Progress 09/13/01 to 01/25/05
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? In 2002, the United States had an estimated annual trade deficit of $8 billion in edible and non-edible fisheries products, including $3.4 billion in shrimp imports. The U.S. seafood trade deficit is the largest for any agricultural commodity, and the second largest, after petroleum, for any natural product. As stated in the current Action Plan for NP 106- Aquaculture, in spite of steady growth in the U.S. aquaculture industry in the 1980s and 1990s, most of which was due to catfish farming: "... the U.S. ranks only tenth in the world in the value of its aquaculture production, and over $1 billion of imported seafood now comes from farm- raised fish and shellfish grown in other countries. There is major potential and opportunity for a substantially larger U.S. aquaculture industry comprised of
multiple fish and shellfish species." The expansion of domestic aquaculture production, including shrimp farming, could help offset this chronic trade deficit. The long-term objective of the project is to technically support and assist the aquaculture and aquafeed manufacturing sector in the U.S. To accomplish this, the project conducts basic as well as applied research that focuses on developing and disseminating new and/or improved aquatic feeds and associated feeding technologies. These feeds and feeding strategies are tailored to the individual requirements of the intended target species and farming system. They are also designed to be economically viable, and environmentally compatible with sustainable development of the aquaculture sector. Nutritional requirements of marine shrimp, in particular the Pacific white shrimp Litopenaeus vannamei, are not well known. There is only conjecture on the relative contributions of feed and pond productivity in meeting these nutritional
needs. The flows and sinks of feed-derived nutrients in shrimp ponds are inadequately understood. The effects of various methods of feed and ingredient processing, formulation, and delivery, and their combination, on the productivity and stability of ponds are unknown, especially as these relate to pond management strategies. The problem is complicated further by the likelihood that feed, feeding strategies, and pond management protocols interact to a considerable extent. However, this may represent an important opportunity because understanding and manipulation of these interactions may generate lower production costs, improve product quality, minimize water use, provide scope for expansion of offshore aquaculture, and mitigate environmental impacts. The importance of responses and interactions between the cultured animal and the culture system is stressed in this project. Several testing facilities have been used; these range from highly controlled indoor laboratory conditions,
to outdoor conditions similar to commercial growout and most recently the zero-water exchange shrimp culture system. Research is designed to test the responses of the culture systems, as well as the responses of shrimp within those culture systems, to experimental feeds, feed management methods, and other manipulations. Shrimp farming in the United States is increasingly dependent upon intensification, disease prevention and control, environmental protection, product quality, cost reduction and profitability, and the use of domesticated shrimp stocks which are disease resistant and genetically improved. These requirements demand the development and use of new, improved processing methods, feed ingredients, and dietary formulations that are not adequately provided by conventional first-generation shrimp feeds and on-farm feed management practices. Since feeds and feeding represent up to half the total operating costs of most semi-intensive and intensive shrimp farming operations, it
follows that the successful development of improved feeds and feeding practices is critical for the future growth and economic viability of the shrimp aquaculture sector in the United States. As noted by H.S. Parker in the December 2001 issue of Agricultural Research: "In Hawaii, an ARS-sponsored project of the Oceanic Institute is probing the nutritional requirements of marine shrimp. This work will yield new feed technologies to produce shrimp more efficiently and with less environmental impact." 2. List the milestones (indicators of progress) from your Project Plan. Note: Some modifications to our FY 2004-2005 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 four trials 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. This project expired 1/25/05. Milestones for FY06 and beyond appear on the report for 5320-31000-007-00D. 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. 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. 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. 1. Tropical agricultural by-product identification. Milestone Substantially Met 2. Tropical agricultural by-product ingredient characterization. Milestone Substantially Met 3. Pellet moisture variation, water stability, and pellet durability (PDI) analyses. Milestone Substantially Met 4. Commercially viable prototype diet formulated for shrimp. Milestone Not Met Other 5. Protocol for attractant testing. Milestone Fully Met 6. Shrimp cultured in
raceways to produce and collect floc. Milestone Fully Met 7. Fractionation of floc in aqueous and solvent phase to obtain fractions (A, B, C. etc.) Milestone Fully Met 8. Role and effects of background algae during the rotifer feeding phase of moi larval development, and impact on hatchery survival and early fingerling growout. Milestone Substantially Met 9. Examine the effects of rotifer enrichment protocols on moi larval development and hatchery survival. Milestone Not Met Other 10. Compare moi production in the Oceanic Institute hatchery with that in the newly opened Pilot Production hatchery facility. Milestone Substantially Met 11. Examine the effect of rotifer density on moi production output in the newer Pilot Production facility. Milestone Not Met Other 12. Methionine and lysine requirement studies with threadfin. Milestone Substantially Met 13. Reduction by half of fish meal from shrimp feeds in low-water exchange systems. Milestone Substantially Met 14. methionine and lysine
effect on fish quality. Milestone Substantially Met 15. Dietary lipid and cholesterol effect on shrimp quality. Milestone Fully 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? This project is being terminated and replaced by 5320-31000-007-00D. Milestones for FY06-FY08 appear on the report for 5320-31000-007-00D. 4a What was the single most significant accomplishment this past year? Improvement of shrimp attractant testing system and methodology : The development of a simpleNBsystem and methodology for testing shrimp attractants and palatability aidsNBhas been improved from the last reporting. A system and methodology were needed to enhance our ability to quickly and reliably screen shrimp attractants, which in turn would allow the development of nutritious, inexpensive feeds containing high levels of plant protein sources in place of fish
