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2 the Thanks to you... march/april 214 global aquaculture The Global Magazine for Farmed Seafood January/February 29 DEPARTMENTS From The Director From The Editor GAA Activities Industry News GAA Calendar Advocate Advertisers 14 Raceway Systems Offer Tools For EMS/AHPN Management Fernando Garcia, Fabrizzio Vanoni, William Long, Dirk Lorenz-Meyer 18 Development Of 1-Monoglycerides Against AHPN Varied Trials Mark Continued Advances Devi Hermsen 22 Caveat Emptor Recommended For EMS/AHPN Management Solutions Stephen G. Newman, Ph.D On the cover: As salmon farming and other forms of mariculture expand, operations must comply with varied laws that deal with rights of use, pollution control and other issues. 24 Beneficial Microbes And Pathogen Control Probiotics Quench Bacterial Quorum Sensing Barbara Weber, Ph.D.; Gonçalo A. Santos, M.S.; Michaela Mohnl, D.I. M.S.; Gerd Schatzmayr, Ph.D. Dr. Christian Lückstädt, Dr. Kai-Jens Kühlmann, Tho Minh Van Rattayaporn Phuthongphan; Erik Van Ballaer Claude E. Boyd, Ph.D. Dr. Adrian G. Hartley-Alcocer; Dr. Eric N. Bink, M.S. Artur N. Rombenso, M.S.; Viviana Lisboa, Ph.D.; Luís André Sampaio, Ph.D. Page 25 Beneficial Microbes And Pathogen Control 28 Dietary Acidifier Potassium Diformate Improves Growth, Survival In Pangasius Probiotics produce antimicrobial compounds and suppress pathogen proliferation by competing for adhesion sites and nutrients, and disrupting quorum sensing. 3 Maximizing Profits Of Larval Shrimp Rearing Trials Seek Best Balance Of Artemia, Artificial Diets Eddy Naessens; Roeland Wouters, Ph.D.; 34 Sustainable Aquaculture Practices Hydrogen Sulfide Toxic, But Manageable 38 Potential Of YY Tilapia Male Technology 42 Mariculture In Rio De Janeiro, Brazil Projects Include Hatchery, Demonstration Unit, IMTA System 46 Legal Issues Affect Offshore Aquaculture Development Twenty Five Years Servicing the Best in the Industry We Get It Done! TM For more information about PFS, please contact: Dan DiDonato- Executive VP of Sales One Main Street Chatham, New Jersey 7928 Phone: ii March/April 214 global aquaculturewww.preferredfreezer.com advocate Katherine Hawes 48 Seafood and Health Changing The Paradigm Vive La Révolution! Roy D. Palmer, FAICD Page 66 Boutique Fish Farms In Brazil Brazil s favorable coastal conditions and affluent market for seafood could support expansion of a niche aquaculture sector: boutique farming. 51 Brazil Survey: Animal Welfare Perspectives Vary Between Producers, Researchers Carlos Eduardo, M. Viegas da Silva, Rubens Nunes, Ana Beatriz Santos de Oliveira, Elisabete Maria Macedo Viegas Scott E. Zimmerman, M.S., CP-FS 54 Preparation, Management Techniques For Certification Audits 58 Food Safety And Technology Temperature Affects Quality, Safety Of Quahog Clams George J. Flick, Jr., Ph.D.; David D. Kuhn, Ph.D. 62 U.S. Seafood Markets Paul Brown, Jr.; Janice Brown; Angel Rubio 66 Marine Fish Farming Boutique Fish Farms Environmentally Conscientious Aquaculture Concept Being Developed In Brazil Daniel Benetti, Ph.D. Dr. George Collings Thomas M. Losordo, Ph.D. 7 Pea Products Offer Good Nutrition, Functional Attributes In Aquafeeds 72 Aquaculturing Engineering Unit Processes In RAS Systems Aeration, Oxygenation global aquaculture advocate March/April 214 1

3 from the director from the editor GLOBAL AQUACULTURE ALLIANCE The Global Aquaculture Al li ance is an international non-profit, non-gov ernmental association whose mission is to further en viron men tally responsible aqua culture to meet world food needs. Our members are producers, pro cessors, marketers and retailers of seafood prod ucts worldwide. All aqua culturists in all sectors are welcome in the organization. OFFICERS George Chamberlain, President Bill Herzig, Vice President Lee Bloom, Secretary Jim Heerin, Treasurer Iain Shone, Assistant Treasurer Jeff Fort, Chief Financial Officer Wally Stevens, Executive Director BOARD OF DIRECTORS Bert Bachmann Lee Bloom Rittirong Boonmechote Rafael Bru George Chamberlain Shah Faiez Jeff Fort John Galiher Jim Heerin Bill Herzig Ray Jones Alex Ko Jordan Mazzetta Robins McIntosh Sergio Nates John Peppel John Schramm Jeff Sedacca Iain Shone Wally Stevens RELATIONSHIP MANAGER Sally Krueger EDITOR Darryl Jory PRODUCTION STAFF Assistant Editor David Wolfe Graphic Designer Lorraine Jennemann HOME OFFICE 4111 Telegraph Road, Suite 32 St. Louis, Missouri USA Telephone: FAX: Website: All contents copyright 214 Global Aquaculture Alliance. Global Aquaculture Advocate is printed in the USA. ISSN Continual Improvement, In Small Steps In an interview at the Global Aquaculture Alliance s GOAL 213 conference, I fielded a question about the organization s role in aquaculture realizing its potential in Africa. In answering the inquiry, I drew from my steadfast belief in the philosophy of continual improvement. We re frustrated teachers, frustrated educators, I said. We really believe that we meet people where they are and move them to a better place. GAA needs to understand where people are in their journeys to be a resource for them. Like any organization, GAA should be judged on not only what it represents, but also what it accomplishes. In 213, the Best Aquaculture Practices program experienced a 31% increase in the number of facilities certified, a 29% increase in the volume of product originating from BAP-certified processing plants and an impressive 57% increase in the volume of product originating from BAP-certified farms. Additionally, the annual output from BAPcertified processing plants neared 1.4 mmt at the end of 213, up 29% from a year ago. Simply put, that s more than 3, mt of product from certified processing plants that wasn t available in the marketplace at the end of 212, and that s 38 processing plants and 14 farms that were not BAP-certified at the end of 212. Continual improvement isn t just a numbers game. It s about expanding one s reach, forging new relationships and, ultimately, advancing a movement. For GAA, working relationships often begin with a memorandum of understanding (MoU), or an agreement that two organizations will work collaboratively to not only promote each other s interests, but also advance responsible aquaculture. For example, in December, GAA inked an MoU with Wuhan Lanesync Supply Chain Management Co. Ltd., one of China s largest food purchasing, distribution, logistics and marketing companies. By teaming up with such an influential company, GAA hopes to encourage more Chinese retailers and foodservice operators to actively participate in the responsible aquaculture movement. For some players, certification is further off; it s a long-term goal. But it s just as important to encourage these players to actively engage in the responsible aquaculture movement. Perhaps that s through improving the accessibility and affordability of thirdparty certification, which is one of the goals of the MoU that GAA signed with the Aquaculture Stewardship Council and GlobalGAP last April. Or perhaps that s through an educational outlet, whether it s GAA s Global Aquaculture Advocate magazine or its annual GOAL conference. GAA strives to deliver accurate, science-based information on responsible aquaculture, bridging the information gap. Case in point: In December, GAA held its first-ever webinar on early mortality syndrome in shrimp, attracting 6 registrants worldwide. For these players, simply receiving accurate, science-based information on a destructive disease will move them to a better place. As I turn the page on my seven-year career as GAA s executive director and look ahead to my leadership role with the Responsible Aquaculture Foundation, I encourage you to find your fit within the responsible aquaculture movement and become more actively involved. Over time, continual improvement is measured not by a trickle of leaps and bounds, but by a series of small steps. Sincerely, Wally Stevens Wally Stevens Executive Director Global Aquaculture Alliance Investment Capital Needed The recently published World Bank report Fish to 23: Prospects for Fisheries and Aquaculture provides perspectives on where the seafood industry is and where it is going in the next three decades. In its foreword, the report states: Feeding an expected global population of 9 billion by 25 is a daunting challenge that is engaging researchers, technical experts and leaders the world over. A relatively unappreciated, yet promising, fact is that fish can play a major role in satisfying the palates of the world s growing middle income group while also meeting the food security needs of the poorest, It will also provide us with better insight into how to actually accomplish our goal of doubling production in a decade. Undoubtedly, aquaculture is the fastest-growing food-producing sector globally, despite the challenges its expansion faces. And it must expand very significantly and rapidly to contribute to the food supply of a growing human population in a sustainable and profitable manner. But our industry will not grow without substantial investment capital. Our farmed seafood industry can offer many attractive opportunities to the global investment sector, be it debt (bank debt, high-yield bonds, private/mezzanine debt) or equity (public or private equity). Investors can see our significant growth potential, given stagnant wild fisheries and increasing global demand for wholesome and quality seafood, with expanding markets for seafood in a fast-growing middle class in Asia and elsewhere, and income growth changing consumer preferences for high-quality protein. Farmed seafood is healthy and sustainable, and can have attractive profit margins. Each of the components of our industry s value chain offers unique opportunities, together with challenges and risks, to potential investors. From an investor s point of view, our industry is large but fragmented, immature and ripe for consolidation, and so is an excellent opportunity for investment portfolio diversification. How much investment is needed? We do not know, but we certainly are looking at substantial numbers if we are serious about significantly increasing aquaculture output in just a few years. To properly address this question requires a major effort on our part to generate more accurate estimates of the capital required, by major species, by global regions and by major technologies. Who will take this initiative? There is only one path to help private investors as well as professional ones from Wall Street and other investment centers discover and consider aquaculture. We must offer competitive financial returns from a well-established, expanding, responsible industry with a solid and developing technological base and a significant and expanding global market demand. As always, we encourage your suggestions for current topics you would like us to cover, as well as your contributions of short articles (~ 1, words). Please contact me at your convenience for details and article guidelines. Your critical comments make our magazine significantly better, and I urge you to continue sending us your comments on how we can best represent and serve our industry. 2 March/April 214 global aquaculture advocate global aquaculture advocate March/April 214 Sincerely, Darryl E. Jory Darryl E. Jory, Ph.D. Editor, Development Manager Global Aquaculture Advocate From an investor s point of view, our industry is large but fragmented, immature and ripe for consolidation, and so is an excellent opportunity for investment portfolio diversification. FOUNDING MEMBERS Agribrands International Inc. Agromarina de Panamá, S.A. Alicorp SAA Nicovita Aqualma Unima Group Aquatec/Camanor Asociación Nacional de Acuicultores de Colombia Asociación Nacional de Acuicultores de Honduras Associação Brasileira de Criadores de Camarão Bangladesh Chapter Global Aquaculture Alliance Belize Aquaculture, Ltd. Bluepoints Co., Inc. Cámara Nacional de Acuacultura Camaronera de Coclé, S.A. Cargill Animal Nutrition Chicken of the Sea Frozen Foods Continental Grain Co. C.P. Aquaculture Business Group Darden Restaurants Deli Group, Ecuador Deli Group, Honduras Delta Blue Aquaculture Diamante del Mar S.A. Eastern Fish Co. El Rosario, S.A. Empacadora Nacional, C.A. Expack Seafood, Inc. Expalsa Exportadora de Alimentos S.A. FCE Agricultural Research and Management, Inc. High Liner Foods India Chapter Global Aquaculture Alliance Indian Ocean Aquaculture Group INVE Aquaculture, N.V. King & Prince Seafood Corp. Long John Silver s, Inc. Lyons Seafoods Ltd. Maritech S.A. de C.V. Meridian Aquatic Technology Systems, LLC Monsanto Morrison International, S.A. National Fish & Seafood Co./ Lu-Mar Lobster & Shrimp Co. National Food Institute National Prawn Co. Ocean Garden Products, Inc. Overseas Seafood Operations, SAM Pescanova USA Preferred Freezer Services Productora Semillal, S.A. Red Chamber Co. Rich-SeaPak Corp. Sahlman Seafoods of Nicaragua, S.A. Sanders Brine Shrimp Co., L.C. Sea Farms Group Seprofin Mexico Shrimp News International Sociedad Nacional de Galápagos Standard Seafood de Venezuela C.A. Super Shrimp Group Tampa Maid Foods, Inc. U.S. Foodservice Zeigler Brothers, Inc. 3

