PROCEEDINGS

OPTIMIZE FOR PROFIT

An Aquafeed.com Technical Workshop

Bangkok, Thailand, March 8, 2006.

SPECIAL SPONSORS

© 2006 copyright Aquafeed.com,LLC. All rights reserved.

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Contents 3. Opening Presentation: Aquaculture in Thailand Dr. Juadee Pongmaneerat, Thai Department of Fisheries 5. Speakers and Summaries

14. Tailoring the feed formulation for maximizing profitability: farm demonstrations with white shrimp in Latin America

Dr. Peter Coutteau, INVE Aquaculture

40. Improving Nutrient Delivery in Aqua Feeds: Implications for Nutritionists and Formulators

Dr. A. Victor Suresh, Bentoli Inc.

49. Formulation software and handling variability Merryl Webster, Format International Ltd

54. The truth about moisture uniformity and equilibration Paul D. McKeithan, Aeroglide Corporation 60. Technology Makes the Difference: Optimizing Size Reduction Technology to Process Better Quality, More Profitable Aquafeeds

Gary Minor, Mill Technology Company

75. Making more profit with New Technologies for Aquafeed Dr. Mian Riaz, Texas A&M University

83. Increasing Aquatic Feed Production through Plant Optimization Galen J. Rokey, Wenger Mfg., Inc.

90. Optimization of formulation and product quality parameters of extruded aquafeeds

Stuart Howsam, Buhler AG

Bonus Papers

96. Replacement of Fish Meal by Poultry By-product Meal and Meat and Bone Meal in Aquafeeds – An Update (2004-2006)

Dr. Y. Yu, National Renderers Association

125. Application of de-oiled soya lecithin in shrimp feeds Dr. Yuyun Mu, Berg + Schmidt Asia Pte Ltd.

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Introduction: Aquafeed in Thailand Dr. Juadee Pongmaneerat, Senior Expert Aquatic Animal Nutrition, Department of Fisheries, Bangkok, Thailand Consumer interest in health, food sanitation and safety has increased because of serious disease outbreaks, such as Bird Flu and Mad Cow disease, as well as problems with drug and chemical residue and undesirable substances. As a result, aquatic-based food, which is safer, healthy and nutritious, is increasingly consumed. Aquaculture has played a great role in aquatic food for both domestic consumption and major economic export products. Moreover, Thai aquaculture development has increased productivity and quality and the safety of food products throughout the production line. Thailand, therefore, has been able to export first quality cultivated marine shrimp since 1991. In spite of disease problems with viruses - namely WSSV, TSV and IHHNV - very high larval stocking density in intensive cultivation has allowed Thailand to produce marine shrimp in the amount of 340,000 tons in the year 2005. 330,000 tons of shrimp is expected for 2006. Total production of freshwater aquaculture in 2003 was about 361,125 tons or 13,171 million Baht. The freshwater species that have the highest economic value are giant freshwater prawns, Nile tilapias, catfish, striped snake-head fish and common silver barbs. Also, freshwater prawns and Nile tilapias are Thailand’s main and most important aquatic animals. The feed used for all of these species needs to be developed for least cost formulation. The Department of Fisheries has set the strategy and policy to develop aquaculture and related technology for sustainable aquaculture and enhance the quality of aquatic food. Thus, food safety and food hygiene have been established as a policy by the government. The Department of Fisheries has also been assigned not only to promote the research and development but also to control the production quality of all processes from farm to table. The standard systems of GAP (Good Aquaculture Practice), CoC (Code of Conduct for Marine Shrimp), GMP and HACCP have been urged to achieve the goal and assure product quality. Implementation of standards, monitoring and surveillance systems, product traceability - the so called TRACE SHRIMP PROJECT, feed quality control, monitoring of drugs and chemical residues, inspection of toxic substances, R&D for reduced-cost, sustainable aquaculture and knowledge transfer to the farmer are also provided. Feed has inevitably played a great role in production costs: some 30-40% of the cost of aquaculture comes from the cost of feed. Today, various feeds are exploited in aquaculture farming, such as fresh, farm-made feed and commercial feeds. Commonly used fresh feeds are trash fish, fishery by-products, poultry by-products and kitchen waste. These fresh feeds are easily spoiled and transmit disease. Standard formulations for nutritionally balanced farm-made feeds that incorporate local raw materials are required, in addition to good processing practices. While commercial feed normally used in intensive and semi-intensive culture systems is cost effective, they reduce profitability. All feeds need profiled and inexpensive ingredients to reduce production costs. The quality of commercial feed is controlled in accordance with the Feed Quality Act B.E. 2525 (1982). The Department of Fisheries supervises the feed for aquatic animals. The quality is inspected from manufacturing factory to farm to ensure that the feed is standard and safe for all consumers. Feeds that must be registered with the Department of Fisheries, are:

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1) Complete aquatic feed 2) Concentrate aquatic feed 3) Premixed aquatic feed and 4) Supplemental aquatic feed. Complete aquatic feed manufactured for commercial use must be registered according to the Feed Quality Act B.E. 2525 (1982). At present, the Department of Fisheries has divided the standards of registered complete aquatic feed as follows: 1) Marine shrimp feed 2) Fresh water shrimp feed 3) Catfish feed 4) Fresh water herbivorous fish feed 5) Fresh water carnivorous fish feed 6) Marine carnivorous fish feed 7) Frog feed and 8) Turtle feed. Today there are 149 feed facilities that have been registered for aquatic feeds, including premix, supplemental feed, concentrate feed and complete aquafeed. In 2006 there are 64 complete feed manufacturing plants: 31 shrimp feed mills, 15 fish feed mills and 18 mills that produce both fish and shrimp feed. It is a good sign that the numbers of feed mills has increased by 21 from the year 2002: 8 shrimp feedmills, 8 fish feedmills and 5 shrimp/fish feedmills. This increase in aquatic feed manufacturing is driven by the epidemic of Bird Flu, which has raised demand for aquatic animals. In addition, because of drug and chemical residue problems, farmers who culture freshwater shrimp have turned to using more commercial feed instead of on-farm mixed feed. The culture of white shrimp has also expanded. Additionally, the Department of Fisheries realizes the importance of system development leading it to the standard of GMP and HACCP. At present, the Department of Fisheries has accredited six aquatic feedmills according to GMP standard and three aquatic feedmills according to HACCP standard; while six aquatic feedmills are pending in the accreditation process (three factories according to GMP and three factories according to HACCP standards). In the near future, CODEX will establish a Code of Practice for Good Animal Feeding and encourage Thailand to use the same standards. Thailand as a food exporting country and regarded as the World’s Kitchen, must inevitably develop and enhance its competitive capability to eliminate trade barriers of the future. It can be thus concluded that the policy regarding aquatic animals is to provide safety for consumers. Feed and materials must be qualified and safe for consumption; therefore feed should be manufactured according to established hygiene and quality standards. Feed must also contains nutrients for aquatic animals with no prohibited substances. The price of feed should be reasonable and also acceptable to the fish farmer. On this occasion, I would like to thank the organizers for giving me the opportunity to present and share academic opinions on the potential of the feed industry and aquaculture, including Thai quality aquatic products. THANK YOU

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Speakers and Summaries Nutritional approaches to aquatic animal health and performance improvements: What can we learn from terrestrial nutrition? The role of nutrition in maintaining health in animals is well known. However, optimizing animal health and performance through nutritional approaches is more than just meeting basic nutritional requirements. This has been understood in poultry and other terrestrial species for many years and nutritional approaches are often adopted to maximize animal health and performance. How can such strategies be used in feeds for fish and shrimp? Dan Fegan Alltech Inc.,Bangkok, Thailand. Email: :dfegan@alltech.com Dan Fegan is Alltech’s regional technical manager for the aquaculture division. Fegan, who is based in Bangkok, is also the current president of the World Aquaculture Society. He has wide experience in commercial shrimp aquaculture and has worked in many countries in Asia and Latin America including Ecuador, the Philippines, Thailand and Malaysia. In Thailand, he worked for nine years with the Aquastar group of companies, a vertically integrated operation working with around 750 individual contract farmers. During recent years he has been heavily involved in health management of commercial farming operations following the outbreaks of first yellowhead disease in 1992/93 and later white spot disease in 1994/95.

Tailoring the feed formulation for maximizing profitability: farm demonstrations with white shrimp Litopenaeus vannamei in Latin America The continuous decrease of shrimp prices during the past years has urged the shrimp farming industry towards improving profitability. Feed constitutes the major cost in (semi) intensive farming and is therefore a traditional target of cost cutting strategies. Reducing the feed cost by using cheaper feed is a strategy which is relatively easy to promote in a feed market where protein level is still often regarded as the single criterion to evaluate feed quality. However, cheaper feeds, obtained by eliminating the most expensive ingredients in the formulation, are not always resulting in improved cost-efficiency. The resulting “nutritional gaps” obviously involve risks that the feed composition no longer covers the nutritional requirements, particularly under more intensive conditions where shrimp are more dependent upon the nutritional inputs from the feed. As a result, cheaper feeds may cost more money to the shrimp farmer than what is saved on the feed formula at the feed mill. Nevertheless, continuous pressure on the shrimp feed producers to reduce feed prices, despite increasing raw material cost, has resulted in a gradual decrease of average nutritional standards of shrimp feed in all shrimp producing regions.

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In this paper we shortly report on the results of an international survey organized in 2002-2003 to analyse representative commercial shrimp feeds in some of the main shrimp producing countries. Also, we will review a number of novel concepts to alleviate nutritional gaps in current shrimp feed formulations. Finally, we will illustrate the applicability of these specialty nutrients to improve profitability under practical farming conditions in semi-intensive farms in Panama and Brazil. Peter Coutteau Ph.D. INVE Aquaculture Nutrition, Hoogveld 91-93, B-9200 Dendermonde, Belgium. Email: p.coutteau@inve.be Dr. Peter Coutteau obtained a Ph.D. in Biological Sciences at the Laboratory of Aquaculture & Artemia Reference Center, University of Gent in 1992 on the filter feeding biology of Artemia and bivalves. He continued his research at postdoctoral level until1997 on lipid nutrition of bivalves, fish and shrimp. In 1997 he joined INVE Technologies NV, the R&D company of the INVE group, where he was responsible for coordinating the research and product development in the aquaculture division till end of 1999. In 1999, he took up the responsibility for the expansion of a new field of research and development of specialty premixes and feeds to improve nutrition and health in on- growing stages of fish and shrimp. Since 2001, Dr. Coutteau has been the Product Manager Farm Nutrition for INVE Aquaculture, where he is responsible for the global coordination of R&D, product development, customer support and fish/shrimp feed mill projects in the unit Farm Nutrition. He has published over 40 refereed papers in scientific journals and over 20 articles in professional aquafeed magazines

Soy proteins in aqua feeds: benefits and shortcomings Aqua feeds today are composed of many sources of nutrients apart from fishmeal. An extensive job has been and is being done evaluating these alternative and supplemental ingredients for the aqua feed business. However, when looking at the supply of protein meals from various sources, one thing becomes very obvious to the viewer; Soy constitutes more than 50 % of all available protein sources. This of course makes soy an interesting protein source for all feed industries including aqua feed. The last year has finally yielded the effect everyone has been discussing in the past few years; fish meal will become a scarce raw material and the price will increase. The aqua feed industry is consuming a larger and larger part of fish meal production. Till last year much of this increase came from other feed industries giving up the use of fish meal for various reasons. For many, especially carnivorous, species, some inclusion of fish meal seems a necessity, the rest coming from both vegetable and animal origins. The future will allocate fish meal to the species which can pay the most ~ needs it the most. Thus demand for alternatives will increase everywhere else. Plenty of ordinary soy products are available, but their protein concentrations are not equal to that of fishmeal. To overcome the CHO content that follows the use of vegetable

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proteins is the first obstacle. SBM can usually be included to a certain extend before starting to cause problems. These problems can then derive from a sheer overload of CHO or it can be caused by more specific anti nutrients (ANF’s), of which Soy have an abundance. Several approaches has been made to increase protein content (reduce CHO’s) and anti nutrients. Of these Soy Protein Concentrates and Enzyme treated products (HAMLET PROTEIN) are the most produced and used in feeds today. These improved products offer a reduced CHO content and as a result, increased protein content. Most known harmful anti nutrients have been reduced or eliminated in these types of product. In future approaches especially the enzyme processing can achieve further improvements in protein level and quality. Characteristics of such a product could be; peptides of various lenght, specific AA composition, solubility or the contrary, vectoring other nutrients etc.etc. An example of such a development is a product with increased P availability showing reduced pollution when replacing fish meal and inorganic P in trout feed. This product is marketed today and the future will bring new products in this line. Lars Andersen Hamlet Protein A/S, P.O. Box 130, Saturnvej 51, DK-8700 Horsens, Denmark. Email: LSA@hamletprotein.dk After gaining his M.Sc. from the Royal Danish Veterinary and Agricultural University in 1990. Lars Andersen became a farm adviser for a farmers’ association in feeding and management until 1997. He then moved to RAG feedmill in Denmark as a product manager, responsible for formulation and quality control. In 2001 he joined Hamlet Protein, where he assumed the position of nutritionist and feed application manager, which he holds today.

Improving Nutrient Delivery in Aqua Feeds: Implications for Nutritionists and Formulators Aquaculture nutritionists and feed formulators recognize the fact that nutrient losses, in the form of leaching in water, occur before and during feed intake by aquatic animals. These losses must be accounted for in aqua feed formulation to achieve more exact optimization of formula cost and animal performance. The problem of leaching losses is the most severe in the case of slow feeding species such as shrimp. As much as 15% of crude protein in the diet may be lost from shrimp feeds before the shrimp gets to eat it. This presentation will primarily discuss the concepts related to nutrient delivery in shrimp and identify the challenges presented to practicing nutritionists, formulators and feed manufacturers in both quantifying and minimizing losses in nutrient delivery efficiencies. It outlines and discusses the following strategies available to minimize the losses: (1) decreasing feed search and consumption time by using feeding effectors (attractants and stimulants); (2) using appropriate feed size; (3) coating the raw materials or feeds with materials that will reduce the rate of leaching; and (4) choosing appropriate feed

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manufacturing technologies for application of ingredients and additives that are prone to leaching. Special focus is on the use of feeding effectors as a tool to minimize leaching losses and how an index of feeding effectors could be developed and used by the formulator to increase flexibility in raw material selection and cost management of shrimp feeds. A. Victor Suresh, Ph.D. Bentoli, Inc. P.O. Box 901149, Homestead, FL 33090-1149, USA. Email: Victor@bentoli.com Dr. A. Victor Suresh received his Bachelor’s, Master’s and Ph.D. degrees in aquatic sciences in India, Thailand and the USA, respectively. After his Ph.D., he joined Ralston Purina International in its R&D division in St. Louis, Missouri, USA. When Ralston Purina spun-off its global farm feeds division into Agribrands International in 1998, he moved to the latter. He was the Director of Aquaculture in Agribrands International when the company was acquired by Cargill, Inc., in 2001. He returned to his home country, India, after the acquisition and became a private consultant to the aquaculture feed industry. His consulting clients included Bentoli, Inc., Cargill Animal Nutrition and the Food and Agriculture Organization (FAO) of the United Nations. He currently consults for Bentoli on a full-time basis. He has authored a number of articles in scientific and trade journals and a few book chapters. He is the founding editor of Aqua Feeds: Formulation & Beyond, a quarterly, international trade magazine for the aqua feed industry.

Formulation software and handling variability Variability is present in the feed manufacturing process and arises from a number of sources, from the raw materials used to the manufacturing process itself. This variability presents a number of challenges and can have a significant effect on the costs of production. This workshop will discuss techniques and tools which are available for the user of formulation software to assess and manage this variability. With these techniques formulators will be able to devise strategies to incorporate these considerations in their planning and thus realize improvements in quality and profitability. Merryl Webster Format International, Format House, Poole Road, Woking, Surrey GU21 6DY, U.K Email: merryl.webster@formatinternational.com Mrs. Webster is the Managing Director of Format International Ltd, the leading international supplier of formulation software to the aquafeed, animal feed, pet food and other industries. In more than 15 years with Format, Merryl has been responsible for the implementation of formulation software around the world, and this has given her a unique insight into its use, potential and the challenges faced by its users. Merryl also has wide

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experience of Format’s clients’ point of view from using this type of software whilst working for a major feed manufacturer and then premix producer prior to joining Format.

Technology Makes the Difference: Optimizing Size Reduction Technology to Process Better Quality, More Profitable Aquafeeds The aquaculture industry relies on size reduction technology to efficiently process quality aquafeed. This workshop will give you an understanding of the three current approaches to the production of aquaculture feed. Traditional Hammermills: Many vendors advocate using a higher tip speed to boost capacity, but increasing the speed can cause problems that adversely affect aquaculture feed, including accelerated component wear and excessive heat generation. Air Swept Classifications: This method generates extreme fineness but also allows oversize particles to pass into finished products. A Multi-impact Process: A new hammermill design reduces particles and ensures uniformity of particle size via a multi-impact process. This technology results in increased throughput capacities and produces the highest quality post-grind of aquaculture feed. Gary Minor Mill Technology Company, PO Box 41483, Minneapolis, MN 55441, USA. Email: gary@milltechnology.com With over 30 years of industry experience, Gary Minor is a recognized authority in hammermill technology. At Jacobson Machine Works, and later at Champion Products, Mr. Minor worked in all aspects of the industry, including supervising the manufacturing process, overseeing quality control, troubleshooting in the field, and selling equipment. These experiences led him to contributing to instrumental modifications of traditional hammermill designs. Inspired by industry needs, Mr. Minor applied his expertise to the creation of new hammermill technology that increases system capacity and efficiency, enhances the quality and fineness of the particle size reduction, and improves overall system performance quality. Mr. Minor is president and CEO of Mill Technology; he founded the company in Minneapolis, Minnesota in 1992.

The truth about moisture uniformity and equilibration Feed moisture uniformity is an important topic in drying aquaculture feed, as it should be. The moisture uniformity from pellet to pellet affects product quality, yield, and cost. There are large paybacks associated with drying the product to a tight tolerance. Skeptics claim that it is not so critical to hold a tight product moisture uniformity specification. Some believe that the product will equilibrate in the holding bins before packaging or

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shipping. This presentation will demonstrate several reasons why moisture uniformity is critical and will provide evidence that moisture equilibration is not what you may think. Paul D. McKeithan Aeroglide Corporation, PO Box 29505, Raleigh, NC 27626-0505, USA, Emai: sales@aeroglide.com Paul D. McKeithan is a market manager for Aeroglide Corporation. Paul has evaluated hundreds of convection driers all over the world. He has also conducted drying theory classes in many different industries and countries. Paul has been published in such magazines as Chemical Engineering and Petfood Industry. Paul has a B.S. in Mechanical Engineering from North Carolina State University.

Making more profit with New Technologies for Aquafeed Extrusion is not new. Extrusion companies continuously trying to bring new ideas in the market to process aquatic feed with better quality, palatability and appearance. Over the last 50 years several enhancements to the extrusion process have taken place. Some of the milestones in the aquatic feed industry are: direct steam injection and preconditioning (1957); twin screw extruders (1978); enhanced preconditioning (1986); increased volumetric capacity (1986); automatic control system (1991); and new generation extruders (1998); retention time controlled DDC (2004); back pressure valve (2004); multi-color and multi-shape aquatic feed (2005). All these new innovation will be discussed and participants will learn how they can use these new technologies to make more profit in aqua feed. Mian N. Riaz Ph.D. Head - Extrusion Technology Program Food Protein Research& Development Center, Texas A&M University College Station, TX 77843-2476 USA E-mal:: mnriaz@tamu.edu Dr. Mian N. Riaz is Head of the Extrusion Technology Program at the Food Protein Research and Development Center, and a graduate faculty in the Food Science and Technology Program at the Texas A&M University, College Station, Texas. He conducts research on extruded snacks, texturized vegetable protein, pet food, aquaculture feed, oilseed processing, biodegradable packing material, and extrusion-expelling of oilseeds. He joined the center in 1992, after graduating from University of Maine with a Ph. D in food science. He published more than 50 papers and articles on extrusion and other related topics. He is also the editor of the book "Extruders in Food Application

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Increasing Aquatic Feed Production through Plant Optimization The various bottlenecks to higher throughputs in an aquatic feed extrusion processing line are reviewed. Possible solutions to these bottlenecks are discussed. Galen J. Rokey Wenger Mfg., Inc., 714 Main, Sabetha, Kansas 66534, USA Email: GalenR@wenger.com Galen Rokey is the Process Manager for the Applications Group within Wenger Mfg., Inc. Prior to this position, he was Manager of the Wenger Technical Center. He has 32 years of laboratory, extrusion process and research experience with Wenger Mfg., Inc. Galen graduated from Kansas State University in 1973 with B.S. degree in the Chemistry Option of Grain Science and Management. A past member of the American Association of Cereal Chemists and the Institute of Food Technologists, Galen has authored numerous publications regarding the extrusion process. He was the recipient of the Alpha Mu Distinguished Service Award in Extrusion Technology from Kansas State University in 1990.

Stuart Howsam Bühler AG, 1/181 Rooks rd. Vermont, VIC 3133, Australia Email: stuart.howsam@buhlergroup.com Stuart Howsam was born in Melbourne, Australia and completed a Batchelor of Chemical Engineering at the University of Melbourne. He joined the Mars Corporation in 1987, initially based at the dry Petfood facility where he completed a number of functions including product and process development work in extrusion, drying, coating and meat preparation systems. He then spent four years in the canned Petfood facility before moving to China in 1994. He spent two years there as R & D manager involved in the design, construction and commissioning of a dry Petfood plant before moving to Argentina where he was also involved in the building of a dry Petfood facility. Returning to Australia in 1998 he joined a team working for Masterfoods Japan involved in the setting up of a technical centre in Tokyo and the development of a number of innovative extruded products. In January 2003 he joined Buhler AG extrusion division where he is responsible for South East Asia sales development and technical development of single screw extrusion systems for the pet and aquafeed markets. He is based in Melbourne. Optimization of formulation and product quality parameters of extruded aquafeeds The focus of the presentation will be on:

Using advanced extrusion systems to produce consistent product quality. Allowing for raw material variation using an SME control system. Extruded shrimp feeds Density control systems

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Bonus Papers Replacement of Fish Meal by Poultry By-product Meal and Meat and Bone Meal in Aquafeeds – An Update (2004-2006) Dr. Y. Yu, National Renderers Association 21/FL., Causeway Bay, Commercial Building, 1-5 Sugar Street, Causeway Bay Hong Kong. Email: nrahkg@nrahongkong.com.hk Research reporting poultry by-product meal (PBM) and meat and bone meal (MBM) as fish meal (FM) replacement in diets for shrimp and fish during the past two years were reviewed for digestibilities and weight gain (WG) response to dietary FM replacement for FM. Nutrients digestibilities and the maximum FM replacement rate are important least cost formulation criteria for selection of protein ingredients and minimizing the variability in growth performance of aquaculture animals. Protein, essential amino acids (EAAs), and energy in PBM were well digested (�80%) by vannamei, hybrid striped bass, large mouth bass, rainbow trout, seabass, and cobia, but to a lesser extent (~70%) by monondon and turbot. Nutrients digestibilities of MBM were reported only for cobia and gibel carp. The average digestibility of MBM measured in earlier trials was about ten percentage points below that of PBM. The maximum FM replacement rate by PBM is 80% for vannamei, fresh water fish, and Coho salmon, but is 50% for warm water marine fish. For MBM, the average maximum FM replacement rate is about 50-60% for most aquatic animals, except for cold water marine fish at 20%. Nutrient digestibilities were generally in agreement with WG except for monondon. Protein blend made from multiple sources of rendered protein meals (e.g. MBM, PBM, feather meal and blood meal) may offer nutrients palatability, and cost complementary benefits as shown with normal feed intake, weight gain, and feed conversion ratio in 100% FM replacement growth trials of rainbow trout (carnivorous) and silver perch (omnivorous). Trials reviewed support the use and value of PBM and MBM as FM replacements in diets for carnivorous and omnivorous aquaculture animals.

Application of de-oiled soya lecithin in shrimp feeds Yuyun Mu (Ph.D), Senior Technical Manager, Berg + Schmidt Asia Pte Ltd, No. 1 International Business Park 1, The Synergy # 09-04, Singapore 609917, Email: yymu@berg-schmidt.com.sg

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Beneficial effects of dietary phospholipids supplementation in terms of development growth and survival are demonstrated in the larval and juvenile stages of various species of shrimp. This could be owing to crucial roles of phospholipids in formation of cell membrane and lipoproteins, in absorption, transport and utilization of dietary lipids including EFA and cholesterol, and in provision of choline, inositol and EFA. Shrimp is incapable of de novo synthesizing PLs to meet the requirement. The published requirements of PLs are variable, depending on shrimp specie and age, test conditions, and the composition and purity of PL sources. The PL requirements of shrimp are mostly in the range of 1-3% in diets. PC and PI are the most active PL fractions of PLs in improving growth and survival of larval and juvenile shrimp. Since de-oiled soy lecithin contains higher levels of PC and PI, its supplementation in feeds offers better options to formulate balanced feeds when taking into account the optimal level and ratio of lipid, EFAs, cholesterol, PLs (particularly PC) required by shrimp as well as the feed properties. It is recommended to supplement 0.5-1.0% de-oiled lecithin in the commercial shrimp feeds for the best cost-effectiveness.

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Tailoring the feed formulation for maximizing profitability: farm demonstrations with white shrimp Litopenaeus vannamei

in Latin America

Peter Coutteau Ph.D. INVE Aquaculture Nutrition, Hoogveld 91-93, B-9200 Dendermonde, Belgium Email: p.coutteau@inve.be Co-authors: Roberto Chamorro1, Alí Vaca1, Carlos del Pozo2 Werner Jost3, Diogo Villaca3, and Jose Domingos3 Alexander Van Halteren4 and Marcos Santos5

1Camaronera de Cocle S.A., and 2Alimentos Larro, Industrias de Nata S.A.; Grupo Calesa, Aguadulce, Panama 3Camanor Produtos Marinhos Ltda, Natal, RN Brazil 4INVE Aquaculture Nutrition, Dendermonde, Belgium; 5INVE do Brazil, Fortaleza, Brazil Summary of Presentation: The continuous decrease of shrimp prices during the past years has urged the shrimp farming industry towards improving profitability. Feed constitutes the major cost in (semi) intensive farming and is therefore a traditional target of cost cutting strategies. Reducing the feed cost by using cheaper feed is a strategy which is relatively easy to promote in a feed market where protein level is still often regarded as the single criterion to evaluate feed quality. However, cheaper feeds, obtained by eliminating the most expensive ingredients in the formulation, are not always resulting in improved cost-efficiency. The resulting “nutritional gaps” obviously involve risks that the feed composition no longer covers the nutritional requirements, particularly under more intensive conditions where shrimp are more dependent upon the nutritional inputs from the feed. As a result, cheaper feeds may cost more money to the shrimp farmer than what is saved on the feed formula at the feed mill. Nevertheless, continuous pressure on the shrimp feed producers to reduce feed prices, despite increasing raw material cost, has resulted in a gradual decrease of average nutritional standards of shrimp feed in all shrimp producing regions. In this paper we shortly report on the results of an international survey organized in 2002-2003 to analyse representative commercial shrimp feeds in some of the main shrimp producing countries. Also, we will review a number of novel concepts to alleviate nutritional gaps in current shrimp feed formulations. Finally, we will illustrate the applicability of these specialty nutrients to improve profitability under practical farming conditions in semi-intensive farms in Panama and Brazil.

