for hatching and isolating individual families, 80- 4'x15" troughs for rearing fry, and 25- 5' circular for holding individual families and mixed tagged families. Also included with this is the necessary infrastructure such as securing adequate water to the tanks and electrical and lighting for rearing and handling of the fish. 1b.Approach (from AD-416) This research will benefit fish farmers, grain farmers and assist in protecting the environment by reducing nutrients in hatchery effluents, and reducing exploitation of ocean fisheries. A series of plant-based feeds will be eveloped, replacing fish meal, and containing the proper balance of essential nutrients. New cultivars of barley and oats will be evaluated as a fish feed ingredient along with alternate crops such as flax, sunflower and safflower. A breeding program will be used to develop new strains of rainbow trout that are better able to utilize plant-based feeds, grow faster and more efficiently and have improved product quality characteristics. Traditional methodologies to determine metabolic scope for a given nutrient or ingredient will be used to evaluate the relative contribution and metabolic efficacy of nutrients supplied in plant-based feeds. Information developed concerning the genetic, biochemical, and physiological responses of rainbow trout to consumption of various plant-derived ingredients will be used as a basis for developing future feed formulations and to develop improved selection parameters for trout breeding programs. Dr. Keshun Liu: Use 4 approaches to address this problem. . 1)evaluate and further process protein-rich by-products of current ethanol or beta-glucan production,. 2)develop modified and novel methods to fractionate grain proteins for trout feed ingredients,. 3)evaluate and process current improved cultivars of barley and oats, such as low-phyate cultivars for making fish feed ingredients, and. 4)select and breed new cultivars of barleys and oats with enhanced composition through collaborating with plant scientist at the ARS, Aberdeen ID (Project # 5366-21000-024-00D). Dr. Frederic Barrows: Solid substrate culture (SSC) has been proven to be an effective method for fungal production on a commercial scale. The effectiveness of this approach is due to the relatively low capital costs and low energy costs. This approach has not been used for the development of animal feeds before, but preliminary studies are quite promising. Utilizing different organisms and different blends of grains, a variety of products will be produced. Dr. Kenneth Overturf: Genetic selection programs for trout have been initiated at the NCCCWA and at the HFCES (through Aberdeen). The goal of the HFCES program is to select fish with an enhanced ability to utilize a plant based feed more efficiently. The goal of the NCCCWA is to select trout for superior growth performance. Improved strains will be reared and analyzed in Idaho. FY99 Program Increase $225,000 Add 1 SY for research on cereal grains & fish FY03 Program Increase $223,537 Add 1 SY FY04 Program Increase $241,566 Add 1 SY FY04 Program Increase $584,232 Add 1 SY FY05 Program Increase $111,600 Replaces 5366-21310-002-00D (12/04). FY06 Program Increase $89,100 3.Progress Report Assessment of the capacity of rainbow trout lines to grow on plant oils. A study is underway to evaluate differences among trout families in growth and metabolism when consuming only plant lipids. Traditionally, trout are fed only lipids from fisheries products. Differences in growth rate among families were observed, with some families apparently able to better utilize plant oils. Laboratory analyses are currently underway to quantitatively evaluate the metabolic ability to convert the plant based omega-3 fatty acid into the fish omega-3 fatty acids. Identification of families with enhanced ability to convert these readily available fatty acids from plants to essential fatty acids may provide a way to cope with the ever increasing price of marine lipids. These studies address the NP106 components of Growth, Development and Nutrition, Problem C, Sustainable Source of Nutrients. Evaluation of Barley Protein Concentrate in the diet of rainbow trout. A protein concentrate was produced from barley (BPC) using a new enzymatic method which also produces ethanol. A patent application has been submitted for this process. A feeding trial was conducted to determine the nutrient digestibility coefficients for macro-nutrients and amino acids of BPC. This information was used to formulate trout feeds with varying levels of BPC in replacement of either fish meal or soy protein concentrate (SPC) and these diets were fed for 90 days to triplicate lots of 30 fish. Results indicate that BPC is a high quality protein, suitable for replacement of either fish meal or SPC. These studies address the NP106 components of Growth, Development and Nutrition, Problem C, Sustainable Source of Nutrients. Genetic mapping of barley beta-glucan mutants. We advanced materials and populations for genetic mapping barley beta-glucan mutants; Started