2005 Annual 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? What does it matter? The United States had an estimated annual trade deficit of $8 billion in edible and non-edible fisheries products in 2002, including $3.4 billion in shrimp imports. This trade deficit is the largest of any agricultural commodity and second only to petroleum for any natural resources product. Expansion of domestic aquaculture production could offset part of this trade deficit and help establish a globally competitive, sustainable (i.e. environmentally non-degrading, technically appropriate, economically viable, and socially acceptable) aquaculture industry in the U.S. Despite considerable scientific advances, optimum feeds, especially those with low or no fish meal, for maximum productivity of Pacific white shrimp (Litopenaeus vannamei) under sustainable culture conditions have not yet been established. Knowledge of the impact of such feeds and the culture environment on product quality and safety of cultured shrimp is limited. A great need exists to address the above problems in support of the U.S. shrimp industry. In Hawaii every year, disposal of numerous by-products that have potential use in the aquatic feed industry (e.g. meat and bone meal, wheat mill run, coffee bean pulp, macadamia nut presscake, molasses) results in economic loss and potential environmental impacts. Identification and nutrient profiling of potential tropical by-products will permit expansion of aquatic feed research to include the use of these potential ingredients. Pacific threadfin (Polydactylus sexfilis), known locally as “moi”, and longfin amberjack (Seriola rivoliana), known locally as “kahala”, are both fast-growing, good-tasting tropical marine carnivorous fish. These fish have been studied at the Oceanic Institute (OI) for the past several years and have shown good market potential. Threadfin and amberjack culture are growing industries in Hawaii and other tropical regions, and there is a need for research on nutrient requirements and feed development, especially those using fish meal alternatives. The effects of feed type, feeding strategy, and culture environment on flesh quality and growth of these fish require further investigation. Research is needed to ensure that the high consumer preference for these animals is maintained under intensive, sustainable culture conditions. Our proposed project takes an integrated approach in addressing the issue of providing the U.S. with healthful, desirable seafood. This project falls under the Aquaculture National Program 106, in the area of Tropical Aquaculture Research, and has particular relevance to the program component concerning growth, development, and nutrition. The research is also closely allied with other aquaculture program components, such as aquaculture production systems, and quality and safety of aquaculture products for consumers. The objectives of this project are in alignment with the NP 106 Mission Statement to “Conduct high quality, relevant, basic and applied aquaculture research to improve the efficiency, profitability, and sustainability of United States aquaculture, and reduce dependence on imported seafood and threatened ocean fisheries.” The potential benefits from attaining these objectives include: an enhanced understanding of vertically-integrated science and technology for production of shrimp, and the model tropical fish, threadfin and amberjack; development of improved feeds with low or no fish meal for tropical species. Improved use of tropical agricultural by-products, feeding technologies, culture management methodology, environmental compatibility, and growth of the U.S. shrimp and tropical marine finfish aquaculture industry; an expanded range of high-quality aquacultured products which will benefit U.S. consumers and food processors, and an increased consumer confidence in the safety of aquatic foods through quality assurance. Anticipated products of the research include lower-cost, low environmental impact aquaculture feeds with low or no fish meal for use in intensive culture systems, using improved growout feed formulations that employ commercially available feed ingredients and selected by-products of the agriculture industry; reduction in reliance of tropical regions on imported ingredients for aquaculture feed production, improved techniques for assessing advanced physical, chemical, and nutritional quality of feed ingredients and finished feeds, development of feeds and feeding practices for maximum productivity of shrimp and two high-value marine finfish, and improved product quality of cultured shrimp, threadfin, and amberjack. The resulting vertically-integrated science and technology can serve as a contribution to the U.S. aquaculture industry. Primary customers of this research are U.S. aquaculture farmers, aquafeed manufacturers, feed ingredient suppliers (including those that generate agriculture by-products), and aquatic food processors who are able to directly apply the results of this research. Equipment manufacturers (feed processing; food quality evaluation) and seafood consumers will benefit from improved technology and increased availability of high-quality, affordable aquacultured products. 