meal. These improvements were accomplished by modifying theNBcontrol diet,NBlengthening the period of shrimp acclimation in theNBtestNBsystem, modifying the trial duration and data collection period for increased accuracy, and broadening the utilization of the test system to include the testing of preferences for different commercial feeds, feed ingredient typesNB(i.e. fish meals)NBadded to or deleted from a feed formulation, as well as the attractants and palatability aidsNBthe system was designed to test. The potential benefits of the improved system and methodology are a reduction in the cost of shrimp diets, which in turn can result in lower cost aquacultured shrimp. 4b List other significant accomplishments, if any. Effectiveness of a low-cholesterol shrimp diet containing no fish meal or fish oil: A low-cholesterol shrimp diet containing no fish meal or fish oil enabled a high rate of sustained shrimp growth, equivalent to that of commercial shrimp feed in low-water exchange
pond-like conditions. Because the marine proteins and lipids traditionally used in shrimp diets can be problematic due to high cost, unreliable supply, and potential bio- security concerns, this research was undertaken to determine whether these ingredients (i.e. fish meal and fish oil) could be eliminated from the formulation without affecting shrimp performance. Two trials were conducted in which shrimp were reared to market size (15-22 g), and in which the growth, feed efficiency, and survival of animals fed an experimental diet containing no fish meal or fish oil and very low in cholesterol (an essential nutrient for shrimp) were compared with that of animals fed two commercial feeds. The outcome of this work could be the development of commercially viable feeds that contain no fish meal or fish oil and very little (if any) cholesterol for shrimp reared under pond conditions. 5. Describe the major accomplishments over the life of the project, including their predicted or actual
impact. A simple yet effective testing system and methodology were developed to enable rapid and reliable evaluation and identification of potential shrimp feed attractants and palatability aids. This testing system and methodology will provide us with a more efficient means of enhancing the attractability and palatability of nutritious, inexpensive aquatic feeds containing high levels of plant protein ingredients in place of fish meal. The potential benefits that could be derived from the information obtained in this simple system will enable the use of a wider range of ingredients that are not currently used in shrimp feed due to their lack of attractability or palatability to shrimp. The information generated will also increase the levels of certain ingredients that can be added to shrimp diets, which is not currently possible without the addition of an attractant or a palatability aid. This new information would provide the feed industry with the potential to reduce the cost of
shrimp diets to the farmer, which in turn would lower the cost of aquacultured shrimp in the domestic and foreign export markets. This accomplishment addresses the problem of insufficient information for determining attractants and palatability aids for shrimp due to subjective factors found in complex culture systems. Such determinations can now be made quickly with this new simplified, quantitative system of testing. Shrimp growout trials in which fish meal was partially replaced with less expensive and more readily available sources of protein indicated that regular poultry by-product meal could replace up to 66.7% of fish meal, and regular feather meal with amino acid supplementation could replace up to 33.3% of fish meal, without any reduction in shrimp growth or survival. This accomplishment addresses the problem of high-cost feed ingredients, because commercial feed nutritionists and feed formulators in feed mills can now use a less expensive poultry by-product meal as a
substitute ingredient for fish meal at half the cost ($260 vs. $600/ton), and get the same growth performance when 2/3 of the fish meal is replaced with poultry by-product meal. The same is true for feather meal ($240 vs. $600 /ton) at the 1/3 level replacement of fish meal. Developed feed processing standards for shrimp feeds in order to improve feed processing techniques, including ingredient particle size reduction, mixing, pelleting, and modified pelleting techniques, for increasing pellet water stability, digestibility and nutrient availability, with consequent improvements in shrimp growth, and less wasted feed and pollution. This accomplishment addresses the need for documented feed processing standards among commercial feed nutritionists and feed manufacturers. We have developed and documented these feed processing standards for shrimp feeds in terms of particle size reduction, mixing, agglomeration, and drying of the finished products. These processing standards can then
be measured by standard quality control testing techniques (particle size analysis , coefficient of variance of a mix, water stability, pellet durability index, energy consumed per ton, pellet leach rates, bulk density) to determine if different processing treatments are improvements over the standard methods used by the aquaculture feed industry. Identified specific shrimp nutrient requirements: magnesium source (magnesium phosphate); cholesterol (0.33% for optimum growth); protein (40% in indoor systems, 30% in outdoor systems). These findings are being used to develop an updated commercially relevant feed for shrimp, which will be made available to shrimp feed manufacturers. Determined that supplemental vitamin and trace mineral premixes are not required in feed for shrimp grown in outdoor systems. Elimination of these premixes will simplify and reduce the cost of shrimp feed production. A peer-reviewed paper on this research has been accepted pending revision. A study in which
the control source of dietary lipid (fish oil) was replaced by several other lipid sources (soy, corn, canola, peanut, olive oil, and lard), showed that growth of shrimp fed the diet containing soy oil was comparable to growth of shrimp fed the control (fish oil) diet, but was lower in shrimp fed the canola oil diet. Partial replacement of fish oil with less expensive soy oil will permit the development of more cost-effective commercial shrimp feeds. Growth of shrimp reared in clear (flow through) water with diets containing two levels of protein (30 and 40%) and four levels of lipid (7, 8.5, 10, and 11.5%) was approximately 35% better with the higher protein level, but dietary lipid level had no effect on growth. This indicated that, under Indoor Controlled Lab (ICL) conditions, diets containing at least 40% protein, but not more than 7% lipid, are required for maximum growth. A trial conducted outdoors in pond water (zero-water exchange) with two levels of protein (30 and 40%) and