4 Join the world s leading aquaculture organization Aquaculture is the future of the world s seafood supply. Be part of it by joining the Global Aquaculture Alliance, the leading standards-setting organization for farmed seafood. Access science-based information on efficient aquaculture management. Connect with other responsible companies and reach your social responsibility goals. Improve sales by adopting GAA s Best Aquaculture Practices certification for aquaculture facilities. Annual dues start at U.S. $15 and include a subscription to the Global Aquaculture Advocate magazine, GAA e-newsletters, event discounts and other benefits. Visit or contact the GAA office for details. Global Aquaculture Alliance Feeding the World Through Responsible Aquaculture GOVERNING MEMBERS Alicorp S.A. Nicovita Alltech Aqua Bounty Technologies Blue Archipelago Berhad Capitol Risk Concepts, Ltd. Cargill Animal Nutrition Chang International Inc Charoen Pokphand Foods PCL Darden Restaurants Delta Blue Aquaculture LLC Diversified Business Communications Eastern Fish Co., Inc. Ever Nexus Sdn. Bhd. Grobest USA, Inc. High Liner Foods Integrated Aquaculture International International Associates Corp. INVE B.V. King & Prince Seafood Corp. Lyons Seafood Ltd. Maloney Seafood Corp. Marine Technologies Mazzetta Co. LLC Megasupply Morey s Seafood International National Fish & Seafood Inc. Novus International Pentair Aquatic Eco-Systems Pescanova USA Preferred Freezer Services Red Chamber Co. Rich Products Corp. Sahlman Seafoods of Nicaragua, S.A. Sea Port Products Corp. Seafood Exchange of Florida Seajoy Thai Union Group Tropical Aquaculture Products, Inc. Urner Barry Publications, Inc. Wuhan Liangzhongxing Supply Chain Management Co., Ltd. Zeigler Brothers, Inc. SUSTAINING MEMBERS Akin Gump Strauss Hauer & Feld Ammon International, Inc. Anova Food Inc. Aqua Star Aquatec Industrial Pecuaria Ltda. A.Z. Gems Inc. BioMar Group Blue Ridge Aquaculture Camanchaca Inc. Channel Fish Processing Co., Inc. Direct Source Seafood DNI Group, LLC DSM Nutritional Products Fega Marikultura P.T. Fortune Fish Co. Gorton s Seafood Great American Seafood Imports Co. H & N Foods International, Inc./Expack Hai Yang International, LLC Harbor Seafood, Inc. Harvest Select International Marketing Specialists ipura Food Distribution Co. Mahalo Seafood LLC Maritime Products International Merck Animal Health Mirasco, Inc. North Coast Seafoods Odyssey Enterprises, Inc. Orca Bay Seafoods Ore-Cal Corp. PSC Enterprise LLC Quirch Foods Rubicon Resources Seacore Seafood, Inc. Seafood Industry Development Corp. Seattle Fish Co. Seattle Fish Co. of New Mexico Seattle Shrimp & Seafood Co., Inc. Slade Gorton & Co., Inc. Solae, LLC Star Agro Marine Exports Ltd. Tampa Bay Fisheries, Inc. Tampa Maid Foods The Fishin Co. The Great Fish Co. United Seafood Enterprises, L.P. ASSOCIATION MEMBERS All China Federation of Industry and Commerce Aquatic Production Chamber of Commerce American Feed Industry Association Asociación Latino Americana de Plantas de Rendimiento Associação Brasileira de Criadores de Camarão Australian Prawn Farmers Association Bangladesh Shrimp and Fish Foundation China Aquatic Products Processing and Marketing Association Fats and Proteins Research Foundation, Inc. Indiana Soybean Alliance Indonesian Aquaculture Society International Fishmeal and Fish Oil Organisation Malaysian Shrimp Industry Association Marine Products Export Development Authority National Fisheries Institute National Renderers Association Oceanic Institute Prince Edward Island Seafood Processors Association SalmonChile Salmon of the Americas Seafood Importers Association of Australasia Seafood Importers and Processors Alliance Soy Aquaculture Alliance Thai Frozen Foods Association Universidad Austral de Chile U.S. Soybean Export Council Washington Fish Growers Association Washington State China Relations Council World Aquaculture Society World Renderers Organization 4 March/April 214 global aquaculture advocate global aquaculture advocate March/April 214 5

5 gaa activities BAP Seeks Comments On New Hatchery Standards Comment Deadline: March 31 A draft of the new Best Aquaculture Practices (BAP) hatchery and nursery standards for finfish, crustaceans and mollusks is now available for public comment on the Global Aquaculture Alliance website. To submit comments, use the electronic form at BAP Standards Coordinator Daniel Lee at or send a fax to The comment period lasts 6 days. The deadline to submit comments is March 31. All properly submitted comments will receive responses and may potentially influence the final standards. The BAP hatchery standards will apply to all aquaculture facilities that produce eggs and/or juvenile aquatic animals for live transfer to other aquaculture facilities. Production facilities can include land-based ponds or tanks, or floating cages and pens. Currently, BAP hatchery standards exist only for shrimp. Once finalized, the new hatchery standards for finfish, crustaceans and mollusks will replace the BAP shrimp hatchery standards and allow companies to pursue four-star designation for species such as salmon, tilapia, catfish and Pangasius. The release of these draft standards is an important advancement for the BAP program, as it provides a consistent, global basis for certifying hatchery and nursery facilities, Lee The new BAP hatchery standards will apply to the production of sea bream and other fish and crustacean species. said. This draft has required substantial input from a specialist technical committee and can be applied to virtually all species. In common with all BAP standards, it is comprehensive and rests on the BAP pillars of community, environment, animal welfare, food safety and traceability. The content of the new hatchery standards was developed by a technical committee led by Dr. John Forster of Forster Consulting Inc. in Port Angeles, Washington, USA. The draft standards were further evaluated by the BAP Standards Oversight Committee, whose membership of varied stakeholders recommended refinements before approving them for release to public comment. First Mussel Plant Attains BAP Certification The Global Aquaculture Alliance s Best Aquaculture Practices (BAP) division is searching for a chairperson to lead the technical committee responsible for developing BAP standards for mollusk farming. The chair of the BAP Mollusk Farm Technical Committee will assist in the appointment of other committee members and then coordinate a review process to convert the existing BAP mussel farm standards to cover other mollusk species. The chair will also process and respond to input received during public comment on the standards. Chair candidates must understand the role that aquaculture Norlantic Processors Ltd. s plant in Newfoundland, Canada, recently became the first mussel-processing plant to attain Best Aquaculture Practices (BAP) certification. Located in Pleasantview on Notre Dame Bay in northeast Newfoundland, the vertically integrated company produces about 45 mt of blue mussels annually, with plans to increase production in the coming years. The company s fresh mussels are marketed to retail and foodservice customers throughout North America under the NPL brand. Along with its processing plant, Norlantic Processors operates three mussel farm sites totaling 728 ha, for which it is pursuing BAP certification in the future. Earning BAP certification is a benchmark for the company s processing plant, said Terry Mills, president of Norlantic Processors. I would like to thank everyone involved in this certification process for our industry in Newfoundland, especially Darrell Green at the Newfoundland Aquaculture Industry Association (NAIA). Our mussel farmers and processors are global leaders in sustainable farming, added NAIA Executive Director Cyr Couturier. Adding the Global Food Safety Initiative-compliant BAP musselprocessing plant standards to the arsenal of third-party certification tools is a testament to this high degree of commitment to demonstrating responsible farming and processing practices. Mussel-processing plants became eligible for BAP certification with the completion of the BAP mussel farm standards in August. The addition of the standards represents a key advancement for the BAP program, as the new standards will be used as a template for broader mollusk farm standards encompassing clams, oysters, scallops and abalone. BAP Seeks Chairperson For Mollusk Committee standards play in promoting responsible aquaculture and in communicating the evolving needs of the marketplace. Much of the work in this position will involve desk-based reviewing, editing and drafting audit clauses and associated implementation guidelines. Applicants should submit resumes via to BAP Standards Coordinator Daniel Lee at or by postal mail to the Global Aquaculture Alliance, 4111 Telegraph Road, Suite 32, St. Louis, Missouri USA. Please indicate if the applicant would be willing to serve as a committee member if not selected as the chair. BAP Program Continues Strong Growth Welcome, New Certif ied Facilities The Global Aquaculture Alliance s Best Aquaculture Practices (BAP) third-party certification program had a fruitful 213. Last year, it experienced a 31% increase in the number of certified facilities, a 29% increase in product volume from BAP-certified processing plants and an impressive 57% increase in the volume of product from BAP-certified farms. The annual output from BAP-certified processing plants totaled 1.42 mmt as of December 31, 213, up from 1.8 mmt at the end of 212. Annual farm output totaled 751, mt at year end, up from 478, mt in 212. At the end of 213, there were 248 BAP-certified seafood plants and 352 certified farms, which joined dozens of additional shrimp hatcheries and feed mills. Much of the growth within the BAP program in 213 came from salmon. Last year, the output from BAP-certified salmon farms more than doubled, while the output from certified salmon plants more than tripled to top 453, mt. Progress also came in standards development in 213. New BAP finfish and crustacean farm standards were completed, opening up the BAP program to include species such as seabass, sea bream, cobia, trout and barramundi. New BAP mussel farm standards were also finalized and will be used as a template to develop Last year, the output from BAP-certified salmon farms more than doubled, while the output from certified salmon plants more than tripled. broader standards for clams, oysters, scallops and other species. The hatchery and nursery standards for finfish, crustaceans and mollusks are also expected to be completed this year. Entering 214, Cooke Aquaculture Inc. North America s largest vertically integrated, independent salmon-farming company attained Best Aquaculture Practices (BAP) certification for its True North processing operations in New Brunswick, Canada; Northeast Nutrition Inc. feed mill and several salmon farm sites in eastern Canada and Maine, USA. Cooke can now market its True North brand salmon with three BAP stars from sales offices in Canada and the United States. BAP certification is a natural continuation of our company s long-term commitment to quality seafood and responsible production, Chief Executive Officer Glenn Cooke said. A healthy marine environment is vital to our operations, and certification through third parties ensures that we remain sustainable in our practices and helps us set goals for improvement. The BAP certification is well known and respected in the marketplace. Cooke Aquaculture will apply for four-star BAP certification once the Global Aquaculture Alliance finalizes its BAP hatchery and nursery standards later this year. In 212, Cooke s Chilean operation, Salmones Cupquelan, achieved BAP certification. Other recently certified facilities are listed in Table 1. Table 1. Additional recent BAP certifications around the world. Facility Location Country Species Farms Kelly Cove Salmon Ltd. (6 farms) Newfoundland and Labrador, Canada Salmon New Brunswick, Nova Scotia Kelly Cove Salmon Ltd. (2 farms) Maine United States Salmon Fuqing Jin Zhuang Aquaculture Co., Ltd. Fuqing City, Fujian Province China Shrimp Kader Exports Private Ltd. Bhimavaram India Shrimp Raoping Qingshan Dai Freshwater Aquaculture Field Chaozhou City, Quangdong Province China Shrimp Thai Union IOM Group 2 (6 farms) Satun Thailand Shrimp Qionghai Zhongpingzi Grobest Qionghai City, Hainan Province China Tilapia Wenchang Longfeng Grobest Wenchang City, Hainan Province China Tilapia Santai Eco Fishery Dongguan, Guangdong Province China Perch Processing Plants Salmones Pacific Star Quellon, Chiloe Chile Salmon DIMEX Los Mochis, Sinaloa Mexico Shrimp Fuqing Dongwei API Fuqing City, Jujian Province China Shrimp Kuliarchar Sea Foods Jhilonja, Cox s Bazar Bangladesh Shrimp Naik Frozen Foods Private, Ltd. Taloja, Maharashtra India Shrimp Thai Union Seafood Singhanakorn, Songkhla Thailand Shrimp Feed Mills Charoen Pokphand Foods Public Co., Ltd. Bangueng, Chonburi Thailand Northeast Nutrition LLC Truro, Nova Scotia Canada 6 March/April 214 global aquaculture advocate global aquaculture advocate March/April 214 7