2.List the milestones (indicators of progress) from your Project Plan. Note: Some modifications to our research became necessary due to insufficient numbers of fingerling threadfin (moi) being available for our planned research tasks. This in turn was due to unexpected mortalities of larval moi in the Institute’s Finfish Department hatchery, and we sought permission from ARS to modify some elements of our research to help address this issue from the point of view of aquatic feeds and nutrition. Formal approval was received from ARS for the following changes: Replace Task 4.2, “Investigate Taurine Synthesis in Shrimp and Fish” under Sub-objective 4, with four trials on nutrition of Polydactylus sexfilis, also known as Pacific threadfin or moi, at the larval stage. Expand Sub-objective 5, “Nutrition for Maximum Productivity and Use of Alternatives to Fish Meal”, to include the four trials noted above on larval moi nutrition. These four trials, outlined below in Sub-objective 4, are also reflected in the text of Question 3 of this report. In addition to research modifications in FY 2004-2005, the tasks in our Research Plan for the coming year (October 2005-September 2006) also reflect the enhanced focus on various aspects of moi feeds and nutrition. Most of this year’s work with moi will take place under Sub-Objective (SO) 5, and consequently, the anticipated milestones associated with this research are listed below under Sub-objective 5. Objective I: Develop Commercially Viable Feeds and Processing Methods Using Tropical Agricultural By-Products Sub-objective 1. Tropical Agricultural By-Products and Ingredients Characterization. Sub-objective 2. Feed Processing. Sub-objective 3. Feed Development and Characterization. Objective II: Establish Nutritional and Biochemical Strategies for Maximizing Larval and Growout Productivity and Reducing Fish Meal in Feeds for Tropical Species Sub-objective 4. Nutritional Biochemistry and Microbiology of Species and Culture Systems. Sub-objective 5. Nutrition for Maximum Productivity and Use of Alternatives to Fish Meal. Objective III: Establish Product Quality of Species Reared on Developed Feeds Sub-objective 6. Product Quality of Cultured Species Reared on Developed Feeds. 15 Months (January 2005 - April 2006) SO = Sub-Objective SO-1. Tropical Agricultural By-Products and Ingredients Characterization: Completed by-product identification. Completed by-product ingredient characterization. SO-2. Feed Processing: Completed pellet moisture variation, water stability, and pellet durability (PDI) analyses. SO-3. Feed Development and Characterization: Commercially viable diet formulated for shrimp. Protocol for attractant testing completed. SO-4. Nutritional Biochemistry and Microbiology of Species and Culture Systems: Shrimp cultured in raceways to produce and collect floc. Fractionation of floc in aqueous and solvent phase to obtain fractions (A, B, C, etc.) complete. Pacific Threadfin (Moi) Hatchery Trials 1 through 4: 1. Examine the role and effects of background algae during the rotifer feeding phase of moi larval development and its impact on hatchery survival and early fingerling growout. 2. Examine the effects of rotifer enrichment protocols on moi larval development and hatchery survival. 3. Compare moi production in the OI finfish hatchery with that in the newly opened Pilot Production hatchery facility. 4. Examine the effect of rotifer density on moi production output in the newer Pilot Production facility. SO-5. Nutrition for Maximum Productivity and Use of Alternatives to Fish Meal: Methionine and lysine requirement studies with threadfin completed. Reduction by half of fish meal from shrimp feeds in zero-water exchange systems. SO-6. Product Quality of Cultured Species Reared on Developed Feeds: Methionine and lysine effect on fish quality evaluated. Dietary lipid and cholesterol effect on shrimp quality determined. 30 Months (April 2006 - July 2007) SO-1. Tropical Agricultural By-Products and Ingredients Characterization: Diet formulation with by-product ingredients completed. SO-2. Feed Processing: By-product processing protocols completed. Lab-scale processing modifications completed. SO-3. Feed Development and Characterization: Physical and chemical analysis of diets completed. Optimal diet for shrimp determined. SO-4. Nutritional Biochemistry and Microbiology of Species and Culture Systems: Fractions A, B, C, etc. tested in shrimp feeds. Chromatographic separation to obtain fractions a, b, c, etc. completed. SO-5. Nutrition for Maximum Productivity and Use of Alternatives to Fish Meal: Fatty acid requirements for fish evaluated. Develop a formulated broodstock diet for Pacific threadfin (moi). Determine effect of diet and enrichments on nutritional biochemistry of s-type rotifers. Determine biochemical changes related to diet during larval development of moi. Develop weaning and nursery diets for moi. Initial determination of digestive enzyme ontogeny regulation in first feeding larval moi. SO-6. Product Quality of Cultured Species Reared on Developed Feeds: Effect of exercise and feed with required lipid on fish quality completed. Effect of dietary fatty acid on fish quality completed. Effect of best attractants and optimum feeding strategies on fish and fish quality determined. 