four levels of lipid (7, 8.5, 10, 11. 5%) indicated there were no significant differences in shrimp growth associated with dietary protein or lipid levels. These results show that shrimp feeds used under Outdoor Microcosm Laboratory (OML) conditions do not need to contain more than 30% protein and 7% lipid. An indoor feeding trial indicated that soy protein concentrate (SPC) could replace up to 25% of the fish meal component in shrimp feed without any reduction in growth or survival, and with the addition of lysine, could replace up to 50% of the fish meal. This is relevant information for U.S. soybean producers. This information was presented at an international shrimp feed and nutrition conference (attendance was sponsored by the American Soybean Association), and was the subject of a peer-reviewed publication (Forster et al., 2002). Identified amino acid balance as the major limitation in the increased use of plant based proteins in shrimp feed, and determined that a methionine
analogue is effective in meeting methionine requirements in shrimp. This information was presented at an international conference (annual meeting of the World Aquaculture Society) and will be the subject of a peer-reviewed publication. Established that mechanical filtration with bead filters, and tank configuration (flat-bottom tanks rather than sloped bottom), improved shrimp growth and survival in an intensive outdoor, zero-water exchange shrimp culture system. This finding provided useful information in the selection of appropriate filtration unit and tank configuration in the intensive culture of shrimp in a zero-exchange system. In a study to examine the effect of salinity level and filtration on growth and product quality of shrimp cultured in intensive, zero-water exchange outdoor systems, juvenile shrimp could be cultivated successfully to market size (~17 grams) at 3 ppt salinity with filtration. This study provided useful information in developing pond management
strategies that can provide a consistent supply of high-quality shrimp grown under these conditions. Established baseline metrics of U.S. commercial shrimp farms that can serve as a reference for improving shrimp production and processing of value-added products. This finding provided useful information to better understand the commercial shrimp operations in the U.S. An outdoor feeding and filtration experiment in a zero-water exchange system, using a high stocking density (208 shrimp/m2) and an Oceanic Institute (OI) prototype 30% protein feed, yielded 21% higher shrimp growth rate, 18% larger shrimp size, and 22% higher survival, compared to shrimp cultured under similar conditions and fed a commercial control diet. Tank diameter and water depth had a significant effect on shrimp biological performance in an intensive zero-water exchange culture system. Deeper and larger diameter tanks yielded better shrimp growth and survival than shallower and smaller diameter tanks. This
finding will be useful in designing appropriate tank system for intensive culture of shrimp. A study to evaluate the effect of two dietary lipid levels and three feeding rates on shrimp growth and product quality in an outdoor zero- exchange culture system indicated that larger shrimp could be produced with higher dietary lipid (7%)NBand higher feeding rate, although no apparent differences in color, flavor, or texture were observed in theNBresulting market-size shrimp (~15 grams). This information will be useful in developing feeds and feeding strategies for improved shrimp growth and product quality. Datasets (e.g. feed types, feeding rates, dietary protein content, culture system and water quality parameters, shrimp growth, and final weights) from outdoor trials were compiled and used in the calibration ofNBshrimp growth model.NB The new production datasetsNBshowed goodNBcalibration results and can be substantially improved by quantifying the contribution of natural productivity to
shrimp growth.NB These new datasetsNBwill help improve our understanding of the complex nature ofNBzero-water exchange culture systems. A study on alternative live feeds for larval shrimp (3-5 days old), was done involving the use of fluorescent microbeads to mark the ciliate Uronema and verify their ingestion by shrimp larvae. The results positively showed that larval shrimp can ingest the ciliate Uronema, which may offer potential as an alternative live feed for larval L. vannamei. This information was published (Decamp et al. 2001) and presented at an international aquaculture conference (annual meeting of the World Aquaculture Society in Beijing, 2002). Published data (Decamp et al., 2003) on how salinity affects the natural microbial community (especially ciliated protozoans) in intensive, outdoor zero-water exchange culture systems showed that different salinity levels (36, 18, and 9) affected the magnitude and peak biomass timing of the ciliate population. This finding will
advance our understanding of ciliates as a potential food source in zero-water exchange outdoor culture systems, including the role of conventional feeds. Demonstrated successful higher density rearing (i.e. an increase in stocking density from 50 shrimp/m2 to 100 shrimp/m2) in outdoor, highly eutrophic zero-water exchange culture systems through better understanding of the interactions of culture water quality, microbial succession patterns, and shrimp growth and health. Shrimp growth rate and final body weight were almost three times greater in this outdoor system compared to growth of shrimp reared indoors in clear water systems and receiving only formulated feed. Demonstrated the technical feasibility of using lower cost shrimp aquafeeds in OIs advanced biosecure, zero-water exchange outdoor culture systems. These improved feed formulations take into account the dietary nutrient requirements of both the cultured shrimp, and that of the living microbial community. This
information was published in Tacon et al., 2002. Laboratory techniques to monitor nutrient pathways in outdoor culture systems using stable isotopes were standardized. These standardized techniques enabled further research on nutrient flows and sinks in shrimp culture systems. Natural productivity in the outdoor zero-water exchange shrimp culture tanks (phytoplankton only) was found to contribute 20-30% of the nitrogen in shrimp muscle tissue. This information was published in Epp et al., 2002. Results from experiments using stable isotope tracers provided an estimation of the significant contribution of the natural microbial community to shrimp amino acid synthesis, suggesting that the natural community either preferentially or essentially provided amino acids required for shrimp growth. A peer-reviewed paper on this work is under review. Established that gamma ray, electronbeam, and X-ray irradiation of feeds provides sterilization with minimum effect on feed quality, and can be
