Dominique P. Bureau Fish Nutrition Research Laboratory, Department of Animal and Poultry Science, University of Guelph

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1 Deconstructing the Fish Meal and Fish Oil Replacement Story in Aquaculture: Focusing on Nutrient Requirements, Characterization of Feed Ingredients and Pragmatic Approaches Dominique P. Bureau Fish Nutrition Research Laboratory, Department of Animal and Poultry Science, University of Guelph Fish meal and fish oil replacement has been the focus of very significant research efforts and hundreds of scientific papers in recent years. Despite years of research, fish meal and fish oil remain very important, quasi essential, components of successful commercial feeds for most fish and crustacean species. This generally has an impact on the feed and production costs for many aquaculture products. The potential to reduce level of fish meal and fish oil in aquaculture feeds is linked with our potential to improve the wholesomeness of our understanding of this issue. Unfortunately, much of research efforts invested so far have had shallow focus and/or inadequate design. Aquaculture nutrition researchers tend to forget that "fish meal and fish oil replacement" is not a true research objective in itself and that fish meals and fish oils are complex ingredients with highly variable chemical compositions and nutritive values. Research and development (R&D) efforts should ideally be a lot more pragmatic and no longer be focused on the "replacement" of one ingredient by other ingredients but rather on "what the animal requires" and "how can we cost-effectively and safely meet the requirements of the animals". Progress is therefore highly dependent on a "balanced" understanding of the nutritional requirements of the animals and the chemical composition and nutritive value of different feed ingredients and feed additives. Characterizing the Composition and Nutritive Value of Feed Ingredients and Additives Over the past five decades, dozens of different protein and lipid sources have been evaluated in hundreds of practical feeding trials. Many of these trials focused on replacing fish meal, fish oil or other high quality protein and lipid sources by putatively more cost-effective protein and lipid sources. What is often overlooked in many trials is that fish meals and fish oils are complex ingredients that are known to vary greatly in chemical composition. The raw material sources and types, seasons, and processing equipment and conditions used in the manufacturing of these ingredients all have great impacts on the chemical composition and nutritive value of these ingredients. Incorporation "20% fish meal in the diet" or "replacing 50% of the fish meal or fish oil of the diet" may mean very different things depending on the type and chemical composition of the fish meal and fish oil used in the study and the fish meal and fish oil levels in the control diet.

2 In many feeding trials, the control diet is formulated with high fish meal levels and/or all essential nutrients are supplied greatly in excess of requirements. The test ingredient is included at graded levels and effect on growth performance is monitored. Under these conditions, the evaluation of the nutritive value of the test ingredients is not very robust nor is it specific enough. For example, a certain level (e.g. 20%) of the test ingredient may be observed to support optimal growth performance in feeds formulated to very high essential amino acid levels (e.g. high fish meal feeds). However, the same level of test ingredient may not be suitable for feeds formulated with low fish meal level and/or lower essential nutrient levels. There is a need to refine methodological approaches so that the focus is on assessment of available nutrient contribution of ingredients to the diet (i.e. the bioavailability of nutrients in ingredients) rather than absence of effect of test ingredients. Studies focusing on the quality and "bio-available" nutrient contribution of feed ingredients have been relatively few and far between. To date assessment of the nutritional value of ingredients has mainly focused on apparent digestibility of proximate analysis components (dry matter, crude protein, lipid, gross energy) and much less emphasis on specific nutrients, with exception of a few key nutrients (e.g., lysine, methionine, phosphorus). There is a need for detailed and accurate characterization of the bio-availability of nutrients in common feed ingredients, the variability in composition and bioavailability of nutrients within ingredient and the various factors (e.g. dietary interactions, biotic factors) that may affect the bioavailability of nutrients in complete feeds. The characterization of the fine chemical composition of different feed ingredients and feed additives should become a priority. Attention should be especially paid to the nutrients and other chemical components present in fish meals, fish oils and other animal products, notably those that may be absent or only at low levels in ingredients of plant origins. R&D efforts should not only focus on the traditional essential nutrients (e.g., essential amino acids, essential fatty acids, minerals, vitamins) but also on more minor nutrients which may play important roles under certain conditions or be conditionally essential at certain life stages (e.g. phospholipids, nucleotides, carotenoids, etc.). Defining Nutritional Requirements and Approaches to Meeting these Requirements Significant efforts have also been invested over the past five decades on the definition of the nutrient requirements of different fish and crustacean species. Despite the thousands of studies published on this topic, it is becoming clear that state-of-the-art is less advanced than what is required by the aquaculture feed industry. A comprehensive review of the literature carried out by an international committee of experts appointed by the US National Research Council to review the nutritional requirements of fish and shrimp recently identified several significant gaps in the definition of nutritional requirements for most commercially important species (NRC, 2011). Globally, there is also need for significant improvements in the focus of studies, the scope and quality of the experimental design and methodological approaches used, and the characterization of the diets and ingredients used. Overall, more systematic efforts need to be invested. It would be recommendable to increasingly focus