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Tailoring the feed formulation for maximizing profitability: farm demonstrations with white shrimp Litopenaeus vannamei in Latin America Peter Coutteau Ph.D., INVE Aquaculture Nutrition Co-authors: Roberto Chamorro1, Alí Vaca1, Carlos del Pozo2; Werner Jost3, Diogo Villaca3, and Jose Domingos3; Alexander Van Halteren4 and Marcos Santos5 1Camaronera de Cocle S.A., and 2Alimentos Larro, Industrias de Nata S.A.; Grupo Calesa, Aguadulce, Panama; 3Camanor Produtos Marinhos Ltda, Natal, RN Brazil; 4INVE Aquaculture Nutrition, Dendermonde, Belgium; 5INVE do Brazil, Fortaleza, Brazil. Introduction The continuous decrease of shrimp prices during the past years has driven shrimp farming towards cost cutting and improving production efficiency. Increased biosecurity measures and selective breeding programmes for optimizing growth and disease resistance are already yielding major benefits on the production efficiency and sustainability of shrimp farming in many regions, particularly for white shrimp Litopenaeus vannamei. However, traditional cost cutting strategies still involve two possibly contradictory measures: increasing stocking densities and reducing feed cost. Reducing the feed cost can be obtained by either improving feeding management or using cheaper feed. It is obvious that the latter strategy is understood as relatively easy in a feed market where protein level is still often regarded as the single criterion to evaluate feed quality. However, cutting costs by eliminating the most expensive ingredients in a formulation is not always resulting in improved cost-efficiency, and as a result may cost more money to the shrimp farmer than the amount that he/she (and/or the feed mill) economizes on the feed formula. Nevertheless, continuous pressure on the shrimp feed producers to reduce feed prices, despite increasing raw material cost, has resulted in a gradual decrease of average nutritional standards of shrimp feed in all shrimp producing regions. The resulting “nutritional gaps” obviously involve risks that the feed composition no longer covers the nutritional requirements, particularly under more intensive conditions where shrimp are more dependent upon the nutritional inputs from the feed. Similar to what happened in the poultry and livestock production, lower margins and increased competition lead to integration into larger farming conglomerates. Under these conditions, shrimp feed producers (whether or not integrated with the farm in the same company) are continuously challenged to tailor their formulations to meet the best cost-efficiency for increasingly larger customers. Shrimp feed formulators explore novel nutritional concepts and/or ingredients on the strict condition of delivering better cost-efficiency to the farmer. The further maturation of the shrimp industry will thus promote innovations in shrimp nutrition and a better understanding of the cost benefit of feed formulations under the wide variety of shrimp culture conditions.

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In this paper we report on the results of an international survey organized in 2002-2003 to analyse representative commercial shrimp feeds in some of the main shrimp producing countries. Also, we will review a number of novel concepts to alleviate nutritional gaps in current shrimp feed formulations. Finally, we will illustrate the applicability of these specialty nutrients to improve cost-efficiency under practical farming conditions in semi-intensive farming of Litopenaeus vannamei in Brazil and Panama. Identifying the nutritional gaps: international shrimp feed survey During 2002-2003 representative samples of commercial shrimp feeds were collected from seven major shrimp producing countries, namely Brazil, Ecuador, India, Indonesia, Madagascar, Mexico and Thailand (Coutteau, 2004). The feed samples were selected among leading feed brands representing the bulk of the feed used by shrimp farmers within these countries. As a result, the survey does not necessarily cover premium feed qualities which are representing only a small market share. Similarly, a limited number of feed samples from a certain country would not allow us to extrapolate the results for the whole country. The shrimp feed samples were analysed using a number of standard feed specifications including crude protein (Kjeldahl method), crude fat (soxhlet method after hydrolysis) and moisture (desiccation). Furthermore, feeds were analyzed on a number of “marker nutrients”, which are regarded as relevant indicators of nutritional feed quality which are generally not specified by the feed producer. Cholesterol Cholesterol is an essential nutrient for penaeid shrimp, which are unable to biosynthesize this key constituent of cell membranes and precursor for steroid and moulting hormones (Akiyama et al., 1992). It has been found to be most effective in different species of shrimp at dietary levels ranging from 0.25% to 0.5% (Duerr and Walsh, 1996; Gong et al., 2000; Chen, 1993). Marine invertebrate meals and oils, such as squid, shrimp, clam, crab, and mussel, are rich sources of cholesterol. However, fishmeal is the major cholesterol source in practical feed formulations for shrimp. Assuming a minimum dietary level of 0.25% cholesterol, fishmeal at 35% inclusion in the diet can deliver around 50% of the requirement. The addition of typical levels of shrimp and squid meal usually provides another 20-30%. As a result, the supplementation of purified cholesterol (0.10-0.15% of the diet) is needed to fully cover the requirement and represents a significant cost in shrimp formulations. Cholesterol level (gas liquid chromatography) was selected as a marker for the use of appropriate levels and qualities of marine ingredients and supplementation of cholesterol. Soluble protein Soluble protein (Kjeldahl, water soluble crude protein) is a diverse group of N-containing water soluble compounds, including proteins, peptides, amino acids, nucleotides, and certain vitamins. These compounds are playing a role in a wide range of physiological activities including the nutritional balance of amino acids, protein digestion, palatability and feed attractiveness, and immune-enhancement (Lee and Meyers, 1997; Cahu et al.,

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1999; Burrels et al., 2001). Although “soluble N-containing compounds” is a complex group for which it is difficult to set a requirement, soluble protein serves as a general indicator for the level and quality of marine ingredients (vegetable proteins generally contain much lower levels of soluble proteins than marine proteins; high quality whole fish meals have a higher soluble protein content than low quality fish meal) and the use of supplementary feeding attractants (which are mostly based on N-containing water soluble compounds). Polar lipids Polar lipids (Folch extraction followed by separation of polar lipid fraction by column chromatography and gravimetric determination) in shrimp feeds are predominantly phospholipids, which play an important role in shrimp nutrition as a component of cell membranes, building blocks for lipoproteins (essential for lipid mobilization in the haemolymph), and constitute a highly available source of choline and inositol (Coutteau et al., 1997). Phospholipids in shrimp feeds usually originate from the use of good quality marine ingredients and/or the use of soybean lecithin. The level of polar lipids is therefore a marker for the use of appropriate levels/qualities of marine ingredients and the supplementation of lecithin. Survey results The survey yielded overall average specifications for protein/fat (expressed on a percent basis) of 41.7/7.3 for feeds for black tiger shrimp (Penaeus monodon) and 35.1/8.9 for Pacific white shrimp (P. vannamei; Table 1; Figure 1). Within those countries producing P. monodon, the feeds sampled in Madagascar and Thailand had higher protein levels than feeds originating from Indonesia and India. White shrimp (P. vannamei) feeds collected from Brazil and Mexico exhibited very similar protein/fat specifications of 36-37/8. In Ecuador only two samples were analyzed for protein/fat specifications which is insufficient to represent the current situation in the market where protein levels are predominantly 22-28% for the semi-intensive farming and 35% for greenhouse production. Devresse (1995) reported average protein/fat (after hydrolysis) specifications of 41.4/7.6 and 33.0/7.0 in SE Asian and Latin American feed samples, respectively. The feeds for black tiger shrimp were based on stronger formulations as shown by the notably higher average levels of all marker nutrients compared to the white shrimp feeds (9.1 versus 5.3% soluble protein; 1.9 versus 1.63% polar lipids; 0.24 versus 0.11% cholesterol). Devresse (1995) found average cholesterol levels (GLC analysis) of 0.18% and 0.12% in SE Asian and Latin American feed samples, respectively. This indicates an overall reduction of cholesterol levels, particularly in black tiger shrimp feeds, over the past decade. Phospholipid levels, measured by Devresse (1995) using TLC-FID Iatroscan and therefore not comparable with gravimetric determination in the current study, were 0.67% and 0.35% for SE Asian and Latin American feed samples, respectively. The limited variation of protein specification (coefficient of varation 6% and 11% for P. monodon and P. vannamei feeds, respectively) confirms that the sector is very sensitive about the protein levels in shrimp feed. Variability among brands is somehow higher on fat (13-18% CV) and moisture content (12-15% CV). However, extreme variations are

17

found in the levels of soluble protein (25-27%), polar lipids (26-38%) and cholesterol levels (34%). It is obvious that these marker nutrients are not taken into account for formulating a great number of commercial shrimp feeds. Extremely low values of polar lipids (1.16% and 0.99% in P. monodon and P. vannamei feeds, respectively) and particularly cholesterol (0.11% and 0.04% in P. monodon and P. vannamei feeds, respectively) were encountered. These values are far below recommended dietary levels of 2% for phospholipids and 0.2-0.25% for cholesterol (Akiyama et al., 1992; Duerr and Walsh, 1996). Developing novel nutritional concepts to fill some of the nutritional gaps Lipid nutrition The cost and availability of cholesterol has shown important fluctuations during the past decade due to large variations in supply and demand in the market for wool grease (lanolin or wool fat), which is a by-product from washing sheep wool from which cholesterol is derived. Furthermore, the need for additional supplementation of cholesterol in shrimp feeds is likely to increase due to the increased pressure on the use of fishmeal as protein source for shrimp feeds. The results of the survey have indicated that dietary cholesterol level is often not considered by shrimp feed formulators due to the cost implications of either supplementing purified cholesterol or significantly increasing the levels of marine ingredients. Cholesterol levels below 0.10-0.15% limit growth in practical feed formulations, even if the other nutrients are formulated to satisfy normal requirements (Coutteau et al., 2002 – Figure 2; Duerr and Walsh, 1996). Moreover, the combination of suboptimal dietary levels of cholesterol and phospholipids may be particularly detrimental to shrimp nutrition due to the interaction between phospholipids and cholesterol requirements (Gong et al., 2000). Recently, a novel nutritional concept has been developed to enhance digestion and absorption of the endogenous cholesterol in shrimp feed ingredients, which reduces the need to supplement purified cholesterol (Coutteau et al., 2002). Growth trials have shown that the supplementation of the cholesterol replacing concept (“Aquasterol”) was equally effective as purified cholesterol in improving growth and food conversion of Penaeus monodon fed a low-cholesterol control diet (Figure 2). Penaeus vannamei farmers in Mexico frequently apply top-coating of Aquasterol on the feed pellets to improve the nutritional status of the hepatopancreas which is monitored by microscopy (Figure 3). The build-up of lipid reserves in the hepatopancreas is considered to be important to reduce the impact of outbreaks of the white spot virus in Mexican farms (G. Chauvet – personal communication). The supplementation of the product to the feed formula increased survival and harvest yield in a farm trial in Ecuador with 24% at stocking densities as low as 5-6/m2 (increase from 362 to 450 kg/ha in 7-10 ha ponds after 85 days of culture; average values of 2 treatment ponds with 1% aquasterol supplemented to the feed versus 4 control ponds without dietary supplementation; F. Jijon -pers. comm.). These field observations suggest that the novel concept, apart from improving cholesterol utilization, has a general effect on lipid absorption and utilization in shrimp.

18

Feeding stimulants and attractants Feeding attractants, defined in a broader sense as compounds attracting the shrimp as well as stimulating them to eat (Lee & Meyers, 1997), are considered of key importance in shrimp nutrition due to their effect on feeding rate and appetite. “Time is money” in shrimp feeding. A fast feeding response in a shrimp pond will reduce the leaching of valuable water-soluble nutrients (including essential vitamins and amino acids) and the accumulation of wasted feed which will in turn affect soil quality and oxygen consumption. Feed attractiveness is measured by the rate at which the feed disappears on the witness feeding tray. Although this technique is difficult to standardize and assumes good feed stability, it has proven to be valuable in comparing the effect of different attractants under field conditions. Ceulemans et al. (2003) concluded from a feeding tray study with P. monodon that the level of fishmeal (25 versus 60% of dietary protein originating from fishmeal) was not a determining factor for feed uptake. However, the addition of selected marine proteins either into the feed mash during processing or, even more effectively, by topcoating with fish oil on the finished pellet, improved feed consumption significantly (Figure 4). Improving feed uptake under a wide variety of practical farming conditions is an area where there is still plenty of room for new developments. Current knowledge of the effects of formulation on feed uptake by shrimp under different conditions of age, crowding, disease status, and environmental conditions is still very limited. Nutrition and Health Penaeid shrimp production is under continuous threat by bacterial and particularly viral infections which have caused disastrous collapses of the industry in all major shrimp producing countries. Shrimp aquaculturists are currently forced to consider genetics, quality of the stocked fry, husbandry procedures and healthy nutrition as the major tools to control disease and ensure the profitability of their business. Current nutritional research efforts are directed towards the identification of requirements for key nutrients affecting the health and immunology of shrimp, such as vitamin C and E, phospholipids, essential fatty acids, trace minerals and pigments. One of the most promising areas of development for strengthening the non-specific defense mechanisms and protect shrimp against disease is the administration of immuno-stimulants. Various compounds have been identified and are mostly derived from the cell envelope of micro-organisms, such as polysaccharides, lipoproteins, and lipopolysaccharides. Preliminary results indicated that the efficacy of various commercially available immuno-stimulants to improve stress and/or disease resistance of fish and shrimp strongly depends on the type of the product and on the supply of adjuvant nutrients that are essential to support the buildup of the immune system. Therefore, the design of specialty premixes to improve disease resistance is based on the selection of the appropriate immuno-stimulants in combination with a balanced supply of key nutrients to support the enhancement of the immune system. Coutteau et al. (2001) demonstrated that the supplementation of a combination of health enhancing nutrients and immuno-stimulants to a high quality feed for Penaeus stylirostris

19

resulted in an enhancement of immuno-competence and resistance to disease after 3 weeks of feeding, as indicated by (1) a reduction of the total number of haemocytes in circulation in the haemolymph (with 26% compared to the control group); (2) a boost of the phagocytosis activity of the haemocytes (33% and 84% increase of basal NBT and stimulated NBT activity, respectively, compared to the control group); and (3) an improved survival in a challenge test (53% less mortality compared to the control group following an experimental infection with Vibrio penaeicida – Figure 5). These results corroborate with findings by a number of laboratory studies showing effects of individual nutrients on health and stress resistance in shrimp. Filling the nutritional gaps: tailoring formulation to optimize cost-efficiency at the farm Shrimp nutrition under farm conditions is influenced by numerous factors, including climatological and environmental conditions, stocking densities, quality of water and pond soil, natural productivity, presence of pathogens in seed and/or environment, genetics, farm and feeding management. Nutritional requirements will largely depend on many of the above factors, although it is difficult to find scientific proves of that under farm conditions. Although many farmers have experimented with commercial feeds to determine the optimal protein level required under their specific farm conditions, the lack of insight in the formulation will prevent them from interpreting the differences in the quality of ingredients and the many other nutrients that may interact with their observations on setting the optimum level of “Kjeldahl nitrogen” in their feed. Shrimp -like all other animals- require a set of digestible nutrients in the right proportions to grow in the most effective way. However, the applicability of least cost formulation of shrimp feeds is still limited due to the complicated interactions between ingredients and feed stability, and the difficulty to cost characteristics such as attractiveness. As illustrated in the previous paragraph, increasing research efforts in the field of shrimp nutrition by the supplying industry of additives and specialty ingredients has resulted in a range of specialty ingredients to fortify the typical raw materials applied in the shrimp feed mill. The relative cost of specialty nutrition and standard ingredients needs to be carefully balanced to reach the best cost/benefit for the farmer. Tailoring formulation to optimize cost-efficiency at the farm: demo at experimental scale in CAMANOR, Brazil Progress in shrimp nutrition has been hampered seriously by the lack of relevance for practical farming of many nutritional studies due to the difficulties to grow shrimp under conditions that are representative for the wide variety of field situations. Still too much of our knowledge on shrimp nutrition is based on evaluating the very restricted growth rates of small shrimp in clear water tanks under stable culture conditions without any possibility of not encountering the feed. The farming conditions are very different and include a variety of sizes, species, natural food organisms besides the feed, feed availability, and daily/seasonal fluctuations in environmental conditions.

20

Pen enclosures in production ponds offer an interesting environment to study nutrition under conditions that are representative of the farming conditions. Shrimp remain in direct contact with the sediment and – provided there is adequate exchange with the rest of the pond water – are in similar water quality conditions and receive natural food organisms as the shrimp in a production pond. The enclosed area and the contained shrimp biomass should be sufficiently large to reduce the effect of the net as an artificial substrate for the periphyton (biofilm) growth. Excess availability of periphyton may mask nutritional deficiencies in the feed which would be apparent under standard pond conditions. To increase the power of experimentation under practical farm conditions, pen enclosures have been evaluated in collaboration with Camanor Produtos Marinhos Ltda, Natal, RN Brazil. A number of feed trials were performed during 2005 at the Peixe Boi shrimp farm located in Porto do Mangue, Rio Grande do Norte state, Northeast Brazil. Experimental units were based on pen enclosure structures (bottom area=100 m2) manufactured with PVC-coated polyester netting (5mm mesh), poles and ropes. 12 pens was installed inside each earthern pond. First results are indicating that the pens are valid testing environments to evaluate cost-efficiency of different feeds under farming conditions. However, further work is required to reveal the effect of various parameters in the testing protocol such as the shrimp size at stocking, the stocking density, and the feeding distribution method on the productivity inside the pens in comparison with that of the pond.

Pen enclosures of 100 m2 installed in shrimp ponds of Camanor Produtos Marinhos Ltda, Natal, RN Brazil.

21

Tailoring formulation to optimize cost-efficiency at the farm : demo at production scale in CAMACO, Panama Due to the complexity of shrimp nutrition, the verification of nutritional concepts requires evaluation under real production conditions at the farm, more than in any other cultivated species. A case study on tailor-made application of specialty nutrition was worked out in collaboration with an integrated shrimp producer in Panama (feed mill Alimentos Larro, Industrias de Nata S.A. and shrimp farm Camaco; Grupo Calesa, Panama; Coutteau et al., 2005).

Grupo Calesa is the owner of CAMACO (Camaronera de Cocle, S.A.) -the leading integrated shrimp producer in Panama- with 1,200 hectares of ponds, molecular biology lab, two maturation facilities (with genetic programs), and two hatcheries; feed mill INASA (Alimentos Larro, Industrias de Nata S.A.) and processing plant ALTRIX.

A shrimp feed line was designed based on standard feed mill ingredients of selected quality and a boost of specialty nutrients to enhance cholesterol and lipid utilization, disease and stress resistance, and feed attractiveness (referred to as “boosted feed”). The formulation was tailored to the conditions of semi-intensive pond farming during a series of preliminary farm evaluations where the effect of graded levels of nutritional boosts were evaluated in terms of cost benefit at harvest. Production data for the optimized formulation in comparison with the standard feed range (premium quality feed of the same protein level, referred to as “control feed”) during the cold and the warm season of

22

2003-2004 are shown in Table 2 and Figure 6 and 7. Significant improvements are observed during the cold as well as warm season in terms of survival (+23/+14% for cold/warm season, respectively), final shrimp size (+5/+11%), harvest yield per ha (+29/+24%), and food conversion (-12/-19%). These results clearly demonstrated that under two different culture regimes (cold season 12/m2 and warm season at 15/m2) the more costly boosted feed formulation significantly improved cost efficiency in terms of cost of feed spent per kg of shrimp produced (-19/-13%) (Coutteau et al. 2005). The optimization of farm profits results from a reduction of production costs (through improving productivity and cost-efficiency) and/or an increase of the value of the end-product (depending mostly on unit weight but also on meat taste, pigmentation, tail yield). The size distribution of harvested shrimp is a crucial factor in the profitability of shrimp farming; particularly in times when shrimp price is significantly decreasing in smaller size classes (Fig. 8). The distribution of shrimp sizes at harvest is affected by many factors including genetics, disease incidence, feeding management (distribution, level, frequency) and feed quality. The importance of nutrition on the homogeneity of shrimp sizes was illustrated recently on production scale at the CAMACO farm where feeds of different nutritional quality (control feed: prime feed based on standard ingredients versus boosted feed: standard feed mill ingredients boosted with specialty nutrients) were compared under similar production conditions in terms of origin of the seed and farm/feeding management. The boosted feed formulation was further specialized by improving digestible protein, available energy and tailoring the nutritional boost to the more stressful conditions of the dry/cold season (lower and more fluctuating temperatures). The evaluation on full production scale involved a total area of 198 ha at a stocking density of 15 per m2 and 491 ha at 12 per m2 (Table 3). The production results recorded by the farm during the second crop of 2005 confirmed earlier data on the importance of feed quality on productivity. The boosted feed formulation yielded 9% and 24% better harvest yield per ha at 15 and 12 per m2, respectively (Table 3). Survival was similar at 15 per m2 (47.4-49.4%) but was drastically improved at the lower density (48.4% for the boosted treatment vs. 41.6% for the control). Growth rate was particularly accelerated due to improved nutrition during the cold season; resulting in 13% and 8% bigger average size of the shrimp fed the boosted feed at harvest. Food conversion improved with 7 and 22%, respectively, at 15 and 12 per m2. At the processing plant, the effect of nutrition was clearly observed by the number of size classes recovered from the farm, ie predominantly 2 size classes for the shrimp fed boosted feed; 3-4 sizes for the shrimp fed the control feed. The shrimp fed the boosted feed showed a less heterogeneous size distribution than the control shrimp (Fig. 9). For shrimp produced at 15/m2, 97% of the shrimp fed the boosted feed were classified in the size classes 50/60+60/70 (whole shrimp counts per kg) whereas this was only 77% for the shrimp fed the standard feed. For shrimp produced at 12/m2, 86% belonged to size classes 50/60+60/70 for shrimp fed the boosted feed versus 58% for the control.

23

This farm data showed that more expensive nutrient-dense feeds containing a well balanced mix of standard and specialty ingredients give superior results at stocking densities of 12-15 per m2 in terms of growth, food conversion, harvested biomass, shrimp value and … profit. The bigger average shrimp size and better size distribution yielded a more valuable shrimp product after processing. Applying current sales prices to estimate the value of the processed whole shrimp produced in each feed group showed an increased value of 4 to 7% for the shrimp fed the boosted feed compared to the control group (Fig. 10). The increased sales value of the end-product (+4 to +7%) adds up to the reduction of farm-gate production cost per unit weight (-11 to -20%) in the improvement of the profitability of the farming operation. References Akiyama, D.M., Dominy, W.G., Lawrence A.L. (1992). Penaeid shrimp nutrition. In: Marine Shrimp Culture: Principles and Practices. Eds.: Fast A.W. and Lester J.I. Elsevier. Pp 535-568. Burrells, C., P.D. Williams, P.F. Forno. (2001). Dietary nucleotides: a novel supplement in fish feeds 1. Effects on resistance to disease in salmonids. Aquaculture 199:159-170. Cahu, C.L.; Zambonino I.Z.; Quazuguel, P.; Le Gall, M.M. (1999). Protein hydrolysate vs. fish meal in compound diets for 10-day old sea bass Dicentrarchus labrax larvae. Aquaculture,171: 09-119. Chen, H.Y. (1993). Requirements of marine shrimp, Penaeus monodon, juveniles for phosphatidylcholine and cholesterol. Aquaculture, 109: 165-176. Ceulemans S., A. Van Halteren, A. Nur, P. Coutteau. (2003). Evaluation of attractants for Penaeus monodon using feeding trays in ponds. ASIAN-PACIFIC AQUACULTURE 2003, september 22-25, Bangkok, Thailand. Coutteau, P., Geurden, I., Camara, M.R., Bergot, P., Sorgeloos, P. (1997). Review on the dietary effects of phospholipids in fish and crustacean larviculture. Aquaculture, 155: 149-164. Coutteau, P., Ceulemans S., Chim, L., Saulnier, D., Lemaire, P. (2001). Improved nutrition enhances immune competence, disease resistance in penaeid shrimp. Global Aquaculture Advocate, Vol 4 (Issue 5), October 2001.

Coutteau, P., Peeters J., Abidin N., (2002). Cholesterol … Indispensable, but not irreplaceable in shrimp feeds. Global Aquaculture Advocate June 2002.

Coutteau, P., 2004. Filling the nutritional gaps in shrimp feed formulation. International Aquafeed, Issue 4, pp. 18-27 (Nov-Dec 2004).

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Coutteau, P., Chamorro R., Del Pozo C., Camargo,R., Dos Santos M., Jijon F.. (2005). Effect of nutrition on productivity and cost-efficiency of semi-intensive farming of Penaeus vannamei in Panama. Global Aquaculture Advocate volume 8 (2), April 2005; 79-78.

Devresse, B. (1995). Nutrient levels in some commercial shrimp feeds and feed ingredients of Asia and Latin America – a comparative analysis. In: Proceedings of Feed Ingredients Asia ’95, Singapore, 19-21 September, 1995. Duerr, E.O. , Walsh.W.Â. (1996). Evaluation of cholesterol additions to a soybean-meal based diet for juvenile Pacific white shrimp, Penaeus vannamei, in an outdoor growth trial. Aquaculture Nutrition 2:111-116. Gong, H., Lawrence, A.L., Jiang, D.H., Gatlin, D.M. III, (2000). Lipid nutrition of juvenile Litopenaeus vannamei I. Dietary cholesterol ans de-oiled soy lecithin requirements and their interaction. Aquaculture, 190: 307-321. Lee P.G., Meyers, S.P. (1997). Chemoattraction and feeding stimulation,. In Crustacean Nutrition, Advances in aquaculture nutrition Volume 6; Ed. Luis R. D'Abramo, Gouglas E. Concklin and Dean M. Akiyama.

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Fig. 1A&B: Composition of representative feeds for Penaeus vannamei and Penaeus monodon in major shrimp producing countries. Data show average, standard deviation and minimum/maximum values for crude protein, crude fat after hydrolysis, soluble crude protein, polar lipids, and cholesterol. See Table 1 for details (Coutteau, 2004). 1A

1B

Mean Mean±SD Min-Max

MONODON VANNAMEI0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

CH

OLE

STE

RO

L (%

)

Mean Mean±SD Min-Max

MONODON VANNAMEI2

4

6

8

10

12

14

16

SO

LUB

LE C

RU

DE

PR

OTE

IN (%

)

Mean Mean±SD Min-Max

MONODON VANNAMEI0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

PO

LAR

LIP

IDS

(%)

26

Fig. 2: Effect of the supplementation of purified cholesterol (85% purity at 1.8 kg/MT) or a cholesterol replacing concentrate (Aquasterol at 15 kg/MT) to a feed containing a sub-optimal cholesterol level (0.1%; originating from the marine ingredients) in Penaeus monodon. Data represent average of group weights from triplicate tanks (Coutteau et al., 2002).

27

Fig. 3: Hepatopancreas tubuli in decreasing nutritional status with further depleted lipid reserves from A to C. Improving lipid absorption through dietary supplementation with Aquasterol promotes stage A.

28

Fig. 4: Results of a feeding tray study in a pond stocked with Penaeus monodon. Data represent consumption of feed after 90 min exposure, averaged over different repetitions in time and space. Treatments: C25: control diet with 25% of dietary protein originating from fishmeal; 25 Attr2 (or 4) INCL2%: 2% of attractant mix 2 (or 4) incorporated during processing of the pellet; 25 Attr4 TOP1%: 1% supplement of attractant mix 4 top dressed with 1% fish oil on the finished feed (Ceulemans et al., 2003).