used beneficially by shrimp farmers in specific-pathogen-free shrimp culture systems. Standardized analytical techniques for the measurement of nutrients in feed ingredients and finished feeds, including the successful publication of "Analytical Procedures Manual for Aquaculture Feeds and Feed Ingredients" within the revised edition of the "AFIA Laboratory Methods Compendium," published by the American Feed Industry Association. This information will be useful to those involved in the analytical and technical aspects of aquaculture nutrition and feed formulation. Results from a series of exploratory trials indicated that the eggs of Pacific threadfin (Polydactylus sexfilis) and greater amberjack (Seriola dumerili) can be effectively and safely disinfected by exposure for 5 minutes to a 1% solution of hydrogen peroxide. Hydrogen peroxide can replace iodine, and sterilization is an important step in increasing hatching rate. These results can be used by aquaculturists as a model for
sterilization of shrimp eggs. In vitro studies conducted with shrimp under outdoor culture conditions established higher specific activity levels for laminarinase in shrimp, an enzyme which may enable them to digest algae from the culture system as a supplemental source of nutrients. These results were recently published in the Journal of the World Aquaculture Society (see Question 7 at the end of this report). The bacterial microflora associated with threadfin and amberjack eggs were characterized using a combination of culture-based, and molecular biology (culture-independent), techniques. The majority of bacteria adhering to the eggs in their early development were culturable on conventional media, suggesting the likelihood that pathogens and other important bacteria can be identified using conventional microbiological methods. In testing the virulence of several bacterial strains to threadfin larvae, most of the strains were not harmful to hatched larvae and may have a role as
probiotic organisms. One of the organisms, a Vibrio sp. strain, was shown to be highly virulent to hatched larvae, although it did not affect their hatching rate. This suggested that bacterial pathogens are able to colonize these eggs, and thereby move between different rearing areas unless they are removed. Threadfin and amberjack eggs were effectively disinfected by exposure to H2O2 or povidone (PVPI) at suitable levels (1,100 and 1,000 mgNBL-1, respectively) without affecting hatch rate or larval survival, but the disinfection efficiency was low at the FDA low regulatory priority limits for both compounds (500 mg L-1 for H2O2, and 100 ppm for PVPI). This suggested that the FDA low regulatory priority limits for these two compounds are too low for threadfin, and illustrate the importance of egg disinfection in restricting pathogen flow in the hatchery. 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? Two oral presentations based on this projects research were given at a scientific meeting: a presentation on the nutritional and sensory qualities of aquacultured amberjack, and a presentation on standardization of a method for fatty acid extraction and analysis of feed and feed ingredients for aquatic animals, were given at
the Aquaculture America annual meeting in New Orleans in January 2005. One of the projects research scientists attended the 2005 Seafood Technology Innovations Conference sponsored by the National Fisheries Institute and the University of Florida Aquatic Food Products Program, held in Orlando, Florida in February. This individual participated in the discussion and exchange of information on the projects research capabilities and technologies on aquatic food quality. This has resulted in a new collaborative project on aquatic food product quality enhancement, and partnership with scientists at the University of Florida Aquatic Food Products program. A new facility, the Product Innovation Laboratory, has recently been completed. This lab provides OI with the capability to conduct R&DNBon theNBimpact of newly developed feeds or other feedNBinputs, as well as the culture environment, onNBthe product quality of aquacultured shrimp and fish.NBNBThe lab has aNBprimary processing area (for
washing, gutting, filleting, skinning, peeling, and trimming), ice machine (for chilling harvested shrimp and fish), fresh and frozen storage sections, a food preparation section,NBsensory evaluation room, portable tables/carts, and small equipment (texture analyzer,NBcolor meter, etc.) required for measurement and assessment of product quality and safety. This facility could also provide other capabilities such as: (a) development of technology for healthy and nutritious innovative seafood products that are responsive to various Hawaii cultures, including expansion of existing, and opening of new, Hawaii markets for aquacultured seafood; (b) utilization of aquaculture processing by-products in foods; (c) development and testing of protocols for seafood safety regulation compliance; and (d) provision of training and short courses, collaboration with industry, and a venue for showcasing aquaculture products. A provisional patent application entitled Heart-Healthy Shrimp (serial
number 60/663,303) was filed in March 2005. 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). Conquest, L., Divakaran, S. Standardization of a method for fatty acid extraction and analysis of feed and feed ingredients for aquatic animals. Aquaculture America Annual Meeting, New Orleans, 17-20 January 2005. Divakaran, S. 2005. Errors in the analysis of aqua feeds and feed ingredients. Aqua Feeds: Formulation and Beyond 2(1):21-23. Obaldo, L., Kamarei, A.R., Huang, A.S. Nutritional composition and sensory qualities of aquacultured amberjack. Aquaculture America Annual Meeting, New Orleans, 17-20 January 2005. Obaldo, L.G., Kamarei, A.R., Huang, A.S. 2004. OI trial: sensory qualities of aquacultured amberjack. Global Aquaculture Advocate 7(1):21- 22.
Impacts
(N/A)
Publications
- Cheng, Z.J., Behnke, K.C., Dominy, W.G. Effect of moisture content, processing water temperature, and immersion time on water stability of pelleted shrimp diets. Journal of Applied Aquaculture. 2002. v. 12(2). p. 79-89.
- Cheng, Z.J., Behnke, K.C., Dominy, W.G. Effect of poultry by-product meal as a substitute for fish meal in diets on growth and body composition of the juvenile Pacific white shrimp, Litopenaeus vannamei. Journal of Applied Aquaculture. 2002. v. 12(1). p. 71-83.
- Cheng, Z.J., Behnke, K.C., Dominy, W.G. Effect of feather meal on growth and body composition of the juvenile Pacific white shrimp, Litopenaeus vannamei. Journal of Applied Aquaculture. 2002. v. 12(1). p. 57-69.
- Decamp, O., Conquest, L., Forster, I., Tacon, A.G.J. The nutrition and feeding of marine shrimp within zero-water exchange aquaculture production systems: Role of eukaryotic microorganisms. In: Microbial Approaches to Aquatic Nutrition within Environmentally Sound Aquaculture Production Systems. C.S. Lee and P. O'Bryen, editors. The World Aquaculture Society, Baton Rouge, Louisiana. 2002. p. 79-86.