6 GAA Signs MoU With Prominent Chinese Foodservice Provider The Global Aquaculture Alliance has entered a memorandum of understanding (MoU) with Wuhan Lanesync Supply Chain Management Co. Ltd. in which the two entities will cross-promote activities and advance responsible aquaculture. Based in Wuhan City, Hubei Province, China, Wuhan Lanesync handles purchasing, distribution, logistics, marketing, processing and other activities for more than 35, restaurants and hotels throughout China. The company also operates more than 2 Xianzhilong retail outlets and seven warehouses. Wuhan Lanesync became a GAA Governing Member in early 213. As part of the MoU, GAA has been designated a supporting partner of the Food Products and Ingredients Expo of China, organized in Wuhan by Wuhan Lanesync for March 21 to 25. The expo will feature more than 5 exhibitors from China and abroad, including China s top 5 restaurant chains, and welcome suppliers and buyers from around the world. GAA will be among the exhibitors and play a role in the conference programs running concurrently with the exhibition. Wuhan Lanesync will assist GAA in connecting with Chinese foodservice operators and retailers and in promoting GAA s Best Aquaculture Practices certification program in the Chinese marketplace. GAA Partners With Australasia Importers The Global Aquaculture Alliance has signed a memorandum of understanding (MoU) with the Seafood Importers Association of Australasia (SIAA) in which the two organizations will work collaboratively to advance responsible aquaculture. In the MoU, SIAA pledged to support the concept of independent, third-party aquaculture certification by working with industry and government to strengthen their understanding of aquaculture certification and with the public to strengthen its understanding of sustainable, responsible aquaculture. Also in the MoU, SIAA members agreed to adopt a general preference for aquaculture products from certified facilities, where applicable. The SIAA will assist GAA in promoting its Best Aquaculture Practices (BAP) certification standards, as well as other globally recognized aquaculture certification standards in Australasia. In return, GAA agreed to keep SIAA informed about updates to the BAP standards, new BAP standards in development, its accredited certification bodies and marketing activities in Australasia. The world, and particularly Australia, are increasingly dependent on aquaculture, as wild fisheries reach sustainable ceilings, SIAA Executive Chairman Norm Grant said. Consumers want to be certain that farms are operating to recognized principles of environmental stewardship and social accountability. International standard holders such as GAA provide credible benchmarks and a pathway to independent certification. AquaStar Fast growth in improved environment! Probiotic strains support gut health. Biodegrading strains and enzymes stabilize water quality and pond bottom. Improved gut health and performance Improved water quality Control of pathogenic bacteria aquastar.biomin.net Naturally ahead 8 March/April 214 global aquaculture advocate global aquaculture advocate March/April 214 9

7 Aidan Connolly Alltech Vice President Takes Mysticism Out Of Aquafeed Production Figures Aidan Connolly said Alltech s annual survey confirmed aquafeed will likely continue as the global feed industry s fastest-growing segment. Global animal feed production is projected to exceed the 1 bmt mark in 214, according to Alltech, one of the world s leading animal health companies. In truth, simply estimating global animal feed production is an elusive task. About four years ago, executives at Alltech, including Aidan Connolly, the company s vice president, identified a void in the availability of reliable, up-todate statistics on animal feed production. The animal feed production industry is fragmented, and state-funded organizations like the Food and Agriculture Organization of the United Nations find it increasingly difficult to gather and maintain figures on feed production due to limited resources. Yet the information is so valuable to the animal feed and food production industries. We identified about 1 organizations that would be interested in this information, said Connolly, who has worked for Alltech for 24 years. So Connolly and his fellow executives put their heads together and came up with a solution get Alltech s 6-plus salespeople to survey the thousands of feed mills they visit annually. The survey results would be used to compile an annual report on animal feed production. On the eve of the International Production & Processing Expo in Atlanta, Georgia, USA, on January 27, Connolly presented the results of Alltech s 214 Global Feed Survey, its third annual report on animal feed production, during a 45-minute webinar. Global animal feed production totaled 963 mmt in 213, up only 1% from 212, according to Alltech, which assessed the compound feed production of 13 countries in December 213 through information obtained from local feed associations and the company s sales team, which visits more than 28, feed mills annually. Connolly attributed the marginal increase in animal feed production to high raw material costs influenced by a series of droughts worldwide, and flattening production in China and the United States, the world s two largest animal feed producers. As shown in Table 1, in 213, China was the world s top animal feed producer at 189 mmt, followed by the United States at 169 mmt, Brazil and Mexico at 67 mmt each, and Spain at 29 mmt. Rounding out the top 1 were India, Russia, Japan, Germany and France. There are 28,196 feed mills worldwide, with Asia representing just less than half of the total at 13,266. North America and Europe followed at 5,736 and 4,886, respectively. The animal feed production industry is leadership profile Steven Hedlund Communications Manager Global Aquaculture Alliance St. Louis, Missouri, USA now valued at about U.S. $5 billion, up from the previous estimate of U.S. $35 billion. Higher feed prices and more accurate survey results fueled the increase, but feed prices are expected to fall in 214, as is the industry s value, said Connolly. Both China and the United States are expected to bounce back in 214, and production in Brazil and Mexico continue to grow, said Connolly. Consequently, global animal feed production is forecasted to surpass 1 bmt this year. According to the 214 Global Feed Survey, the annual 4-mmt production of aquafeed represents only about 4% of global animal feed production. However, production is up from 34.4 mmt in 212 and 29.7 mmt in 211. Aquafeed manufacturing is expected to be the fastest growing segment again this year, said Connolly. The number is still quite small just 4% of the world s animal feed production, Connolly continued. But it s much more consequential in China and throughout Asia. By region, Asia is by far the world s largest aquafeed producer, at 31 mmt, more than three-quarters of the total. Europe, Latin America and North America followed at 3.8, 3. and 2. mmt, respectively. By country, China is the Table 1. Top 1 feed-producing countries. Country Annual Total (mmt) Pigs Dairy Beef Calves Layers Broilers Turkeys Aquaculture Pets Horses China United States Brazil Mexico Spain India Russia Japan Germany France world s largest aquafeed producer, at 23 mmt. During the webinar, Connolly addressed the challenge of compiling statistics on aquafeed production. Alltech adjusted figures in its 214 survey results after learning its 213 figures were too high. Many, many people try to evaluate the numbers for aquafeed globally, and most of them give up [because] it s too difficult, explained Connolly. It s extremely difficult because there may be political reasons for [inflating] the numbers. And aquaculture is quite tricky because a lot of feed is produced seasonally, so you can get a number that is not really representative of what happens during the year. In a one-on-one interview after the webinar, Connolly addressed aquafeed in more depth. He said to expect aquafeed prices to generally trend downward in 214, citing declining raw material costs as a result of increased production. Connolly also shared his thoughts on the need for aquaculture to better tell its story and on the emergence of third-party certification. Unfortunately, some people like to tear apart aquaculture, he said. It s very important to protect the brand of aquaculture. Aquaculture has a great story to tell, said Connolly, but it doesn t always get told. Its attributes are numerous: Fish, especially oily fish, are high in hearthealthy omega-3 fatty acids. Fish convert feed protein to food protein more efficiently than any other animal. Aquaculture is a more sustainable means of growing the world s seafood supply than wild fisheries production. In addressing the emergence of thirdparty certification programs such as Best Aquaculture Practices, Connolly encouraged transparency throughout the aquaculture production chain, including feed mills. All other proteins face the issue of traceability, he said. The question is, How do I convince consumers that the industry is looking out for their interests? Consumers are skeptical. But third-party [certification] programs can alleviate that skepticism. Evaluating aquafeed globally is extremely difficult because there may be political reasons for [inflating] the numbers. And aquaculture is quite tricky because a lot of feed is produced seasonally. 1 March/April 214 global aquaculture advocate global aquaculture advocate March/April

8 Leadership and Learning Help make aquaculture s journey toward greater production and sustainability possible by joining the 3-plus seafood professionals and thought leaders from around the world who attend GAA s annual Global Outlook for Aquaculture Leadership conference. Through GOAL, GAA strives to carry out its mission of responsible aquaculture by providing a venue at which leadership development, cooperation and education are encouraged Back In Vietnam In 214, GOAL returns to Ho Chi Minh City, Vietnam, for the first time since 25. Vietnam is an aquaculture hotbed, with seafood exports topping U.S. $6 billion annually. Located in downtown Ho Chi Minh City, the 47-room Sheraton Saigon features 2,5 square meters of meeting space, including a 1,8-square-meter ballroom. To access the GOAL 214 registration page, scan the QR code to the left. Why Attend GOAL? GOAL features three days of information and analysis on the farmed seafood value chain, with a plenary session in the morning and breakout sessions in the afternoon. On Day 1, aquaculture production data and fish health management issues such as early mortality syndrome are highlighted. Day 2 addresses the challenges and opportunities facing aquaculture, including aquafeed sustainability, social responsibility, and innovation and technology. On Day 3, the marketplace is discussed with the participation of many of the world s leading retailers, representing tens of thousands of outlets and hundreds of millions in buying power. GOAL also features networking and socializing opportunities, and aquaculture tours. GOAL is attended by a cross-section of representatives from industry, retail and foodservice, government, academia, the investment community and the NGO community. Since its inception in 21, GOAL has evolved into a must-attend event for many top-level seafood executives and aquaculture thought leaders. Who Attends GOAL? Africa, 1% Southeast Asia, 9% North America, 46% Middle East, 4% Australia, 1% Europe, 33% Latin America, 5% 12 March/April 214 global aquaculture advocate global aquaculture advocate March/April