developing mapping populations of barley mutants; Finished modifications of beta-glucan and amylose measurement protocols. Developed flanking DNA markers for three lpa genes in barley. Those markers will greatly help the breeding selection of lpa trait. Identified an endosperm specific promoter in barley. Beta-glucan modification and lpa incorporation can expand the barley marketing and provide cost-effective by-product for fish feed development and reduce water pollution. These studies address the NP106, Component 1, Problem C. Genomic Resources and Sustainability and Environmental Compatibility of Aquaculture. Development of nutrient-enriched ingredients from grains and grain by-products. A key co-product of ethanol production is distillers dried grains with solubles (DDGS) and this product is now abundant and competitively priced. Several experiments were conducted to investigate. 1)particle size distribution of DDGS and its relation to other quality parameters,. 2)relationship in quality parameters between raw corn and DDGS, and. 3)nutrient enrichment of DDGS through additional processing. Factors affecting sieving efficiency were also investigated. In addition, a separate study was conducted to investigate distribution of functional lipids in a barley kernel and effects of storage. NP106, Component 1. 4.Accomplishments 1. Evaluation of balancing amino acid profile in plant-protein based diet formulations for rainbow trout to reduce total dietary protein. Current diets for rainbow trout may be over formulated with protein to meet individual amino acid requirements. Researchers at Hagerman ID, found that when diets are formulated for available amino acids, instead of crude protein, growth rate can be maintained and total dietary protein can be reduced. Synthetic amino acids are supplemented to provide a better amino acid balance than currently suggested in the literature. Supplementing with synthetic lysine, methionine and threonine reduced total dietary protein by 11% and increased protein retained as growth by 35%. The impact of this research will be to reduce both feed cost and nitrogenous waste released into the environment. These studies address the NP106 components of Growth, Development and Nutrition, Problem D. Nutrient Use and Feed Evaluation and were conducted at the Hagerman Fish Culture Experiment Station. 2. Importance of Potassium, Magnesium, Sodium and Inositol Supplementation to Plant Based Diets for Trout. Because supplies of fish meal are severely limiting expansion of aquaculture, replacement of fish meal with plant proteins is necessary but this results in the loss or reduction of other nutrients besides just protein. Researchers at Hagerman, ID, found that when both macro-minerals and inositol were removed from a plant-based diet, weight gain, protein retention and energy retention were all significantly decreased. Removing only the macro-minerals from the diet resulted in decrease of both protein and energy retentions, indicating that weight gain would also be reduced during a longer study. This study demonstrates the importance of considering the supplementation of potassium, magnesium, sodium and inositol to plant based diets for trout, and may provide a critical link to the development of fish meal free aquaculture diets. These studies address the NP106 components of Growth, Development and Nutrition, Problem C, Sustainable Source of Nutrients and were conducted at the Hagerman Fish Culture Experiment Station. 3. Analysis of variation between families of rainbow trout in carbohydrate utilization. Energy is an expensive nutrient in the feed and identification of families of trout that can use the relatively inexpensive carbohydrates for energy would be beneficial. Determining the level of variation within rainbow trout and determining how the fish partition energy from carbohydrates is an important step in the formulation of sustainable alternative protein diets for fish. Researchers at Hagerman ID, measured the expression of genes involved in carbohydrate regulation and plasma glucose levels in families of rainbow trout fed either a low or high carbohydrate diet. These studies determined that there were significant differences in the ability of distinct rainbow families, that typically consume mainly protein and to a very limited extent carbohydrates as an energy source, to utilize diets that were fortified with high levels of carbohydrates. Expression of certain genes used in this study are now being evaluated for use as selective markers for development of rainbow trout strains that have an enhanced potential for growth on plant based diets. These studies address the NP106 components of genetic improvement, Problem C. Genomic Resources and were conducted at the Hagerman Fish Culture Experiment Station. 