- Divakaran, S., Obaldo, L.G., Forster, I.P. Note on the methods for determination of chromic oxide in shrimp feeds. Journal of Agricultural and Food Chemistry. 2002. v. 50. p. 464-467.
- Obaldo, L.G., Divakaran, S., Tacon, A.G.J. Method for determining the physical stability of shrimp feeds in water. Aquaculture Research. 2002. v. 33(5). p. 369-377.
|
Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? The United States currently imports over $9 billion worth of seafood and fisheries products annually. The resulting $6.5 billion trade deficit (including $3.1 billion in shrimp imports in 2001) is the largest of any food and agriculture commodity and the second largest after petroleum, among other natural products (H.S. Parker, Agricultural Research, December 2001 issue). The expansion of domestic aquaculture production, including shrimp farming, could help offset this trade deficit. The long- term objective of the project is to technically support and assist the aquaculture and aquafeed manufacturing sector in the U.S. To accomplish this, the project conducts basic as well as applied research that focuses on developing and disseminating new and/or improved aquatic feeds and associated feeding technologies. These feeds and feeding strategies are tailored to the individual requirements of
the intended target species and farming system. They are also designed to be economically viable, and environmentally compatible with sustainable development of the aquaculture sector. Nutritional requirements of marine shrimp, in particular the Pacific white shrimp Litopenaeus vannamei, are not well known. There is only conjecture on the relative contributions of feed and pond productivity in meeting these nutritional needs. The flows and sinks of feed-derived nutrients in shrimp ponds are inadequately understood. The effects of various methods of feed and ingredient processing, formulation, and delivery, and their combination, on the productivity and stability of ponds are unknown, especially as these relate to pond management strategies. The problem is complicated further by the likelihood that feed, feeding strategies, and pond management protocols interact to a considerable extent. However, this may represent an important opportunity because understanding and manipulation of
these interactions may generate lower production costs, improve product quality, minimize water use, and mitigate environmental impacts. The importance of responses and interactions between the cultured animal and the culture system is stressed in this project. Several testing facilities are being used; these range from highly controlled indoor laboratory conditions, to outdoor conditions similar to commercial growout and most recently the zero-water exchange shrimp culture system. Research is designed to test the responses of the culture systems, as well as the responses of shrimp within those culture systems, to experimental feeds, feed management methods, and other manipulations. The major areas of research includes shrimp nutritional requirements, nutrient flow and sinks within pond systems, feeds and ingredient processing technology, ingredient characterization, feeds management, pond management, statistical and economic modeling, and production system models. 2. How serious is
the problem? Why does it matter? Shrimp farming in the United States is increasingly dependent upon intensification, disease prevention and control, environmental protection, product quality, cost reduction and profitability, and the use of domesticated shrimp stocks which are disease resistant and genetically improved. These requirements demand the development and use of new, improved processing methods, feed ingredients, and dietary formulations that are not adequately provided by conventional first-generation shrimp feeds and on-farm feed management practices. Since feeds and feeding represent up to half the total operating costs of most semi-intensive and intensive shrimp farming operations, it follows that the successful development of improved feeds and feeding practices is critical for the future growth and economic viability of the shrimp aquaculture sector in the United States. As noted by H.S. Parker in the December 2001 issue of Agricultural Research: "In Hawaii, an
ARS-sponsored project of the Oceanic Institute is probing the nutritional requirements of marine shrimp. This work will yield new feed technologies to produce shrimp more efficiently and with less environmental impact. U.S. imports of shrimp, exceeding $3 billion annually, are by far the largest contributor to the ballooning fisheries trade deficit. So, a viable domestic shrimp aquaculture industry is a high national priority." 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? This research falls under the Aquaculture National Program 106 (100%), and in particular, is related to the program component concerning growth, development, and nutrition. In addition, the research is closely allied with other aquaculture program components such as genetic improvement (through improved growth and feed efficiency), integrated aquatic animal health management (through improved nutrition), reproduction and early development
(through the development of larval and broodstock feeds), aquaculture production systems (through the development of static-water or zero exchange shrimp production systems), sustainability and environmental compatibility of aquaculture (through the use of zero exchange culture systems and thereby minimizing water resource use), and information and technology transfer (through the development of an electronic newsletter and web page containing practical information and databases on aquatic nutrition and feed technology). 4. What were the most significant accomplishments this past year? 4A. Single Most Significant Accomplishment During FY 2003 To define optimum feed and culture management practices in a high stocking density, zero-water exchange shrimp culture system, we needed to conduct a growth experiment during which a prototype feed (30% protein) developed by the Oceanic Institute (OI) was compared with a commercial feed. The OI culture system tanks (circular, flat-bottomed,
1.52 m diameter and 1.15 m3 volume) were equipped with Aquamats' to augment natural productivity and bead filters to remove solid and toxic wastes, and were stocked with 1.8-g shrimp at very high density (208 shrimp/m2). Shrimp fed the OI prototype feed averaged 21% higher growth, 22% higher survival rate, and 18% larger final weight compared to shrimp fed the commercial diet, reflecting an average yield of 3 kg/m2 or 4.8 kg/m3 for the OI feed, vs. 2.1 kg/m2 or 3.3 kg/m3 for the commercial feed, or a 45% higher yield for the OI feed. This finding represents a significant improvement in our understanding of diet and system interactions, and provides a benchmark for future development of feeds, feeding strategies, and culture management practices for intensive shrimp production in an environmentally friendly system. 4B. Other Significant Accomplishments All of the work described below was conducted at the Oceanic Institute in Waimanalo, Hawaii. Research was needed to establish