9 production ems/ahpn update Shrimp raised in round tanks or concrete raceways after transfer from hatcheries gain weight and health before being stocked in ponds for final growout. Raceway Systems Offer Tools For EMS/AHPN Management Summary: Highly controlled tank and raceway systems can help farmers raise postlarval shrimp to larger sizes before stocking in growout ponds. The higher densities and zero water exchange of typical raceways demand increased biosecurity, high-quality feeds, probiotics and careful transfer methods. Raceways have recently been implemented to lessen the impacts of early mortality syndrome. In Mexico, no EMS outbreaks were seen in raceways utilizing lowered stocking densities, extended raceway culture and shortened pond growout. The approach targets smaller shrimp and more growout cycles. Fernando Garcia Epicore BioNetworks Inc. 4 Lina Lane Eastampton, New Jersey 86 USA Fabrizzio Vanoni William Long Dirk Lorenz-Meyer Epicore BioNetworks Inc. Shrimp raceway technology has been refined over the last eight years to improve production system efficiency. In parallel, producer interest has increased, as raceway systems can mitigate the adverse effects of poor weather and diseases. In the early days, the raceway concept started with the objective of acclimatizing animals to growout system conditions. The concept has since evolved to control animal health and water quality conditions, to optimize and improve feeding efficiency, to improve survival during the first days of culture and to prevent exposure of the animals to disease vectors that can result in early outbreaks in ponds. Raceway Requirements Shrimp are transferred from hatcheries at postlarval stages of P.L. 6 to P.L. 12, depending on farm salinity, and instead of being directly stocked into ponds, they are stocked in different-shaped tanks at farms at higher densities than in growout ponds. The raceways typically operate with zero water exchange. The higher stocking densities and zero water exchange demand higher technology, more infrastructure, increased biosecurity, and additional controls and tools. Raceways require filtration systems, efficient aeration systems, good water quality, the use of microorganisms, siphoning, temperature control, greenhouse structures and better feeds all with the objective of keeping a stable environment for strong, healthy animals. Another benefit of these systems takes advantage of the compensatory growth that occurs when Litopenaeus vannamei are finally stocked in growout ponds. Compensatory growth reduces cycle length and improves feed conversion. Key Factors Once adequate raceway infrastructure is in place, a very important factor learned from experience in the different countries 14 March/April 214 global aquaculture advocate global aquaculture advocate March/April

10 Table 1. Average results for shrimp raised in raceways at different stocking densities and for different culture periods. Density (shrimp/l) Days Survival (%) Average Weight (g) Raceways with zero water exchange employ filtration systems, aeration and other equipment to maintain a stable rearing environment where the technology has been used is that the staff in charge of the raceways should have a hatchery background in order to maintain the required level of control. The application of probiotics, microorganisms that have health benefits when consumed, is a key component in these systems. The protocols for the different probiotics must be adjusted depending on the biomass, water quality conditions and animal conditions. To deal with organics, for example, 1 to 3 ppm of probiotic should be added every 72 hours. To deal with toxics, 2 to 5 ppm of probiotic should be added every 48 hours. For pathogens in water, 5 to 1 ppm of probiotic should be added until the pathogens are controlled. To deal with pathogens in the gut, 1-4 g probiotic/kg of feed should be added. The feeds used in these systems have to be of hatchery quality. Trying to use growout-quality feeds impacts water quality and subsequently results in bacteriological problems that can lead to poor production performance and weak animals. The last key component is having the tools and expertise to properly transfer the animals from the raceways to the ponds. Animals must be healthy and strong before transfer. Otherwise, all that was gained in the previous stage can be lost. A lot of technology has been developed in this area. At the beginning, transfers were made with wet animals for short distances. Today, transfers are made to ponds up to 2 km away from the raceway units using specially designed pumps that minimize animal stress and mortalities. The trend today is to lower densities in order to achieve higher weights in raceways. Biomasses of up to 7 kg/m 3 have been achieved. Average results for weight and survival at different stocking densities and culture periods are summarized in Table 1. severe. Production dropped approximately 5% in 213. Based on previous experience with white spot syndrome (WSS), most farms tried to achieve large-size animals after the EMS outbreak, as they used to do after WSS affected farms. However, with EMS disease, pond populations continue to decrease and reduce final survival rates. According to information supplied by Proaqua Mexico, a leading supplier of specialized aquaculture equipment and feeds, no EMS outbreaks have been reported in raceways, regardless of the time spent in the raceways. However, mortalities develop once animals are stocked in ponds. With this experience and taking advantage of the large raceway infrastructure in Mexico, a few groups changed the production strategy for the second cycle. They lowered stocking densities, extended the days of culture in the raceways and then shortened growout cycles in the ponds to target smaller shrimp and more growout cycles. Some of these farms stocked raceways at an average of 1.5 shrimp/l for 55 days, achieving 4.5-g weight and 8% survival. When stocked in growout ponds at an average of 6.5 animals/m 2, the shrimp reached 16 to 18 g in 3 days of culture. The harvested animals reflected an average feed-conversion ratio (FCR) of.6 and average survival of 85%. The farms managed to run four cycles in growout ponds with this approach. They produced over 3, kg/ha during 213 with fewer days of culture and lower FCR. PENTAIR AQUATIC ECO-SYSTEMS SPARUS PUMP WITH CONSTANT FLOW TECHNOLOGY Oversized strainer basket and volute. Saltwater-rated 316 stainless internal fasteners, and heavy-duty mechanical seal. 2 NPT suction and discharge ports. Implementation Elsewhere Several attempts to transfer this concept to countries in Southeast Asia have yielded mixed results. Farmers have tried building raceways at farms, but with insufficient biosecurity, tools and technology, By operating the pump s motor at the minimum they encountered problems with size variation, survival and transfers. rate, Constant Flow Technology saves energy speed needed to achieve the user-defined flow 3 hp totally enclosed fan cooled motor, Raceways In Mexico Alternative ideas were put in place. while automatically delivering the exact flow with permanent magnets. The impacts of early mortality syndrome/acute hepatopancreatic necrosis Stocking in cages within ponds with no soil rate needed, even as system conditions change. contact for the first 3 days reportedly (EMS/AHPN) in Mexico have been avoided EMS outbreaks. Also, segregating young animals within a 2 to 3% section of ponds with nets and then releasing the THE NEW, shrimp to the remaining pond areas after 2 COMPLETELY to 3 days has been reported to help. Raceway infrastructure and methods implementation of raceways has been A successful experience with the RENOVATED vary regionally. reported in Malaysia with good production performance, successful transfer of the Your #1 source for products, PENTAIRAES.COM! Local adaptions are typically needed animals to growout ponds and successful system design and training. to achieve success. harvest in a shorter pond cycle. There is still room to improve by adjusting feeding regimes, probiotic protocols and temperature conditions in order to achieved better weights in the raceways. Online Orders: PentairAES.com Phone Orders and Tech Advice: Apopka Blvd., Apopka, Florida 3273, USA 16 March/April 214 global aquaculture advocate global aquaculture advocate March/April Pentair Aquatic Eco-Systems, Inc. All Rights Reserved. The world s first aquaculture duty pump to deliver a CONSTANT user-defined flow rate Pump motor speed self-adjusts to maintain the constant flow rate setting, even as system conditions change IP55-rated enclosure for robust service life in wet locations and harsh conditions Ask about how the Pump Affinity Law can save you money! NEMA IP55 rated enclosure for robust service in wet locations. Integrated wiring compartment for easy access and quick installation.

11 production Development Of 1-Monoglycerides Against AHPN Varied Trials Mark Continued Advances This image from an electron microscope shows the membrane alteration caused by 1-monoglycerides, which disturbed homeostasis and led to the death of the E. coli bacteria cell. Photo courtesy of Dr. Morten Hyldgaard, Aarhus University. Summary: 1-monoglycerides have long been known for their antibacterial and antiviral effects in the human pharmaceutical and animal husbandry industries. As a sustainable substitution for the preventive use of antibiotics, these molecules are being evaluated in the fight against early mortality syndrome in shrimp. The 1-monoglycerides prevent the bacteria that cause EMS from taking up energy and water, and basically starve the pathogens. Including 1-monoglycerides in feed also prevents the bacteria from multiplying. Infection rates were most strongly reduced in shrimp fed liquid 1-monoglycerides. In the human pharmaceutical industry and livestock husbandry, the capacity of 1-monoglyceride molecules is well known in the fight against a broad range of bacteria and viruses. In practice, these molecules are used as antipathogenic compounds in a sustainable substitution for the preventive use of antibiotics. However, in aquaculture, these molecules were not well known until recently. The catastrophic consequences of the spread of early mortality syndrome/acute hepatopancreatic necrosis (EMS/AHPN) in shrimp, caused by a virulent Vibrio bacterium, formed a challenging arena in which 1-monoglycerides entered the aquaculture market. After an extensive research project with different formulations of 1-monoglycerides, the first EMS-free cycle was reached at a shrimp farm with chronic AHPN problems in May 213 in Thailand. Although this outcome was promising, much was still unknown, and farmers were asked to participate in additional research trials. Comprehensive Solutions Although multiple pathogens found closely related to AHPN are currently being investigated, the major causative agent is a virulent strain of Vibrio parahaemolyticus. This bacterium has close similarities to V. cholera and V. harveyi, giving researchers a head start in finding possible solutions to protect the shrimp sector against disastrous mortality rates. Devi Hermsen Framelco Ruisvoorn 5, NL-4941 S.B. Raamsdonksveer, The Netherlands ems/ahpn update Vibrio parahaemolyticus is spread via many different vectors, such as phytoplankton, zooplankton or detrimental pond residues. The chitin exoskeletons of shrimp are also suspected to serve as carriers for Vibrio bacteria. With so many vectors involved, eradication of Vibrios is not possible nor desired, since Vibrios also have beneficial purposes in pond ecosystems alongside other organisms. Addressing a problem as complex as AHPN requires a comprehensive approach. Although its underlying mechanisms are still in the initial stages of understanding, knowledge is rapidly increasing thanks to close collaboration among stakeholders. The adoption of a holistic approach with a focus on lowering concentrations of infection vectors in shrimp ponds, preventing colony formation inside the culture animals and counteracting the toxin produced by V. parahaemolyticus has been proposed to restore balance and reduce the impacts of AHPN. One of the proposed methods to prevent AHPN is finding a way to disrupt bacterial attachment to the stomach walls of shrimp before biofilm encapsulation can protect the bacteria against antipathogenic compounds. Feeding 1-monoglycerides is believed to counteract V. parahaemolyticus colony formation by preventing invading bacteria from multiplying. Specific types of monoglycerides are also known for emulsifying characteristics that can increase the permeability of early-stage biofilms. This combination is thought to be the strength of 1-monoglycerides against AHPN progression. 1-Monoglyceride Effects 1-monoglycerides are based on a novel technology of esterifying a fatty acid on the alpha position of a glycerol molecule. The result is a new type of molecule with specific characteristics that is neither a fat nor an acid. These molecules are non-corrosive, work independently of ph and are very heat stable thanks to the covalent bonds inside the cores of the molecules. This makes these molecules suitable for feed additive applications. For many years, 1-monoglycerides have been known for their antibacterial and antiviral effects in the human pharmaceutical industry as well as animal husbandry. They cannot be classified as antibiotics, because the molecules do not literally kill pathogens. The 1-monoglycerides prevent bacteria from taking up energy and water, and therefore basically starve the pathogens to death. Fundamental research performed by the Research and Development Department of Framelco and the Pharmaceutical Department of the University of Lisbon showed that the homeostasis of FRA Shrimp Protect liquid 1-MONOGLYCERIDES FRA Shrimp Protect liquid 1-MONOGLYCERIDES TO REDUCE EMS-IMPACT Based on a patented technology, Framelco developed FRA Shrimp Protect liquid: a specific formula containing several short chain and middle chain 1-monoglycerides. Feeding this product to shrimp reduces the infection rate and thereby lowers mortality caused by EMS. An in vivo challenge trial showed mortality reduction of 8% after 7 days of using FRA Shrimp Protect liquid incorporated in the shrimp feed (inclusion rate.8%). (213, University Arizona, Dr. Lightner) FRAmelco.com REDUCES EMS-IMPACT Prevents high infection rate «Lowers mortality caused by EMS «Increases profit «Antibacterial «Antiviral «Product characteristics» Heat stable» Active in GIT and blood stream» Non GMO» Available in liquid form» Available in 25kg cans, 25kg drums, 1kg IBC, and bulk» Produced in GMP+ certified facilities Framelco is an innovative company specialized in the development, manufacturing and marketing of feed additives worldwide. We dedicate ourselves to provide the industry additive solutions that meet the current and future demands, allowing both aquaculture and agriculture to increase productivity, sustainability and profitability. 18 March/April 214 global aquaculture advocate global aquaculture advocate March/April % 1 Mortality % 86.7% Pos. control 6.7% FRA Shrimp Protect liquid.% Neg. control