3 the research efforts on the 15 or so fish and crustacean species (e.g., carp species,, tilapias, Pangasid catfish, Atlantic salmon, Penaeid shrimp, etc.) that represent more 80% of the global farmed fish and crustacean production. While improving the accuracy of estimates of essential nutrient requirements and improving the characterization of feed ingredients are critical factors, determining how this information can be translated into meaningful and robust nutritional and feed formulation guidelines is equally important. It is a very complicated issue that is too often overlooked by aquaculture nutritionists. Aquaculture feeds are characterized by the wide nutritional specification to which they are formulated to. This is expected given the very large number of fish and crustacean species produced around the world using feed-based production systems. However, the protein, lipid, starch and digestible energy contents of feeds can significantly vary not only as a function of species and life stages for which they are formulated (trout vs. tilapia feed, starter vs. grower vs. finisher feed), but also as a function of a myriad of other factors, such as production systems, farmers' or feed manufacturers preferences, environmental constraints, and socio-economical conditions (e.g., fish price, access to credit, degree of risk). Most fish feed manufacturers have to serve a large client base cultivating numerous fish and invertebrate species in very different production systems (ponds vs. cages, marine vs. freshwater environment, etc.) and socio-economical contexts (small farmers vs. large vertically integrated corporations). These factors, as well as, the multitude of opinions with regards to optimal levels and modes of expression of essential nutrient requirements limits the ability of manufacturers to meaningfully translate scientific advances into practical and cost-effective feed formulation guidelines. There is clearly a need for more consideration of how the information may be potentially used when designing research trials. Contrasting the response of animal to increasing essential nutrient levels in different dietary matrices (e.g. diets with different digestible energy levels), and different species and life stages may allow to gain knowledge on the impacts of these different factors on essential nutrient utilization and requirements and enable the development of more robust feed formulation guidelines and models. Keeping an Eye on the Prize In defining the focus and objectives of R&D efforts, aquaculture nutrition researchers and feed manufacturers should keep the perspectives of aquaculture producers in mind. They ideally should first focus on generating information needed to meaningfully address key economical and production issues (growth, feed efficiency, disease resistance, product quality, etc.). The focus also should be on generating information needed to be able to adapt feed formulations to an ever more competitive and demanding market and to stricter environmental constraints and consumer demands.

4 Increasing collaboration between feed manufacturers, ingredient suppliers, fish producers, and research organizations has been instrumental in improving the quality and relevance of fish nutrition research in the past few decades. Many aquaculture feed manufacturers are investing heavily in research and development activities and have established own research facilities to test their commercial feed formulations, determine the effect of feed composition/nutritional specifications and feed ingredients on growth and feed efficiency of animals grown under commercial-like conditions. This has probably resulted in improvement of the quality of feed available to aquaculture producers. However, limited amount of information from these efforts trickles down to the global aquaculture nutrition community since the information generated is generally proprietary and is closely guarded from public disclosure for competitive advantage. Nonetheless, a healthy, arm-length, relationship with different industry stakeholders can truly help commercial relevance of academic research efforts in aquaculture nutrition and help this field meaningfully progress to address current and future challenges, including those related to fish meal and fish oil replacement.