production parameters in order to maintain a suitable environment for optimum shrimp growth and microbial population in intensive, low/zero-water exchange culture systems. A study examined tank diameter, water depth, temperature, aeration, mixing, and filtration under outdoor culture conditions. This investigation determined that tank diameter and water depth had a significant effect on shrimp biological performance in an intensive zero-exchange culture system, indicating that deeper and larger diameter tanks yielded better shrimp growth and survival than shallower and smaller diameter tanks. This information enhanced the knowledge of shrimp production systems with higher efficiency. Research was needed to investigate whether minimizing microbial contamination in shrimp feeds plays an important role in producing a healthy population of shrimp. A shrimp feed formulated at OI and a commercial shrimp feed of similar nutritional composition were sterilized by autoclaving, gamma ray,
electron-beam and X-ray irradiation. Results confirmed that gamma ray, electron-beam and X-ray irradiation of feeds provided sterilization of feeds with minimum effect on feed quality. The shrimp industry could benefit by using radiation sterilization of feeds to minimize feed contamination for the production of healthier shrimp which yield higher returns. Research was needed in feed processing to resolve the problem of high- moisture feed mix sticking to conveyor walls, elbows and bends in the pelleting equipment in currently used feed mill designs. A reduction in moisture content helped resolve some of the problems of high-moisture feed sticking to transfer conveyors in the OI feed lab. By reducing the moisture level from 30% to 22-24%, an improved production rate was achieved with no loss in pellet water stability. When combined with a proposed rearrangement in equipment configuration, this modification has the potential to streamline certain feed processing procedures for the
aquatic feed manufacturing industry. To further develop cost-effective feeds for shrimp culture, there was a need for improved understanding of how culture water affects protein and energy requirements in shrimp. Growth and survival of shrimp were compared over ten weeks, using two sources of water (flow-through well water, and static water with high accumulations of suspended particulate organic matter), and diets containing one of four lipid levels (7, 8.5, 10, 11.5%) and one of two protein levels (30% or 40%). Although dietary lipid level had no impact on shrimp growth in both water systems, in the flow-through system shrimp fed the 40% protein diets grew better than those fed the 30% diets, while in the static water system, shrimp growth did not appear to be affected by protein level. This information provides justification for developing more cost effective feeds containing lower levels of lipid and protein for use in culture systems with low or zero water exchange, and
supports the desirability of this type of culture environment over flow-through systems. C. None D. None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Shrimp Nutrient Requirements, Feed Processing and Analysis: Shrimp growout trials in which fish meal was partially replaced with less expensive and more readily available sources of protein indicated that low-ash poultry by-product meal could replace up to 45%, and feather meal could replace up to 15%, of fish meal without any reduction in shrimp growth or survival. Continued progress in identifying specific shrimp nutrient requirements: magnesium source (magnesium phosphate); cholesterol (0.33% for optimum growth); protein (40% in indoor systems, 30% in outdoor systems). Determined that supplemental vitamin and mineral premixes are not required in feed for shrimp grown in outdoor systems. A study with shrimp larvae 3-5 days old, involving the use of fluorescent
microbeads to verify their ingestion of protozoan organisms, proved that larval shrimp can ingest the ciliate Uronema, which may offer potential as an alternative live feed for larval L. vannamei. Developed improved feed processing techniques, including ingredient particle size reduction, mixing, pelleting, and extrusion processing, for increasing feed nutrient digestibility and availability, with consequent improvements in shrimp growth and reduced feed wastage and pollution. Identified the potential methods for sterilizing shrimp feeds by electron-beam and X-ray irradiation for use in specific-pathogen- free shrimp culture systems. Standardized analytical techniques for the measurement of nutrients in feed ingredients and finished feeds, including the successful publication of "Analytical Procedures Manual for Aquaculture Feeds and Feed Ingredients' within the revised edition of the 'AFIA Laboratory Methods Compendium', published by the American Feed Industry Association. Shrimp
Culture Technology Development: Demonstrated successful higher density rearing (i.e. an increase in stocking density from 50 shrimp/m2 to 100 shrimp/m2) in outdoor, highly eutrophic zero-water exchange culture systems through better understanding of the interactions of culture water quality, microbial succession patterns, and shrimp growth and health. Shrimp growth rate and final body weight were almost three times greater in outdoor culture systems where natural productivity is present, compared to growth of shrimp reared indoors in clear water. Natural productivity in the outdoor zero-water exchange shrimp culture tanks (phytoplankton only) was found to contribute 20-30% of the nitrogen in shrimp muscle tissue. Mechanical filtration with bead filters, and tank configuration (flat- bottom tanks rather than sloped bottom) improved shrimp growth and survival in an intensive outdoor, zero-water exchange shrimp culture system. Developed stable isotope methodologies for tracking nutrient
pathways in outdoor systems. Demonstrated the technical feasibility of using lower cost shrimp aquafeeds in OI's advanced biosecure, zero-water exchange outdoor culture systems. These improved feed formulations take into account the dietary nutrient requirements of both the cultured shrimp, and that of the living microbial community. Established baseline economics of shrimp farming under zero-water exchange culture conditions. 6. What do you expect to accomplish, year by year, over the next 3 years? Year 1: FY 2003 Shrimp Nutrient Requirements Determine the requirements of Litopenaeus vannamei shrimp for the following nutrients: the highly unsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA); and methionine. Feed Nutrient Flow and Sinks Measure rates of natural productivity that are related to feed inputs, determine nutritional value of the floc, and determine the toxicity of ammonia and nitrite to juvenile shrimp L. vannamei, in the outdoor zero- water
exchange shrimp culture system. Feeds Management Determine the effect of feeding rate on shrimp productivity and culture system performance in the outdoor zero-water exchange shrimp culture system. Pond Management Determine the effect of water filtration, temperature, aeration, and mixing on shrimp productivity and culture system performance in the outdoor zero-water exchange shrimp culture system; identify the bacteria colonizing the digestive tract as well as the live prey of intensively vs. extensively-reared marine fish larvae; and compare the diversity and stability of the gut microflora in marine fish larvae under these different rearing conditions. Ingredient Characterization Develop a rapid in vitro method of measuring digestibility that is applicable to shrimp feeds; analyze shrimp feed ingredients, feeds, and floc for cumulative toxins (tetrachlorobenzodioxin, mercury, arsenic, selenium, and cadmium). Feeds and Ingredient Processing Develop a standard reference shrimp diet