50556065707580859095

100

C25 25 Attr2INCL 2%

25 Attr4INCL 2%

25 Attr4 TOP 1%

Feed

con

sum

ed b

y sh

rimp

afte

r 90

min

exp

osur

e (%

)

29

Fig. 5: Cumulative mortality (%) in the course of the experimental infection for Penaeus stylirostris after being fed for 3 weeks either on a diet supplemented with a mixture of immunostimulants and health enhancing nutrients (“AQUASTIM”) or on a control diet without supplement (“CONTROL”). Data represent average and standard deviation of two (non-infected controls) or four (challenged shrimp) tanks each initially stocked with 25 shrimp. Different superscripts indicate significant differences (Student t-test, P<0.05; Coutteau et al., 2001).

0

5

10

15

20

25

30

35

0 24 48 72 96 120TIME AFTER INFECTION (h)

CU

MU

LATI

VE M

OR

TALI

TY (%

)

CONTROL non-infected CONTROL infectedAQUASTIM-S non-infected AQUASTIM-S infected

-53%

a

b

30

Fig. 6: Effect of a tailor-made nutritional boost on weekly shrimp growth in semi-intensive production of Penaeus vannamei in the CAMACO farm (Grupo Calesa, Panama) during the warm season (Trial 2: april-september) (see Table 1 for detailed production data).

0.30.40.50.60.70.80.91.01.11.21.3

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16WEEKS AFTER STOCKING

WEE

KLY

GR

OW

TH (g

)

CONTROL FEED

BOOSTED FEED

31

Fig. 7: Effect of a tailor-made nutritional boost on feed cost per kg of shrimp produced in semi-intensive production of Penaeus vannamei in the CAMACO farm (Grupo Calesa, Panama) during the cold season (Trial 1: October 2003 - January 2004) and the warm season (Trial 2: April - September 2004) (see table 2 for detailed production data; Coutteau et al., 2005).

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

TRIAL 1 (12/m2-cold season)

TRIAL 2 (15/m2 - warm season)

CO

ST

EFF

ICIE

NC

Y

(USD

FEE

D C

OS

T/K

G S

HR

IMP

PR

OD

UC

ED

)

CONTROL FEEDBOOSTED FEED

-19%

-13%

32

Fig. 8. Whole shrimp price in function of size (example taken from C.F. Woodhouse, Fish Farmer International July 2005).

WHOLE SHRIMP PRICE IN FUNCTION OF SIZE

0

1

2

3

4

5

6

40/50 50/60 60/70 70/80 80/100 100/120KG COUNT

US

D/K

G

33

Fig. 9: Effect of feed formulation on the size distribution of head-on shrimp at the CAMACO farm (Grupo Calesa, Panama) during the cold season at stocking density of 12 and 15 per m2 (July 2005-January 2006). See text and Table 3 for details.

SHRIMP SIZE DISTRIBUTION AT PROCESSING (CAMACO CROP II-06 - farming 15/m2)

0%

10%

20%

30%

40%

50%

60%

70%

40/50 50/60 60/70 70/80 80/100COUNT (PCS/KG HEAD-ON)

PER

CEN

TAG

E

BOOSTED FEEDCONTROL FEED

0%

10%

20%

30%

40%

50%

60%

40/50 50/60 60/70 70/80 80/100COUNT (PCS/KG HEAD-ON)

PER

CEN

TAG

E

BOOSTED FEEDCONTROL FEED

SHRIMP SIZE DISTRIBUTION AT PROCESSING (CAMACO CROP II-06 - farming 12/m2)

34

Fig. 10: Effect of feed formulation on the production cost and value of whole processed shrimp in the CAMACO farm (Grupo Calesa, Panama) during the cold season at stocking density of 12 and 15 per m2 (July 2005-January 2006). Cost and value per unit weight of shrimp produced on the boosted feed are expressed relatively to those fed the control feed. See text and Table 3 for details. CAMACO FARM – CROP II-2006 – FARMING AT 12/M2 CAMACO FARM – CROP II-2006 – FARMING AT 15/M2

70

80

90

100

110

Production cost(USD/LBS)

Shrimp value(USD/LBS)

% C

HA

NG

E

CONTROL FEED BOOSTED FEED

-11%+7%

70

80

90

100

110

Production cost(USD/LBS)

Shrimp value(USD/LBS)

% C

HA

NG

E

CONTROL FEED BOOSTED FEED

-20% +4%

35

Table 1: Composition of representative feeds for Penaeus vannamei and Penaeus monodon in major shrimp producing countries. Data based on the analysis of n feed samples collected during the period 2002-2003 for crude protein (CP), crude fat after hydrolysis (CF), moisture, soluble crude protein (Sol CP), polar lipids (PL), and cholesterol (CHOL). Data represent average from n analyzed samples, coefficient of variation (CV%), and extreme values (min/max). P. MONODON CP CF Moisture Sol CP PL CHOLAVERAGE 41.7 7.3 9.2 9.1 1.90 0.24 CV% 6% 13% 15% 25% 26% 34% MIN 37.8 5.0 6.7 5.3 1.16 0.11 MAX 48.2 8.5 11.9 14.3 2.95 0.41 n 25 21 25 25 23 25 AVERAGE PER COUNTRY INDIA (n=8) 40.2 6.5 10.7 7.4 1.45 0.18 INDONESIA (n=3) 41.5 7.6 8.3 10.6 2.59 0.24 MADAGASCAR (n=6) 42.5 8.2 8.0 10.0 1.94 0.22 THAILAND (n=8) 42.8 7.7 9.1 9.6 1.95 0.32 CV% PER COUNTRY INDIA 4% 10% 7% 15% 4% 29% INDONESIA 3% 8% 11% 32% 12% 19% MADAGASCAR 5% 1% 10% 24% 23% 30% THAILAND 8% 10% 10% 19% 25% 23% P. VANNAMEI CP CF Moisture Sol CP PL CHOLAVERAGE 35.1 8.9 9.3 5.3 1.63 0.11 CV% 11% 18% 12% 27% 38% 34% MIN 26.5 5.6 7.5 2.7 0.99 0.04 MAX 39.1 11.5 11.4 8.0 3.28 0.21 n 17 17 17 17 14 20 AVERAGE PER COUNTRY (includes few samples from Panama, Germany, Surinam) BRAZIL 36.3 8.4 9.8 5.4 1.40 0.11 ECUADOR 32.8 10.5 8.9 5.4 2.91 0.13 MEXICO 36.8 8.2 9.0 4.3 1.40 0.09 CV% PER COUNTRY BRAZIL (n=8) 1% 20% 11% 22% 5% 20% ECUADOR (n=2; except CHOL n=5) 22% MEXICO (n=3) 3% 16% 23% 6% 21% 11%

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Table 2: Evaluation of a tailor-made nutritional boost on semi-intensive production of Penaeus vannamei in the CAMACO farm (Grupo Calesa, Panama) during the cold season (Trial 1: October 2003 – January 2004) and the warm season (Trial 2: April –September 2004) (Coutteau et al., 2005). TRIAL 1: 12/m2 cold season - 3 ha ponds - seeding october 2003 - harvest january 2004

CONTROL

FEED BOOSTED

FEED % CHANGE

BOOSTED/CONTROL # ponds 3 13 Total pond area (ha) 9 39 Culture period (days) 99 101 Survival (%) 34 42 +23% Final weight (g) 11.7 12.3 +5% Harvest yield (kg/ha) 479 617 +29% FCR 2.77 2.16 -12% avg weekly growth (g) 0.83 0.85 +2% TRIAL 2: 15/m2 warm season - 3-6 ha ponds - seeding april 2004 - harvest august/september 2004

CONTROL

FEED BOOSTED

FEED % CHANGE

BOOSTED/CONTROL # ponds 10 9 Total pond area (ha) 47 55 Culture period (days) 128 124 Survival (%) 42 48 +14% Final weight (g) 17.9 19.8 +11% Harvest yield (kg/ha) 1,124 1,395 +24% FCR 2.66 2.16 -19% avg weekly growth (g) 0.98 1.11 +13%

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Table 3: Evaluation of a tailor-made nutritional boost on semi-intensive production of Penaeus vannamei in the CAMACO farm (Grupo Calesa, Panama) during the cold season at 15 per m2 (Trial 3: July – December 2005) and 12 per m2 (Trial 4: August 2005 – January 2006). TRIAL 3: 15/m2 cold season - seeding July 2005 - harvest December 2005

CONTROL

FEED BOOSTED

FEED % CHANGE

BOOSTED/CONTROL # ponds 39 27 Total pond area (ha) 117 81 Culture period (days) 119 122 Survival (%) 49.4 47.4 -4% Final weight (g) 14.9 16.8 +13% Harvest yield (kg/ha) 1096 1196 +9% FCR 2.20 2.06 -7% avg weekly growth (g) 0.88 0.97 +10% TRIAL 4: 12/m2 cold season - seeding August 2005 - harvest Januari 2006

CONTROL

FEED BOOSTED

FEED % CHANGE

BOOSTED/CONTROL # ponds 46 70 Total pond area (ha) 202 289 Culture period (days) 120 124 Survival (%) 41.6 48.4 +16% Final weight (g) 14.4 15.6 +8% Harvest yield (kg/ha) 727 904 +24% FCR 2.40 1.88 -22% avg weekly growth (g) 0.84 0.88 +4%

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Improving Nutrient Delivery in Aqua Feeds: Implications for Nutritionists and Formulators A Victor Suresh, Bentoli, Inc. Introduction The term “nutrient delivery” relates to the fact that only a part of the nutrients in the feed reach the shrimp’s mouth and that the other part is lost due to physical deterioration of feed or leaching of water soluble nutrients from the feed. Shrimp nutritionists, feed manufacturers and farmers have long recognized the importance of keeping the feed stable in water for several hours so that wasting of nutrients due to physical deterioration is minimal. Shrimp farmers in Asia insist that feeds remain stable in water for at least three hours. By using binders and advanced pelleting technology, it is now possible in Asia to produce shrimp feeds that are water stable for four hours or more. However, even the most stable shrimp feed would leach out nutrients. For the practicing shrimp nutritionist, formulator and feed manufacturer, two major challenges are presented:

(a) How to quantify and account for leaching loss of nutrients when formulating a diet? (b) What strategies are available to minimize the leaching loss?

Leaching Loss Several authors have observed the loss of dry matter, crude protein, crude fat and ash in shrimp feed pellets immersed in water (Smith et al. 2002; De Muylder & Hillion 2003; Carvalho & Nunes, 2006). The reported losses range from 5-12% for dry matter within the first four hours of immersion in water. While all components of a feed are subjected to the action of water, the ones that are rapidly lost from the feed are water soluble components. These include simple sugars and starches, salts, amino acids and water-soluble vitamins. Smith et al. (2002) and Carvalho & Nunes (2006) reported nearly similar patterns of protein leaching in commercial shrimp feeds for the black tiger shrimp, Penaeus monodon and the Pacific white shrimp, Litopenaeus vannamei, respectively (Figure 1). Their data show that most protein losses in shrimp feed occurs within the first two hours of immersion in water. Nearly 15% of the crude protein in the feed is lost within the first three hours in the water. Highly water soluble cations such as sodium and potassium are lost up to 95% within the first hour of immersion in water (Figure 2: De Muylder et al. 2005). There are two paradoxes in the leaching of nutrients from shrimp feeds. The first paradox relates to the fact that certain water-soluble components of food serve as chemical signals indicating the presence of food to aquatic organisms. Particularly effective are nitrogenous compounds such as amino acids, short-chain peptides and biogenic amines. These compounds are commonly referred to as attractants and are required in the feed to facilitate rapid identification of the feed. A certain level of leaching of these compounds is, therefore, desirable. However, leaching is not a selective process. Along with attractants, other water-soluble nutrients that are not known attractants such vitamin C and phosphorus are also lost.

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02468

1012141618

0 2 4 6 8

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% protein loss

P. monodon feed (Smith etal. 2002)L. vannamei feed (Carvalho &Nunes, 2006)

Figure 1: Leaching loss of crude protein from two commercial shrimp feeds

0

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60

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100

0 0.5 1 1.5 2hours of leaching

% remaining DMCaMgNaKPAsh

Figure 2: Leaching loss of macro minerals from shrimp feeds (Source: De Muylder et al. 2005) The second paradox is that while water solubility of nutrients is undesirable from a leaching perspective, it may be desirable from a nutrient bioavailability perspective in shrimp. The shrimps are known to have a simple gut with no stomach or acid secretion. So, nutrients that are soluble in water may have higher bioavailability in shrimp, though it has been proven only in the case of phosphorus. Phosphorus forms that are highly soluble in water have the highest bioavailability in shrimp and abalone (Table 1).

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Table 1: Relationship between water solubility of phosphorus source and the availability of phosphorus from the source in shrimp and abalone P Source % P Loss After 1 h* Apparent P

Availability in Abalone*

Apparent P Availability in Shrimp**

Mono Sodium Phosphorus

33.4% 66.5% 68.2%

Mono Calcium Phosphorus

33.2% 72.6% 46.3%

Dicalcium Phosphorus

8.2% 27.9% 19.1%

* Sales et al. 2003 ** Davis & Lawrence 1997

The above two paradoxes present additional challenges to the practical nutritionist, formulator and feed manufacturer in designing cost-effective feeds for shrimp. Minimizing Leaching Loss Within the perimeter of the paradoxes presented above, there are a number of strategies to minimize leaching losses in shrimp feed: (1) Decrease the time between feeding and feed intake by shrimp. This can be done by

incorporating one or more concentrated feeding effectors in the feed that will minimize the searching time and accelerate feed consumption rate for shrimp. This strategy is explored more in detail elsewhere in this chapter.

(2) Size the feed appropriately to optimize both feed intake and leaching. A scientific assessment of whether smaller shrimp feeds result in more or faster feed consumption is essential. Smaller feeds are not only expensive to make due to manufacturing costs, they also cost more in terms of leaching losses. Due to high surface area to volume ratio, smaller feeds leach more rapidly than larger feeds. Furthermore, the process of manufacturing crumbles subjects the feed to mechanical stress and makes it more prone to leaching. Alternatives to crumbling such as spheronization to make intact micro-particles are available and should be explored.

(3) Coat the pellets or the water-soluble nutrient sources so that leaching losses are minimized in the water, yet there is no interference with digestion and bioavailability. Millamena et al. (1998) and Alam et al. (2002) have demonstrated that it is possible to coat extremely water-soluble crystalline amino acids such as L-lysine with carboxymethyl cellulose (CMC), gelatin and casein and achieve good growth rate in shrimp fed with diets containing the coated amino acids. Leaching of choline chloride can be reduced by 75-78% by coating it with CMC (Fady Raafat Michael, Kagoshima University, Japan. Personal communication). De Muylder & Hillion (2003) showed that top coating of pelleted shrimp feeds with 2% fish oil can substantially minimize leaching losses of dry matter and lipids, but not ascorbic acid. De Muylder et al. (2005) reported that a method combining chelation and coating reduced the leaching of macro minerals such as sodium, potassium and phosphorus. Improved performance

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of shrimp fed a diet incorporating the modified minerals indicated that the minerals were available to the shrimp even after modification.

(4) The method of application of nutrient sources may determine its leaching rate. De Muylder & Hillion (2003) showed that the use of ascorbic acid in the mixer with other ingredients before pelleting resulted in far lower leaching rates compared with spraying the ascorbic acid on the exterior of the feed pellets. So, water soluble nutrients are best applied during feed manufacturing than at the farm as a top coating. The only exception to this is attractants that can be sprayed on the pellet so they can leach out and indicate the presence of feed.

Use of Feeding Effectors to Minimize Leaching Losses “Feeding effectors” is a broad term that refers to chemoattractants, feeding incitants and stimulants that play key roles in the identification and consumption of food by aquatic animals (Smith et al. 2002). Decreasing the time between placing the feed in the water and feed intake by shrimp would help to lower the leaching losses. This can be achieved by incorporating a concentrated attractant in the feed that will minimize the searching time for shrimp. When the attractant also has stimulant properties, feed intake will be accelerated. Table 2 shows that topical application of a commercial feeding effector increased feed intake in shrimp in the first hour after feeding.

Table 2: Feed consumption by shrimp (P. monodon) given a feed with or without a top coating of a commercial feeding effector (Aqua Savor ™, Bentoli, Inc. USA) in check trays in commercial production ponds over a 1-week period in November 2003. Location of the trial: Ongole, Andhra Pradesh, India. Pond No. Feed Feed given (g) Feed consumed

(g) Higher consumption (%) in relation to control

1 Control 75 35 Control +

Feeding Effector

75 55 27%

2 Control 90 40 Control +

Feeding Effector

90 70 33%

3 Control 80 30 Control +

Feeding Effector + Oil

80 50 25%

4 Control 50 30 Control +

Feeding Effector + Oil

50 35 10%

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The importance of incorporating feeding effectors in shrimp feeds has long been recognized and commercial shrimp feeds have always been formulated with a number of ingredients that are known, natural sources of feeding effectors to the shrimp. These include fishmeal, fish solubles, fish hydrolysates, squid meal, squid liver paste or meal, shrimp head meal, shrimp meal and various bivalve meals. Yeast and yeast-derivatives have also been recognized as feeding effectors. However, inclusion levels of these ingredients are based on experience and mostly through specifying minimum levels in the feed. In spite of the vast amount of work that has been done on feeding effectors (see Lee & Meyers 1997 for a review), clear guidelines or objective criteria on inclusion levels do not exist. As a result, cost optimization based on feeding effectors in the ingredients or finished feed is not possible today. There is a need to develop an objective index of “feeding effectors” or at least “attractability” in ingredients and finished feeds so that feeds could be formulated based on target “feeding effector” or “attractability” values. It is also desirable to develop one or few laboratory methods that can be used to measure the values. Achupallas (2004, Personal Communication) proposed that a simple measure of protein solubility in water could be used as an index of “attractability” in raw materials and finished feed. This idea follows the facts that nitrogenous compounds are known attractants to shrimp and those nitrogenous compounds that readily dissolve in water are likely to be the most effective attractants. Table 3 presents the protein solubility values of some raw materials commonly used in shrimp feeds. Preliminary testing indicated that these values are probably valid indicators of “attractability” in the Pacific white shrimp, L. vannamei (Nunes et al. 2006). High levels of protein solubility corresponded with high levels of positive choices for the ingredient in Y-maze choice trials (Table 4). Table 3: Total protein and water-soluble protein contents of selected raw materials used in shrimp feeds (Data source: Achupallas, Unpublished) Raw Material Total protein (%) Soluble protein (%) Fish meal, Prime 67 13 Fish soluble (USA, Menhaden) 31 29 Fish soluble (Peru, Anchovy) 45 41 Fish protein hydrolysate 70 50 Squid liver meal 46 17 Aqua Savor®* 70 60 * Commercial product manufactured by Bentoli, Inc., USA Even while objective indices and values for “feeding effectors” in ingredients are needed, recent publications (Smith et al., 2005; Williams et al. 2005; Nunes et al. 2006) indicate the need to revise our understanding of ingredients that were previously thought to be key feeding effectors in shrimp feeds. Smith et al. (2005) found that squid meal was not a feeding stimulant in the black tiger shrimp, P. monodon and that feed intake declined with increasing inclusion of squid meal in the feeds. Squid liver meal was, however, an effective attractant in L. vannamei feeds (Nunes et al. 2006). Both Smith et al. (2005) and Nunes et al. (2006) found betaine to be a less

44

effective attractant. Smith et al. (2005) observed that P. monodon showed a significantly greater preference for feeds containing crustacean and krill meals (Table 5). Williams et al. (2005) found that the crustacean meal contained a growth factor that was present predominantly in the insoluble protein constituent of the meal. Table 4: Attractability of nine commercial ingredients to L. vannamei. Each comparison represents the response of one animal submitted simultaneously to two attractants. Total of 45 comparisons for each attractant. Data for detection and feeding represent time in seconds. (Source: Nunes et al. 2006) Attractant + choices (%)1,2 % rejection3 Detection5 Feeding5 %CP6 SP/CP7 Put8 Cad8 Hist8

CON 20.0f 22.2 -4 -4 46.7 66.2 851.4 0.0 0.0 VDB80 35.6ef 37.5 381b 80b 79.8 13.2 97.9 0.0 0.0 VDB68 40.0def 27.8 408b 345ab 68.1 10.1 0.0 0.0 0.0 CAA 66.7ab 0.0 313ab 495a 79.6 77.9 0.0 222.3 140.2 CFSP 73.3a 3.0 308ab 374ad 30.9 13.7 0.0 567.7 0.0 SLM 62.2abcd 0.0 256ab 364ab 41.5 23.8 910.2 145.9 0.0 Bet 42.2cde 15.8 321ab 134bcd 70.3 0.5 0.0 8.2 0.0

DFSLH 53.3abcde 8.3 321ab 288ab 89.2 14.0 696.4 1040.3 95.4 DFSHH 46.7bcde 19.0 363b 254ab 88.9 14.2 873.9 1380.0 167.7 WSPH 60.0abcd 0.0 202a 406ac 72.1 19.2 0.0 483.7 410.0 X2 P <0.001 -4 -4 -4 -4 -4 -4 -4 -4

1Positive choice (%) = (number of choices/number of comparisons) x 100; 2Values in the column which do not share a same superscript are statistically different between them by the z-test (P<0.05); 3Rejection (%) = (number of rejections/number of positive choices) x 100; 4Not applicable; 5Comparisons against the control diet (neutral gelatin + soybean meal); 6%Crude protein: N x 6.25, total N determined by auto-analyzer C, N, H; 7%Soluble protein: Bradford (1976) bovine serum albumine as standard; 8Putrescine, Cadaverine and Histamine in mg/kg by ionic chromatography. CON = Gelatin control; VDB80 = Vegetable Dried Biomass, 80% crude protein; VDB68 = Vegetable Dried Biomass + glutamate + betaine, 68% crude protein; CAA = Aqua Savor®, a commercial attractant/stimulant product made by Bentoli, Inc; CFSP = Condensed Fish Soluble Protein; SLM = Squid Liver Meal; Bet = Betaine; DFSLH = Dried Fish Soluble, Low Histamine; DFSHH = Dried Fish Soluble, High Histamine; WSPH = Whole Squid Protein Hydrolysate Conclusions

1. Aquaculture nutritionists and feed formulators need to account for nutrient losses through leaching in cost and performance optimization of aqua feeds.

2. In feeds for slow feeding species such as shrimp, the losses of water soluble nutrients in the first 1-2 hours represent significant opportunities for feed cost management.

3. While less water-soluble nutrients or protecting the nutrients from leaching are possible options for a formulator, it is necessary that the formulator consider whether the less soluble forms are as bioavailable to the animal as the more soluble ones.

4. There is a need to reevaluate the importance of feed size in feed intake in species like shrimp. Leaching rates increase with decreasing feed size. Crumbling also increases leaching rate.

5. Feeding effectors (attractants and stimulants) are useful tools to minimize nutrient delivery losses because they help to increase the rate of feed consumption.

45

6. Emerging research on feeding effectors indicate the need to critically reevaluate ingredients such as squid meal that were previously considered to be feeding effectors in shrimp.

7. The development of an easy-to-use and objective index of “feeding effectors” will help formulators and nutritionists to assess and value ingredients considered to be in rich in feeding effectors. This approach will also help in formulating diets based on a target level of “feeding effector.”

8. Protein solubility may have the potential to indicate the quantity of “feeding effectors” in an ingredient. Research is currently underway to validate this hypothesis.

Table 5: Daily feed intake by the black tiger shrimp, P. monodon, when provided a choice of the base, control feed and a feed containing the test ingredient. Source: Smith et al. 2005. Daily Feed Intake Base feed (%) Test feed (%) Control, Base Feed 50.4 49.71 Test Feeds Squid meal 2.5% 61.1 38.9 Squid meal 5% 48.3 51.7 Crustacean meal 1% 33.5 66.5 Crustacean meal 2.5% 45.4 54.6 Crustacean meal 5% 10.2 89.8 Krill meal 1% 65.7 34.3 Krill meal 2.5% 31.3 68.7 Krill meal 5% 19.3 80.7 Fish Hydrolysate 0.5% 47.2 52.8 Fish Hydrolysate 1% 57.6 42.4 Fish Hydrolysate 2% 50.4 49.6 Krill Hydrolysate 0.5% 59.8 40.2 Krill Hydrolysate 1% 57.4 42.6 Krill Hydrolysate 2% 65.5 34.5 Betaine 0.5% 43.8 56.2 Betaine 1% 63.1 36.9 Betaine 2% 60.5 39.5 1 – The shrimp were given a choice between two trays containing the same control, base feed. References Alam, M.S., Teshima, S., Yaniharto, D., Koshio, S. & Ishikawa, M. 2002. Dietary amino acid

patterns and growth performance of juvenile kuruma prawn, Marsupenaeus japonicus. Comparative Biochemistry & Physiology, Part B, 133: 289-297.

Carvalho, E.A. & Nunes, A.J.P. 2006. Effects of feeding frequency on feed leaching loss and grow-out patterns of the white shrimp Litopenaeus vannamei fed under a diurnal feeding regime in pond enclosures. Aquaculture 252:494-502.

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Davis, D.A. & Lawrence, A.L. 1997. Minerals. Pages 150-163 In: L.R. D’Abramo, D.E. Conklin & D.M. Akiyama (Eds.) Crustacean Nutrition. Advances in World Aquaculture, Volume 6. The World Aquaculture Society, Baton Rouge, USA.

De Muylder, E. & Hillion, B. 2003. Nutrient leaching: inclusion of extra nutrients in shrimp feed. International Aqua Feed, November-December, 31-33.

De Muylder, E. Tu, H.T. & Van Speybroeck, M. 2005. The influence of macrominerals (Ca, P, Mg and K) in the diet on growth of Litopenaeus vannamei at low salinities. World Aquaculture 2005, Book of Abstracts, p. 152. World Aquaculture Society, Baton Rouge, LA, USA.

Lee, P.G. & Meyers, S.P. 1997. Chemoattraction and feeding stimulation. In: Crustacean Nutrition (D’Abramo, L.R., Conklin, D.E. & Akiyama, D.M. eds), pp. 292–352. World Aquaculture Society, Louisiana State University, Baton Rouge, LA, USA.

Millamena, O.M., Bautista, M.N., Rayes, O.S. & Kanazawa, A. 1998. Requirement of juvenile marine shrimp, Peneaus monodon Fab. for lysine and arginine. Aquaculture, 164: 95-104.

Nunes, A.J.P., Marcelo V.C., Andriola-Neto, F.F., Oliveira, G. & Lemos, D. 2006. Measure of feeding stimulation of commercial attractants for the white shrimp, Litopenaeus vannamei through behavioral bioassays and ingredient chemical profile. Abstracts, AQUA 2006, Annual Meeting of the World Aquaculture Society, Florence, Italy, May 9-13, 2006.

Sales, J., Britz, P.J. & Viljoen, J. 2003. Dietary phosphorus leaching and apparent phosphorus digestibility from different inorganic phosphorus sources for South African abalone (Haliotis midae L.). Aquaculture Nutrition 9:169-174.

Smith, D.M., Burford, M.A., Tabrett, S.J. Irvin, S.J. & Ward, L. 2002. The effect of feeding frequency on water quality and growth of the black tiger shrimp (Penaeus monodon). Aquaculture 207:125-136.

Smith, D.M., Tabrett, S.J., Barclay, M.C. & Irvin, S.J. 2005. The efficacy of ingredients included in shrimp feeds to stimulate intake. Aquaculture Nutrition 11:263-272.

Williams, K.C., Smith, D.M., Barclay, M.C, Tabrett, S.J. & Riding, G. 2005. Evidence of a growth factor in some crustacean-based feed ingredients in diets for the giant tiger shrimp, Penaeus monodon. Aquaculture 250:377-390.