12 bacterial cells is severely affected by 1-monoglycerides through their altering of membrane stability and blocking of the membrane uptake facilitator called aquaporin-3 (Figure 1). Treated bacteria are not able to take up sufficient glycerol, a main energy source, and therefore cannot multiply to build up a secure presence in the new host. Since colony formation is inhibited, the bacterial quorum sensing that leads to further biofilm and toxin production is effectively down regulated. 1-Monoglyceride Formulations Two types of formulations were tested in Thailand: one containing short-chain 1-monoglycerides and the other containing both short- and middle-chain 1-monoglycerides. Both formulations were tested in dry powder form using a carrier added to the shrimp feed by coating. Five farms were selected two with chronic AHPN problems, two with occasional outbreaks and one with no AHPN history. Results from a full growout cycle showed the formulation containing both short- and middle-chain 1-monoglycerides performed better. Mortality was strongly reduced, and early harvesting was not needed. Two subsequent AHPN-free cycles were achieved at a farm with previously chronic AHPN problems. However, shrimp in some ponds still showed poor shell quality, indicating impaired health status likely caused by the presence of a high pathogen load. The pond with no AHPN history showed better growth performance, although not significantly. The synergetic relationship between short- and middle-chain 1-monoglycerides was confirmed by an additional trial performed by an independent research laboratory and an in vivo challenge trial. Vibrio-challenged shrimp with short- and middle-chain 1-monoglycerides in their diets showed a 16% reduction in mortality compared to that of shrimp that received solely short-chain or middle-chain monoglycerides. Continued Studies Even with this improved formulation, the full prevention of AHPN observed in the original pilot studies could not be consistently achieved in subsequent field trials. On average, mortality reduction of 2 to 3% was observed in the field, with only some areas where mortality was prevented completely. A small-scale challenge trial showed a strong shift from green colonies to yellow colonies in agar cultures of shrimp tissue derived from animals fed 1-monoglycerides via enriched Artemia. The water solubility of short-chain 1-monoglycerides and practical difficulties associated with coating this formulation on feed pellets might explain the inconsistent results. More research was needed to increase the potential strength of the 1-monoglycerides. A large-scale field trial at the Research Institute for Aquaculture in Vietnam found that more consistency could be reached by incorporating the 1-monoglyceride molecules into the feed mash before pellet pressing. The experiment showed that shrimp treated with dietary 1-monoglycerides performed better than the control groups in growth and general survival. Yet it became clear that negative external factors like changes in climate or water quality could in some cases overshadow the capacity of the molecules. An extra experiment was carried out to investigate the effects of 1-monoglycerides in relation to those of antibiotics. One treatment group continuously received dietary 1-monoglycerides combined with an additional short-term curative treatment with antibiotics when early AHPN symptoms revealed. These shrimp were compared with a treatment group that received antibiotics when needed without 1-monoglycerides, and a negative control with no 1-monoglycerides or antibiotics. Relative Intensity (A.U.) Figure 1. Membrane recovery after osmotic shock: disturbed homeostasis. Survival (%) Control 1- M.G. Treatment Figure 2. Survival of shrimp challenged by virulent Vibrio parahaemolyticus and receiving dry or liquid 1-monoglyceride dietary formulations. Mortality (%) Time (seconds) Dry T1 Dry T2 Liquid T1 Liquid T2 Positive Liquid 1-Monoglyceride Negative Control Treatment Control Figure 3. Mortality of shrimp fed a formulation containing both short-chain and middle-chain 1-monoglycerides. Results showed that animals were more receptive to antibiotic treatment when 1-monoglycerides were present in their diets. The treatment group that received 1-monoglycerides and short-term antibiotics reached full harvest size in good health, while the group with solely antibiotic treatment needed early harvest. The control group performed worst. It was proposed that the 1-monoglycerides inhibited biofilm production and better exposed the harmful bacteria to chemical treatment. The ban on antibiotics in many countries, however, demands an approach without these chemical substances. Bioavailability: Dry Versus Liquid 1-Monoglycerides In the search for ways to increase the strength of the 1-monoglycerides, a challenge trial was conducted together with the University of Arizona Department of Veterinary Science and Microbiology, led by Dr. Donald Lightner. Experimental diets were composed containing combinations of several 1-monoglycerides and several microingredients. Dry and liquid formulations were also tested. To compensate for the absence of varying external factors, the trial was performed with a high infection rate. The results were surprising. Due to the high infection rate, all shrimp fed dry 1-monoglyceride formulations died from AHPN within three days. The shrimp fed liquid formulations, however, resisted the challenge and stayed alive (93.3%) until the end of the study (experimental day 17, challenge day 7). See Figure 2. In humans or livestock, 1-monoglycerides are normally applied in dry form by attaching the molecules to a carrier. Inside the digestive tract, the 1-monoglycerides are released from this carrier and then perform the antipathogenic activity. It was concluded that the fast rate at which feed passes through shrimp does not allow full release of the molecules and therefore reduces their bioavailability. This might explain insufficient results from earlier field trials. The best-performing liquid formulation reduced mortality with 8% compared to that of the positive control group (Figure 3). Shrimp received a.8% dietary inclusion of liquid 1-monoglycerides once a day. In this treatment group, the few dead shrimp represented a 6.7% mortality rate. But after inspection, it turned out these individuals likely died due to cannibalism instead of AHPN. Histological analyses of sampled shrimp performed at the Aquaculture Pathology Laboratory at the University of Arizona showed infection rates were strongly reduced in shrimp fed liquid 1-monoglycerides. Complete eradication of Vibrio parahaemolyticus was not possible, but the minimal presence of this bacterium in shrimp tissue did not necessarily lead to AHPN development during the trial. Several field trials are being run to investigate the long-term effects of liquid 1-monoglycerides. Preliminary data show promising results. Most ponds have not shown AHPN outbreaks so far. In ponds at heavily infected farms, AHPN development cannot always be prevented, but has been delayed for weeks. Concluding data from several stocking cycles are expected in the next two months. JOIN GAA The World s Leading Aquaculture Organization Aquaculture is the future of the world s seafood supply. Be part of it by joining the Global Aquaculture Alliance, the leading standards-setting organization for farmed seafood. Access information on efficient aquaculture management. Connect with other responsible companies and reach your social responsibility goals. Improve sales by adopting GAA s Best Aquaculture Practices certification. Visit or contact the GAA office for membership details. Food & Feed Safety Kits Bioo Scientific Corporation is a global leader in the food and feed safety testing industry, committed to improving the safety of the world's food and feed supply by developing accurate, affordable tools that enable streamlined, sensitive screening for pathogens, toxins, and drug residues in aquaculture products. 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13 production ems/ahpn update While adequate aeration is necessary to ensure no stress from low dissolved-oxygen levels, high levels of nutrients can offset this by allowing blue green algae to proliferate. AHPN is caused by a common bacterium, but wholesale disinfection of aquatic ecosystems is not a sustainable practice. Caveat Emptor Recommended For EMS/AHPN Management Solutions Summary: Disease is a result of interrelated interactions among the host, the environment and the pathogen. As with other diseases, early mortality syndrome/acute hepatopancreatic syndrome in shrimp does not involve a simple disease process, so a single, all-encompassing solution is unlikely. The development of polymerase chain reaction testing to detect the bacteria that cause EMS is important, but until further testing confirms its specific identification of the EMS Vibrio, confirmation by bioassay of presumptive positives to ensure pathogenicity is a prudent intermediate step. Caveat emptor is Latin for let the buyer beware, a phrase with which most of us are familiar. While some might take this as a blanket admonition that no one can be trusted, I think it prudently advises all of us, among other things, not to look for easy fixes to complex problems. Governments make certain practices illegal, and where impacts on consumers can be substantial, they enforce labeling requirements, as an example, that ensure buyers understand there may be risks associated with the use of some products. Nonetheless, this adage remains very much a truism in today s world. Disease Solutions With every major disease outbreak in shrimp farming, there have been those who are understandably drawn to the large profit potential that a widespread solution would offer. Yet I am not aware of a single instance where the tools offered significantly impacted the disease process in the long run and remained in common use. Tools that provide long-lasting and broadreaching solutions are not the norm for controlling diseases in farmed shrimp. Many compounds kill bacteria and viruses, and more are being found all the time. Disinfectants chemicals and ionbased compounds, among others join a variety of plant extracts, chlorine, ozone and other bacteria as documented solutions to kill many different types of bacteria, including the Vibrio implicated in early mortality syndrome (EMS) or acute hepatopancreatic necrosis (AHPN). It is important to recognize that killing bacteria under controlled conditions in a lab is not equivalent to controlling the processes that allow the disease to occur at farms. Stephen G. Newman, Ph.D. President and CEO AquaInTech Inc nd Place Southwest Lynnwood, Washington USA If killing the bacteria was all that was needed to prevent or stop infection, the use of chlorine might have prevented the AHPN problem from developing in the first place. Some have speculated that the overuse of some of the routinely used disinfection tools may have created niches that have allowed bacteria such as the etiologic agent of AHPN to evolve and thrive. Wholesale attempts at disinfection of large swatches of aquatic ecosystems could be useful in some cases, but in the big picture, this is not a sustainable practice. Understanding Disease Process The aquaculture community is still very much in the early stages of understanding the disease process of AHPN. The pathology is well documented, and scientists have been able to isolate the pathogen and infect shrimp to produce the characteristic pathology. But even though scientists can reisolate the bacterium and repeatedly get a pathognomonic disease process, the disease process in the field may not be as simple. Laboratory environments are usually highly controlled, whereas the field is subject to a myriad of variables. Disease is a result of interactions among the host (shrimp in this case), the environment and the pathogen. Host genetics, species, life stage and pathogenfree status are all contributing factors. The environment in which animals are produced is constantly changing and quite complex, with stress all too often being the norm. The presence of nutrients that encourage the growth of one bacterial species over others, a myriad of metabolic by-products and end products, and the physical and chemical parameters of the water are also elements of disease susceptibility. The pathogen itself may have a preferred mechanism for producing disease that affects its virulence. There is little reason to believe that EMS/AHPN has a simple disease process. There is no evidence that the Vibrio behind AHPN is an obligate pathogen that produces disease merely by its presence. There are threshold levels below which one does not see acute disease, and it is likely present in many ecosystems where no acute disease is observed. This is normal for most diseases. A simple, single all-encompassing solution for AHPN is unlikely. Caveat emptor! Pathogen Detection Several recent articles purport that the availability of a tool to detect the pathogen responsible for AHPN will solve the problem. Unfortunately this is misleading, and while detecting the pathogen is of critical importance in learning how to live with it, this is a tool for detection and not a solution. This tool, known as polymerase chain reaction (PCR) allows specific sequences of DNA to be copied, making it easy to detect very low levels of DNA. This technology utilizes a specific DNA sequence that allows ready detection of the organism of interest and no others. The amplified sequence should be unique to the specific organism for which one is screening. The etiologic agent of EMS is a variant of a common marine bacterium, Vibrio parahaemolyticus. This organism is nearly ubiquitous in marine environments, and there are innumerable strains, with more being characterized all of the time. While PCR is a very valuable tool, until it is in widespread use, and testing confirms its specific identification of the EMS Vibrio, there remains the specter that it will not detect just the pathogenic form. Caveat emptor! Perspectives Caution is in order for the time being. A number of PCR probes are currently available and in use, and there is the possibility of false positives. The mere presence of a PCR-reactive isolate could cause panic in areas where there has been no clearly identified disease. Confirmation by bioassay of presumptive positives to ensure pathogenicity is a prudent intermediate step to take until more definitive data are available. It is important to bear in mind that even though EMS/AHPN appears to have been present in farms for at least four years, it is still spreading, and we are still in the early phases of understanding what is occurring. If it is similar to other pathogens, you can expect the solution to EMS will not be a simple one. And since it is caused by bacteria, not a virus, it is not likely that we will see the development of tolerance to AHPN any time in the near future. Let the buyer beware is a wise approach to take toward quick solutions. 22 March/April 214 global aquaculture advocate global aquaculture advocate March/April