for U.S. industry applications; continue development of a simplified method, involving less equipment and fewer steps, of processing shrimp feeds with good water stability using a CPM pellet mill; develop a simple testing protocol for use in evaluating naturally-occurring attractants in shrimp feeds; and investigate the use and effects of genetically modified organism (GMO)products in shrimp feeds, following standard protocols which have been approved for working with these products. Statistical, Economic, and Production System Models Generate shrimp growth and feeding models using new production system datasets representing the effect of feed input, temperature, and other water quality variables, as a means of developing an overall production system model, using the aquaculture simulation software AquaFarm(. Technology and Information Transfer Continue with publication in peer-reviewed journals and trade publications; hold a fifth Industry Liaison Committee meeting; and continue work
on developing a detailed construction plan for the Aquatic Feeds Research and Pilot Production Facility (Feed Mill). Year 2: FY 2004 Determine shrimp methionine (+cysteine) requirements, which will allow more effective utilization of alternative protein sources. Continue studies on the effect of alternate lipid sources on shrimp performance in pond water, and examine the effect on product quality. Investigate feeding methodology in a high density pond system for shrimp culture, as a means of achieving better productivity and more effective feed utilization. Determine the chemical and biochemical changes caused by shrimp feed and feed ingredients on water quality, production of nutrients in the outdoor zero-water exchange shrimp culture system, and in the shrimp themselves. Evaluate alternative diets for marine shrimp and finfish larvae (particularly the use of cultured ciliates) in model and commercial-scale rearing systems, for their potential as a cost-effective, reliable
alternative to Artemia for larval shrimp, and possibly finfish. Reformulate the standard reference diet, developed in 2003 for use in the outdoor zero-water exchange shrimp culture system, with alternate ingredients using least-cost formulation programs, to produce commercially viable feeds without compromising essential nutrients. Continue studies on commercial binders, pellet stability, durability, palatability, and feed attractants, in line with ingredient changes resulting from least-cost formulation efforts. Assist the shrimp feed industry in feed formulation, feed production research, ingredient testing, and binder research. Continue planning the design and construction of the Aquatic Feeds Research and Pilot Production Facility (Feed Mill) in Hilo, Hawaii. Continue unit-process modeling work from 2003 to calibrate physical and biological unit-process models utilizing datasets from the project, including further development and refinement of unit-process models in addition to
parameterization. Year 3: FY 2005 Investigate the coupling of energy and protein/amino acid requirements in pond water with shrimp density and feeding rate, in order to improve the productivity of the outdoor zero-water exchange shrimp culture system. Continue ongoing research from 2004 to identify factors that result in enhanced or reduced shrimp growth, and if crashes occur in the outdoor zero-water exchange culture system, investigate the cause to help prevent future episodes of system instability. Investigate the effects of applying various nutrients (fertilizers) at varying rates to the outdoor zero-water exchange shrimp culture system. In particular, determine how fertilization rate affects water chemistry parameters and microbial community development, as well as shrimp growth and productivity, and combine this information with the results from further studies on the role of floc biomass, composition, and productivity on shrimp performance, to develop management
practices that maximize shrimp productivity and survival in the zero-water exchange culture system. Continue ongoing work from 2004 on the standard reference shrimp diet by producing it on a commercial scale with the cooperation of U.S. feed manufacturers, and comparing the reference diet with commercial shrimp feeds under farm conditions. Continue efforts to reduce feed costs be selecting ingredients and other feed additives (binders, attractants etc.) and modify feed production techniques. Integrate unit-process models into system models using the software AquaFarm(, with emphasis on modeling of natural food resources, the contribution of these resources to shrimp nutrition, and impacts of the culture environment and water quality on shrimp performance. 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? Established an Industry Liaison Committee (ILC) for the project with nationally/internationally respected experts representing the various sectors of the aquatic feed manufacturing and shrimp farming industries, including commercial feed producers, feed equipment manufacturers, animal nutrition specialists, and commercial shrimp farmers. The primary objective of the ILC is to provide guidance in identifying and prioritizing needs of the shrimp farming and feeds industries for improved technologies, products, and services. By holding regular annual meetings with this committee, industry needs and paths of technology transfer are kept current and focused. The fourth meeting of the ILC was held in May 2002 between project scientists and the ILC members. The ILC members submitted their written report in mid-July 2002, expressing overall support for the project's objectives, and offering constructive recommendations in all areas of
the project's research. In addition, during the reporting period (October 2001-September 2002), project staff authored or co-authored 6 publications in scientific journals and conference proceedings, 5 papers in non-scientific trade publications, and made 7 presentations at national and international aquaculture conferences. Information on the use of poultry by-product meals and meat and bone meals has been transferred to industry through direct dialogue and presentations at conferences (notably the World Aquaculture Society meeting). Information on commercially-available feed binders has been transferred to binder manufacturers, as well as the feed producers that use binders in their feed formulations. In addition, the project has assisted commercial feed manufacturers by testing their aquaculture feeds and feed ingredients in growout trials on site at the Institute, and by formulating and producing finished aquaculture feeds. 8. List your most important publications in the popular
press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). Dominy, W.G., Fraser Dominy, S. Make the most of mixing. World Grain. 2002. v. 20(3). p. 18-21. Forster, I., Decamp, O., Conquest, L., Tacon, A.G.J. The nutrition and feeding of shrimp within experimental, zero-water exchange aquaculture production systems. Presented at the Annual Meeting of the World Aquaculture Society, Beijing, People's Republic of China, 23-27 April 2002. Obaldo, L.G. Feed texture--Potential quality control method. Global Aquaculture Advocate. 2002. v. 5(2). p. 58-59. Obaldo, L.G. Texture characteristics of pelleted shrimp feeds. Presented at Aquaculture America 2002 - The National Conference and Exposition of the World Aquaculture Society. San Diego, California, 27-30 January 2002. Obaldo, L.G., Ernst, D.H. Design and modeling zero-exchange shrimp production. Global Aquaculture Advocate. 2002. v. 5(3).