Acknowledgements: Thanks are to Dr Alberto JP Nunes of Instituto de Ciências do Mar (Labomar), Brazil and Dr David Smith of CSIRO Marine Research, Australia for access to unpublished data.

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Formulation software and handling variability Feed manufacturers can reduce variability in finished products by modeling nutrient variability and applying statistical process control techniques in formulation and production. Merryl Webster, Format International Some years ago, Format International embarked on a serious investigation of the sources of nutrient variation and error in feed production. Prompted at the time by the difficulties facing nutritionists of reliably and consistently producing feed mixtures which conformed to specification, we undertook to try to understand the impact of variability in raw materials and to find a way to improve formulation techniques. The project resulted in the development of new software tools which assist formulators in assessing the risk of variation and which offer some strategies for reducing its impact. Additionally several action points came out of the exercise which can be applied in any conventional feed manufacturing plant which is concerned to minimize the adverse impact of unintentional variation in feeds. Formulators often assume that the vast majority of finished product variability is accounted for by the true variability of the raw materials in use. Everyone knows that ingredients vary from load to load — we understand many of the reasons — and formulators normally follow a programme of sampling, testing, and regular matrix updating in order to have the best possible prediction of current nutritional profiles prior to preparing a new formulation. Netting out sampling and analytical error In this connection, it is worth distinguishing between ‘variability’ and ‘error’ or ‘uncertainty’. Raw materials are truly variable; however, when sampled and tested, error, or uncertainty, is introduced which further can complicate the picture. Laboratories usually report standard deviations which contain both the true variation and the sampling and analytical error. This inflates the true picture and the other errors must therefore be addressed before the impact of the true variation can be handled in any predictive model. We took reported ingredient standard deviations and netted out sampling and analytical error in order to use the ‘true’ variability in a predictive model. When ingredient standard deviations alone are used to predict final product variability the results vastly understate the actual observed variation measured through finished product testing. In fact, the ingredient variability, per se, accounts for only 25% of the observed finished product variability. So where is all the other variability coming from if it cannot be explained by the ingredients alone? Production process error When we start looking at the production process, we immediately realise that there are weighing errors — deviations between the requested quantities of ingredients and the quantity actually delivered onto the scales.

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After raw material variation, the next major source of error stems from the automated weighing and batching process. Weighing errors are related not only to the size of the weigh scale, but also to the ‘in-flight’ weight of material, to particle size and flow characteristics, and possibly to other unexplored factors. When analysing the effects of weighing error, it is necessary to assign an individual error to each raw material and this is best expressed as a standard deviation in kilos or pounds. Since a weighing error is incurred for each ingredient in the batch, then the greater the number of ingredients in the mixture, the greater the weighing errors. Producing larger batch sizes will incur proportionately smaller weighing errors than production of small batches—given the same size scales. It follows that logical designs for feed plants will incorporate weighing scales of different capacities to cope with the need to weigh accurately very different quantities of constituent materials into a batch. This is very largely observed in practice, except that there is still the odd plant which tries to weigh (accurately!) 5 kg of salt onto a scale of maximum capacity 2500 kg. Multiplicative error Previous published work took an additive approach to the analysis of product variance, as though the impact of ingredient variation was independent of the weighing system. Whilst this simplification might have been valid or tolerable if weighing errors were very small, it is not appropriate in a dynamic weighing system where errors are not small. In fact, raw material variation and weighing errors interact in a multiplicative way which amplifies the potential variation in the composition of the product. While this statement is not immediately intuitive, it does make sense when thought through. For example, the quantity of protein from soybean meal delivered into a mixture of feeds is a function not only of the protein concentration in the meal, but also of the quantity weighed onto the scale. Thus variation is amplified through the interaction between ingredient variability and weighing errors. Let us assume that we can obtain good predictions of the plant weighing errors (and most computer controlled batching systems should be able to readily generate such data), and that we have good measurement of the ingredient variability, most commonly expressed as a standard deviation. Can we now obtain a better prediction of the likely variability of a given finished product formulation? Can we explain more than 25%? The multiplicative model we developed which predicts the standard deviation of the nutrient attributes of the finished feed incorporates: ● Reported ingredient standard deviations; ● Weighing errors by ingredient; ● Product batch size; and ● Sampling-analytical error. We find it is able to account for around 85% of the observed variability in finished products, making it much more useful, then, than considering raw material variation in isolation. It is now

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possible to look at proposed formulations, prior to production, in order to ‘risk assess’ them for compliance with the intended nutritional specification. Formulators can see the probability that their formulas will fall within the intended nutritional or legal constraints, and can make informed decisions which balance product consistency with cost effectiveness. Error in a sample For example, taking a test ration for which we know the mean and standard deviations for the nutrients ‘oil’ and ‘protein’, and the other inputs, we can compute the expected standard deviation on oil and protein in the finished product. From that, we can calculate the probability of meeting the specification. In the screen image extract (Figure 1), we have an expected mean protein level and mean fat level both of 40% resulting from a formula where the constraints were set at minimum 40% and maximum 40% for both nutrients. We have also said that +/- 5% variation from the lower and upper bounds is ‘acceptable’, giving us a target range of 38% to 42%. We can see that the probability of being within our target range is 61% for Protein, and 78% for Fat. Since we would have liked a probability of 90% and 80% respectively, these are flagged on screen as errors.

Figure 1: Risk assessment for Protein and Fat in Test diet, showing higher than acceptable probability of being outside nutrient specification

Once formulators and plant managers have a good predictor of the ‘normal’ or ‘expected’ finished product variability, they are better able to recognise the abnormal and know when to intervene. NIR-based segregation at intake Another point of note generated by our original work was the identification of the benefits of rapid testing, using near-infrared reflectance analysers (NIR) at the point of ingredient intake. Now, NIR testing has become much more affordable and much more common, and it continues to strengthen in appeal and reliability. For example, deliveries of an ingredient — say wheat — may be NIR-tested at intake for protein, and then ‘streamed’ or segregated into different bins of low-protein and high-protein based on an expected mean. Figure 2 shows the results of fractionating 132 deliveries of Wheat into either

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“High CP” or “Low CP” bins. Deliveries below an expected mean of 11.2% were streamed into the “Low CP” bin and deliveries above went into the “High CP” bin. This results in two bins of material, which of course are different in mean protein level, but much more importantly, which are individually much less variable. The standard deviation of the chosen nutrient is around half that in materials streamed on intake, compared with the full population of all deliveries of that material. This is an easy way of ‘manufacturing’ reduced-variability materials. An interesting further consequence of segregation of wheat according to protein content (into low-protein and high-protein lots) is that it results in two materials which also have significantly different contents of fat and fibre, but not ash. Figure 3 shows the distribution of oil content in the two bins of materials – Low CP and High CP – which result purely from separation according to protein content.

Crude Protein contents of Wheat

0

5

10

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25

30

9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5

Crude Protein %

Num

ber o

f Sam

ples

Low CPHigh CP

Figure 2: NIR fractionation of wheat deliveries according

to protein content

Oil Contents of Wheat

0

5

10

15

20

25

0.1 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2

Ether Extract %

Num

ber o

f Sam

ples

Low CPHigh CP

Figure 3: NIR fractionation of Wheat deliveries on protein content also generates differences in oil content

52

The ramifications of variations in other nutrients associated with variation in the chosen nutrient are not always appreciated by the industry at large. One reason is the lack of data on covariance properties of raw materials, owing to the fact that few samples are exhaustively tested for all nutrients on the same sample. It is still more common to concentrate on testing only the ‘critical’ nutrients. Less nutrient testing ahead? Recent trends are for even less testing to be carried out on conventional nutritional composition of ingredients, owing to the necessity of spending more of the lab budget on microbiological testing and other safety issues. This is a concern. Another point of concern is that few plants accurately determine the variability of materials within a given load or delivery. Standard operating procedures may specify that the load is sampled at several different points, but almost everyone then amalgamates those samples into one, which is then tested. This technique loses any measure of within-batch variability, which may well be just as large as that between deliveries. Why does that matter? Well, it matters to those manufacturing plants which pursue rapid updates of matrix and reformulation following testing of materials at intake (or, rather belatedly, as the materials are being drawn out of the bins on the way to the scales). The danger is that the testing protocols may cause the compositional matrix to oscillate unnecessarily with consequent repercussions on formulation stability and on logistical supply issues. Predicting nutrient variability in products The proper prediction of the variability of nutrients in a product requires information on material weighing errors, on ingredient variation, on sampling and analytical errors, and on batch size. Weighing errors and ingredient variation interact in a multiplicative way and account for around 85% of the variability in finished products. The utility of predicting the standard deviation of nutrients for a formula increases dramatically when applied to formulation software tools. The tools allow the user to: ● Specify the desired probability of meeting a nutrient level; ● Quantify the value of variable versus consistent materials; ● Assess the cost benefits of streaming ingredients and of rapid testing; and thus ● Make informed decisions on investment strategies. Perhaps most important of all, such software allows the formulator to understand what is normal variation and what is abnormal — and thus when to relax and when to panic!

53

The truth about moisture uniformity and equilibration Paul McKeithan, Aeroglide Corporation Everyone knows that feed moisture uniformity is important and most plants monitor the uniformity of the product. However, not everyone understands how critical it is to have a good pellet to pellet uniformity. Here are a number of reasons why you need to take a closer look at your product moisture uniformity. Product Quality: Product stability is one of the main reasons you monitor moisture content. You are actually targeting a certain water activity in the product. Water activity is defined as “a measure of the energy status of the water in a system or the degree to which water is “bound” and, hence, its availability to act as a solvent and participate in chemical and biochemical reactions and growth of microorganisms.”1 It is important to monitor this water activity to know that the feed is at a stable state and, therefore, does not have enough free water to create microbial growth. However, it is not as practical to measure water activity in a production environment so moisture content is generally measured instead. For a given product moisture content can be related to the water activity. With this knowledge you can target feed moisture that is safe from mold and an acceptable quality for your customers. Production Economics: If you are producing dried extruded feed and you are not focusing on your drying uniformity, you may be wasting a significant amount of money. You are aware that non-uniform drying can cause mold growth in your packaged product due to wet pellets, or result in wasted money due to excessive energy used to over-dry some pellets, but you may be overlooking the biggest cost of non-uniform drying. Drying your product non-uniformly is like throwing product out the exhaust stack. This is because your feed product is sold based on the weight of product shipped out your door. One of the key specifications for feed products is the moisture content. Suppose that your finished feed cannot contain over 10% moisture, and that your drier is only drying the product to +/- 3% moisture on a wet weight basis. This means that you must dry your product to 7% moisture on average in order to ensure that no product is over the 10% moisture maximum. The result is that you are sending 3% less product to packaging than if you dried to only 10% moisture. This lost production is going out the drier exhaust stack in the form of water vapor. If your drier could be made to dry more uniformly, you could raise your discharge moisture and get more production out of your dry feed line with no additional cost or ingredients. The value of this lost production can be staggering. Consider a 15 ton/hour dry feed line that is producing feed overdried by just 3% moisture. If the line runs 24 hours per day for 350 days a year, the lost production is:

yeartonsyeardaysdayhrtph /3780%3/350/2415 =×××

1 Publication no. W-1999-1214-01F. 2000 American Association of Cereal Chemists, Inc. Understanding the Importance of Water Activity in Food, A.J. Fontana, Jr.

54

That’s an additional 3,780 tons of product per year that could be sold simply by drying more uniformly. Most common causes of poor moisture uniformity: First, the product must be consistently extruded. Once an extruder is set-up and allowed to run in a steady state production it will usually produce a consistent feed. The feed that leaves the extruder is high in moisture and temperature and therefore willing to release its moisture quickly. The drying curve is linear at this time; meaning that the product will lose its moisture as if it was just a cup of water. It is critical that each piece of product travels the same process path as it goes from a soft wet feed to the stable dry feed. Any difference along this path is a source of inconsistent product moisture. Some of the more common areas for this moisture difference occur in the drier and are caused by but not limited to: inconsistent bed loading, uneven airflow, and uneven product retention time. Tips to improve moisture uniformity: The drying process is often where the most product moisture inconsistency can be introduced so it is important to have this process evaluated. An experienced drier expert will be able to adjust the drier to achieve its best potential moisture uniformity for that particular drier configuration and design. The drier expert should also conduct a detailed moisture uniformity check. Most daily process moisture samples are taken after the drying process in a mixed product stream. This sample would represent a masked sample. Taking a sample every so often is fine for production records, but for product quality or economics, a more discrete set of moisture samples must be taken. It is important that you understand your piece to piece product moisture uniformity. Moisture Equilibration: You may be one of the many feed producers who feel that piece to piece variations are not important since you believe that product will equilibrate in the bins or after packaging. This is not the case! The following graph shows typical results from tests conducted using extruded feed to monitor the rate of equilibration. This particular test placed several samples of 9mm extruded feed taken at two different moisture contents in a sealed bag. The wet sample was approximately 12.5 % moisture wet weight basis. The dry sample was approximately 5.5% moisture wet weight basis. These samples were mixed and packaged together. The combined average moisture content, similar to a typical production moisture sample, was approximately 9.2%. At different intervals the samples were analyzed and the results were telling. The equilibration curve may surprise you. The graph below shows the equilibration curve for 7 hours. There is some equilibration but it is clear the curves do not meet at the average. Even after several days the product maintained a 3% moisture difference. So, although the average moisture sample was 9.2%, after 7 hours the bag still contained product with moisture content over 11%. This higher moisture content could cause mold growth yielding product unacceptable to your customers. So with this in mind, when you are performing a moisture tolerance investigation, you should check your product after the drying stage and prior to any mixing. Because this can be difficult, let’s review some important considerations.

55

Equilibration Curve1 kg wet with 1 kg dry feed mixed in plastic lined bag

4.005.006.007.008.009.00

10.0011.0012.0013.0014.00

0 2 4 6 8

Time (hours)

Moi

stur

e (%

ww

b)

Dry Product (%wwb)Wet Product (% wwb)Average (%wwb)

The vast majority of extruded feed driers are horizontal conveyor driers. However, a few producers have installed some vertical semi-continuous batch driers on extruded feed lines. Different methods are required. Typically, taking samples from a conveyor drier is much easier due to better access and is continuous rather than a semi-continuous mode of operation. Collecting product moisture samples from a conveyor drier On a conveyor drier, you will want to investigate cross machine moisture variations, as well as transient moisture variations in time. You may also want to take samples from the first or second bed in a two or three pass drier to help in pinpointing the source of the variations. Figure 1 shows where to take the discharge samples.

56

Figure 1: Side view of a two-pass Horizontal Convection Conveyor drier showing the sample

location. The samples should be taken as a matrix in the vertical plane of the product prior to discharge from the bed. The bed of product can typically be accessed through the end doors of the drier. Figure 2 shows the discharge product sample matrix.

Discharge Sample Location

Product Feed

0.3m

1/2 of Depth

1/2 of Bed Width

Sample Locations

0.3m

57

Figure 2: Location grid for moisture samples taken on a Horizontal Convection Conveyor Drier. Collecting product moisture samples from a vertical drier Taking samples from a vertical drier is considerably more difficult since you have very limited access to the decks of product. Also, you will need to take more samples in a “set” of samples since the vertical drier is a batch operation rather than a true continuous operation. This means that you are now looking to measure moisture variations across the whole deck of product rather than just across the bed as in a conveyor drier. Unfortunately, these difficulties make it very challenging to take a proper set of samples from the vertical drier. You will need to find a safe and effective way to collect these samples as different vertical configurations present different challenges to access the product prior to mixing. You should remember that a moisture gradient can exist through the depth of the product as well as across the width and length of the deck. Remember that you should not take samples at the discharge because this will be a mixed sample that can mask the piece to piece moisture content.

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Figure 3: Front cross section of a Vertical Convection Drier. So why should you care: As pointed out in this article, there are many compelling reasons to care about product moisture uniformity. With a tight moisture tolerance the economical savings are great. You are now efficiently utilizing your cheapest product ingredient - water, without fear of producing an unstable and non-saleable product. And with new evidence that shows product moisture equilibration is at best dampening the effect of significant moisture uniformity, the key is having the right drying equipment and operating it properly.

Product sample location. Last heating level prior to cooling.

59

0

Mill Technology CompanyMill Technology Company

Optimizing Profit in the Size Reduction Industry

Mill Technology Company

2006

60

Mill Technology Co.Mill Technology Co.© Copyright 2006 Mill Technology Company

About Mill Technology Company

Leading, Global Manufacturer of Hammermill Equipment

Over 30 years of Hammermill experience

Customer service focused

Products target the Aquatic Animal and Pet Food Industries

Based in Minnesota, USA

61

Mill Technology Co.Mill Technology Co.© Copyright 2006 Mill Technology Company

A Sample of our Installed Base Is Diverse and Multinational Demonstrating Our Capabilities to Work in All Regions of the Globe

Friskie’s Pet Food• San Paulo, Brazil

Pet Food App•Sydney, Australia

Aquatic China

Feed App• 6 US Plants

Nelson & Sons• Utah, USA

Pet Food App•Missouri, USA

Industrial App• USA, UK

Mill Technology Company

Corporate headquartersSample Installed Mill Sites

6 Shrimp App•Thailand•Vietnam•Indonesia

Burris Feed• 3 Mill System

62

Mill Technology Co.Mill Technology Co.© Copyright 2006 Mill Technology Company

CoarseHog

Extra FineCosmetic

Talc

Mill Technology fills a void in the Hammermill Industry, Particularly in the Quality Grind Application

PinLarge Hammermills

Quality Grind Roller Mills

Mill Technology’s equipment falls into the Quality Grind Application area. Through better technology these products improve productivity and

efficiency increasing the value provided to our clients.

TraditionalHammermill

Air Classified

Mill TechnologyAltima Hammermill

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Mill Technology Co.Mill Technology Co.© Copyright 2006 Mill Technology Company

What Industries are presently using Pre/Post Mix Grinding?What Industries are presently using Pre/Post Mix Grinding?

Pet Food

Aquaculture Shrimp Feed Manufacturers

Salmon Feed Manufacturers

Trout Feed Manufacturers

Manufacturers requiring high quality finished product

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Mill Technology Co.Mill Technology Co.© Copyright 2006 Mill Technology Company

Why are these types of Feed Manufacturers using this method?

Because they require…

High Quality Product

Consistence of Quality

Quality Products at Competitive Prices

65

Mill Technology Co.Mill Technology Co.© Copyright 2006 Mill Technology Company

Tradition Hammermill Full Screen

66

Mill Technology Co.Mill Technology Co.© Copyright 2006 Mill Technology Company

Air Swept Classification

A common misconception…

It’s not the need for super fine

products it’s the lack of larger

particles

67

Mill Technology Co.Mill Technology Co.© Copyright 2006 Mill Technology Company

Advantages of using a Pre/Post Mix Grinding System

Efficiently increase plant tonnage

Remain competitive while expanding

Improvements in product and service quality

Increased production efficiencies

Greater mix homogeneity

A decrease in coefficient of variation (CV)

Improvements in grind quality

Improvements in the starch gelatinization

Better steam and moisture absorption in the conditioner

An increase in pellet mill performance and efficiencies

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Mill Technology Co.Mill Technology Co.© Copyright 2006 Mill Technology Company

Theory of Altima Hammermill Pulverizing Operations

The greater the velocity differences, between an impact force, and the products

exploding against a zero velocity impact device the greater the reduction

capability

69

Mill Technology Co.Mill Technology Co.© Copyright 2006 Mill Technology Company

9 Rules of the Post Grind Hammermill

1. The hammermill must operate at 1500 or 1800RPM.

2. The hammermill should have bi-rotational capability.

3. The hammermill must have multitude quadrants, impact plates and screens.

4. The hammermill must have internal de-acceleration chambers.

5. It should have maximum impact forces. (Hammers)

6. The hammers should be easily and quickly changed.

7. The top section should be designed with additional cutting surface.

8. The top section should be closed, therefore not allowing assisting air to discharge without effecting or assisting the grind operation.

9. The screen carriages should be strong enough to maintain hammer to screen clearance.

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Mill Technology Co.Mill Technology Co.© Copyright 2006 Mill Technology Company

Impact of Air Flow

Good air comes in through the mill assisting the grind processBad air bleeds in from places other then the mill reducing the amount of good

air that can assist the grinding process

71

Mill Technology Co.Mill Technology Co.© Copyright 2006 Mill Technology Company

Proper System Configuration for Air Assisted Hammermill Operation

72

Mill Technology Co.Mill Technology Co.© Copyright 2006 Mill Technology Company

Post Grind Percentages

03.6

10.819.3

28.7

38.5

48.6

58.467

7582.2

88.292.4 94.8 96.6 98 99.2 100100

0

20

40

60

80

100

120

Micron

Perc

ent

704497.8

352248.9

176124.45

8862.23

4431.11

2215.56

117.78

5.53.89

2.75 1.381.94

Mesh25

3545

6080

120170

230355

400

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Mill Technology Co.Mill Technology Co.© Copyright 2006 Mill Technology Company

Mill Technology is Changing The Way Our Customer’s Perceive Value

Our product line’s performance continues to set new standards in productivity and efficiency

Double capacity

Consume half the energy

Spend pennies on the dollar in consumable parts

Controlled particle size distribution with less deviation

Achieves a great degree of fineness then conventional hammermills

74

Making More Profit With New Technologies for Aquafeed Mian N. Riaz, Ph.D., Extrusion Technology Program, Food Protein Research & Development Center, Texas A&M University Introduction Extrusion is not new and the basic principles of extrusion are the same since it was introduced to the feed industry. On the other hand, extrusion companies continuously are trying to bring new technologies in the market that can be adopted with extruders to process a good quality aquafeed. Currently an aquafeed producer can use these new technologies, which will allow the person to make floating, sinking, slow sinking and high fat aqua feed at the same extruder without changing the configuration. These new technologies have increased the flexibility of extruders, together with one of the most important resulting benefits, minimizing down time required for configuration changes. These new technologies allow feed processor an option to choice a wide range of shape, density, texture and color of the pellet. New Technologies for Aquafeed: In this article, the author will try to summarize some of the new technologies, which are presently available in the markets to improve the process of aquafeed production. 1. Retention time controlled device for preconditioner 2. Conical co-rotating twin-screw extruder 3. Back pressure valve for density control 4. Post extrusion pressure chamber devices to control bulk density 5. Mid barrel valve for controlling shear stress and SME 6. Multi-color and multi-shape die design Retention time controlled Device for Preconditioner A new innovation is the retention time controlled (RTC) preconditioner, which allows the operator to control and adjust the preconditioner retention time online. This system gives the operator the following benefits: • Continuous control of conditioning cylinder retention time. • Simplified startup sequence and reduced off-spec product during startup. • Constant discharge rate of feed during shutdown or product changeover. • Increase retention time on current conditioning cylinders. • Time and temperature documentation for process verification records for the

production of clean feeds. This system requires two key components to be added to a conventional preconditioner. First, a metering device must be mounted at the discharge of the conditioning cylinder. It acts as a “choke” point enabling the conditioning cylinder to be filled to a much higher level. This allows the operator to make use of a greater percentage of the conditioner’s free volume. The operator enters the retention time and the desired production rate. Raw materials are metered into the conditioning cylinder by a feeding device. To maintain

75

proper control this feeding device must operate in a gravimetric mode. The other critical component is that the preconditioner must be mounted on load cells to measure the weight of feed held in the cylinder. Based on the dry feed rate and the weight of material in the cylinder, the control system then is able to accurately determine the instantaneous retention time in the preconditioner. Based on the retention time and feed rate set points, the controller sets the discharge feeder speed to deliver the appropriate rate to the extrusion system. This feature allows the process retention time to be adjusted depending on the product characteristics. For example, some formulations may require additional retention time to allow for complete hydration of the mash. This system allows for online retention time adjustment. The retention time is adjusted during the process without the operator needing to shut down and make any hardware adjustments to the beater or paddle configuration. In addition this feature simplifies the startup, shut down and product changeover sequence. It allows better utilization of raw materials and avoids cross contamination between recipes and products. During startup, the raw material is metered into the conditioning cylinder and is mixed with steam and/or water to begin the hydration and cooking process. The discharge feeder remains off until the mash within the conditioning cylinder has been held for the desired retention time. Then the discharge feeder begins delivering the conditioned mash to the extruder. This dramatically reduces the material wasted during startup procedures for standard conditioning cylinders. The operator no longer must discard the mash while waiting for the conditioning cylinder to reach the operating temperature and moisture content. Obviously some material will still need to be discarded; however, the total amount can be dramatically reduced. During shutdowns or product changeovers, the discharge feeding device continues to deliver the conditioned mash at the specified rate. Thus, the extruder continues operating at its optimum until the conditioning cylinder is virtually empty. In traditional systems, the extrusion rate slowly decreases once the raw mash is no longer metered into conditioning cylinder. This reduces the amount of waste material and the amount of off-spec or contaminated product. Retrofitting a current conditioning cylinder with the loss-in-weight controls can increase the retention time and possibly allow an increase in production capacity. The feeder mounted at the conditioning cylinder discharge acts as a restriction and allows the cylinder to be filled to a higher level. Thus, current conditioning cylinder that operates with a fill level of 40 percent may be able to reach a fill level of 60 or even 70 percent. This would greatly increase the amount of retention time. Also, by more fully utilizing the conditioning cylinder’s volume, an increase in production capacity may be realized if there are not downstream process restrictions. Finally, this system allows process documentation of the times and temperatures the mash was subjected to during processing. This is especially useful for those concerned with pathogen destruction and food safety concerns. Since the retention time is one of the user inputs for the control system, the operator can document with certainty that the mash was held at a given temperature for a specified period of time.