5 Back to the Future- Creating new opportunities, meeting new challenges in fish nutrition Dr. Simon Davies Professor of Fish Nutrition, University of Plymouth Modern intensive aquaculture presents unique challenges and great opportunities for research at both fundamental and applied levels. The expansion of this industry requires solutions in feed technology based on sustainable use of raw materials and a shift towards more natural sources of nutrients in keeping with consumer expectations for transparency and safety of the food chain. In the second decade of 21 st century we are revisiting some previous efforts of the 1970 s in finding novel proteins and biotechnological derived products for inclusion in formulated feeds for fish and shrimp. However we now have an elaborate tool box of modern techniques embracing molecular biology such as genomics, proteomics and metabolomics for advancing our knowledge and development in key areas. Nutritional solutions must now go beyond the attainment of achieving good growth and production but also reinforce health and disease resistance to promote the concept of the robust fish. Fish within intensive rearing systems may be subjected to various stressors and this may lead to both production related pathologies (skin, fin erosion, deformities) and increased susceptibility to infectious diseases. The redefining of fish nutrition will require assessing the main nutrient requirements for various species, but also place emphasis on innovations in micronutrient sources such as vitamins and minerals. The latter are especially important and recent work on selenium and zinc are highlighted with respect to organic selenium and zinc (Selplex and Bio-plexed zinc) showing significant improvements in bioavailability and metabolic function in fish as shown by elevated enzyme activities and gene expression for trout fed these additives. Attention is given to functional glycomics with an emphasis on yeast cell wall constituents and their complex polysaccharide and sugar moieties (e.g. Bio-Mos) conferring unique cellular signalling properties and modulation of resident and pathogenic intestinal microbiota in fish. This leads to an associated improvement in gut integrity (morphology) with consequent effects on gut mediated immunology for the both innate and specific immune systems. Yeast is one major example of a novel single cell protein concentrate for inclusion in aquafeeds in our suite of alternative sources to replace fish meal. A commercial British biofuel derived yeast protein concentrate was tested for carp with promising results which are discussed in context and scope for the future.

6 Building new aquafeeds: Feeding for health and performance in Tra catfish (Pangasiaodon hypophthalmus) Dr. Le Thanh Hung Dean of the College of Fisheries, Nong LAM University Tra catfish (Pangasianodon hypophthalmus) has been cultured in the Mekong Delta, Vietnam, where, because of the extremely high stocking densities used, the bacterium Edwardsiella ictaluri commonly causes severe losses. A 10-week feeding trial was conducted to evaluate the effect of Actigen (Alltech Inc.) on the performance and non-specific immune response in Tra catfish. A total of 1600 Tra catfish (average weight = g) were cultured in hapas (1 x 1 x 1 m) submerged in concrete tanks, with a stocking density of 100 fish/hapa (nursery cage). Fish were acclimated to tanks for 2 weeks before the trial began. There were 4 treatment groups (4 replicates each). Fish were fed a commercial feed (28% protein) twice daily that contained 0%, 0.04%, 0.08%, or 0.12% Actigen. After 10 weeks of feeding, blood was sampled from 5 fish per replicate and white blood cell count and serum lysozyme activity determined. Also after 10 weeks of feeding, 25 fish/hapa were challenged with E. icaturi (10 6 cfu/ml) or ammonia (150 mg total ammonia/l). Challenge responses were measured as follows: E. icaturi challenge cumulative mortality 14 d post challenge; ammonia challenge survival rates at 24 and 48 h post challenge. Final fish weight was greater (P<0.05) in fish fed the 0.08 or 0.12% Actigen treatments compared with the control. Specific growth rates (SGRs) increased with increasing Actigen in diet. Fish fed Actigen (0.08 or 0.12%) had SGRs greater (P<0.05) than controls. Feed intake and FCR tended to improve with increased Actigen supplementation; these values in response to 0.12% Actigen differed P<0.05 compared from the control. Survival rates did not differ (P>0.05) between treatments. Total leukocyte counts increased with Actigen supplementation; counts were highest (P<0.05) for the 0.08 and 0.12% treatments. Actigen (0.12%) enhanced (P<0.05) lysozyme activity. Fish began to die 4 d post E. icaturi challenge. Survival rates 14 d post challenge were lowest in the control and increased with Actigen supplementation. Survival rates were greater (P<0.05) with 0.12% Actigen compared with the control. Survival rates in response to ammonia challenge increased (P<0.05) with Actigen supplementation (0.08 or 0.12%). In conclusion, feed supplemented with 0.08% or 0.12% Actigen improved Tra catfish weight, feed intake, FCR, measures of nonspecific immunity, and survival rates in response to E. ictaluri infection or ammonia challenge.