p. 56-58. Obaldo, L.G., Divakaran, S., Uchida, R.S. NIR accurate method for determining chemistry of shrimp feed ingredients. Global Aquaculture Advocate. 2001. v. 4(5). p. 58-59. Obaldo, L.G., D.H. Ernst, D.H. Shrimp zero-exchange culture system: Preliminary results, modeling and analysis. Presented at the 4th International Conference on Recirculating Aquaculture. Roanoke, Virginia, 18-21 July 2002. Obaldo, L.G., Ernst, D.H. Modeling zero-exchange shrimp farming system: a review. Poster presentation. Presented at the Annual Meeting of the World Aquaculture Society. Beijing, People's Republic of China, 23-27 April 2002. Obaldo, L.G., Ernst, D.H. Modeling zero-exchange shrimp farming system using AquaFarm(. Poster presentation. Presented at the Annual Meeting of the World Aquaculture Society. Beijing, People's Republic of China, 23-27 April 2002. Obaldo, L.G., Martinez-Cordero, F., Leung, P.-S., Divakaran, S. Economic analysis of alternative feeding in a zero exchange shrimp
aquaculture system. Presented at the Annual Meeting of the World Aquaculture Society. Beijing, People's Republic of China, 23-27 April 2002. Nagano, N., Decamp, O. Ingestion of the first-feeding larval stage of the Pacific white shrimp Litopenaeus vannamei on ciliated protozoa. Poster presentation. Annual Meeting of the World Aquaculture Society. Beijing, People's Republic of China, 23-27 April 2002. Tacon, A.G.J., Forster, I.P. No single solution: Effluent management requires tailored policies that reflect production diversity. Global Aquaculture Advocate. 2002. v. 5(2). p. 30-31. Scientific Publications Cheng, Z.J., Behnke, K.C., Dominy, W.G. Effect of moisture content, processing water temperature, and immersion time on water stability of pelleted shrimp diets. Journal of Applied Aquaculture. 2002. v. 12(2). p. 79-89. Cheng, Z.J., Behnke, K.C., Dominy, W.G. Effect of poultry by-product meal as a substitute for fish meal in diets on growth and body composition of the juvenile
Pacific white shrimp, Litopenaeus vannamei. Journal of Applied Aquaculture. 2002. v. 12(1). p. 71-83. Cheng, Z.J., Behnke, K.C., Dominy, W.G. Effect of feather meal on growth and body composition of the juvenile Pacific white shrimp, Litopenaeus vannamei. Journal of Applied Aquaculture. 2002. v. 12(1). p. 57-69. Decamp, O., Conquest, L., Forster, I., Tacon, A.G.J. The nutrition and feeding of marine shrimp within zero-water exchange aquaculture production systems: Role of eukaryotic microorganisms. In: Microbial Approaches to Aquatic Nutrition within Environmentally Sound Aquaculture Production Systems. C.S. Lee and P. O'Bryen, editors. The World Aquaculture Society, Baton Rouge, Louisiana. 2002. p. 79-86. Divakaran, S., Obaldo, L.G., Forster, I.P. Note on the methods for determination of chromic oxide in shrimp feeds. Journal of Agricultural and Food Chemistry. 2002. v. 50. p. 464-467. Obaldo, L.G., Divakaran, S., Tacon, A.G.J. Method for determining the physical stability of
shrimp feeds in water. Aquaculture Research. 2002. v. 33(5). p. 369-377.
Impacts
(N/A)
Publications
- Cheng, Z.J., Behnke, K.C., Dominy, W.G. Effect of moisture content, processing water temperature, and immersion time on water stability of pelleted shrimp diets. Journal of Applied Aquaculture. 2002. v. 12(2). p. 79-89.
- Cheng, Z.J., Behnke, K.C., Dominy, W.G. Effect of poultry by-product meal as a substitute for fish meal in diets on growth and body composition of the juvenile Pacific white shrimp, Litopenaeus vannamei. Journal of Applied Aquaculture. 2002. v. 12(1). p. 71-83.
- Cheng, Z.J., Behnke, K.C., Dominy, W.G. Effect of feather meal on growth and body composition of the juvenile Pacific white shrimp, Litopenaeus vannamei. Journal of Applied Aquaculture. 2002. v. 12(1). p. 57-69.
- Decamp, O., Conquest, L., Forster, I., Tacon, A.G.J. The nutrition and feeding of marine shrimp within zero-water exchange aquaculture production systems: Role of eukaryotic microorganisms. In: Microbial Approaches to Aquatic Nutrition within Environmentally Sound Aquaculture Production Systems. C.S. Lee and P. O'Bryen, editors. The World Aquaculture Society, Baton Rouge, Louisiana. 2002. p. 79-86.
- Divakaran, S., Obaldo, L.G., Forster, I.P. Note on the methods for determination of chromic oxide in shrimp feeds. Journal of Agricultural and Food Chemistry. 2002. v. 50. p. 464-467.
- Obaldo, L.G., Divakaran, S., Tacon, A.G.J. Method for determining the physical stability of shrimp feeds in water. Aquaculture Research. 2002. v. 33(5). p. 369-377.
|