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Although a loss-in-weight conditioning cylinder offers many benefits, it is not required for all situations. In situations where long production runs on a single product occur, the additional cost for this system may not yield sufficient economic benefits to offset the additional capital costs. However, for those systems in which frequent product changeovers occur, or wide variation in raw materials exists, the additional capital investment could quickly be recouped from the reduction in product waste, increased product quality, and increased in product capacity. Conical co-rotating twin-screw extruder Most of the aquafeed industry uses single screw extruder to produce floating fish feed. Twin screw extruders are also used when more than 17% fat is required in the pellet or size of the pellet is less than one mm. At the same time the cost of the twin screw extruder is almost double than the single screw extruder. Extruder manufacturer came up with new design of twin screw extruder which is called conical co-rotating twin screw extruder. This is latest technology which is available to aqua feed industry to make floating and sinking feed without changing any configuration. This extruder design allows for positive compression in the barrel and reduces possibility of back feeding. Positive compression yields an efficient manner of imparting mechanical energy into the extrudate. The conial design of this extruder causes the material to be kneaded and sheared along the screw profile. In traditional twin screw extruders, the melt is kneaded and sheared by shear locks, mixing lobes, or cut-flight screw elements. This “profile kneading” eliminates the need for special screws and locks to provide the appropriate cooking. Extruder shafts and screws can be machined from a single piece of steel, resulting in lower manufacturing costs. Maintenance and down time are also reduced, because a screw profile change is not needed for each different product. Back Pressure Valve Final product characteristics such as density can be controlled by extruder die restriction. Extrusion industry has developed the revolutionary back pressure valve (BPV) to adjust die restriction while the extrusion system is in operation. By changing the restriction at the discharge of the extruder during operation, the product density can be varied by up to 25 percent without changing the screw configuration or the final die. The variable-opening BPV is mounted on the end of the extruder prior to the final die. Specific Mechanical Energy (SME) and extrusion pressure are process parameters controlled by valve positioning. The BPV provides internal control of shear stress and SME for regulation of important product properties:

• bulk density • size and uniformity of cell structure • starch gelatinization • shape definition • water and fat absorption

77

The BPV also reduces the need for altering the extruder configurations between different product families. An integral part of the BPV is a by-pass feature to divert product from the die/knife assembly for service and start-up/shutdown procedures which also improves sanitation in this area. Post Extrusion Pressure Chamber Another new technology which is available in the market from several extruder manufacturers is an enclosed chamber which surrounds the die/knife assembly and permits control of pressure external to the extruder and die (often referred to as an EDMS or External Density Management System.) Desired pressures are maintained in the knife enclosure by a special airlock through which the product discharges. Compressed air or steam can be used to generate the required pressure in the chamber. As pressure increases, the water vapor point increases which reduces product “flash-off expansion” and thus increases density. Expanded or partially expanded products which normally exit the extruder die at a bulk density that is lower than desired, can be “densified” with this post-extrusion pressure chamber (EDMS) around the die/knife assembly. One particular challenge in the aquatic feed industry is to produce a fully-cooked feed of sufficient bulk density to sink rapidly and still absorb the required oil during the coating step. It is reflected that EDMS pressure adjustment was independent of extrusion processing parameters to control product density in a suitable range for 17-24 percent total oil in the final products. No other extruder operating parameters were changed except the chamber pressure to achieve this flexibility in product characteristics. The pressure chamber can be coupled with a BPV to provide additional process controls: adjust SME on-line for control of critical product properties, divert off-spec product during startup from the pressure chamber, accurate control of product density external to the extruder and die, no extruder configuration changes required to make expanded or dense feeds, and increase extruder capacity over vented configurations by 25 to 50 percent. Mid-Barrel Valve Extrusion companies came up with the idea to install a valve in the middle of the extruder barrel to serve as an adjustable restriction device for controlling shear stress and SME during extrusion of aquafeed. The name of this valve is mid-barrel valve (MBV). The MBV can be adjusted from a setting that adds little or no restriction to a setting that can almost completely restricts the passage of the extrudate, and has demonstrated SME increases of 100 percent or more. Insertion of this on-line valve can greatly enhance the flexibility of the extrusion process without the downtime associated with configuration changes. A mid-barrel valve can also be connected to the extruder control system to automatically adjust and maintain the SME valve to its desired set-point in order to make floating or sinking aquafeed.

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Manufacturing Multi-Color and Multi-Shape Feeds Dry expanded feed can be a variety of shapes and colors – either as a single shape or color, sold alone in a package, or as more commonly found in today’s marketplace, a mixture of shapes and colors sold together in a package. The trend towards products of this type versus the less interesting single shape and color varieties becomes evident when surveying the products available in the local supermarket. A recent trip to the local supermarket for this purpose reveals that roughly 75% of dog food products and 80% of cat foods are mixtures of shapes and colors as compared to the single shape and single color varieties. Some products are a relatively simple blend of different shapes all having the same color and made from the same formulation. While there are some inherent challenges to making products of this type, of greater interest are products that are blends of different colors. Products having different colors are inherently products of identical formulations, where the coloring agent is often the only formulation difference. There are at least three options available to the pet food manufacturer for manufacturing multi-color and multi-shape products. These options are a blending bin system, a multi-color/multi-shape extrusion die assembly, or multiple extruders each producing a different colored product and blending the products on-line. The blending bin system is the most commonly used method to manufacture multi-color products. With this system, products of different colors are manufactured by extruding, drying, coating, and cooling, and then storing each color separately in a storage bin. After the requisite number of colors has been manufactured, the products are mixed together by metering products out of the bins at the desired ratios, blending them together, and packaging the blended product. The multi-color/multi-shape extrusion die assembly is an extruder attachment recently introduced by an extrusion company as an extruder accessory that can be retrofitted to existing twin screw extruders. It allows the continuous and simultaneous production of a blend of colored products at the extruder die. These colors then pass together through the dryer and coating system, and on to packaging. The final method of manufacturing multi-color products is to set up a processing plant with multiple extruders, each making a different single product color. These products are then blended by passing them through a common dryer that is serving multiple extruders, or by passing the product from each extruder through its own dryer and then blending the products of different colors together as they pass through the coating system. The blending bin system and the multiple extruder system are relatively well-known in the industry and therefore do not require extensive additional discussion here. However, the multi-color/multi-shape extrusion die assembly is a new and innovative solution to manufacturing multi-color products and merits further discussion. The multi-color/multi-shape extrusion die assembly is specifically for twin screw cooking extruders. This revolutionary design allows the continuous and simultaneous production of multiple color and multiple shape products. Liquid dyes are injected into the extrusion

79

die assembly using a high pressure metering pump. In the extrusion die assembly, the liquid dyes are homogenously mixed with the extrusion cooked base formulation in separate mixing chambers and then extruded through a die arrangement where the products are shaped. Any number of shapes can be manufactured as long as the shapes can be balanced for uniform die flow for each color and shape combination. The system is designed for the simultaneous production of either two colors. While using the multi-color/multi-shape extrusion die assembly for the production of multi-color products has some flexibility limitations not present in other systems, these limitations do not prevent the assembly’s use in manufacturing the vast majority of multi-color/multi-shape pet food products currently on the market. Using the multi-color/multi-shape extrusion die assembly requires the use of water soluble dyes to color the products. Dyes that are not water soluble, such as iron oxide, are not compatible with the high pressure pumps that are readily available. A further requirement of this system is that the products be of the same density. In addition, the blending ratio of products of different colors is fixed. For example, utilizing a 2 color system, you can make a blended product that is a 50/50 blend of each color. As already mentioned, the multi-color/multi-shape extrusion die assembly can be retrofitted to an existing twin screw extruder. It is available in three models to match twin screw extruders that are in use today. By employing simple modifications, the multi-color/multi-shape extrusion die assembly is installed at the discharge end of the extruder. The die can be assembled and set-up off line, then attached to the extruder in less than one hour. After completion of production of the multi-color product, the multi-color/multi-shape extrusion die assembly can again be removed from the extruder and the extruder returned to production of normal single-color products in less than one hour. The multi-color/multi-shape extrusion die assembly can then be disassembled and cleaned while the extruder is employed in production of single color products. When a pet food manufacturer makes a choice between the systems or methods they employ in manufacturing their products, they must consider the advantages and disadvantages to each method. There are a number of factors that should be weighed when determining which of the three methods for manufacturing multi-color/multi-shape pet foods is best suited for your particular situation. These factors include cost, space requirements, ease of implementation, ease of use, flexibility and efficiency – both from the standpoint of energy use and from the standpoint of waste generation. Due to its inherent characteristics, the multi-color/multi-shape extrusion die assembly has advantages in equipment cost, space requirements, ease of implementation, ease of use, energy use and waste generation. However, the multi-color/multi-shape extrusion die assembly has less flexibility compared to the other methods. Although the other methods, the blending bin system and utilization of multiple extruders, have much greater flexibility, it comes at a price, not only in terms of capital outlay, but also in space requirements, difficulty in implementation, and high energy use. These are the latest technologies which are available presently in the market from different extruder manufactuers. These technologies can help to overcome some of the

80

variation we see every day in the aquafeed raw material from differnet sources. These technologies can reduce the down time for configuration changes required in the extruders for different densities products. At the same time these technologies offer a wider range of the raw material uses as well as producing wider range of the feed products.

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Extrusion Technology Program Food Protein R&D Center

Texas A&M University The Extrusion Technology Program at Texas A&M University offers 4 Courses in the area of extrusion every year:

Aquaculture Feed Extrusion, Nutrition & Management Feeds & Pet Food Extrusion Snack Food Processing: Extruded Snacks and Tortilla Chips Texturized Vegetable Protein & Other Soy Products

We conduct specialized training for the industry in the area of Aquafeed, Pet food, TVP, and Extruded Snacks. Our Pilot Plant is equipped with 8 different types of extruders. We also work for the industry for R&D in the development of their products. Our 13th Annual Aquaculture Feed Practical Short Course will be held on September 24-29, 2006 and 17th Annual Feeds and Pet Food Practical Short Course will be on January 28 - February 2, 2007. During these courses, we will be demonstrating four different types of extruders making different kinds of feeds. Please visit our website for more information about these courses: www.tamu.edu/extrusion All of our courses are "hands on"; that is, we conduct equipment demonstrations in concert with each day's lectures to facilitate the learning experience and allow for 'one-on-one' interaction with highly qualified experts from industry. Come to one of our short courses on the campus of Texas A&M University, College Station, Texas, and see how the Food Protein R&D Center has successfully trained attendees from across the world with practical instruction for 25 years. Established in 1939, the Food Protein Research and Development Center is one of the oldest land-grant agricultural research and service programs in the nation. It specializes in process development for diverse agricultural crops and animal products into food, feed, and industrial ingredients. Basic research and testing technology development and training projects are conducted for private industry, trade associations, and state, federal, and international agencies. The Food Protein R&D Center is part of the Texas A&M University System through the Texas Engineering Experiment Station and managed as a center within the Artie McFerrin Department of Chemical Engineering.

Table 1: Global Aquaculture Production by Region Global Region Seafood Production (million tons)

Asia 37.0 Europe 2.0

Americas 1.2 Africa 0.3

Table 3: Final Product Bulk Density Correlation with Buoyancy Properties

Pellet buoyancy

Sea water @ 20ºC

(3% salinity)

Fresh water@ 20ºC

Fast sinking

> 640 g/l > 600 g/l

Slow sinking

580-600 g/l 540-560 g/l

Neutral buoyancy

520-540 g/l 480-520 g/l

Floating <480 g/l <440 g/l

Table 2: Buoyancy Properties of Feed for Common Aquatic Species Floating Slow-sinking Sinking Alligator Bluefin Tuna Cod Carp Flatfish Flounder Catfish Mai mai Halibut Eel Salmon River crab Frog Sea bream/bass Sea bream/bassKoi Artic Char Abalone Milkfish Tilapia Sea urchin Tilapia Trout Shrimp Trout Yellowtail Turbot

Increasing Aquatic Feed Production through Plant Optimization

Galen J. Rokey, Wenger Manufacturing Inc.

There are many different aquatic species that are now farmed or cultured. The number of species and the tonnage of the annual “farmed” harvest continues to increase as sustainable aquaculture gains support. It is estimated that more than 30 percent of the 100 billion dollar (US) global seafood market is from aquaculture. This equates to over 40 million ton of seafood produced each year from aquaculture. World demand is expected to increase by at least three percent annually over the next few years. Roughly 90 percent of aquaculture production occurs in the Asian region of the world. Global aquaculture production by region is shown in Table 1. Fresh water aquaculture accounts for 58 percent of the output and marine aquaculture accounts for 42 percent of the output. The various aquaculture species (both marine and fresh water) can be categorized according to the buoyancy properties of their feed (Table 2). To achieve the level of buoyancy required, specific bulk density ranges have been established for each feed for the environment in which the feed is being fed (Table 3). The floating/sinking properties change with water temperature and salinity. The recipe, the hardware, and the operating parameters for the extrusion, drying, cooling, and coating processes are adjusted to meet necessary buoyancy properties and fat levels in the final product. Power inputs, measured as SME (specific mechanical energy), and moisture levels are the major operating parameters that are controlled during the

83

extrusion process to yield the desired bulk density of the final product. In an effort to meet increased aquaculture feed production requirements, it is important to maximize throughputs in an existing production line. Conducting a simple line audit can easily identify bottlenecks to higher throughputs. Bottlenecks that potentially limit throughputs of an existing extrusion line are as follows:

1) Preconditioning capacity 2) Available extruder power 3) Extruder volumetric capacity 4) Die open area 5) Down time 6) Upstream/downstream unit operations

Preconditioning Capacity The preconditioning step initiates the heating process by the addition of steam and water into the dry mash. Uniform and complete moisture penetration of the raw ingredients significantly improves the stability of the extruder and enhances the final product quality. Objectives of a preconditioning step are to continuously hydrate, heat, and uniformly mix all of the additive streams together with the dry recipe. The preconditioning process is simple. Raw material particles are held in a warm, moist, mixing environment for a given time and then are continuously discharged into the extruder. This process results in the raw material particles being hydrated and heated by the steam and water in the environment.

Dual shaft, intermeshing preconditioners have improved mixing in comparison to the single shaft preconditioners and have a longer average retention time of up to one and one-half minutes for a similar throughput. Dual shaft, intermeshing preconditioners have beaters that can be changed in terms of pitch and direction of conveying. This feature of adjustable beaters is not found on many conditioning devices. Of all the preconditioners available today, the differential diameter/differential speed preconditioners (DDC) are the most sophisticated. The DDC has the best mixing characteristics combined with the longest average retention times of those available (Figure ). DDC preconditioners offer retention times of up six minutes for given throughputs comparable to the 15 to 45 seconds possible in single preconditioners or multiple-stacked single conditioners (sometimes referred to as dual conditioners). The two shafts of a DDC preconditioner are counter-rotating so that material is continuously interchanged between the two intermeshing chambers for maximum mixing.

Figure 1: DDC (Differential Diameter Cylinder) Preconditioner

84

Un-preconditioned raw materials are generally crystalline or glassy amorphous materials. These materials are very abrasive until they are plasticized by heat and moisture within the extruder barrel. Preconditioning prior to extrusion will plasticize these materials with heat and moisture by the addition of water and steam prior to their entry into the extruder barrel. This reduces their abrasiveness and results in a longer useful life for the extruder barrel and screw components.

Extruder capacity can be limited by energy input capabilities, retention time, and volumetric conveying capacity. While preconditioning cannot overcome the extruder’s limitations in volumetric conveying capacity, it can significantly contribute to energy input and retention time. Retention time in the extruder barrel can vary from as little as five seconds to as much as two minutes, depending on the extruder configuration. Average retention time in the preconditioner can be as long as five minutes. For some high moisture processes, the energy added by steam in the preconditioner can be as much as 60 per cent of the total energy required by the process.

To increase preconditioner capacity or to compensate for inadequate preconditioning, one or more of the following steps can be employed:

1) Increase preconditioner size 2) Increase existing preconditioner fill by one-time adjustment of beater

configuration 3) Add automatic Retention Time Control (RTC )system 4) Increase energy inputs in the extruder

Available Power to the Extruder When power is the limitation to more throughput, the options to remove this bottleneck are more obvious. Factors to consider include the following:

1) Install larger extruder drive motor (more available kW) 2) Check with extruder manufacturer to determine maximum allowable installed

power based on system design limitations 3) Factor in the effects of removing other bottlenecks (improved preconditioning,

etc.) Extrusion systems in the industry are available with power trains of over 2000 kW. Lack of power is the most common bottleneck to higher production rates for existing process lines. An extrusion system operating at or above full load for most products cis an indication that power is the limitation to higher throughputs. Volumetric Capacity of the Extruder Volumetric capacity is based on the free volume geometry of the extruder screw and the screw speed. Plotting the screw speed (revolutions per minute) versus potential output (kilograms per hour) indicates screw performance or efficiency (Figure 2). In most cases, actual output is lower than the potential volumetric capacity due to backwards pressure or leakage flow. However, when the extruder is designed with a cooled, grooved inlet feed throat and barrel sections, the output can be higher than the expected, calculated volumetric capacity of the screw.

85

Figure 2: Screw Speed versus Output

0

1000

2000

3000

4000

5000

6000

200 400 600 800 1000 1200 1400Extruder screw speed (rpm)

Ext

rdue

r fee

d ra

te (k

g/hr

)

Figure 3: Peripheral die openings

A bottleneck due to volumetric capacity is usually manifested by the extruder operating in a “choked” or full condition. Barrel fill will be great enough to plug or partially plug the barrel steam and water injection ports. In extreme cases, in-feed material will visibly fill the extruder inlet and over-flow the throat. Force-feeding devices are some times disguised as a tool to increase volumetric capacity, but their main function is to eliminate product bridging in the extruder inlet due to poor mixing in the preconditioning stage. The volumetric capacity for an extrusion line can be increased in one or more of the following ways:

1) Install a larger extruder screw diameter 2) Increase extruder screw speed 3) Configure the extruder with screw geometries designed for maximum conveying

efficiency 4) Utilize grooved barrel liners 5) Control extruder barrel temperatures with heating/cooling systems

Open Area of Die Assembly A specific die open area is required to develop the proper back pressure and barrel in the extruder during processing. This open area requirement remains rather constant for a product having a distinct buoyancy. If the die area is insufficient, products may over-expand and extruder loads are excessive as a result of increased barrel fill. Increasing the die open area to increase throughput potential is a straight forward relationship. Many die design techniques are employed to increase the number of die openings and the total die open area. The most common arrangement of die orifices is on the die face which is axially positioned with the extruder center line. A substantial increase in the number of die orifices can be realized when they are arranged on the

86

periphery of the die extension in a pattern that is radial to the extruder centerline (Figure 3). Downtime and Usable Product Reduced down time is often overlooked as a bottleneck to higher plant capacities. An extrusion line that has a throughput of ten ton per hour loses a potential of five ton of product for every 30 minutes of downtime. A certain amount of downtime is unavoidable due to scheduled maintenance, product change-over, and other plant functions such as fumigation and sanitation. Many feed manufacturers believe they operate their lines 24 hours a day, seven days a week, and are surprised to look at end-of-the-year production records which can indicate up to 20 percent actual downtime. Practices that can be implemented to reduce downtime are as follows:

1) Production schedules adjusted for minimum product switch-over time 2) Hardware tools installed that have quick-change features 3) Control systems designed for compressed startup/shutdown modes 4) Production personnel trained to reduce downtime 5) Preventative maintenance programs implemented 6) System hardware designed for maintenance and cleaning accessibility

In addition to downtime reduction, increasing usable product is a significant opportunity where off-spec product may run as high as eight percent of total production. Considerations for increased levels of usable products include the following:

1) Automated retention time control in preconditioners to reduce startup/shutdown wastes

2) Screw element and liner designs to give positive conveyance 3) High extrude speeds and variable speed drives to shorten process response times 4) On-line, automated control of SME and recipe analysis 5) Automated extruder control systems that compress startup/shutdown modes 6) Experienced and trained production personnel to control process 7) Process flows that handle the product gently 8) Systems to recycle under-processed material and off-spec product

Upstream/downstream Processing It is easy to focus on the extrusion operation and the potential bottlenecks, however, most bottlenecks occur along the process flow in areas other than the extruder. An audit to increase plant production levels should include an evaluation of each unit operation along the entire flow. Potential bottlenecks could be found in one or more of the following areas:

1) Grinding/sifting 2) Storage 3) Conveying 4) Drying/cooling 5) Coating 6) Packaging

87

All unit operations along the process line must be properly sized to avoid a flow bottleneck. As each bottleneck is identified and eliminated, a new, secondary bottleneck will likely appear. A different bottleneck may be identified for each product that is manufactured in a given process line. This auditing process can continue indefinitely, but at each step it is necessary to do a cost/benefit analysis to determine if the economics are favorable.

88

Wenger Aquatic Feed Systems . . . versatility to cover the water column.

binders. Special applications that require even up to 5 days of

water stability are possible.

Wenger’s time-tested extrusion equipment allows you to

incorporate up to 45% total fat for high energy feeds like

salmon pellets. Naturally, we also offer one of the broadest

lines of equipment on the market, including single- and

twin-screw extruders, dryers, coolers and blenders

with capacities ranging from 0.1 to 22 tons/hour.

From top to bottom, shrimp to catfish, we’re ready

to fill your specific aquatic feed specifications.

There are nearly 20,000 species of fish inthe world. Fortunately, Wenger Aquatic Feed Systems

offer the versatility to feed them all, not to mention crawfish,

frogs, shrimp and eels, too. Wenger extruders produce a

full range of feeds for both fresh and salt water species

with products that range in pellet sizes from 0.6 to 50 mm.

Unique extruder features also permit precise control of

finished product density, so you can produce

floating, fast-sinking or slow-sinking feeds as

needed. Durability of feeds for bottom dwellers

has shown stability of up to 24 hours without

USA 816 891 9272 / EUROPE 32 3 232 7005 / ASIA 886 4 2322 3302 / WWW.WENGER.COM C E R T I F I E DISO 9001:2000

Optimization of formulation & quality of extruded aquafeed

Stuart HowsamBuhler AG

2 | VICTAM 2006 - Buhler | | © Bühler

Why Extrusion

• improving water stability and texture• High water absorbtion but low solubility• Matrix of texturized proteins and gelatinized starch• Minimal leaching of nutrients

• Correct pellet density and water absorbtion

• Flexibility in least cost formulation

• Accurate sizes from 1mm up to 25mm

• Reduction of anti-nutritive compounds

• Producing less fines, less breakage

To improve the overall feed conversion by

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Quality issues associated with extrusion of aquafeeds

Fat level fluctuation

Bulk density variation

Moisture variation

Liquid flow fluctuation

Feed flow fluctuation

Degree of cook

Type of cook

Quality Issue

Raw material specification variation

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Quality issues associated with extrusion of aquafeeds- Solutions

• Specific mechanical energy (SME) control systems

• Specific Thermal energy (STE) control

• Density control systems

• Extruder control systems

• Example – shrimpfeeds

5 | VICTAM 2006 - Buhler | | © Bühler

95°C

110°C

120°C

130°C30 Wh/kg

20 Wh/kg

10 Wh/kg

Product backup before die

Conventional, one-step extrusion process:

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SME control systems – an example

90

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Specific Mechanical EnergySME vs Position of SME-Valve

Features of SME-Module

1. No change of screw configuration

2. Few types of different screw elements

3. Screw wear compensation

4. No addition of water

5. No variation of screw speed

15

20

25

30

35

0 20 40 60 80 100

SME valve position [%]

SME

[Wh/

kg]

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ECOtwin – BCTB, Submenu SME Control

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SME control system- Effect of oil addition

0

5

10

15

20

25

30

0 3 10

% oil addition

SME

Wh/

kg

0

100

200

300

400

Bul

k de

nsity

g/l

SME Bulk density

• Trial date 20.10.04

• 29% protein, 7% fat in premix

• Total flow = 630kg/hr

• Screw rpm = 800

• Soya oil addition

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SME control system- Effect of oil addition

05

10152025303540

3 6 9

% oil addition

SME

Wh/

kg

0

100

200

300

400

Bul

k de

nsity

g/l

SME Bulk density05

10152025303540

3 6 9

% oil additionSM

E W

h/kg

0

20

40

60

80

100

% g

elat

iniz

atio

n

SME % gelatinization

3% 6% 9% oil add’n

•Trial date 23.3.05•Petfood formulation•Total flow = 525kg/hr•Screw rpm = 570•Soya oil addition

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Effect of SME on product quality

Trial performed to determine effect of SME on quality of shrimp feed

Observations:• Shrimp feed produced using low shear extrusion (25Wh/kg) was more stable after 4 hours in water than feed produced using medium shear extrusion (35Wh/kg)(figs 1 & 2)

• Shrimp feed produced using a pellet mill was less stable in water (fig 3)

Conclusions:• SME can influence the water stability and appearance of aquafeeds

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Combination of SME and STE control

Retention time [sec.]

Gel

atin

isat

ion

(DSC

) [%

]

0

20

40

60

80

100

0 20 40 60 80 100

~25% water

15

20

25

30

35

0 20 40 60 80 100SME valve position [%]

SME

[Wh/

kg]

• Total energy input ET = SME + STE

• product requires a certain energy (ET) for cooking but the ratio of SME to STE may vary

• STE may be adjusted by:• adjustment of steam flow• adjustment of preconditioner residence time

• SME may be adjusted by:• altering screw configuration• altering dieplate configuration• using an SME control valve

91

13 | VICTAM 2006 - Buhler | | © Bühler

Two step preconditioning

Raw Material

Conditioned Material

Mixing unit

Retention unit

Steam and

liquids High rpm

Low rpm

A two stage preconditioner will allow:

• A high rpm mixing section for good mixing• A separate low rpm retention section with residence time adjustable via a variable speed drive

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Combination of SME and STE control- example

440450Bulk density (g/l)

38.423Preconditioner fill (%)

3434Preconditioner residence

time (s)

126.5SME valve pos’n (%)

36.636.9SME (Wh/kg)

3934Die pressure (bar)

88Steam in (%)

1010Water in (%)

1000600Feed rate (kg/h)

17.5.04

16:50

17.5.04

16:42

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Conditioned material in:

Bulk density 500 g/l

Extrudate out:

Bulk density250- 650 g/l

Steam additionVapor venting

Density controlCooking extruder

Forming extruder

Density control systems

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Example 3: The temperature in the cooking zone reaches 135°C. By means of vacuum, the temperature is lowered to 80°C in the venting zone in order to avoid temperatures above 100°C at the die.

Density Control - Temperature ProfilePr

oces

s Te

mpe

ratu

re

140°C

80°C

100°C

120°C

312

Example 1: The temperature in the cooking zone reaches 138°C by friction. By partial venting of the steam, the temperature decreases to 105°C in the venting zone. Due to the friction, it rises again to 118°C at the die.

Example 2: Due to marginal mechanical energy dissipation in the cooking zone, the temperature reaches only 110°C. With live steam injection at the venting spout, the steam pressure rises the dough to a final temperature of 140°C at the die.

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ECOtwin – BCTB, Submenu Density control

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SME and density control systems combined

100

110

120

130

°C

2 4 6 8 10s

neutral

venting

vacuum

steaminjection

0.1

1.3

2.1

2.8

steam pressure in bar

90

140

25 Wh/kg

30 Wh/kg

40 Wh/kg

92

19 | VICTAM 2006 - Buhler | | © Bühler

Effect of density and SME control systems on Bulk Density

1 Positive pressure operation with steam addition for low BD product.

2 Pressure neutral operation with steam return to preconditioner.

3 Negative pressure operation with vacuum system for high BD product.

300

400

500

600

700

800

0.4 0.8 1.2 1.6 2 2.4 2.8

degassing pressure [bar]

bulk

den

sity

[g/l]

3 21

SME-valve open (15 Wh/kg)

SME-valve closed

(35 Wh/kg)

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Cooking, venting and compression zone

Backfill due to die resistance

Backfill at high torque

Crumbs in venting zone

Hot meal infeed

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Extrusion control systems

22 | VICTAM 2006 - Buhler | | © Bühler

Quality issues associated with extrusion of aquafeeds

Fat level fluctuation

Bulk density variation

SME control system

Density control system

Moisture variation

Liquid flow fluctuation

Feed flow fluctuation

Degree of cook

Type of cook

Extruder control system

Extruder control system

Extruder control system

SME/STE control

SME/STE control

Quality Issue Solution

Raw material specification variation

SME control system

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Example- Shrimpfeeds

• Pellet sizes from 0.8mm to 2.3mm

• Bulk density 630-700 g/l

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Example- Shrimpfeeds

• Shrimp locate feed by smell and taste• Shrimp may take hours to locate and digest feed• Shrimp grind the food externally before digesting it• They are bottom feeders• Shrimpfeeds are expensive

Requirement –

• Low to medium protein content, i.e. 30-40%• Good conversion rate, i.e. waste remains in pond• fast water absorbtion, high water stability• Good sensory properties• 100% sinking

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25 | VICTAM 2006 - Buhler | | © Bühler

Example- Shrimpfeeds

• Through use of SME, density and extruder control systems and consequent improved control of the cooking process the following benefits are achieved:

• 100% sinking product• Improved digestibility• No binders required in recipe• Improved water stability• Improved water quality• Reduced mortality• Ability to process a wider range of raw materials

26 | VICTAM 2006 - Buhler | | © Bühler

Extruder operating costs

8.69.511.9Total

0.60.60.6Personnel

0.40.30.3Waste

0.81.21.6Interest

1.31.92.5Amortization

1.01.02.0Wear

2.12.11.7Thermal energy

2.42.43.2Electrical energy

Single screw extruder

ECOtwin extruderTwin screw extruder

Costs (USD/tonne)

Assumptions•50,000t/a•10 tonnes/hr•Period of amortization – 8 yrs•Interest rate 8%•Raws cost 600 USD/t

•capital cost of:•single screw extruder USD 500k•twin screw extruder USD 1000k•ECOtwin extruder USD 750k

•Utilities cost EU average

27 | VICTAM 2006 - Buhler | | © Bühler

Aquafeed Production costs

Typical example:

Marine feed plant 40‘000 t/y

Raw Material84.6%

EE Extrusion0.5%

Wear Parts0.4%

EE Drying - Coating0.3%

TE Drying0.7%

EE Grinding, Others0.6%

TE Extrusion0.2%

Packaging, Others1.3%

Personnel4.3%

Financing Costs7.1%

28 | VICTAM 2006 - Buhler | | © Bühler

•SME control• SME control permits running at optimum process conditions, independent of screw configuration, resulting in lower overall mechanical energy consumption.