14 production Beneficial Microbes And Pathogen Control Probiotics Quench Bacterial Quorum Sensing Barbara Weber, Ph.D. Biomin Research Center AT-343 Tulln Technopark 1 Austria Gonçalo A. Santos, M.S. Michaela Mohnl, D.I. M.S. Gerd Schatzmayr, Ph.D. Biomin Research Center A surplus of organic matter in culture systems provides excellent growth conditions for opportunistic pathogens. Beneficial bacteria mineralize organic matter and thereby help to reduce disease risk. Summary: A range of alternative products is available to improve animal health and water quality, and control pathogen loads. Beneficial bacteria, or probiotics, produce antimicrobial compounds and suppress pathogen proliferation by changing the ph of animals gastrointestinal tracts and competing for adhesion sites and nutrients. Probiotic bacteria can also disrupt the quorum sensing communication that allows bacterial populations to coordinate biofilm formation, virulence and production of antimicrobials and exoenzymes. This quorum quenching renders bacteria less virulent and more vulnerable. Bacterial pathogens are a major concern for commercial aquaculture. Most pathogens relevant to aquaculture are opportunistic and constitute part of the normal microflora on animal surfaces and in their guts, and in water and soil. As long as water conditions, animal health and the microflora are stable, the likelihood of opportunistic pathogens to cause disease is low. However, when conditions change, water quality deteriorates, and animal health is compromised. Opportunists take their chance, proliferate quickly and cause infectious disease. Thus, to control the level of opportunistic pathogens, maintenance of a stable environment and water quality is crucial. Proper management techniques are a prerequisite. In addition, measures like vaccinations and antibiotics are applied, but they also have disadvantages. Vaccines are not available for every 24 March/April 214 global aquaculture advocate species and disease. They are very specific to distinct diseases, mainly those viral in nature, and not effective in shrimp. The efficacy of vaccines depends on many variables, such as the immune status and size of the animal, formulation, and the administration route and regime. Additionally, vaccination is expensive, labor intensive and stressful for the animals. In the past, antibiotics were often used for growth promotion as well as disease prevention. However, uncontrolled use of antibiotics led to a rise in resistance phenotypes among bacterial pathogens. The continuous selective pressure not only triggered resistances, but also transmission of these resistances into the environment. Consequently, the application of antibiotics as preventative measures is controversial. Today a range of alternative products is available to improve animal health, water quality and control pathogen load. The use of beneficial bacteria, or probiotics, is one way to maintain stable conditions and prevent opportunistic pathogens from taking over. Water Quality Management Farmed aquatic animals discharge feces, urine and uneaten feed into their culture water. When this organic matter accumulates, levels of toxic compounds increase, and water quality subsequently deteriorates. Thus, excess amounts of organic matter and toxic compounds need to be removed. Bacteria can aid this process. The use of plants or microorganisms like bacteria to remove waste products is called bioremediation. A surplus of organic matter provides excellent growth conditions for opportunistic pathogens and sets the stage for infectious disease. Beneficial bacteria such as Bacillus species mineralize organic matter and thereby help to reduce the burden. Nitrogen compounds such as nitrite, nitrate and ammonium ions originate from decomposing waste and animal excretions. When these compounds exceed certain levels they are toxic. Ammonium ions interfere with neuronal processes, and prolonged exposure to elevated nitrite levels causes slow suffocation, especially when oxygen is limited. Although unproblematic at low concentrations, prolonged exposure to nitrate can cause weight loss and render animals susceptible to infectious disease. To avoid such complications, beneficial bacteria are introduced to culture systems. These species perform nitrification and/or denitrification (Figures 1 and 2) and thereby lower

15 NITRIFICATION N + NH 4 NH 2 OH - NO 2 - NO 3 Oxic Organic Ammonia Hydroxylamine Nitrite Nitrate Suboxic NH 2 OH NO - NO 2 - NO 3 Nitrous Nitric Nitrite Nitrate Oxide Oxide DENITRIFICATION Figure 1. Bacterial nitrification and denitrification. Remaining Compound (%) Aerobic N 2 Anaerobic Aerobic Anaerobic Aerobic Anaerobic Atmosphere Water Column Bottom Hours 24 Hours 48 Hours Figure 2. In vitro nitrogen compound bioremediation. In the laboratory, cells were cultured with and without oxygen for two days in the presence of high ammonium, nitrate or nitrite levels. Growth (OD65) Ammonium Nitrate Nitrite Luxl AHL Synthase Negative Control Promoter LuxR Positive Control Growth Inhibition Acyl-Homoserine Lactones LuxR Figure 3. Basic quorum sensing mechanism in gram-negative bacteria. Targets for quorum quenching are labeled with black stars. Quorum Quenching No Effect 8, 7, 6, 5, 4, 3, 2, 1, Growth Light Figure 4. Quorum quenching example. Growth inhibition by default leads to less Q.S.-related activity (light production). LuxR Receptor (Transcription Factor) Dimerization Target Genes Gram Negative mrna Light (RLU) ammonium, nitrate and nitrite levels in the water. Bacterial nitrification is the oxidation of ammonium to nitrate via hydroxyl amine and nitrite. Denitrification is the reduction of nitrate to nitrous oxide and finally to nitrogen gas, which returns into the atmosphere. Nitrite is an intermediate in both processes. To efficiently remove all three compounds, a mix of bacteria capable of nitrification and/or denitrification is advantageous. Sulfur compounds like hydrogen sulfide are another problem. Hydrogen sulfide is generated during anaerobic degradation of organic matter at the bottoms of ponds. Hydrogen sulfide interferes with aerobic respiration and thus leads to the suffocation of animals. Black sludge, which occurs when hydrogen sulfide and iron react to form iron sulfide, indicates the presence of hydrogen sulfide. Hydrogen sulfide is used by several bacterial species. Of special interest are those in the Thiobacillus and Paracoccus genera. Thiobacillus species, for example, simultaneously remove nitrate and hydrogen sulfide in a process called sulfoxidizing denitrification and convert the compounds to non-toxic sulfate and nitrogen gas. Beneficial Bacteria Versus Pathogens Pathogen antagonism is an essential feature of probiotic bacteria. In general, antagonism is achieved through competitive exclusion, which encompasses several mechanisms: production of antimicrobial substances like bacteriocins, production of organic acids that reduce ph in animals gastrointestinal tracts and thereby prevent the proliferation of pathogens, competition for adhesion sites and competition for nutrients. Recently, a new approach has emerged in terms of pathogen antagonism: Disruption of quorum sensing, a bacterial cell-tocell communication mechanism. Bacterial Talk Quorum sensing (Q.S.) describes the regulation of gene expression according to population density. Q.S. is mediated by small signal molecules that are continuously produced. As bacterial populations grow, signal molecules accumulate and upon reaching a threshold, changes in gene expression are induced. This mechanism allows bacterial populations to coordinate group activities, such as biofilm formation, virulence and production of antimicrobials and exoenzymes. These processes put a metabolic burden on the cells and are only worth initiating when the success rate is high enough. In terms of virulence, efficient and timely expression of virulence factors ensures that the infection is successful. Researchers have identified and isolated various kinds of signal molecules. The most common signal molecules are acylated homoserine lactones (AHL) for gram-negative bacteria and small peptides for gram-positive bacteria. Modifications such as variation in the length of the carbon side chain of AHLs confer species specificity. Independent of gram classification, numerous species respond, detect or produce the universal signal molecule AI-2. Genera such as the Vibrios employ signal molecules that are specific for a group of related bacteria. All these molecules provide information on the environment, species composition, presence of competitors for nutrients and metabolic status of the community. The basic system for AHL signaling consists of a synthase for AHL production and a cognate transcription factor for AHL detection (Figure 3). Upon binding of the AHL, the detector will activate/deactivate target genes. More sophisticated multichannel systems were discovered in Vibrios. Here, different signals are detected by their cognate receptors in the membrane, and a regulatory cascade activates/deactivates target genes. Each signal has a specific channel, and information from each channel is converted into the same signaling cascade. Quorum Quenching When communication is sabotaged, group activities cannot be coordinated, and traits that are regulated by Q.S. do not function properly. In the best case, quorum quenching renders bacteria less virulent and more vulnerable. Several steps in Q.S. systems are targets for quorum quenching (Figure 3). When synthesis of signal molecules is repressed, cells are silent and unable to talk. If the target is signal detection, stability of the receptor is affected, or structurally similar molecules block access to the receptor binding site, but do not activate the receptor. Signal molecules can be destroyed by enzymatic degradation, and thus, messages never arrive at receiver cells. Nature has already developed a variety of inhibitors produced by plants, bacteria and algae. For example, garlic contains compounds that block information flow, and the red macroalgae Delisea pulchra produces halogenated furanones that mimic signal molecules and prevent access to the receptor. Bacteria have developed mechanisms to destroy and degrade AHLs. Several Bacillus species produce enzymes that hydrolyze the lactone ring of the AHL. Other species, like Ralstonia or Variovorax, produce enzymes that target the amide bonds of AHLs. Beneficial bacteria that not only target the growth of pathogens, but possess these enzymes can control pathogens in two ways. Growth is directly inhibited by the production of antimicrobial substances. Communication is also sabotaged to keep the pathogen under control. Quenching Systems During the last few decades, researchers have developed a plethora of indicator strains that express traits like light production, fluorescence or pigment production in the presence of Q.S. signal molecules. These indicator strains are employed for detection but also to investigate quorum quenching. Because beneficial bacteria produce antimicrobial substances, it is important to carefully determine whether the quorum quenching effect is in fact connected to a lack of communication or whether the effect is due to growth inhibition (Figure 4). 26 March/April 214 global aquaculture advocate global aquaculture advocate March/April