45 Months (July 2007 - October 2008) SO-1. Tropical Agricultural By-Products and Ingredients Characterization : (Specific milestones not anticipated at this stage for this sub-objective.) SO-2. Feed Processing: (Specific milestones not anticipated at this stage for this sub-objective.) SO-3. Feed Development and Characterization: Shrimp diet recommended to industry. Commercially viable diet formulated for fish. Attractants identified. SO-4. Nutritional Biochemistry and Microbiology of Species and Culture Systems: Fractions a, b, c, etc. tested in shrimp feeds. Structural elucidation of fractions a, b, c, etc. competed. Feeding trials to establish sparing effect of sulfur amino acids completed. SO-5. Nutrition for Maximum Productivity and Use of Alternatives to Fish Meal: Lysine requirement studies completed (amberjack). Protein:energy ratios in diets for fish optimized. Fish meal and oil reduced by half in fish diets. SO-6. Product Quality of Cultured Species Reared on Developed Feeds: Optimum protein:energy ratios effect on fish quality determined. Reduced fish meal / fish oil effect on fish quality completed. 60 Months (October 2008 - January 2010) SO-1. Tropical Agricultural By-Products and Ingredients Characterization : Optimal by-product inclusion in diet determined. SO-2. Feed Processing: Recommend diet/process to industry. SO-3. Feed Development and Characterization: Optimal diet for fish determined. SO-4. Nutritional Biochemistry and Microbiology of Species and Culture Systems: Pilot-scale evaluation of shrimp growth-promoting compounds completed. SO-5. Nutrition for Maximum Productivity and Use of Alternatives to Fish Meal: Effects of exercise and lipid level on fish established. Fish meal and oil reduced by 2/3 in fish diets. SO-6. Product Quality of Cultured Species Reared on Developed Feeds: Correlation of fish quality with protein/energy ratios and amino acids completed. Correlation of fish quality with dietary lipid, fatty acids, and exercise completed. Quality of shrimp and fish reared on commercially viable feeds established. 4a.What was the single most significant accomplishment this past year? Effectiveness of an inexpensive form of methionine for use in shrimp diets: Different forms of methionine (including L-methionine, DL-methionine and HMTBA [(2-hydroxy-4-methylthio) butanoic acid]) were shown to be equal in their ability to meet the needs for dietary methionine of shrimp grown in low-water exchange pond-like conditions. A trial was conducted in order to find an inexpensive form of methionine (HMTBA) that could be used to improve the nutritional quality of diets containing high levels of soybean meal, which is an inexpensive source of protein but contains a low level of methionine. In this trial, the growth rate of shrimp fed a control diet containing low methionine (high soybean meal) was compared with the same diet supplemented with a fixed level of each of three forms of methionine. Use of HMTBA could enable formulation of commercial shrimp feeds with higher levels of soybean meal in replacement of fish meal, while meeting the nutritional needs of the animal. 4b.List other significant accomplishments, if any. Identification of bioactive growth-promoting compounds in shrimp culture systems: In ongoing research to isolate bioactive compounds in the microbial community (floc) of shrimp culture systems that are beneficial to shrimp nutrition, wet floc was collected from shrimp raceways, freeze-dried, fractionated with aqueous and organic solvents, and the components analyzed, indicating 12% dry matter with 32% crude protein and 2% crude fat. The research was undertaken in an effort to find new ways of augmenting shrimp growth by isolation, identification, and quantification of growth-promoting bioactive compounds in the floc from shrimp culture systems, and to use the methods established in the course of this work for future shrimp growout trials.   In the work to date, new methods have been developed to fractionate the floc and isolate various components via HPLC and GC analysis, including carotenoid pigments, bioactive sterols, peptides and phenolic antioxidants, amino acids, fatty acids, and vitamins. This research has the potential to assist feed manufacturers in developing improved feeds for better shrimp performance. Effect of dietary lipid and cholesterol on quality of cultured shrimp: An experiment in which shrimp were fed a diet containing a very low level of cholesterol and a high level of docosahexaenoic acid (DHA) resulted in a lower level of cholesterol and a higher level of DHA in the tail muscle tissue of market-size shrimp, compared to shrimp fed a commercial control diet.  Research was needed to determine whether the nutritional quality of cultured shrimp could be improved through modifications in the feed formulation, as a means of providing heart-healthy seafood to consumers who are aware of the importance of diet in overall health.  The trial was conducted with 9 g shrimp (initial weight), fed with experimental and control diets and grown in outdoor culture tanks (1100 liters functional volume, 75 animals/tank, 2 L/min flow rate) to the market sizes of 20 g and 27 g, followed by evaluation of the effects of diet on cholesterol and DHA content of shrimp tail muscle tissue.  Results clearly showed that shrimp product quality can be improved (i.e., positive quality attributes enhanced, and negative attributes reduced) by substantially manipulating the ingredients incorporated into the diet. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. Two academic prototype shrimp feeds (one for indoor culture, one for outdoor) were developed which provide excellent performance, often outperforming commercial feeds. The commercially viable versions of these feeds are being formulated and tested, after which they will be moved to pilot scale for commercial applications. The problems addressed by this accomplishment include the traditional use in academic feed formulations of ingredients that are costly and not available year-round or in large quantities, or contain too many ingredients in the formulation, which increases the time required for manufacturing, thereby increasing the inventory, need for storage space, number of purchases, and number of tracking and accounting entries and payments. The objective is to simplify the formulation and use common feed ingredients currently available to the feed industry at the minimum cost. Different forms of methionine (including L-methionine, DL-methionine and HMTBA [(2-hydroxy-4-methylthio) butanoic acid]) were shown to be equal in their ability to meet the needs for dietary methionine of shrimp grown in low-water exchange pond-like conditions. Use of HMTBA could enable formulation of commercial shrimp feeds with higher levels of soybean meal in replacement of fish meal, while meeting the nutritional needs of the animal. In ongoing research to isolate bioactive compounds in the microbial community (floc) of shrimp culture systems that are beneficial to shrimp nutrition, wet floc was collected from shrimp raceways, freeze-dried, and fractionated into aqueous and solvent phases. Analysis of the components indicated a composition of 12% dry matter, 32% crude protein, and 2% crude fat. In the course of this research, new methods were developed to fractionate the floc and isolate various components via HPLC and GC analysis, including carotenoid pigments, bioactive sterols, peptides and phenolic antioxidants, amino acids, fatty acids, and vitamins. This research has the potential to assist feed manufacturers in producing improved feeds for better shrimp performance. An experiment in which shrimp were fed a diet containing a very low level of cholesterol and a high level of docosahexaenoic acid (DHA) resulted in a lower level of cholesterol and a higher level of DHA in the tail muscle tissue of market size shrimp, compared to shrimp fed a commercial control diet.  Results clearly showed that shrimp product quality can be improved (i.e., positive quality attributes enhanced, and negative attributes reduced) by substantially manipulating the ingredients incorporated into the diet. Note: In the accomplishments listed above, the potential beneficiaries include U.S. aquaculture farmers, aquafeed manufacturers, feed ingredient suppliers (including those that generate agriculture by-products), and aquatic food processors who are able to directly apply the results of this research. Equipment manufacturers (feed processing; food quality evaluation) and seafood consumers, will also benefit from improved technology and increased availability of high-quality, affordable aquacultured products. 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 presentation on methionine in feeds for Pacific white shrimp, Litopenaeus vannamei, was given at the annual meeting of the World Aquaculture Society in Bali, Indonesia, in May 2005. Project staff requested and received approval to have aquacultured shrimp, Pacific threadfin, and amberjack included in a revised color poster entitled Hawaii & Pacific Islands Seafood, produced by the State of Hawaii, Department of Business, Economic Development and Tourism, Ocean Resources Branch. The poster presents photographs of all the major seafood species in the islands, detailed species information, cooking instructions, yield % of round weight, buying and cooking tips, and nutritional information. The project was given 5,000 copies, on which “Oceanic Institute” stickers can be placed prior to distribution to interested individuals and organizations in the aquaculture and seafood industries. The Oceanic Institute’s Nutrition Department (which is responsible for this project) will be represented by two staff members when they attend the American Feed Industry Association conference, expo and Aquaculture Committee meeting, to be held in Kansas City, Missouri in September of 2005. One of them will also help staff the AFIA Aquaculture Committee booth at the expo that will explain to booth visitors about the OI Nutrition Department’s research and activities, and how it supports and relates to the feed industry. Both AFIA and its Aquaculture Committee members are important recipients of the Nutrition Department’s research and activities. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Divakaran, S., Forster, I., Velasco, M. 2004. Limitations on the use of shrimp Litopenaeus vannamei midgut gland extract for the measurement of in vitro protein digestibility. Aquaculture 239:323-329. Divakaran, S., Moss, S.M. 2004 In vitro evidence of laminarinase activity in the digestive gland of juvenile Pacific white shrimp Litopenaeus vannamei. Journal of the World Aquaculture Society 35(4):546-550. Kamarei, A.R., Trygstad, C. 2004. Designing clinical trials to substantiate claims. Food Technology 58(10):28-35.