•Reduced power consumption

SystemDescriptionBenefit

•SME control• Running at optimum SME means a lower overall wear rate of screw and barrel.

•Reduced wear

•SME control•Density control•Automatic control system

• Automatic control systems allows faster startups and shutdowns.• Slurry systems allow recycling of waste material.• Vented steam from density control system is re-cycled.

•Less waste

•SME control•Density control

• Optimal cooking conditions means consistent quality.• SME and density control produces consistent quality independent of raw material variations.• Density control allows much greater control of BD, less offspecproduct.

•Quality improvements

•SME control•Density control

• TSE process with SME and density control operates over a wider range of raw material specifications than SSE.

•Raw material savings

Economic considerations

29 | VICTAM 2006 - Buhler | | © Bühler

Thankyou

94

Bühler AGExtrusion SystemsCH-9240 Uzwil, SwitzerlandT +41 71 955 3797F +41 71 955 2481E-Mail: es.buz@buhlergroup.comwww.buhlergroup.com

The Buhler twin-screw extrusion systems set standards in performance, flexibility and ease of operation: a reliable platform for exciting new products, advanced formulations and aquaculture diets.

Buhler Extrusion Systems are your reliable partner for aquaculture diets.

New Dimensions in Twin-Screw Extrusion Technology.

RZ_ES Ins.aquaculture_A4.indd 1 10.10.2005 13:44:01

Replacement of Fish Meal by Poultry By-product Meal and

Meat and Bone Meal in Aquafeeds –

An Update (2004-2006)

Dr. Y. Yu National Renderers Association nrahkg@nrahongkong.com.hk

Abstract

Research reporting poultry by-product meal (PBM) and meat and bone meal (MBM) as fish meal (FM) replacement in diets for shrimp and fish during the past two years were reviewed for digestibilities and weight gain (WG) response to dietary FM replacement for FM. Nutrients digestibilities and the maximum FM replacement rate are important least cost formulation criteria for selection of protein ingredients and minimizing the variability in growth performance of aquaculture animals. Protein,

essential amino acids (EAAs), and energy in PBM were well digested (≧ 80%) by

vannamei, hybrid striped bass, large mouth bass, rainbow trout, seabass, and cobia, but to a lesser extent (~70%) by monondon and turbot. Nutrients digestibilities of MBM were reported only for cobia and gibel carp. The average digestibility of MBM measured in earlier trials was about ten percentage points below that of PBM. The maximum FM replacement rate by PBM is 80% for vannamei, fresh water fish, and Coho salmon, but is 50% for warm water marine fish. For MBM, the average maximum FM replacement rate is about 50-60% for most aquatic animals, except for cold water marine fish at 20%. Nutrient digestibilities were generally in agreement with WG except for monondon. Protein blend made from multiple sources of rendered protein meals (e.g. MBM, PBM, feather meal and blood meal) may offer nutrients palatability, and cost complementary benefits as shown with normal feed intake, weight gain, and feed conversion ratio in 100% FM replacement growth trials of rainbow trout (carnivorous) and silver perch (omnivorous). Trials reviewed support the use and value of PBM and MBM as FM replacements in diets for carnivorous and omnivorous aquaculture animals.

96

Introduction Animal proteins are considered essential dietary component for carnivorous

aquatic animals, and are desirable protein source for omnivorous species. Fish meal (FM) has been the prime choice among all animal proteins for its protein quality and the palatability. However, for various reasons the supply of FM will be insufficient in meeting the demand from feeding of aquatic and terrestrial species. Poultry by-product meal (PBM) and meat and bone meal (MBM) are potentially suitable replacements for FM in aquafeeds due to their resemblance to FM in nutritional composition but are much lower in cost. Early studies conducted by National Renderers Association (Yu 2006) and others (Allan and Rowland 2005, Tan et al. 2005, Tidwell et al. 2005) have demonstrated that a large portion of FM can be replaced by PBM and MBM without impairing the growth of fish and shrimp.

For effective use of PBM and MBM as FM replacements, aquafeed nutritionists need to know the digestibility coefficients of key nutrients preferably measured from the same species as the diet being formulated for. This improved precision in formulation will not only give a more consistent and predictable growth of the aquatic animal but also a more accurate estimate for of feed cost of production. Provision of reliable knowledge on the maximum rate of FM substitution by PBM and MBM without a negative effect on weight gain another important variable in aquafeed formulation for avoiding erratic growth performance, yet reducing the dependence on FM.

The objective of the present paper is to present the latest (2004 to 2006) findings in digestibility and growth response of fish and shrimp when fed diets with PBM and MBM as FM replacements.

Nutritional Composition of Poultry By-product Meal (PBM) and Meat and Bone Meal (MBM)

Nutrients and amino acids composition of PBM, MBM and FM used in several digestibility and growth trails in China (Xue and Yu, 2005) is listed in Table 1. The FM Sample was taken from a leading aquafeedmill in S. China, and was identified as Peruvian origin. Both PBM and MBM were supplied by US renderers, and were considered of high quality as evident from the above average (NRC, 1993) content of essential amino acids (EAA, Table 1), data in Table 1 implies the inappropriateness of substituting FM with PBM or MBM on simple equivalent crude protein basis. Nutritionists should use the analyzed nutrients and EAA values of PBM, MBM, and FM for diet formulation. Recent advancement in NIR technology could provide these values to nutritionists in a very short turn around time (e.g. ten minutes) at a reasonable cost.

97

Table 1. Nutrient composition (%) of fish meal, meat and bone meal and poultry by-product meal used in shrimp digestibility and growth trials

MBM1 PBM2 FM3

Dry matter 96.6 97.5 92.6 Crude protein 54.0 65.6 62.9 Crude fat 12.7 12.5 11.1 Essential Amino acids Arginine 3.33 4.01 3.20 Histamine 1.43 1.72 1.61 Isoleucine 1.93 2.69 2.40 Leucine 3.66 4.85 4.41 Lysine 3.27 4.42 4.41 Methionine 1.29 1.59 1.6 Phenylalanine 2.07 2.70 2.43 Threonine 2.1 2.71 2.50 Valine 2.44 3.13 2.63 Cystine 0.61 0.74 0.59

Tyrosine 1.39 1.92 1.91 1Meal and bone meal (US) 2Poultry by-product meal (US) 3Fish meal (Peruvian)

Requirements: Energy, Protein and Amino Acids

Requirements of energy, protein and amino acids of most aquatic species are interrelated, and should be evaluated simultaneously for one particular species. Literature search has indicated that only few species have reliable requirements researched (e.g. trout, carp, tilapia and hybrid striped bass) (NRC, 1993). Nonetheless, the best estimates of requirement of protein and amino acids are given in Table 2 (Bureau, 2000). Gross requirement of EAAs can be estimated from the EAAs profile of the carcass. Relative importance for feedmills in meeting the requirements of one particular species is firstly total protein, secondly, EAAs as % feed, and lastly EAAs as species, formulation precision can be improved by using digestible protein and EAAs for requirements and ingredients. Crystalline amino acids should be used in most fish diets if ended, but the efficiency of utilization of these EAAs may be lower in shrimp

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feeds as compared with fish or poultry feeds. Coated EAAs for slow rate of releasing may be desirable for shrimp feeds. Table 2. Protein and amino acids requirements of selected fishes and shrimp

Salmon Catfish Carp Tilapia Bass Milkfish Monondon Vannamei Eel Protein % (Juvenile) 40-45 32-36 31-38 30 40-45 40 40 35 44 Arginine % Protein 4.2 4.3 4.4 4.1 4 5.6 5.8 5.8 4.5 % Feed 1 1.2 .9 1.44 1.06 1.3 1.68 2.32 2.03 1.53

Histamine % Protein 1.6 1.5 2.4 1.7 1.5 2. 2.1 2.1 2.1 % Feed .52 .36 .72 .43 .5 .6 .84 .73 .72

Isoleucine % Protein 2 2.3 3 3.1 2.7 4 3.4 3.4 4 % Feed 1.23 .54 .81 .78 .9 1.2 1.36 1.19 1.35

Leucine % Protein 3.6 3.5 4.7 3.4 4.6 5.1 5.4 5.4 5.3 % Feed 1.22 .72 1.17 .86 1.5 1.53 2.16 1.89 1.8

Lysine % Protein 4.8 5 6 4.6 4.3 4 5.3 5.3 5.3 % Feed 1.8 1.08 1.98 1.29 1.4 1.2 2.12 1.86 1.8

Methionine + cystine % Protein 2.4 2.3 3.5 3.2 2.1 4.8 3.6 3.6 3.2 % Feed .67 1.35 1.1 .67 .7 1.44 1.44 1.26 1.1

Phenylalanine + Tyrosine % Protein 5.3 4.8 8.2 5.6 4.3 5.2 7.1 7.1 5.02 % Feed 1.41 .54 2.25 .95 1.4 1.56 2.84 2.48 1.98

Threonine % Protein 2 2.1 4.2 3.8 2.4 4.9 3.6 3.6 4 % Feed .93 1.08 1.35 .95 .8 1.47 1.44 1.26 1.35

Tryptophan % Protein .6 .5 .8 1.0 .6 .6 .8 .8 1.1 % Feed .2 .45 .27 .25 .2 .18 .32 .28 .36

Valine % Protein 2.2 3 4.1 2.8 2.4 3 4. 4 4 % Feed 1.3 .64 1.26 .7 .8 .9 1.6 1.4 1.35

1 On a 90% dry basis

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Digestibility Trials Poultry By-Product Meal (PBM) Since 2004 nutrients and EAAs digestibilities of PBM have been measured in shrimp (monondon and vannamei), hybrid striped bass, largemouth bass, rainbow trout, seabass, turbot and cobia (Table 3). Poultry by-product meal was typically mixed with a base mix ( FM being the only protein source) at a ratio of 3:7. A typical base mix formulae and analysis used for shrimp trials in China ( Xue and Yu, 2005) is given in Table 4. Under the similar experimental condition, EAAs digestbilities of PBM were significantly (p< .05) higher (87 vs 71%) for vannamei than monondon. For comparative purpose, nutrients and EAAs digestibilities were also measured for FM in vannamei (Table 5). Both protein meals were well digested (~85%) by vannamei. The digestibility of leucine, lysine, methionine, phenylalanine and threonine in PBM was however notably higher than that in FM. Digestibilities of nutrients and EAAs were high (~89%) and similar among the finfish tested except for turbot which had an average EAAs digestibilities of 68%, or about 24% lower than other fish listed in Table 3. Nutritionists should recognize the specis specific of EAAs digestibilities when formulating diets.

With similarity in nutrients composition (Table 1) and digestibility (Table 3), PBM should be considered as one of the most suitable substitute for FM in aquafeeds.

Table 3. Nutrients and amino acids digestibility (%) of poultry by-product meal

measured in various aquatic species

Shrimp1

Nutrient monondon vannamei

Hybrid striped bass2

LM bass3

Rainbow trout4 Seabass5 Turbot5 Cobia6

Dry matter 47.9 63.2 - 82.6 65.1 - - 80.9

C. protein7 77.6 84.2 60.5 81.5 82.5 83.2 66 90.9

Energy 72.8 84.0 - 85.2 74.5 - - 90.6

Essential Amino Acids

Arginine 71.6 85.7 86.7 91.2 - 91.2 75.4 94.2

Histidine 77.6 89.0 100 93.1 - 95.1 45.4 91.3

Isoleucine 70.6 90.8 89.9 85.8 - 88.7 57.4 92.2

Leucine 74.9 89.4 88.5 88.6 - 89.1 80.8 92.9

Lysine 81.9 92.5 89.1 90.8 - 97.4 86.1 91.8

Methionine 83.7 95.0 95.2 71.3 - 93.3 77.6 92.5

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Table 3 (continued)

Nutrient Shrimp1 Hybrid striped bass2

LM bass3

Rainbow trout4 Seabass5 Turbot5 Cobia6

Phenylalanine 68.4 89.0 87.8 87.5 - 84.9 65.7 91.4

Threonine 69.0 85.1 97.7 86.1 - 86.8 83.0 93.2

Valine 62.0 81.1 89.7 83.0 - - - 92.2

Crystine 63.6 76.0 - 50.4 - 64.5 43.2 -

Tryosine 58.8 88.1 89.4 96.2 - - - 93.2

Avg. EAA8 71.1 87.4 91.4 84 - 87.9 68.3 92.5 1Xue and Yu, 2005 5Davies and Serwata, 2005 2Gaylord and Rawles, 2005 6Zhou et al., 2004 3Portz and Cyrino, 2004 7Crude protein 4Cheng et al., 2004 8Average essential amino acids

Table 4. Formulae and nutrient composition (%) of base mix used in shrimp

digestibility trials.

% Formulae

Fish meal 33 Soybean meal 8 Peanut bran 20 Squid meal 3 Blood meal 3 Fish oil 1 Soy oil 1 Soy lecithin 1.5 Wheat flour 24.8 Zeolite 2 Premix 2.5

Analysis Dry matter 89.9 Crude protein 43.7 Fat 8.0 Gross energy(MJ/kg) 18.2 Ash 11.8 Total Phosphorus 1.7

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Table 5. Apparent nutrients and amino acids digestibility of poultry by-product meal and fish meal in shrimp (vannamei)

Poultry by-product meal Fish meal

Dry matter 63.2 64.3

Crude protein 84.2 80.7

Energy 84.0 85.2

Essential amino acids

Arginine 85.7 89.7

Histidine 89.0 91.2

Isoleucine 90.8 88.5

Leucine 89.4 70.4

Lysine 92.5 85.4

Methionine 95.0a 80.5b

Phenylalanine 89 77.3

Threonine 85.1 79.2

Valine 81.1 81.5

Crystine 76.0 78.9

Tryosine 88.1 89.2

Average EAAs1 87.4 82.9

Xue and Yu, 2005, China 1Average essential amino acids Meat and Bone Meal (MBM) Only two digestibility trials were reported during the past two years on MBM. The limited data in Table 6 indicates that protein in MBM was well digested (83%-87%) by gibel carp and cobia while dry matter and energy digestibilities were somewhat lower than protein perhaps due to the relatively high ash content (Table 1). Digestibility of EAAs averaged 91% for MBM by cobia, which was consistent with the digestibility of protein (87%), and was comparable with 93% of PBM (Table 3). Results suggest that the quality of MBM used in Cobia trial was exceptional well, and once

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again illustrate the importance of using the analyzed EAAs value for diet formulation rather than using the average book value (e.g. NRC, 1993). Previous studies (Yu, 2006) indicate that nutrients and EAAs digestibilities of MBM are generally lower than PBM and FM by about 10% measured from various aquatic species. For improved precision on utilization of MBM in diets for shrimp and fish, more trials on digestibilities of MBM should be conducted. Table 6. Nutrients and amino acids digestibility of meat and bone meal measured in

gibel carp and cobia gibel carp1 Cobia2

Dry matter 63.1 60.4 C. protein3 83.3 87.2 Energy 78.4 90.4 Amino Acids Arginine - 93.1 Histidine - 88.2 Isoleucine - 91.1 Leucine - 92.3 Lysine - 84.5 Methionine - 92.6 Phenylalanine - 91.5 Threonine - 91.6 Valine - 91.4 Crystine - - Tryosine - 91.6

Avg. EAAs4 - 90.8 1Xie and Yu, 2005 3Crude protein 2Zhou et al., 2004 4Average essential amino acids

Growth Trials Poultry By-product Meal (PBM) A total of seven growth trials were reported in the past two years (shrimp, largemouth bass, hybrid striped bass, sunshine bass, black rockfish, cuneate drum, and grouper). Details of each trial (e.g. FM replacement rates, FM content in control diet, initial weight (IW), specific growth rate (SGR), weight gain(WG), feed intake (FI), feed conversion ratio (FCR), body composition, and trial length) are provided in Table 7. Survival rate is not listed because no significant relationship has been demonstrated with dietary PBM substitution for FM. Since PBM and FM contain comparable level of

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crude protein, dietary substitution of FM by PBM has been frequently done on equal weigh basis for growth trial as illustrated in formulae of a typical substitution trial with monondon in China (Table 8)(Xue and Yu, 2005). This practice is strongly discouraged as it ignores the possible variability in EAAs content and their digestibilities as illustrated in this paper. Test diets for FM substitution growth trials should be formulated on a digestible nutrients basis (Allan and Rowland, 2005).

FM substitution rates of all seven trials ranged from 15 to 100%. Among all growth response variables, WG is considered as the most important economic variable for aquaculture producers in this paper, and therefore was selected for analysis of response trend to FM substitution, and the maximum replacement rate (WG begins to decline sharply beyond the maximum replacement rate). All WG values in Table 7 were standardized to “Relative WG as % of FM Control diet” (i.e. WG of FM control being 100%), and were plotted against replacement rates as in Figure 1-8. Shrimp (monondon) fed PBM substituted diets gained more weight (up to 6%) till the replacement reached 75%. At 100% FM replacement, WG was identical to that FM control, and supplementation of crystalline methionine resulted in no further improvement in WG. Shrimp body composition was not affected by PBM replacement up to 100% (Table 7). The growth response of monondon to PBM substitution for FM is not in full agreement with digestibility data in Table 3, even though the source of PBM was the same for digestibility and growth trials. According to Australian workers (Allen et al. 2000), protein and EAAs digestibilities of FM by monondon were in the range of 80-90%, which are much higher than 59-78% of PBM reported by Xue and Yu (2005) (Table 3). One probable reason for better WG of shrimp fed PBM was the difference in actual EAAs content in PBM and FM. This may also explain the zero response in WG to methionine supplementation. High quality PBM could meet EAAs requirements of monondon adequately. Other possible explanation could be the increased FI resulting from PBM substitution for FM. When similar trials are conducted with vannamei, WG response to FM substitution with PBM was positive (i.e. better than FM control) up to 80% replacement rate (Figure 2). These results imply the maximum FM replacement rate with PBM in shrimp diets is about 80%. Weight gain response of largemouth bass and sunshine bass was apparently not affected by PBM substitution for FM even up to 100% (Figure 3 and 4). The PBM replacement also had no effect on FI, FCR, and body composition (Table 7). However, for hybrid striped bass, PBM needs to be supplemented with crystalline lysine and methionine for equal WG at 100% replacement of FM (Figure 5). Additional supplementation of threonine and leucine according to “Ideal Protein” failed to further

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improve the WG (Figure 5). This trial indicated that crystalline EAAs are effective EAAs source for hybrid striped bass, and the ideal protein may need further refinements.

Compared with fresh water fish, fewer studies have been conducted with marine fish on FM substitution with PBM in the past two years. Among the three species evaluated, grouper maintained WG up to 90% of FM control at 100% replacement rate

(Figure 6). At high rates of replacement (≧ 80%), FI and FCR deteriorated slightly

(Table 7). Two levels (30 and 50%) of FM replacement were tested with cuneate drum (Figure 7). Weight gain was reduced by 10%, and appeared to be related with FCR or protein quality. High rates of replacement may require supplementation of crystalline EAAs. Replacement work with black rockfish has been preliminary (Xie and Yu, 2005a). At 15% and 16% FM replacement rate, WG response was variable (Figure 8). It appears that WG response of marine fish to FM replacement by PBM to be more variable than that of fresh water fish. Future trials with multiple replacement rates are warranted as mariculture in Asia is growing rapidly and has a very large production potential in the coming decade.

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Table 7. Response of fish and shrimp to fish meal substitution with poultry by-product meal in growth and body composition.

Growth Body composition (%)7 FM1replacement rate (%) IW2

(g) SGR3 Wt. gain4 (g)FI5

(g/fish) FCR6

Moisture CP Lipid Ash Reference

Shrimp (monondon)

0 (37% Peruvian FM)8 0.2 4.25 2.28 7.8 3.42 76.0 17.2 .5 4.2 Xue and Yu (2005)

25 0.2 4.23 2.38 8.0 3.37 78.3 15.7 .6 3.7 (56D)9

50 0.2 4.41 2.51 7.9 3.13 78.6 15.4 .5 3.9

75 0.2 4.51 2.70 7.8 2.88 79.2 15.2 .5 3.8

100 0.2 4.28 2.60 8.4 3.22 77.9 16.0 .7 3.6

100+Met.10 (0.16%) 0.2 4.23 2.44 8.8 3.59 81.3 13.7 .3 3.3

Largemouth bass

1. 0 (30% Herring meal) 3.1 3.30 41.6 62.4 1.5 71.3 17.2 7.3 3.7 Tidwell at al. (84D)

50 3.1 3.20 39.6 59.4 1.5 70.6 17.3 7.2 3.8 (77D)

2. 0 (30% Herring meal) 6.9 2.7 504 80.3 1.6 73.8 16.1 6.8 3.5

75 6.9 2.8 50.7 81.1 1.6 74.1 16.6 6.8 3.7

100 6.9 2.8 50.7 86.2 1.7 73.8 16.5 7.2 3.2

1FM = Fish meal 5FI = Feed intake 9Trial length in days 13 % body Weight/day

2IW = Initial weight 6FCR = Feed conversion ratio 10Met = Crystalline methionine

3SGR = Specific growth rate 7On wet basis 11 Muscle ration = fillet mass x100/fish mass

4Wt. gain = Weight gain 8Percent fish meal in control diet 12 Intraperitoneal tat ratio = peritoneal fat mas. x 100/fish meal

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Table 7 (continued)

Growth Body composition (%) FM1replacement rate (%) IW2

(g) SGR Wt. gain (g)

FI (g/fish) FCR

Moisture CP Lipid Ash Reference

Hybrid Striped bass

0 (56.6% Menhaden)8 75 188a 314 1.67a 42.1a 11 6.612 Gaylord and Rawles (2005)

100 75 136b 290 2.13b 40.4b 6.3 (70D)

100 + Lys (1.16%) 75 141b 293 2.08b 40.1b 6.1

100 + Lys + Me + (0.57%) 75 181a 311 1.72a 42.0a 6.2

100 + Lys + Me + Thr (0.31%) 75 160ab 307 1.92ab 40.7b 5.5

100 + Lys + Me + Thr + Leu (0.47%) 75 175a 313 1.79a 41.1ab 6.3

Sunshine bass

0 (30% Menhaden) 36 2.05 338 603 1.78 74.9 20.7 3.2 1.4 Muzinic et al. (2006)

33 36 2.01 322 549 1.72 74.9 19.8 5.0 1.3 (112D)

67 36 1.99 315 552 1.75 74.9 20.1 3.7 1.3

100 36 2.05 339 578 1.70 74.9 19.8 3.8 1.3

Black rockfish

1. 0 (72.5% White FM) 7.8 2.73 2.34 1.01 67.8 15.9 10.1 4.2 Xie and Yu (2005a) 15 7.8 2.77 2.26 0.98 68.4 16.3 9.6 4.3 (56D)

2. 0 (64.5% White FM) 7.8 2.68 2.41 1.08 66.5 17.2 10.9 4.3

16 7.7 2.32 2.15 1.05 67.9 16.3 9.6 4.3

Cuneate drum

0 (35% Herring meal) 27 2.21 67 2.03 1.05 73.7 15.7 6.4 3.8 Wang et al. (2005)

30 27 1.97 55 1.88 1.07 73.1 16.0 7.0 3.9 (56D)

50 27 1.98 57 1.95 1.10 73.4 15.7 6.9 3.8

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Table 7 (continued)

Growth Body composition (%) FM1replacement rate (%) IW2

(g) SGR Wt. gain (g)

FI (g/fish) FCR

Moisture CP Lipid Ash Reference

Grouper

0 (40% Anchovy FM) 5.2 2.89 24 25.4 1.06 74 14.9 4.9 4.0 Tan et al. (2005)

20 5.2 2.81 23 23.5 1.02 74 14.5 4.8 4.1 (60D)

30 5.2 2.90 24 25.2 1.05 75 14.0 4.5 3.9

40 5.2 2.78 22 22.4 1.02 74 14.9 4.7 4.0

60 5.2 2.76 22 24.0 1.09 74 14.4 4.8 4.1

80 5.2 2.68 19 21.1 1.11 73 15.1 5.0 4.1

100 5.2 2.62 19 21.5 1.13 75 14.0 4.3 3.9

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Table 8. Formula and nutrient composition (%) of control and experimental diets

used in Shrimp (monondon) growth trials. %FM1replaced by PBM2

0 25 50 75 100 100+AA3

Formula Fish meal 37 28 19 9 0 0 PBM2 0 9 18 27 36 35 SBM4 12 12 12 12 12 12 Peanut bran 16 16 16 16 16 16 Squid meal 3 3 3 3 3 3 Zeolite 2 2 2 2 2 2 Soy lecithin 1.5 1.5 1.5 1.5 1.5 1.5 Fish oil 1 1 1 1 1 1 Soy oil 1 0.9 0.8 0.7 0.6 0.6 Wheat flour 24 25 25 26 26 26 Na2HPO4 1.6 1.6 1.6 1.6 1.6 1.6 Methionine 0 0 0 0 0 0.16 Other 1 1 1 1 1 1

Analysis Dry matter 89.0 90.0 90.0 89.0 89.0 90.0 Crude protein 44.2 44.1 43.7 43.6 43.0 43.0 Crude fat 8.0 8.3 8.6 8.6 8.7 8.3 Ash 10.5 10.2 9.7 9.4 8.9 Total P5 1.5 1.5 1.6 1.5 1.5 1.5 Gross energy

(MJ/kg) 18.1 18.5 18.6 18.7 19.1 19.1

1Fish meal 2Poultry by-product meal 3Amino acid (methionine) 4Soybean meal 5Phosphorus Meat and Bone meal Compared with PBM, fewer growth studies were reported for MBM (Table 9). Chinese workers (Tan et al. 2005) measured growth response of vannamei to MBM substitution for FM (Figure 9). Weight gain was not affected up to 60% of replacement, but had a 7% reduction in WG at 80% replacement. Feed conversion ratio also deteriorated by 9% at high level of replacement (Table 9). Weight gain response suggests that the maximum FM replacement rate by MBM is 60%.