16 production Dietary Acidifier Potassium Diformate Improves Growth, Survival In Pangasius Improvement in the protein-efficiency ratios of juvenile Pangasius may have been a direct result of the effects of potassium diformate on stomach ph. Summary: Alternatives to antibiotics as growth promoters for fish are sought worldwide. As examined in various studies, potassium diformate can improve the growth and health status of Pangasius when the dietary acidifier is included in feed. In a commercial-scale trial, Pangasius that received dietary potassium diformate had greater weight gains, feed conversion, survival and protein efficiency than the fish in the negative control group. If chosen properly, acidifiers like potassium diformate can also positively affect digestion processes in fish. Aquaculture production has increased rapidly within the last 3 years, with the vast majority of this production expansion occurring in Asia. Pangasius production was a major driver of this trend. Farming of Vietnamese Pangasius, Pangasianodon hypophthalmus, has become increasingly attractive for the food industry worldwide. According to the latest United Nations Food and Agriculture Organization data, Vietnam is currently the world s leading Pangasius producer, raising over 1.15 mmt in 213. Especially in Southeast Asia, farmers are looking for possibilities to economize Pangasius culture. Dr. Christian Lückstädt ADDCON GmbH Kaiserstr. 1a Bonn, Germany Dr. Kai-Jens Kühlmann ADDCON Asia Co. Ltd. Bangkok, Thailand Tho Minh Van ADDCON Asia How Chi Minh City, Vietnam Management Concerns However, high stocking densities and suboptimal water quality can impair fish health and growth performance. In the past, antibiotic growth promoters were often used to help overcome these limitations. The routine use of antibiotics is a subject for much debate in the animalfarming and feed and food industries. Public opinion and regulatory authorities in most exporting countries often focus on the misuse of antibiotics in aquaculture, and public attention has shifted toward production methods. Consumers are increasingly turning to fish from sustainably managed sites, so alternatives to antibiotics are sought worldwide in a variety of forms. Feed Additives, Dietary Acidifiers The growth and health status of fish can be improved through the inclusion of feed additives in high-quality feeds. This strategy will likely be one of the main factors in the future success of Pangasius farming in Southeast Asia. Among these additives, acidifiers have been increasingly used in diets for many fish species over the last decade. Successful testing has been performed with salmon, rainbow trout, African catfish, European seabass, Asian seabass, milkfish, tilapia and shrimp. Dietary acidifiers, including potassium diformate, have been used in Pangasius culture for the last five years. The four acidifiers used include a blend consisting of ammonia formate and ammonia propionate, a mixture of free formic and propionic acid and their salts, sodium butyrate and potassium diformate (KDF). KDF is one of the most frequently used dietary acidifiers in aquaculture, with reports on its use available from the Americas, Europe, Africa, Asia and Australia. In general, the results show significantly improved performance parameters in Pangasius fed dietary acidifiers. Commercial-Scale Trial One such trial demonstrated the effects of potassium diformate under Table 1. Performance data for Pangasius reared for 32 weeks with or without.2% dietary potassium diformate (KDF). Control.2% KDF Difference (%) Feed-conversion ratio Mortality (%) Protein-efficiency ratio commercial conditions in the Mekong Delta area. Added to a commercial catfish diet with 28.% crude protein at a.2% dosage, it was given to fish of approximately 2 g initial body mass for 32 consecutive weeks. More than 54, fish were stocked in one control pond and one treatment pond, each with a size of roughly 5, m 2. Thus, the stocking density was around 51 fish/m 2. During the trial, the water temperature in both ponds ranged 26 to 33 C. The fish were kept and fed according to normal pond management. At the end of the trial, a subsample of harvested fish indicated Pangasius in the treated group had greater weight gains than the fish in the negative control group 1.3 versus.84 kg. Similarly, feed conversion, survival and protein efficiency were improved in the treated group (see Table 1). Due to the design of the study with no replicates, no statistics could be established. Improved Digestion The noted 8% improvement in the protein efficiency ratio, which has been previously reported in other fish species, may have been a direct impact of the acidifier on stomach ph. The main author reported last year that this direct impact on protein digestion is often overlooked. A recent meta-analysis for potassium diformate found significantly improved weight gain and feed efficiency in tilapia at levels that can be already described as growth promotion. These results may not have stemmed only from the established antibacterial effects. Since acidifiers, if chosen properly, affect buffering capacity and/or stomach ph, they also have impacts on the digestion processes in the gastric tract especially in the stomach. This view is supported by research from 211 and 212 by Spanish scientists Manuel Yufera and fellow researchers, who looked into the impacts of stomach ph and pepsin activity in marine fish. In addition to the performance parameters, it was also interesting to investigate the additive s impacts on protein efficiency, as despite its major influence on the sustainability of fish production, such data are still scarce. Production Impacts In an attempt to quantify the impacts of KDF on Pangasius production on average, the three available sets of data from two commercial trials and one laboratory study were evaluated. The average impacts of.2% dietary KDF on production parameters against the negative control are displayed in Table 2. All analyzed data showed numeric or significant improvements in the relevant performance parameters, particularly weight gain, survival and productivity. In addition, KDF showed comparable improvements in Pangasius growout, as previously documented in tilapia in the main author s 212 meta-analysis. The protein efficiency was improved by an average of 4.4%. This is partly supported with a significantly increased carcass yield in one of the studies. Furthermore, Ei Lin Ooi and co-workers reported in 21 on the strong antimicrobial effects of a dietary acidifier against E. ictaluri in challenged fish. It can be therefore be concluded that the use of dietary acidifiers, including potassium diformate, can be an effective option for sustainable aquaculture that results in improved health and performance of Pangasius. 28 March/April 214 global aquaculture advocate global aquaculture advocate March/April Weight Gain Feed-Conversion Ratio Protein- Efficiency Ratio Table 2. Performance parameters of Pangasius fed.2% dietary potassium diformate as difference from control. Productivity Index Difference to control (%)

17 production Maximizing Profits of Larval Shrimp Rearing Trials Seek Best Balance Of Artemia, Formulated Diets Eddy Naessens Roeland Wouters, Ph.D. Rattayaporn Phuthongphan Erik Van Ballaer INVE Aquaculture Hoogveld Dendermonde, Belgium Multiple trials showed that partial replacement of Artemia in larval shrimp diets did not reduce quality or yield, but total replacement did. Summary: Easily digested, live Artemia offer important animal health benefits and improved performance when included in feeding regimes for larval shrimp. However, pilot- and commercial-scale trials have confirmed that Artemia can be partially replaced in shrimp feeding. Adding a dry feed product to partially replace Artemia in larval diets significantly reduced the consumption of Artemia cysts without compromising the overall postlarvae yield and quality. Cost analysis of the commercial production runs also revealed important savings when applying the replacement protocol. However, total replacement of Artemia showed reduced performance. Much has been said and written about brine shrimp s key role in the development and sustainability of shrimp and marine fish fry production. Artemia cysts have been at times both glorified and cursed by various stakeholders in the aquaculture industry. Several institutes and companies have researched the viability of a complete substitution of Artemia by a formulated diet. Although some claim to have found the perfect alternative, the fact remains that shrimp seed producers agree that live Artemia remain indispensable for an optimal production process. Many confirm that one can grow shrimp larvae without Artemia, but admit that doing so will inevitably compromise the survival, growth rate or overall condition and health of the animals. Artemia Issues Artemia is a natural product obtained by harvesting natural bodies of water. The ability of suppliers cyst harvests and inventories to meet market demand therefore relies on the unpredictability of Mother Nature. Every year, this impacts availability, quality and price. Also, the Artemia resource is limited. Artemia harvest data from the last 25 years indicates that during years with favorable conditions, the natural annual production peaked around 3, mt. All of those cysts were consumed every year by the aquaculture industry. On the other hand, according to the forecasts of the United Nations Food and Agriculture Organization, World Bank and other entities, the yield from seafood production is expected to double during the next 15 years. It is obvious that such growth can only be realized when more fry can be produced with less Artemia. Other reasons to search for Artemia alternatives include the biosecurity concerns inherent to any live and fresh feeds, and the well-documented fact that due to their very specific habitats, some strains of Artemia have an incomplete nutrient profile to sustain optimal development of marine organisms. In addition, the obligatory cyst hatching and nauplii preparation required for Artemia production make them less user-friendly than an offthe-shelf product. Artemia Benefits Live Artemia offer important benefits when compared to formulated diets. One is the ease with which Artemia are digested by their prey. Once the nauplii are captured, autolysis sets in and substantially assists shrimp larvae s own still incomplete digestive systems. Another important advantage is the fact they are alive. Artemia nauplii swim around in the water column. They don t sink and eventually pollute the water like leftover formulated diets tend to do. This is especially important in one-phase culture systems that are still the preferred rearing technique in Asia. Other benefits include the fact that Artemia nauplii are the perfect biocarriers for enrichment with nutrients and health components that improve the overall yield of larviculture of fish and shrimp. Additionally, persistent live factor characteristics such as attractability and palatability warrant the exceptional dietary status of Artemia. Artemia nauplii are the perfect biocarriers for enrichment with nutrients and health components that improve the overall yield of larviculture of fish and shrimp. 3 March/April 214 global aquaculture advocate global aquaculture advocate March/April