4. Identification of an aleurone/endosperm specific gene promoter in barley. Tissue-specific gene promoters control the location of target genes to give precise modification of a plant for specific traits that are potentially very useful in understanding genetic contribution. As such, more information is needed, especially on the identification of tissue-specific gene promoters. Researchers at Aberdeen, ID, investigated whether the low phytic acid 1 candidate gene is endosperm specific so that a tissue-specific gene promoter could be identified. The qRT-PCR profile confirmed that expression of the gene is dramatically enhanced in developing seeds compared to other tissues and sequence characterization revealed the promoter region. These studies address the NP106 components of Growth, Development and Nutrition, Problem C, Sustainable Source of Nutrients 5. Modifications of both beta-glucan and amylose detection protocols. Beta-glucan content is an important component of barley and a prime focus of barley breeders, but anlayses are expensive and slow. ARS researchers at Aberdeen, ID, have developed a modification for this process that is faster and reduces chemical cost by 32% of the original cost, saving thousand of dollars each year. The amylose detection protocol modification allows multiple assays of phosphorus, beta-glucan, and amylose for a single sample preparation and significantly reduces the labor cost for those nutritional trait analyses. These efforts address the NP106 components of Growth, Development and Nutrition, Problem C, Sustainable Source of Nutrients. 6. Quality and nutrient enrichment of distillers dry grains with solubles (DDGS). Increased production of ethanol from corn has resulted in large quantities of DDGS and it is believed the quality of the material is affected primarily by the quality of the raw material.ARS researchers at Aberdeen, ID, found that the important characteristics of chemical composition, particle size distribution (PSD), and color varied greatly among DDGS. Although the composition of the original DDGS had little effect on how particle size and other attributes were distributed, distributions of color and nutrients were mostly linked to PSD. DDGS was further fractionated to produce materials of different chemical composition. Fractionating DDGS not only enhances nutritional values but also would expand market share. These studies address the NP106 components of Growth, Development and Nutrition, Problem C, Sustainable Source of Nutrients. 8. Distribution of healthful lipids in a barley kernel and effects of storage. Barley is a rich source of healthful lipids, such as vitamin E (tocopherols and tocotrienols) and phytosterols, and the location or distribution of these nutrients in the grain is unknown. ARS researchers at Aberdeen, ID, found that oil, phytosterols and tocopherols were concentrated in the outer layers but tocotrienols were located lower in the germ layer and higher in the endosperm region of the barely kernel. Storage caused no change in the amounts of oil and tocopherols but significant changes in sterols and tocotrienols. This information is essential for the development of functional lipid products from barley and to maintain product stability. These studies address the NP106 components of Growth, Development and Nutrition, Problem C, Sustainable Source of Nutrients 5.Significant Activities that Support Special Target Populations NONE 6.Technology Transfer Number of Active CRADAs 1 Number of the New MTA (providing only) 4 Number of Invention Disclosures Submitted 1 Review Publications Pierce, L., Palti, Y., Silverstein, J., Barrows, F.T., Hallerman, E.M., Parsons, J.E. 2008. Evaluation of family growth response to fish meal and plant-based diets in rainbow trout (Oncorhynchus mykiss). Aquaculture. 278:37-42. Liu, K.S. 2008. Particle Size Distribution of Distillers Dried Grains with Solubles (DDGS) and Relationships to Compositional and Color Properties. BioResource Technology 99:8421-8428 Liu, K. 2008. Measurement of Wheat Hardness by Seed Scarifier and Barley Pearler and Comparison with Single-Kernel Characterization System . Cereal Chemistry. 85(2):165-173 Liu, K.S. and F.-H. Hsieh. 2008. Protein-Protein Interactions during High-Moisture Extrusion for Fibrous Meat Analogues and Comparison of Protein Solubility Methods Using Different Solvent Systems J. Agric. Food Chem. 56: 2681-2687. Hu, G., Jackson, E.W., Bonman, J.M. 2007. Expansion of pcr-based marker resources in oat by surveying genomic-derived ssrs from barley and wheat. Agronomy Society of America, Crop Science Society of America, Soil Science Society of America Meeting. Crop Sci. 47:2004-2012 (2007). Barrows, F., Gaylord, T.G., Sealey, W.M., Haas, M.J., Stroup, R.L. 2008. Processing soybean meal for biodiesel production; effect of a new processing method on growth performance of rainbow trout, Oncorhynchus mykiss. Aquaculture.283:143-147. Barrows, F., Gaylord, T.G., Sealey, W.M., Smith, C.E., Porter, L. 2008. The effect of protein source and vitamin premix on growth efficiency of rainbow trout, Oncorhynchus mykiss.. Aquaculture 283:148-155. Barrows, F., Gaylord, T.G., Stone, D.J., Smith, C.E. 2007. Effect of protein source and nutrient density on growth efficiency of rainbow trout (Oncorhynchus mykiss). Aquaculture Research.38:1747-1758