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Weight gain response of juvenile gibel carp to MBM replacement for FM is depicted in Figure 10 (Xie and Yu, 2005b). The WG response was negative linearly from 20 to 100% replacement rates. The most probable reason could be the size and age of the fish as previous studies with larger size showed no negative effect in WG up to 50% replacement rate (Figure 11). Meat and bone meal replacement had no effect on body composition of gibel carp (Table 9). The maximum replacement rate for juvenile and adult gibel carp is 20% and 50% respectively. The WG response of the two marine fish (cuneate drum and black rockfish) is presented in Figure 12 and 13 (Wang et al. 2006, Xie and Yu, 2005a). These preliminary studies showed that both fish did not respond well beyond 15 to 30% of MBM replacement, and multiple replacement rates studies should be conducted is order to determine the maximum MBM replacement rate. Rendered Protein Blend 1. Rainbow trout (Rahnema et al. 2005): In a 70 days growth trial, rainbow trout were fed either FM control diet or FM replacement (100%) diets (Feather meal and meat meal or plant protein based commercial diets. Growth performance was not significantly (P<.05) different between feather meal and meat meal mixture ( FeM + MM), and FM control (Table 10). Feed conversion ratio was higher for the former and could be due to wastage during feeding. Carcass sensory evaluation was also not different between FM control and FeM + MM group. Data from this trial suggests that growing rainbow trout utilize nutrients from FeM + MM, and FM equally well for weight gain, but do not accept plant based diet readily as compared with FM or animal protein based diet (Table 10). 2. Silver perch (Allan and Rowland, 2005): Multiple rendered proteins (meat and bone meal, poultry by-product meal, blood meal, and feather meal) were used as FM replacement, and as dietary protein source for least cost digestible nutrients based diet formulation (Table 11). One of the FM replacement diet had near identical digestible nutrients content as the FM control diet and the other replacement diet had reduced levels (20%) of digestible protein and EAAs (Low Pro Diet). Growth performance and cost of weight gain of the 187 days commercial scale trial are given in Table 12. There were no significant (P < .05) difference between FM control and the least costed FM replacement diets. On a digestible nutrients formulation base, rendered protein meals were well utilized by silver perch, and thus allowing the reduced use of FM, reduced feed cost for fish production, and maintaining the normal growth rate of silver perch (Table 13). This trial serves as a good model for evaluating the potential use of any new ingredients in aquafeeds.

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Table 9. Response of fish and shrimp to fish meal substitution with meat and bone meal in growth and body composition.

Growth Body composition (%)7 FM1 replacement rate (%)

IW2 (g) SGR3 Wt. gain4 (g)

FI5 (g/fish) FCR6

Moisture CP8 Lipid Ash Reference

Shrimp (vannamai) Tan et al., 0 (40% Anchovy meal) 0.9 5.86 8.0 1.37 (2005)(56D)9

20 0.9 6.03 8.6 1.42 30 0.9 5.82 4.4 1.39 40 0.9 6.16 8.1 1.32 50 0.9 5.78 8.2 1.41 60 0.9 5.82 8.4 1.44 80 0.9 5.46 8.1 1.49

Black rockfish 1. 0 (72.5% White FM) 7.8 2.73 2.3413 1.01 67.8 15.9 10.1 4.2 Xie and Yu,

15 7.7 2.68 2.22 1.00 68.3 15.9 9.7 4.1 (2005a) (56D) 2. 0(64.5% White FM) 7.8 2.68 2.41 1.08 66.5 17.2 10.9 4.3

16 7.8 2.23 2.11 1.07 67.8 16.3 10.2 4.5 Gibel carp

0 (43.2% White FM) 3.6 170 277 1.63 71.4 14.5 7.9 2.3 20 3.6 155 259 1.67 71.0 14.4 8.2 2.4

Xie and Yu, (2005b) (56D)

40 3.6 129 244 1.89 70.9 14.5 8.1 2.4 60 3.6 129 257 1.99 71.1 14.5 7.9 2.3 80 3.6 105 226 2.15 71.2 14.6 7.9 2.4 100 3.6 102 237 2.32 70.9 14.1 9.3 2.3

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Table 9 (continued)

1FM = Fish meal 5FI = Feed intake 9Trial length in days 13 % body Weight/day

2IW = Initial weight 6FCR = Feed conversion ratio 10Met = Crystalline methionine

3SGR = Specific growth rate 7On wet basis 11 Muscle ration = fillet mass x100/fish mass

4Wt. gain = Weight gain 8Crude protein 12 Intraperitoneal tat ratio = peritoneal fat mas. x 100/fish meal

Growth Body composition (%)7 FM1 replacement rate (%)

IW2 (g) SGR3 Wt. gain4 (g)

FI5 (g/fish) FCR6

Moisture CP Lipid Ash Reference

Cuneate drum 0(35% Herring meal) 27 2.21 67 2.03 1.05 73.7 15.7 6.4 3.8 Wang et al. 10 27 1.79 49 2.09 1.35 74.2 15.6 5.8 4.0 (2005)(56D)30 27 2.00 57 1.94 1.07 73.3 15.6 6.8 3.9 50 27 1.62 40 2.03 1.40 77.6 13.2 5.4 3.4

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Table 10. Growth response and carcass quality of rainbow trout to different dietary protein source (70 days)

Rahnema, Borton and Shaw, 2005. J. Appl. Anim. Res. 27:77-80 Table 11. Formulation and composition (%) of test diets for silver perch

Test Diet Control High Pro1 Low Pro2

Formulation Fish meal 5 0 0

Meal and bone mal 36.88 37.48 29.39 Poultry by-product meal 0 10.25 10.58 Blood meal 0 1.65 0 Feather meal 0 5.00 5.00 Soybean meal 0 5.00 0 Canola meal 5.00 0 0 Peanut meal 5.00 5.00 0 Lupins 7.36 0 0 Field peas (dehulled) 10.39 0 0 Corn gluteu meal 5.19 0 0 Methionine 0.27 0.15 0 Composition Crude protein 38.2 39.4 31.4 Crude fat 10.3 9.3 13.3 Dig.3 protein 31.1 31.7 25 Dig. energy (MJ/Kg) 13.3 13.7 14 Dig. lysine 1.9 1.9 1.5 Dig. M + C4 1.4 1.6 1.2 Dig. leucine 2.6 2.5 1.9 Dig. isoleucine 1.3 1.3 1.1

Dig. arginine 2.6 2.5 1.8 Dig. histidine 0.8 0.8 0.6 Dig. P + T5 2.5 2.5 1.9 Dig. valine 1.5 1.7 1.4 Dig. threonine 1.3 1.4 1.1 Dig. P6 0.8 0.7 0.6

1High protein test diet 3Dig. = Digestible 5P + T = Phenylalanine + Tyrosine

2Low protein test diet 4M + C = Methionine + Cystine 6Phosphorus

Plant based Fish meal Feather and meat meal Initial wt. (g) 914 924 912 Feed intake (g) 1239a 1546b 2124c

Daily gain (g) 12.7a 16.3b 15.5ab Feed/gain 1.38a 1.33b 1.93b

Carcass quality Tenderness 5.12a 6.09b 5.53ab Juiciness 5.35a 6.28b 5.79ab

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Table 12. Production response of silver perch to different combination of protein sources.

Test Diet Control High Pro1 Low Pro2

Final Weight (g/fish, 187D) 461.3 453.3 432.7 Growth rate (g/fish/day) 2.1 2.1 2.0 Feed/gain 1.6 1.7 1.7 Survival % 91.8 94.0 93.3 Production (MT/ha) 6.4 6.3 6.2 Ingredient cost (AU$/kg fish) 0.94 0.96 0.96

Allan and Rowland, 2005. Aqua. Res. 36:1322 1High protein test diet 2Low protein test diet Table 13. Progress on formulation and feed cost reduction – Silver perch 1995 2005 2006

%Fish meal in diet 27 5 0

%Rendered animal proteins <5 ~30 ~50

%Daily growth rate (g/fish) 2.0-2.3 2.0-2.4 2.0-2.4

Feed cost/kg fish (AU$) 1.8 1.1 0.94 Allan and Rowland, 2005. Aqua. Res. 36:1322 Recommendations for Application of Poultry By-product Meal and Meat and Bone Meal in Aquafeeds The recommended digestion coefficients of protein, energy, and EAAs, and the maximum FM replacement rate for shrimp (vannamei), fresh water (warm and cold water) and marine (warm and cold water) fish are given in Table 14 for PBM, and Table 15 for MBM. These values are useful in formulating least east diets utilizing PBM and MBM as FM replacements while guarding the normal growth performance of aquaculture animals. All digestion coefficients were discounted by 5% as a safety margin. Feed nutritionists should use the analyzed nutrients and EAAs values of all ingredients available for feed formulation. While digestibilities and the maximum FM replacement rates are higher for PBM than MBM, nutrients requirement specifications of the feed will determine the optimum use rate of the two protein meals. Generally, Diets with a relatively high digestible protein (DP) requirement (20% and above) are more likely to use PBM and MBM while low DP requirement diets will more likely select plant source ingredients. Data in Table 13 and 14 may be revised when new and reliable data becomes available.

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Table 14. Formulation recommendation for poultry by-product meal (PBM) in

aquafeeds. Fish

Fresh water Marine

Shrimp

(vannamei) Warm water

Cold water

Warm water

Cold water

Digestibility (%)1 Protein 80 78 78 83 65 Energy 80 81 71 86 753

Essential Amino acids

Arqinine 81 85 822 88 72 Histidine 85 92 852 89 43 Isoleucine 86 83 792 86 55 Leucine 85 84 822 86 77 Lysine 88 85 872 90 82 Methionine 90 79 902 88 73 Phenylalanine 85 83 812 84 62 Threonine 81 87 802 86 79 Valine 77 82 802 87 - Cystine 72 48 532 61 41 Tyrosine 84 88 862 89 -

Maximum fish meal replacement rate (%) 80 80 802 50 20-804

1Digestibility coefficient x 0.95 (discount) 2Rainbow trout (Hardy and Cheng, 2002) 3Gilthead seabream (Lupatsch et al. 1997) 4Coho salmon (Higgs et al. 1979)

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Table 15. Formulation recommendation for meat and bone meal (MBM) in aquafeeds.

Fish Fresh water Marine

Shrimp

(vannamei) Warm water

Cold water

Warm water

Cold water

Digestibility(%)1

Protein 78 79 853 83 864

Energy 66 75 792 86 874

Essential Amino acids

Arqinine - 702 74 89 90

Histidine - 72 92 84 75 Isoleucine - 71 82 87 86 Leucine - 74 90 88 86

Lysine - 72 89 80 86 Methionine - 78 88 88 94 Phenylalanine - 71 88 87 87

Threonine - 73 92 87 85 Valine - 73 92 87 85

Cystine - 64 - - 61 Tyrosine - 77 92 87 87

Maximum fish meal replacement rate (%) 60-70 40-50 50-60 50-60 20 1Digestibility coefficient x 0.95 (discount) 2Silver perch (Allan et al. 2000) 3Rainbow trout (Bureau, 1998) 4Rockfish (Lee. 2002) Conclusion Poultry by-product meal and MBM are high protein animal source dietary ingredients for carnivorous and omnivorous aquatic animals. Recent research have indicated that PBM resembles FM in nutritive value (composition, digestibilities, FI, FCR and body composition of fish and shrimp) and could replace most of FM (up to 80%) in shrimp and several economically important fish diets without causing a reduction in WG. Meat and bone meal should be mainly considered for its cost advantage over FM as its nutritive value is slightly lower than FM and PBM. When

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diets for rainbow trout (carnivorous) and silver perch (omnivorous) were formulated with specification of digestible nutrients, multiple rendered animal proteins (MBM, PBM, feather meal, and blood meal) were selected on a least cost basis up to 50% of diets, which supported a normal FI, WG, and FCR as compared with the control diet formulated with FM. Fewer FM replacement trials have been conducted for marine fish as compared with fresh water fish, but mariculture has been growing rapidly in Asia. With the declining supply of FM and raw (trash) fish, the future growth of the industry will depend on the availability of quality commercial aquafeeds made with PBM and MBM as FM replacement. Decision on ingredients selection and their inclusion rates when formulating aqua diets should largely be based an accurate nutrients composition, digestibility, palatability, and the risk of anti-nutritional factors.

5060708090

100110

10 20 30 40 50 60 70 80 90 100Fish meal replacement %

PBM

PBM+Met. China, 2005

Relative w

t. gain %FM

control

Fig. 1. Weight gain response of shrimp (monondon) when fed FM substituted diets with graded levels of poultry by-product meal (PBM)

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PB M – W hite Shrim p (L .vannam ei)

50

60

70

80

90

100

110

120

10 20 30 40 50 60 70 80 90 100 Fish meal replacement %

PBM (FD) Texas, 1998

PBM (PFG) + SBM (1:2), 1998

PBM(PFG), OI , Hawai i , 2002

China, Qingdao 2002

Relative W

t. gain % of FM

Control

F ig . 2 . W eight gain response of w hite shrim p w hen fed FM substitu ted diets w ith graded levels of PB M

5060708090

100110

10 20 30 40 50 60 70 80 90 100Fish meal replacement %

Trail 1Trail 2Keutucky,US 2005

Relative w

t. gain %FM

control

Fig. 3. Weight gain response of largemouth bass when fed fish meal (FM) substituted diets with graded levels of poultry by-product meal (PBM)

5060708090

100110

10 20 30 40 50 60 70 80 90 100Fish meal replacement %

Keutucky, US, 2006

Relative w

t. gain %FM

controlFig. 4. Weight gain response of sunshine bass when fed FM substituted diets with poultry (turkey) by-product meal

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5060708090

100110

10 20 30 40 50 60 70 80 90 100Fish meal replacement %

PBM

PBM + Lys

PBM + Lys + Met

PBM + Lys + Met + Thr

PBM + Lys + Met +Thr + Leu

US 2005

Relative w

t. gain %FM

control

Fig. 5. Weight gain response of hybrid striped bass when fed FM substituted diets with poultry by-product meal (PBM)

50

60

70

80

90

100

110

10 20 30 40 50 60 70 80 90 100

Fish meal replacement %

China, 2005

Relative w

t. gain %FM

control

Fig. 6. Weight gain response of grouper when fed FM substituted diets with poultry by-product meal (PBM)

5060708090

100110

10 20 30 40 50 60 70 80 90 100Fish meal replacement %

China, 2005

Relative w

t. gain %FM

control

Fig. 7. Weight gain response of cuneate drum when fed FM substituted diets with poultry by-product meal (PBM)

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5060708090

100110

10 20 30 40 50 60 70 80 90 100Fish meal replacement %

Trial 1

Trial 2

China, 2005

Relative w

t. gain %FM

control

Fig. 8. Weight gain response of black rockfish when fed FM substituted diets with poultry by-product meal(PBM)

5060708090

100110

10 20 30 40 50 60 70 80 90 100Fish meal replacement %

China, 2005

Relative w

t. gain %FM

control

Fig. 9. Weight gain response of shrimp (vannamei) when fed FM substituted diets with meat and bone meal (MBM)

50

60

70

80

90

100

110

10 20 30 40 50 60 70 80 90 100Fish meal replacement %

China, 2005

Initial wt. = 3.6g

Relative w

t. gain %FM

control

Fig. 10. Weight gain response of juvenile gibel carp when fed FM substituted diets with meat and bone meal (MBM)

120

5060708090

100110

10 20 30 40 50 60 70 80 90 100Fish meal replacement %

Wuhan, China 2001

Xian Hanbo 2002

MBM – Gibel Carp

Relative w

t. gain %FM

control

Fig. 11. Weight gain response of gibel carp when fed FM substituted diets with graded levels of MBM

50

60

70

80

90

100

110

10 20 30 40 50 60 70 80 90 100Fish meal replacement %

Relative w

t. gain %FM

control

Fig. 12. Weight gain response of cuneate drum when fed FM substituted diets with meat and bone meal (MBM)

5060708090

100110

10 20 30 40 50 60 70 80 90 100Fish meal replacement %

Trial 1

Trial 2

China, 2005

Relative w

t. gain %FM

control

Fig. 13. Weight gain response of black rockfish when fed FM substituted diets with meat and bone meal (MBM)

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References Allan, G.L. and S.J. Rowland., 2005. Performance and sensory evaluation of silver

perch (Bidyanus bidyanus Mitchell) fed soybean or meat meal-based diets in earthen ponds. Aquaculture Research 36:1322-1332

Allan, G.L., S. Parkinson, M.A. Booth, D.A.J. Stone, S.J. Rowland, J. Frances, and R.

Warner-Smith., 2000. Replacement of fish meal in diets for Australian silver perch, Bidyanus biyanus: I. Digestibility of alternative ingredients. Aquaculture 186:293-310

Bureau, D.P. (2000). Use of rendered animal protein ingredients in fish feed. Fish

Nutrition Research Laboratory Research Report. Department of Animal and poultry Science, University of Guelph, Canada.

Bureau, D.P., C.Y. Cho, H.S.Bayley and A.M. Harris, 1998. Apparent digestibility of

amino acids of feather meals, and meat and bone meals for salmonids. Fats and Protein Research Foundation, Inc. Bloomington, IL.

Cheng, Z.J., R.W. Hardy and N.J. Huige, 2004. Apparent digestibility coefficients of

nutrients in brewer’s and rendered animal by-products for rainbow trout (Oncorhynchus mykiss (Walbaum)). Aquaculture Research 35:1-9.

Davies, S.J., and R.D. Serwata, 2005. Reduction of fish meal in commercially

important temperate/marine fish species with selected animal protein concentrate bends with respect to protein, energy and amino acid digestibility. Director’s Digest No. 332 Fats and Protein Research Foundation, Inc. Bloomington, Illinois

Gaylord, T.G. and S.D. Rawles, 2005. The modification of poultry by-product meal for

use in hybrid striped bass Morone chrysops x M. saxatilis Diets. J. World Aquaculture Society 36(3): 363-374

Hardy, R.W. and Z.J. Cheng, 2002. Effect of poultry by-product meal supplemented

with L-lysine, DL-methionine and L-histidine as a replacement for fish meal on the performance of rainbow trout, Director’s Digest. No. 317. Fats and Protein Research Foundation, Inc. Bloomington, IL

Higgs, D.A., J.R. Markents, D.W. Maequrrie, J.R. McBride, B.S.Dosanjh, C. Niehols

and G.Hoskins, 1979. Envelopment of practical dry diets for Coho salmon, Oneorhynchus Kisutch, using poultry by-product meal, feather meal, soybean meal and rapeseed meal as major protein sources. Proc. World Symp. On Finfish Nutrition and Fishfeed Technology 2:191- 215

Lee, S.M., 2002. Apparent digestibility coefficients of various feed ingredients for

juvenile and grower rockfish (Sedates schlegeli). Aquaculture 207: 79-95 Lupatsch, I., G.W. Kissil, D. Sklan, and E. Pfeffer, 1997. Apparent digestibility

coefficients of feed ingredients and their predictability in compound diets for gilthead seabream, Sparus aurata L. Aquaculture Nutrition 3:81- 89

Muzinic, L.A., K.R. Thompson, L.S. Metts, S. Dasgupta and C.D. Webster, 2006. Use

of turkey meal as partial and total replacement of fish meal in practical diets for sunshine bass. Aquaculture Nutrition 12:71- 81

National Research Council. (1993). Nutrient Requirements of Fish. National Academy

of Science, Washington, D.C.

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Portz, Li. And J.E.P. Cyrino, 2004. Digestibility of nutrients and amino acids of different protein sources in practical diets by largemouth bass Micropterus salmoides (Lacepede, 1802). Aquaculture Research 35:312- 320

Rahnema, S., R. Borton and E Shaw, 2005. Determination of the effects of fish vs. plant

vs. meat protein-based diets on the growth and health of rainbow trout. J. Appl. Anim. Res. 27:77-80

Tan, B.P., K.S. Mai, S.X. Zheng, Q.C. Zhou, L.H. Liu and Y. Yu, 2005. Replacement of

fish meal by meal and bone meal in practical diets for the white shrimp Lifopenaeus vannamei (Boone). Aquaculture Research 36:439- 444

Tan, B.P, S.X. Zheng and Y. Yu, 2005. Replacing fishmeal with poultry by-product

meal: Practical diets for grow-out culture of grouper Epinephelus coioides. International Aquafeed. July- August: 26-29

Tidwell, J.H., S.D.Coyle, L.A. Bright and D. Yasharian, 2005. Evaluation of plant and

animal source proteins for replacement of fish meal in practical diets for the largemouth bass Micropterus salmoides. J. World Aquaculture Society 36(4):454- 463

Wang, Y., J.L.Guo, D.P. Bureau and Z.H. Cui, 2006. Replacement of fish meal by

rendered animal protein ingredients in feeds for cuneate drum (Nibea miichthioides). Aquaculture (In press)

Xie, S.Q. and Y. Yu, 2005a. Utilization of several alternative protein sources for black

rockfish (Sebastes schlegeli Higendorf). Research Report No. 46. National Renderers Association, Inc., Hong Kong, China

Xie, S.Q. and Y. Yu, 2005b. Meat and bone meal replacement in diets for juvenile gibel

carp ( Carassius auratus gibelio): Effects on growth performance, phosphorus and nitrogen loading. Research Report No.48. National Renderers Association, Inc., Hong Kong, China

Xue, M. and Y. Yu, 2005. Digestion and growth response to dietary fish meal

substitution with poultry by-product meal of shrimp (L. monondon and L. vannamei). Research Reoprt No. 49. National Renderers Association, Inc., Hong Kong, China

Y. Yu, 2006. Use of poultry by-product meal, and meat and bone meal in aquafeeds.

Proceedings of Asia Aquafeed Conference (In press) Zhou, Q.C., B.P. Tan, K.S. Mai and Y.J. Liu, 2004. Apparent digestibility of selected

feed ingredients for juvenile cobia Rachycentron canadum. Aquaculture 241:441- 451

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Application of de-oiled soya lecithin in shrimp feeds Yuyun Mu (Ph.D), Berg + Schmidt Asia Pte Ltd Introduction Lipids refer to compounds that are relatively insoluble in water but are soluble in organic solvents. They could be differentiated according to their polarity. With respect of such classification, some lipids such as triacylglycerols, wax esters, and sterol esters are called nonpolar lipids because of nonpolar hydrocarbon groups (Higgs and Dong, 2000). By contrast, other lipids such as phospholipids (PLs) along with glycolipids (e. g., shingomyelin and glyceroglycolipids) are classified as polar lipids owing to polar groups as the part of the basic structure (Hertrampf, 1991; Higgs and Dong, 2000). PLs consist of glycerol in which positions 1 and 2 are esterified with two fatty acids and position 3 with phosphoric acid and nitrogenous base (Akiyama et al., 1992). PLs are classified into different types according to nitrogenous base moiety. For example, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) have choline, ethanolamine, serine and inositol as their nitrogenous base, respectively (Tacon 1987; Higgs and Dong, 2000). The beneficial effect of dietary PL on growth and survival of larval and juvenile stages of many species of marine fish and shrimp has been well documented (Teshima, 1997; Coutteau et al., 1997; ). At first, these findings appeared surprising as de novo synthesis of PLs has been demonstrated in crustaceans (Chapelle et al., 1985; Teshima et al, 1986c; Kanazawa and Koshio, 1994). Since there was such apparent discrepancy, substantial research efforts have been done to explain the effect of PLs and to find out PL requirement. The findings available are beneficial in better understanding to nutritional significances and application of PLs in shrimp aquaculture. Soya lecithin: the effectively commercial sources of PLs. As one of the essential elements of biological membranes, PLs present in all cells of animals and plants. Some of important sources for commercial use and research work in aquaculture originate from plants like soya beans, sunflower seeds, rape seeds, ground –nuts and maize, and also from animals like poultry eggs, bovine brain, bonito eggs , ovine brain and carpet shells(Hertrampf, 1991). Gill (1998) showed PL contents of a number of aquafeed ingredients (Table1), ranging from the lowest level of 0.50% for tuna meal to the highest one of 16.26% for oyster meal. It is obvious that the levels of PLs in aquafeed ingredients are variable. However, when counting for the concentration and cost of PLs, commonly used aquafeed ingredients may not be the most economic sources. PLs or lecithin is a natural component of soybean. After oil extraction, lecithin gums are obtained from crude soy oil which contains 2.5-3.0% PLs. Through carefully heating and steaming, lecithin gums are separated from oil owing to its swelling capacity, and these gums are further dried under a vacuum to remove moisture and to obtain crude soya lecithin (Hertrampf, 1991; Russett, 2001). In fact, crude soy lecithin is a complex mixture of acetone insoluble phospholipids combined with varying amount of other substances such as triglycerides, fatty acids, sterols, glycolipids and carbohydrates.

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Table 1: Approximate phospholipids content in aquafeed ingredients (Gill, 1988) Ingredients Phospholipid level, %Clam meal 1.27 Egg powder 2.14 Fish meal 2.47 Krill meal 2.03 Oyster meal 16.26 Shark liver oil 2.50 Shrimp head meal 3.82 Shrimp meal 1.02 Shrimp liver meal 6.36 Squid liver oil 1.53 Squid meal 3.39 Squid oil 0.76 Tuna meal 0.50 Yeast, tourla 1.27

Typical compositions of fluid and de-oiled soya lecithin are shown in Table 2. PL concentration in fluid soya lecithin may be different. Usually, fluid soya lecithin contains 60-63% PL as acetone insoluble. It shall contain a minimum of 50% acetone insoluble according to U.S. Federal Codex and a minimum of 60% according to the European E 322 regulation, respectively. De-oiled lecithin usually contains 95-97% acetone insoluble. Table 2: Typical compositions of fluid and de-oiled lecithin (Li and Peisker, 2005)

Ingredients Fluid soy lecithin De-oiled soy lecithin

PLs as acetone insoluble, % 63 97 Soybean oil, % 36 2 Moisture, % 1 1 Fat, % 93 90 Kcal/ kg 7600 7000 Fatty acid, g/100 g fat 68 54 Relative fatty acid composition of total fatty acids, % Stearic acid 5 5 Palmitic acid 16 20 Oleic acid 18 9 Linoleic acid 54 59 Linolenic acid 7 7 Major PLs PC, % 14 23 PE, % 12 20 PI, % 9 14 Phosphatidic acid (PA), % 5 8 Choline, % 2.1 3.1 Inositol, % 1.8 3.4

The effectiveness of PLs may be affected by PL fraction and components. Akiyama et al. (1992) reported that for larval P. japoniocus, PL containing choline (i.e. PC) and inositol (i.e.