18 Daily Relative Contribution (%) Daily Relative Contribution (%) Z 1 Z 1 Control Total Feed Artemia Formulated Feed Z 1 /Z 2 Z 2 Z 2 /Z 3 Z 3 Z 3 /M 1 M 1 M 2 M 3 P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 P 9 P 1 Stage Artemia Replacement Artemia Formulated Feed Total Feed Z 1 /Z 2 Z 2 Z 2 /Z 3 Z 3 Z 3 /M 1 M 1 M 2 M 3 P 1 P 2 P 3 P 4 P 5 P 6 P 7 P 8 P 9 P 1 Stage Figure 1. Cumulative consumption and relative formulated feed/artemia content in two feeding protocols in a pilot-scale feeding trial. Dry Matter (g/mt/day) Dry Matter (g/mt/day) Replacement Trials INVE Technologies N.V., the research and development company of INVE Aquaculture, has carried out trials to evaluate the effects of Artemia reduction on the hatchery culture performance of Litopenaeus vannamei shrimp by partially and fully replacing the Artemia with commercial larval feeds, most notably a series of improved formulations that has been recently introduced as the Frippak Fresh Gold range. Pilot Scale A series of pilot-scale experiments was executed at INVE Aquaculture s test center in Thailand. Control cultures in all tests consumed approximately 3 kg Artemia cysts/million P.L. 1. Their performance was compared to cultures in which only about half of this amount was fed and to cultures where Artemia content was reduced to zero. In the control runs, the Artemia were supplemented with traditional microencapsulated larval shrimp diets formulated with fresh and natural ingredients (TRAD). The tanks with reduced Artemia input were supplemented with the new partial Artemia replacement (NPAR). Zero-Artemia treatments were fed 1% formulated diets, either NPAR or a mixture of commercially available industry-standard formulated diets (MIX). Relative Survival (%), Dry Weight Survival Dry Weight Diet (type) TRAD NPAR MIX Mixture Artemia (kg) Figure 2. Relative performance of larval cultures fed various combinations of Artemia and formulated diets. Postlarvae Count 1,2 Southeast Asia (P.L. 5 ) Count (P.L./g) Length (mm) 1, Latin America (P.L. 1 ) TRAD NPAR Postlarvae Length Southeast Asia (P.L. 7 ) Latin America (P.L. 1 ) TRAD NPAR Figure 3. Postlarvae counts and lengths in Asian and Latin American commercial-scale trials. The progression and ratios of the live Artemia and artificial diets are pictured in Figure 1. The animals showed similar larval development and survival in the control and treatments with partial Artemia replacement. Lengths and postlarvae counts were not significantly different (P >.5). In setups with zero Artemia, significantly slower growth rates were recorded. Except for the tanks that received the new Artemia replacement, survival was also significantly reduced (Figure 2). Commercial Scale Following the pilot-scale trials, further validation was sought in industrial systems. Commercial-scale trials were carried out in four production units, two in Latin America and two in Southeast Asia. In the Americas, the culture systems employed a two-phase Southeast Asia: Production Of 1 Million P.L. 8 Feed Component TRAD NPAR TRAD NPAR Compound Diets Requirement (g) Cost (U.S. $) protocol with transfer at P.L. 4/5. Larvae were cultured in either flat-bottom or parabolic rectangular tanks, and postlarvae were transferred to flat-bottom raceways. In Asia, the feeding regimes used a cocktail of partial Artemia replacements and other diets, including flakes. The culture systems used one-phase protocols. In the American systems, the Artemia replacement ratio was 6%, with 4% replacement in the Asian trials. The results obtained in all four systems showed no adverse impacts of the reduced Artemia on yield (survival, development and growth) and quality of the postlarvae at harvest. On the contrary, faster development and growth were registered (Figure 3). Survival was similar to that in the control feeding regimes for all treatments. Aside from the similar to better culture yields, a key advantage of partial Artemia replacement is its beneficial impacts on larval production cost. Cost analysis of the commercial production runs revealed important savings when applying the replacement protocol (Table 1). 32 March/April 214 global aquaculture advocate global aquaculture advocate March/April NPAR TRAD Other diets Artemia 9 3,82 1, ,655 1, Total cost Benefit (%) 22.4 Latin America: Production Of 1 Million P.L. 12 Feed Component TRAD NPAR TRAD NPAR Compound Diets Requirement (g) Cost (U.S. $) NPAR TRAD Other diets Artemia 75 4,917 3, 1,487 1,956 1,244 1, Total cost Benefit (%) 29. Table 1. Feed requirements and costs for production of 1 million postlarvae. The results obtained in all four systems showed no adverse impacts of the reduced Artemia on yield (survival, development and growth) and quality of the postlarvae at harvest, but significantly reduced the larval production cost.

19 production Hydrogen Sulfide Toxic, But Manageable sustainable aquaculture practices Claude E. Boyd, Ph.D. Department of Fisheries and Allied Aquacultures Auburn University Auburn, Alabama USA Hydrogen sulfide in sediment, which mainly results from sulfate reduction by microorganisms, can diffuse into overlying water and enter the water column. Summary: Hydrogen sulfide, which can form in pond bottom sediment, is toxic to aquatic animals because it interferes with reoxidation of cytochrome a 3 in respiration. The main practices for lessening the risk of hydrogen sulfide toxicity are conservative feeding to avoid wasted feed on pond bottoms, plenty of aeration to prevent low dissolved-oxygen levels and provide a flow of oxygenated water across the soil-water interface, and liming to prevent acidic sediment and water. Sulfur is an essential element for plants, animals and bacteria. It is present in natural waters and water of aquaculture systems, mainly as the sulfate ion. In humid regions, sulfate concentrations in water usually are 5-5 mg/l, but in arid regions, concentrations often exceed 1 mg/l. Seawater contains 2,7 mg/l of sulfate, on average. Although sulfate is rarely applied to aquaculture systems specifically for increasing ambient concentrations, it is present in feed and a few water quality amendments. Issues In Aquaculture The main sulfur-related issue in aquaculture is the occasional presence of toxic concentrations of hydrogen sulfide. Sulfide can occur in water because it is a metabolite of Desulfovibrio and certain other bacteria found in anaerobic zones usually in sediment. These bacteria use oxygen from sulfate as an alternative to molecular oxygen in respiration. There are three forms of sulfide (H 2 S, HS - and S 2- ), and they exist in a ph- and temperature-dependent equilibrium. The effect of ph on the distribution of the three forms at 25 C is shown in Figure 1. As ph increases, the proportion of hydrogen sulfide declines, and that of HS - rises until the two forms have roughly equal proportions at ph 7. At greater ph, HS - is the dominant form, and there is no S 2- until the ph is above 11. Hydrogen sulfide is toxic to aquatic animals because it interferes with reoxidation of cytochrome a 3 in respiration. This effect is caused almost entirely by H 2 S, while HS - is essentially non-toxic. Even if it is toxic, S 2- is not an issue, because it does not occur at ph values found in aquaculture systems. Hydrogen Sulfide Concentration The concentration of hydrogen sulfide must be estimated from total sulfide concentration, because methods for determining sulfide in water typically measure the total concentration of the three sulfide forms. The proportions of H 2 S at different ph values and temperatures provided in Table 1 can be used for estimating hydrogen sulfide concentration. To illustrate, suppose the ph is 7.5 at 26 C in freshwater with a sulfide concentration of.5 mg/l. The factor for these conditions is.238. Multiplying the factor by the sulfide concentration of.5 mg/l gives an H 2 S concentration of.119 mg/l. In seawater of the same temperature and ph, the concentration would be less by a factor of.9. Sulfide In Sediment Hydrogen sulfide formation in sediment is mainly the result of sulfate reduction by microorganisms. Sulfate reduction occurs at a lower oxidation-reduction (redox) potential than is necessary for the reduction of iron and manganese by microorganisms. Thus, ferrous iron and manganous manganese usually are present in zones where hydrogen sulfide is produced. Iron, manganese and other metals quickly react with hydrogen sulfide to form highly insoluble metallic sulfides that precip- 34 March/April 214 global aquaculture advocate global aquaculture advocate March/April

20 Table 1. Factors for estimating hydrogen sulfide concentration from measured concentrations of total sulfide. For seawater, multiply the factors by.9. Temperature ( C) ph itate. This process usually lessens the hydrogen sulfide concentration in sediment, but over 1 mg/l of hydrogen sulfide has been reported in some sediments. Hydrogen sulfide in sediment can enter overlying water by diffusion. It also can be mixed into the water column by biological activity and sediment disturbances by seine hauls and strong water currents caused by wind or mechanical aeration. If the rate at which hydrogen sulfide enters the water exceeds the rate of its oxidation, there will be a detectable concentration of this potential toxin in the water column especially in the layer a few centimeters above the sediment-water interface. Toxicity The 96-hour lethal concentration 5 (LC5) values for hydrogen sulfide to freshwater fish species range 2-5 μg/l, and much lower concentrations stress fish and make them more susceptible to disease. A measure of toxicity, LC5 reflects the concentration of a compound in water that killed 5% of the test animals in a specified period of time, e.g., 96-hour LC5. Ideally, freshwater fish should not be exposed to more than 2 μg/l of hydrogen sulfide for long periods. Shrimp and other marine species tend to be more tolerant of hydrogen sulfide than freshwater species are. Values for 96-hour LC5s of hydrogen sulfide to marine species range 5-5 μg/l. Nevertheless, hydrogen sulfide concentration probably should not exceed 5 μg/l in aquaculture ponds with brackish water of full-strength seawater. As with freshwater fish, elevated concentrations of hydrogen sulfide increase the susceptibility of marine organisms to disease especially Vibriosis in the case of shrimp. Studies in laboratory soil-water systems conducted at Texas A & M University suggested that high sulfide concentrations in sediment pore water did not affect shrimp, provided the soilwater interface remained aerobic, and the dissolved-oxygen concentrations in the water column were at 7% saturation or greater. Studies also have shown that the risk of hydrogen sulfide toxicity increases with lower sediment and water ph. Measurement Total sulfide concentration measurement is a complex task by standard laboratory methods, but aquaculturists can use hydrogen sulfide kits for easier total sulfide analyses. The kits provide relatively reliable data. Of course, estimation of hydrogen sulfide concentration from total sulfide concentration requires data on water temperature and ph (Table 1).The presence of hydrogen sulfide often can be detected by its extremely strong, rotten egg odor. Measurable hydrogen sulfide in water usually means a low dissolved-oxygen concentration in the water or at the sediment-water interface, and aeration should be increased. Management As mentioned above, aerator-induced water currents can disturb sediment, favoring the mixing of hydrogen sulfide into the water, but the positive benefits of oxygenation by aeration far outweigh this effect. Nevertheless, aerators should be installed in a manner that minimizes the disturbance of sediment. The main practices for lessening the risk of hydrogen sulfide toxicity are conservative feeding to avoid wasted feed on pond bottoms, plenty of aeration to prevent low dissolved-oxygen levels and provide a flow of oxygenated water across the soil-water interface, and liming to prevent acidic sediment and water. Between crops, pond bottoms should be thoroughly dried. Sediment should be removed from ponds where it is too deep to dry thoroughly, and acidic pond bottoms should be limed. Some products are sometimes applied to ponds because they can potentially alleviate hydrogen sulfide problems. These include potassium permanganate application to water at a concentration six to eight times the hydrogen sulfide concentration permanganate can oxidize sulfide. Iron compounds such as ferrous oxide have been applied to sediment at rates of 1 kg/m 2 or more to encourage precipitation of hydrogen sulfide in sediment pore water as iron sulfide. Sodium nitrate added to the water column can help maintain oxygenated conditions at the soilwater interface and lessen the opportunity of hydrogen sulfide diffusion into the water. Probiotics often are added to ponds with the belief that they lessen the risk of hydrogen sulfide toxicity. Sulfur-oxidizing bacteria already are present in ponds, and it is doubtful that probiotic treatments are effective for removing hydrogen sulfide. Zeolite is sometimes claimed to absorb hydrogen sulfide, but the treatment rate necessary for this to be effective would be far too great to be affordable. Mole Fraction H 2 S HS ph Figure 1. Effects of ph on the relative proportions of H 2 S, HS -, and S 2-. S 2-36 March/April 214 global aquaculture advocate global aquaculture advocate March/April

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