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PI) are most beneficial, that PL containing essential fatty acids are most effective, and that the position of the essential fatty acids affects PLs’ effectiveness (Kanazawa, 1983). Similarly, Teshima (1997) reported that PC and PI fractions of soybean lecithin as a PL source were effective for juvenile P. japonicus when supplemented to casein-base diets, while PE fraction was ineffective in improving the development, growth and survival. Clearly, soy lecithin is the most effective source for PLs in aquaculture since it either in fluid or powder form offers a high PL concentration and contains PC, PE and PI as the major components of PLs (Table 2). Furthermore, de-oiled soya lecithin is better than crude one in views of quality stability, PL concentration and application. Fluid soya lecithin is difficult to handle owing to its viscosity and may be variable in quality and PL concentration depending on processing. On the contrast, de-oiled lecithin is consistent in compositions, stable in quality and more easily in handling because of further removal of oil residue from crude soya lecithin. Biological functions of PLs The membrane PL source

PLs are the major lipid components of biological membranes. The functional properties, physical uniformity and fluidity of the membrane are determined by the followings: 1). the levels and types of constituent PLs; 2) the fatty acid compositions of the PLs, especially at position 2 of the glycerol backbone; 3) the interactions of the PLs with cholesterol and either enzymatic or structural proteins; and 4) the specific pairing of long-chain polyunsaturated fatty acids with ∆9 (18:1) monounsaturated fatty acids in the sn-1 position, especially in PE (reviewed by Higgs and Dong, 2000). It have been reported that early life stage of fish and shrimp are not capable of synthesizing PL at a rate sufficient to meet the requirement for formation of new cell component during the initial short period of rapid growth (Kanazawa, 1993; Geurden et al 1995).The resistance to salinity stress and consequently survival increased in postalrval Penaeus japonicus (Camara et al., 1996; Kontara et al., 1998) and in postlarval P. vannamei (Coutteau et al., 2000) due to dietary PL supplement. Cells must adapt to establish a new equilibrium between the environment and the physcochemical properties of their membranous structure in order to survive the new conditions. PLs involve in such adaptation owing to their influences on uniformity and fluidity of the membrane. Within cell membranes, phospholipids are organized in the form of bilayers which ensure constant renewal and regeneration of the cell materials. Generally, PC is the most active component (Kanazawa, 1993; Teshima, 1997). This may be explained by its specific need for PC as the major constitute of polar lipids incorporated into membranes as well as of lipoproteins (Coutteau et al., 1997; Coutteau et al, 2000). Without PLs in membranes, there is neither cell respiration occurring in the mitochondria nor mobility of the membranes. It remains unclear whether essential fatty acids (EFAs), which are preferentially esterified to the sn-2 position in PL, are assimilated as a single entity or separately from the PL molecules (Coutteau et al., 2000). The latter may have important implications for possible direct effect of the EFA content in dietary PL on the composition and function of PL in membranes and /or lipoproteins. Roles in digestion, absorption and transport of neutral lipids

Since dietary neutral lipids such as cholesterol and /or triglycerides easily enter the midgut gland of crustaceans (Teshima and Kanazawa 1983) regardless of the presence of PL in the

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diet, the role of PL in emulsification, digestion and absorption of neutral lipids is questionable. However, PLs have hydrophilic and hydrophobic properties. PL (lecithin) may be required as a surfactant for efficient emulsification and digestion of dietary lipids such as triglycerides and cholesterol in the early stage of crustaceans (Conklin et al., 1980). Exogenous and endogenous lipids shall be incorporated into chylomicrons or lipoproteins for the transportation via the blood or lymphatic system. PLs are not only the important components of the above lipid transport vehicles, and also have the influence on the assembly and stability of lipoproteins. Teshima et al (1986c) hypothesized that dietary PL may provide specific lipid classes as substrate for the formation of lipoprotein in shrimp which are the main mediators of lipid transport in the hemolymph of shrimp, and contain polar lipids as the main lipid component. In crustaceans, dietary PLs accelerate mobilization and transport of triglycerides and cholesterol among body tissues and organs, thus enhancing utilization of dietary lipids and cholesterol. (Coutteau et al. 1997; Teshima, 1997). For example, Kontara et al. (1998) reported that beneficial effects of dietary PL in P. japonicus may be attributed by better mobilization of the lipid from midgut gland or gut into the hemolymph, which resulted in enhanced lipid deposition in the tissues as well as an increase of the energy available for growth. Based on the previous observation that dietary PL deficiency resulted in the decreases both in body retention rates of dietary lipids and cholesterol in juvenile P. japonicus and in body concentrations of steryl esters, free sterols, PC and PI in the larval P. japonicus, Teshima (1997) concluded that shrimp is incapable of effectively utilizing dietary lipids, especially cholesterol for growth and survival in the absence of supplemental soybean lecithin. Effects on body lipid composition

PLs affect lipid deposition in tissues of shrimp which may be owing to their functions in lipid transport. Teshima et al., (1986a) found that increased levels of PL in juvenile P. japonicus body, in particular PC and cholesterol due to the supplementation of 3% soybean lecithin in the diet. Similarly, larval P. japonicus fed the diet containing 3% soybean lecithin had high tissue levels of sterol ester, free sterol, PC and PI compared to larvae fed on a PL deficient diet (Teshima et al., 1986e); Levels of both neutral lipids (i.e., triglycerides and cholesterols) and polar lipids (mainly PC) in hepatopancreas and hemolymph in juvenile P. japonicus were increased owing to dietary PL supplementation (Teshima et al., 1986b). The increase in polar lipid content in the whole body of juvenile P. merguiensis was found due to the increase of PC in the diet (Thongrod and Boonyaratpalin 1998). Dietary inclusion of soybean PC resulted in an increased retention of lipid n-3 highly unsaturated fatty acids (HUFA) in shrimp tissue compared to that of shrimp fed a PL-free diet containing identical levels of total lipid and n-3 HUFA in larvae P. vannamei (Coutteau et al., 2000). It is concluded that, larvae and juveniles of shrimps supplemented with dietary PLs increase the retention and levels of polar and neutral lipids in hemolymph, muscles and tissues, in particular PC, cholesterol and n-3 HUFA (Chen and Jenn, 1991; Kontara et al, 1998; Coutteau et al., 2000;). It has been reported that dietary PL type influences the proportions of n-3 HUFA in the tissues of larval and juvenile shrimps. The highest and lowest proportions of n-3 HUFA were found in larval P. japonicus receiving soybean PC and PI, respectively (Teshima et al., 1986e). A higher proportion of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) was observed in juveniles P. japonicus fed the diet containing 3% soybean lecithin (Teshima et al., 1986a). The improved efficiency of dietary EFA because of supplemented PL is likely the basis for the positive interaction between EFA and PL requirement (Coutteau et al., 1997).

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Such positive interaction may contribute to improved resistance of shrimp to adverse growing conditions such as osmotic stress and salinity stress (Russett, 2001) and also to reduced requirements for n-3 HUFA (Kontara et al., 1997). Benefits of dietary phospholipids in shrimp Provision of energy, phosphate, EFA, choline and inositol

Phospholipids are polar lipids and may be more easily emulsified and less susceptible to limited secretion of hypothetical bile salt, thus being highly digestible in larval and juvenile crustaceans. It has been postulated that dietary PLs may serve as the effective and direct source of nutrients like metabolic energy, available phosphate, choline and inositol for early stages of crustaceans (Hertrampf, 1991; Coutteau, 1997). Coutteau et al. (1997) noted that PLs are superior sources of energy and EFA to neutral lipids for the growth and survival of larval shrimp whose digestive capacity may not be fully developed . This may be applicable to the post-larval and juvenile shrimp. Crustaceans, including penaeid shrimp, have only a limited capacity to synthesize the linoleic and linolenic familities of fatty acids, and possess little or no ability to chain-elongate and desaturate n-3 and n-6 PUFA to the n-3 and n-6 HUFA (Kanazawa et al., 1979; Kayama et al., 1980; Teshima et al., 1992). Penaeids are dependent upon the dietary supply of the EFA: LA, LNA, EPA and DHA (D’Abramo, 1997). Therefore, in shrimp, dietary needs for EFAs shall be met by all of LA, LNA, EPA and DHA (D’Abramo, 1997). PLs may improve the efficiency of EFAs supplied as neutral lipid and thus reduce requirements for n-3 HUFAs (Kontara et al., 1997). The fatty acid compositions of PL appear critical for their nutritional functionality in crustaceans since saturated PC has been found to be ineffective in P. japonicus (Kontara et al, 1998). PC provides a bio-available and time-released source of choline (Li and Peisker 2005). Choline is the precursor of acetylcholine that is the most important neurotransmitter. Without supplementary PC, the formation of acetylcholine may be endangered in shrimp as in mammals. Choline and inositol in the presence of PLs are more effective than artificially synthetic choline and inositol for aquatic animals (Hertrampf, 1991). For example, dietary choline present in choline chloride is not as effective as soy lecithin in reducing molt death syndrome in juvenile lobsters (Teshima, 1997). Beneficial effects of PC and PI on growth and survival in shrimp are not replicated by supplementing equal levels of artificial choline and inositol, respectively (Teshima 1997). Interaction with cholesterol

Cholesterol is the important and essential nutrient because of its roles as a cell constitute, a lipoprotein component, a metabolic precursor of adrenal and reproductive hormones, vitamin D and bile acids. (Tacon 1990; Akiyama et al., 1992; Russet, 2001). So, cholesterol is involved in complete molt, growth and survival of shrimps. PLs are believed to emulsify cholesterol for better absorption in crustaceans owing to their surfactant property. The beneficial effects of cholesterol improved by supplemental PLs are predominantly attributed to improved mobilization of cholesterol from digestive tract to hepatopancreas, hemolymph and muscles (Coutteau et al., 1997; Teshima, 1997). The interaction between cholesterol and PLs on weight gain has been investigated with various species. With varying lecithin levels in the diet, 0.5% cholesterol was required for the optimal growth of P. penicillatus (Chen and Jenn, 1991) and that of P. monodon (Chen 1993). Similarly, with larval and post-larval P. monodon, Paibulkichakul et al. (1998) did not

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observe the interaction between lecithin and cholesterol on growth and survival. However, Gong (1999) found the significant interaction between cholesterol and PLs. For the optimal growth of juvenile P. vannamei, 0.4%, 0.2% or 0.1% cholesterol was required in the diet without PLs, with 1.5 or 3.0 % of supplemental de-oiled soya lecithin, or with 5% of supplemental de-oiled soya lecithin, respectively. The decreased requirement of cholesterol owing to supplementary lecithin may be owing to increased absorption, transport and utilization of cholesterol in crustaceans. Improvements in growth, feed conversion and survival

Supplementation of dietary PLs (lecithin or purified PC) results in significant improvements in growth, feed conversion and survival of a lot of shrimp species (Hertrampf, 1991, Akiyama, 1992; Teshima, 1997). The reported species includes P. japonicus (Teshima and Kanazawa 1983; Teshima, 1986a; Camara et al., 1996), P. pencillatus (Chen and Jenn 1991), P. monodon (Piedal-Pascual, 1985, 1986; Chen, 1993; Paibulkichakul et al., 1998), P. chinensis (Kanazawa, 1993), P. vannamei (Camara, 1994; Coutteau et al., 1996). PL deficiency may lead to difficulty in larval development of crustaceans (Coutteau et al., 1996; Coutteau et al., 1997). Dietary PL inclusion is able to improve tolerance to osmotic shock and salinity in postlarval P. japonicus (Teshima et al, 1986a , 1986b; Kontara et al., 1998), in zoeal, mysid and postlaval P. monodon (Paibulkichakul et al., 1998), in postlarval P. vannamei (Coutteau et al., 2000) and to prevent incomplete molting and molt death syndrome in larvae and post-larvae of lobster (Teshima, 1997). Such inclusion is also beneficial in improving the efficiency of dietary EFA and in increasing the retention of n-3 HUFAs in tissues (Coutteau et al., 1997; Teshima, 1997; Russett, 2001). The improvements in utilization of energy and essential nutrients like EFA and cholesterol are contributed to better growth and higher survival of shrimp fed soy lecithin. Effects on reproduction of broodstocks

Supplementation of soy lecithin is effective in improving reproductive performances of parent shrimp. Bray et al. (1989) demonstrated that soybean lecithin effectively improved nauplii production, hatch ratio, sperm counts. Cahu et al. (1994) found that less than 2% PLs in broodstock diets resulted in depressed spawning frequency and egg numbers per spawn in P. vannamei. Lecithin is reported to be suitable for successful maturation and spawning of pond reared tiger shrimp (Millamena et al. 1986 in review of Hertrampf, 1991). Requirements and application of phospholipids in shrimp Requirements of phospholipids

Although crustaceans can synthesize PLs, the biosynthesis can not meet metabolic requirement for PLs during larval and juvenile stages. Shrimp have the essential requirement of PLs for growth and survival. The requirements of main cultured shrimp species are shown in Table 3. The optimal levels of PLs are dependent upon the species, age and PL fraction. In general, Larval stages of crustaceans are very sensitive to dietary PL levels and PL requirement decreases with age or developmental stage of shrimp (Coutteau et al., 1997, Teshima 1997; Russett, 2001). PC and PI are more effective in promoting growth and survival of larval and juvenile shrimp and are regarded as active PL fractions (Hertrampf, 1991; Teshima, 1997; Coutteau et al., 1997). For most shrimp species, the estimated PL requirements are mostly in the range of 1 to 2% PL plus PI for larvae and that of 1.0-1.5% PC plus PI for juveniles (Table 3; Coutteau et. al., 1997), respectively. Supplementation of PC and PI is applicable to decrease PL requirements (Hetrampf, 1991; Teshima 1997).

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Inclusion of soy lecithin in commercial feeds As the most importantly commercial sources of PLs, soy lecithin is used in shrimp feeds to meet the PL requirement. The younger the shrimp, the more soy lecithin has to be included in the feeds (Hertrampf, 1991; Akiyama et al., 1992). In commercial feeds, crude soy lecithin is often dosed at 1 to 2 % of the diets (Akiyama et al., 1992), contributing about 0.25-0.50% PC plus PI. Such lower dosages of crude soy lecithin may be not able to provide sufficient amount of PLs to shrimps, especially that of PC and PI or to larval and juvenile shrimps. Table 3: Requirements of phospholipids in shrimp Species, stage, wet weight

Phospholipid source (purity)

Optimal level (%)

Reference

P. chinensis Juveniles, 0.71 g

SL: 42% PC, 20% PI, 6% PE, 4% LPC and others

2.0 Kanazawa (1993)

P. Japonicus, Larvae, Zoa I/II Juveniles 1g Juveniles, 5g

SL: PC 24%, PE 30%, PI 18% and other PLs. SL: 35% PC, 18% PI Soy PC: 95% PC

3.0

3.0 1.5

Teshima and Kanazawa (1983) Teshima et al (1986a) Camara (1994)

P. merguiensis Juveniles, 0.1 g

SL: 60% PLs 1.0-2.0 Thongrod and Boonyaratpalin (1998)

P. monodon Juveniles, 0.09 g Juveniles, 0.45 g Zoeal, mysid and postlarval

SL: 63% PL Soy PC: 80% PC, 20% LPC SL

2.0 1.25

1.0-1.5

Piedad-Pascual (1986) Chen (1993) Paibulkichakul et al, 1998

P. penicillatus Juveniles, 1 g

Soy PC: 80% PC, 20% LPC

1.25 Chen and Jenn (1991)

P. stylirostrics SL (ICN Biochemicals) 1.5 Bray et al. (1990) P. vannamei Juveniles, 2 mg

Soy PC: 95% PC SL: 86% PL

1.5 6.5

Coutteau et al (1996) Coutteau et al (1996)

* PL, phospholipids; SL, soy lecithin; PC, phosphatidylcholine; PI, Phosphatidylinositol; PE, phosphatidylethanolamine; LPC, lysophosphatidylcholine; PS, phosphatidylserine;

Balanced formulation with de-oiled soy lecithin

For the maximum growth and survival of shrimp, feeds shall be optimum and balanced in terms of lipid nutrition, e. g. dietary levels of crude lipids, all EFA, PLs (particularly PC and PI), cholesterol and the ratio of n-3 to n-6 fatty acids. However, the optimal levels of dietary lipids in shrimp feeds are less than 8% (Akiyama et al., 1992). This is most likely owing to more mortality and reduced growth caused by higher lipid levels in feeds. As compared with crude soy lecithin, de-oiled soy lecithin contains less lipid content, and more PLs and PC plus PI (Table 2). Thus, inclusion of de-oiled lecithin in shrimp feeds is helpful to save the formula space for the optimum levels and/or ratio of dietary EFA, PLs and cholesterol under the constraint of the dietary lipid level. To fully meet the theoretical PL requirements discussed above, 4.0-9.0% standard fluid soy lecithin (63% PL as A.I.) or 2.5-5.5 % de-oiled lecithin (95-97% PL as A.I.) should be dosed

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in the larval shrimp feeds. Similarly, PL requirements of juvenile shrimp shall be fulfilled by 4.0-6.5% standard fluid soy lecithin (63% PL as A.I.) or 2.5-4.0 % de-oiled lecithin (95-97% PL as A.I.). Supplementation of fluid soy lecithin to reach optimal dietary PL levels very likely results in excess of the dietary lipid level limit (8%). In another word, it is impossible to reach optimal levels of crude lipid, EFA, cholesterol and PLs in the shrimp feeds by including fluid soy lecithin in feeds. When considering the cost-effectiveness and PL contributions from other feedstuffs, it is recommended to supplement de-oiled soy lecithin in shrimp feeds at 0.5 to 1.0%. Cost-saving effects of de-oiled soy lecithin

De-oiled soy lecithin provides more biologically-active source of LA, LNA, choline and inositol as compared with neutral lipids, and synthetic choline and inositol (Hertrampf, 1991; Coutteau et al., 1997). Supplementation of de-oiled soy lecithin is in combination with neutral lipids to meet EFA requirements. Furthermore, since there is positive interaction among PL, protein, EFAs and cholesterol, supplementation of PLs may be beneficial in reducing dietary requirements for protein, EFA, cholesterol (Knotara et al., 1997; Teshima, 1997; Russett, 2001). Artificial choline is poor available owing to the fast-soluble in rearing water and inositol is very expensive. Dietary inclusion at 1.0 Kg de-oiled soy lecithin/MT feeds contributes dietary choline by about 36 mg /Kg feed and dietary inositol by about 38 mg /Kg feed (Hertrampf, 1991), respectively. Inclusion of de-oiled lecithin will be able to saves aqua feed cost by directly increasing availability of dietary EFA, protein /amino acids, choline and inositol. Improvement in shrimp feed properties

De-oiled soy lecithin is easily handled, accurately dosed and homogeneously mixed during the feed processing as compared with crude soy lecithin (Hertrampf, 1991; Coutteau et al., 1997; Russett, 2001). Dietary inclusion of de-oiled soy lecithin is effective in improving water stability of shrimp feeds, decreasing the leaching of water-soluble nutrients (B vitamins and trace minerals) and other feed additives (amino acids and attractants). PLs are able to protect vitamins A and E from oxidation during the processing and storage of feeds, to stimulate feed consumption, and to increase utilization of dietary nutrients (Hertrampf, 1991; Coutteau et al., 1997; Russett, 2001; Li and Peisker 2005). These properties of PLs are of particularly crucial for larval and juvenile feeds. Summary Beneficial effects of dietary phospholipids supplementation in terms of development growth and survival are demonstrated in the larval and juvenile stages of various species of shrimp. This could be owing to crucial roles of phospholipids in formation of cell membrane and lipoproteins, in absorption, transport and utilization of dietary lipids including EFA and cholesterol, and in provision of choline, inositol and EFA. Shrimp is incapable of de novo synthesizing PLs to meet the requirement. The published requirements of PLs are variable, depending on shrimp specie and age, test conditions, and the composition and purity of PL sources. The PL requirements of shrimp are mostly in the range of 1-3% in diets. PC and PI are the most active PL fractions of PLs in improving growth and survival of larval and juvenile shrimp. Since de-oiled soy lecithin contains higher levels of PC and PI, its supplementation in feeds offers better options to formulate balanced feeds when taking into account the optimal level and ratio of lipid, EFAs, cholesterol, PLs (particularly PC) required by shrimp as well as the feed properties. It is recommended to supplement 0.5-1.0% de-oiled lecithin in the commercial shrimp feeds for the best cost-effectiveness.

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Reference Akiyama, D. M., Dominy, W.G., Lawrence, A. L. 1992. Penaeid shrimp nutrition. In: Marine Shrimp Culture: principles and Practices. Edited by A. W. Fast and L.J. Lester. Elsevier Science Publisher. Pg: 535-568. Bray, W. A., Lawrence, A.C., Leung-Trujillo, J. R. 1989. reproductive performance of Ablated Penaeus stylirosris fed a soy lecithin supplement. Journal of the World Aquaculture Society 20: 19A Cahu., C., Guillaume, J.C., Stephan, G., Chim, L. 1994. Influence of phospholipids and highly unsaturated fatty acids on spawning rate and egg and tissue composition in Penaeus vannamei fed semi-purified diets. Aquaculture 126: 159. Camara, M.R. 1994. Dietary phosphatidylcholine requirements of Penaeus japonicus Bate and Penaeus vannamei Boone (Crustacea, Decapoda, Penaeidae). Ph.D. Thesis. Univ. of Ghent, Belgium 173 pp. Camara, M.R., Coutteau, P, Sorgeloos, P. 1996. Dietary phosphatidylcholine requirements in larval postlarval Penaeus japonicus Bate. Aquacult. Nutr. 3, 39-48. Chapelle, S., Abdul Malak, N., Zingelstein, G. 1985. Incorporation of w-6polyunssaturated fatty acids into phospholipids of the crab Carcinus maenas. Biochem. System Ecol. 13: 459-465. Chen, H.Y., Jenn, J.S. 1991. Combined effects of dietary phosphatidylcholine and cholesterol on the growth, survival and body lipid composition of marine shrimp, Penaeus penicillatus. Aquaculture 96, 167-171. Chen, H. Y. 1993. Requirements of marine shrimp Penaeus monodon juveniles for phospatidylcholine and cholesterol. Aquaculture, 109:165-176.

Conklin, D.E., D’Abramo, L.R., Bordner, C.E., Baum, N.A. 1980. A successful purified diet for the culture of juvenile lobster: the effect of the lecithin. Aquaculture 21 243-249.

Coutteau, P., Kontara, E. K. M., Sorgeloos, P. 2000. Comparison of phosphatidylcholine purified from soybean and marine fish roe in the diet of postlarval Penaeus vannamei Boone. Aquaculture 181. 331-345. Coutteau, P., Camara, M.R., Sorgeloos, P. 1996. The effects of different levels and sources of dietary phosphatidylcholine on the growth, survival, stress resistance and fatty acid composition of post-larval Penaeus vannamei. Aquaculture 147: 261-273.

Coutteau, P., Geurden, I., Camara, M.R., Bergot, P., Sorgeloos, P. 1997. Review on the dietary effects of phospholipids in fish and crustacean larviculture. Aquaculture 155. 149-164. D’Abramo, L. R. 1997. Tryacylglycerol and fatty acids. In: L.R., D’Abramo, D.E., Conklin, D.M., Akiyama (eds), Crustacean Nutrition, Vol. 6, World Aqauculture Society, Baton Rouge, LA. Pp 71-84.

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Gill, C. 1998. Shrimp feeds: update on phospholipids from raw materials. Feed International, March. Pg 12. Gong, H. 1999. Effects of dietary phospholipids, cholesterol and choline chloride on growth, survival and tissue lipid fractions of litopenaeus vannamei juveniles. Wildlife and Fishieries Science. Texas A & M Ph.D. Dissertation. Henderson, R.J., Tocher, D. R. 1987. the lipid compositions and biochemistry of freshwater fish. Prog. Lipid Res. 26, 281-347.

Hertrampf, J. W. 1991. Feeding aquatic animals with phospholipids: II. Fishes. Lucas Meyer Publication No. 8 Lucas Meyer, Hamburg, 31 pp.

Higgs, D. A., Dong, F.M. 2000. Lipid and fatty acids. In> Encyclopedia of Aquaculture. Editor Stickney, R.R., John Wiley & Sons, Inc.. New york. Pp476-496. Kanazawa, A. 1983. Panaeid Nutrition. In: Proceeding of the second International Conference on aquaculture Nutrition: Biochemical and Physiological approaches to shellfish nutrition. Special Publication No .2. edited by P.C. Langdon and D. E. Conklin. World Maricuklture Society. Louisiana State University. Baton Rouge, Louisiana, USA. Pg 87-105. Kanazawa, A. 1993. Essential phospholipids of fish and crustaceans. In: Fish Nutrition in Practice, Edited by Kaushik, S.J., luquet, S. J.. Biarritz (France) June 24-27. INRA, Paris. pp. 519-530. Kanazawa, A. 1997. Effects of docosahexaenoic acid and phospholipids on stress tolerance of fish larvae. Aquaculture 155, 129-134. Kanazawa, A., Koshio, S. 1994. lipid nutrition of the spiny lobster Panulirus japonics (Decapoda Palinuridae): a review. Crustaceana. 67:226-232. Kayama, M., Hirata, M., Kanazawa, A., Tokiwa, S., Saito, M. 1980. Essential fatty acids in the diet of prawn: II. Lipid metabolism and fatty acid composition. Bull. Jpn. Soc. Sci. Fish. 46:483-488. Kontara, E.K.M., Coutteau, P. , Sorgeloos, P.1997. Effects of dietary phospholipids on requirements for and incorporation of n-3 highly unsaturated fatty acids in post larval Penaeus japonicus Bate. Aquaculture, 158: 305. Kontara, E.K.M., Djunaidah, I.S., Coutteau, P., Sorgeloos, P. 1998.Comparison of native, lyso, and hydrogenated soybean phosphatidylchoiline as source for p[hospholipids in the diets of postlarval Penaeus japonicus. Arch. Anim. Nutr. 51, 1-9. Li, Y. D., Peisker, M. 2005. Soybean lecithin in animal nutrition. Feed Magazine, 1-2. Pg 28-34. Paibulkichakul, C., Piyatiratitivorakul, S., Kittakoop, P., Viyakarn, V., Fast, A. W., Menasveta., P. 1998. Optimal dietary levels of lecithin and cholesterol for black tiger prawn Penaeus monodon larvae and postlarvae. Aquaculture 167: 273-281. Piedal-Pascual, F. 1985. lecithin requirement of Penaeus monodon juveniles.. In: Y. Takaki, J.H. Primavera and J.A. Llobrera (Eds.) Proceedings of the first international conference on

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the culture of penaeid prawn / shrimps. Iloilo , Philippines, 1984. Southeast Asian Fisheries Development Centre, Aquaculture Department, Manila, Philippines. Page 181 Piedal-Pascual, F. 1986 Effect of supplemental lecithin and lipid sources on the growth and survival of Penaeus monodon juveniles. In: Proceedings of the first Asian Fisheries Forum. Asian Fisheries Society. Pg 615-618.

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Tacon, A. G. J. 1990. Standard methods for the nutrition and feeding of farmed fish and shrimp. Vol. 1; the essential nutrients. Argent Laboratories Press, Washington.

Teshima., S. 1997. Phospholipids and sterols. In: Crustacean Nutrition. Edited by L.R., D’Abramo, D. E. Conklin, D.M., Akiyama. Pp 85-107. Teshima., S., Kanazawa, A. 1983. Digestibility of dietary lipids and cholesterol in the prawn. Bull. Jpn. Soc. Sci. Fish 49:963-966. Teshima., S., Kanazawa, A., Koshio, S. 1992. Ability for bioconversion of n-3 fatty acids in fish and crustaceans. Oceanis 18, 67-75. Teshima., S., Kanazawa, A., Kakuta., Y. 1986a. Effects of dietary phospholipids on growth and body composition of the juvenile prawn. Bull. Jpn. Soc. Sci. Fish 52: 155-158. Teshima., S., Kanazawa, A., Kakuta., Y. 1986b. Effects of dietary phospholipids on lipid transport in the juvenile prawn. Bull. Jpn. Soc. Sci. Fish 52: 159-163. Teshima., S., Kanazawa, A., Kakuta., Y. 1986c. Role of dietary phospholipids in the transport of 14 C tripalmitin in the prawn. Bull. Jpn. Soc. Sci. Fish 52:519-524. Teshima., S., Kanazawa, A., Kakuta., Y. 1986e. Growth, survival and body lipid compositions of prawn larvae receiving several dietary phospholipids. Mem,. Fac. Fish., Kagoshima Univ. 35: 17-27.

Thongrod, S., Boonyaratpalin, M. 1998. Cholesterol and lecithin requirement of juvenile banana shrimp, Penaeus merguiensis. Aquaculture 161: 315-321.

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