7 Improving fillet texture and flesh quality of Atlantic salmon through dietary optimization Dr. Turid Mørkøre Associate Professor, Norwegian University of Life Sciences Aquaculture is the fastest growing animal food-producing sector with a growth rate of approximately 7% per annum since A major challenge for a continued growth of global fish farming industry is adequate supply of feed raw materials at acceptable prices. Among the farmed species, Atlantic salmon has a high global economic importance in cool-water aquaculture. Salmon is appreciated for its pink and firm flesh that is rich in healthy omega-3 long chain polyunsaturated fatty acids and a wide array of highly bioavailable macro and micronutrients. In salmon feed, fish meal and fish oil have traditionally been the main ingredients, but continued growth in an efficient, safe and sustainable manner requires less dependency on marine feed resources. Fortunately, salmon has high ability to utilize plant and other alternative feed ingredients for body growth, and several studies have shown that salmon can be a net producer of marine proteins. But how are the new plant based feeds affecting the fillet quality as perceived by the processing industry and consumers? These are issues that will be addressed along with some considerations related to the possibility to counteract quality deviations such as soft texture, liquid leakage and deviating fillet appearance (including deformities), through certain dietary supplementations (i.e. amino acids, vitamins and minerals).

8 Replacing fish meal An imperative that aquaculture must successfully address Dr. Shuichi Satoh Department Leader of Marine Bioscience, Tokyo University of Marine Science & Technology Fish meal is a major protein source in aquafeed especially for carnivorous species. Increasing demand, unstable supplies and high prices of fish meal (FM) with the expansion of aquaculture have made it necessary to search for alternative protein sources. Plant protein sources such as defatted soybean meal and corn gluten meal are good candidates as FM substitutes, however those ingredients contain antinutritive substances such as phytic acid and also lack essential nutrients such as taurine. We conducted experiments to determine the effects of combined taurine, phytase, and enzyme complex (Allzyme SSF, Alltech Inc.) added to low-fm and non-fm diets on the performance of red sea bream. In our first experiment, low-fm diet (20% FM) formulated with defatted soybean meal and corn gluten meal, and taurine was supplemented to low-fm diet at 0.2%. Phytase (1000 FTU/g) or Allzyme SSF (0.05 or 0.1%) was also supplied with taurine. These diets were fed to juvenile red sea bream (14.3 g) for 12 weeks; performance and digestibility were compared with a control, FM-based diet. Growth was lowest in fish fed low-fm diet, was improved by taurine supplementation, and improved more by additional supplementation with phytase. However, these dietary groups could not attain the growth of the FMbased group. Conversely, supplementation with both taurine and SSF improved growth to the same level as the FM-based diet. Phytase or SSF supplementation also improved the digestibility and absorption of protein and phosphorus. These results suggest that combined supplementation with taurine and SSF improves the growth performance of red sea bream fed low-fm diet. In our second experiment, low (20 or 10% FM) and non-fm diets were formulated and supplemented with taurine and SSF. The test diets were fed to 1-kg red sea bream for 12 wk. Temperatures were o C. In the first 4 wk, the temperature was somewhat higher than optimal for red sea bream, thus fish growth was impaired. In the second 4-wk period, temperature was optimal (21 25 o C), and fish growth improved. The lowest growth was observed in fish fed non- or 10% FM diet. Growth was improved markedly with SSF; weight gain was similar to or exceeded that of fish fed the FM-based diet. However, in the third 4-wk period ( o C), although fish grew well, the positive effect of SSF supplementation was not obtained. These results suggest that SSF supplementation to low- or non-fm feed improves the growth of red sea bream when temperatures are optimal. Our results demonstrate that combined supplementation with taurine and Allzyme SSF may improve the growth of red sea beam fed low- or non-fm diet when the water temperatures are higher than 21 o C.

9 Sustainable shrimp farming: Solutions for meeting the biosecurity challenge Dr. Nyan Taw Senior Technical Advisor and Manager, Blue Archipelago Berhad For sustainable shrimp farming one of the major factors is farm biosecurity. Farm biosecurity begins with design and construction of farm. During late 1980s shrimp farms were design and constructed based on technology available at the time. The system was pond base with water flow through system. The system worked well until appearance of bacterial outbreaks. Due to this reservoirs were added to manage and control bacteria problems. However, during mid1990s viral especially WSSV appeared. Again the design has to be changed to enable to treat incoming water before use in culture ponds (Nyan Taw, 2005, 2008 & 2011). With biosecure farm design and construction, biosecure operation system needs to follow (Nyan Taw 2010). Biofloc, a very recent technology seem a very promising for stable and sustainable production as the system has self-nitrification process within culture pond water with zero water exchange (Yoram, 2000, 2005a&b). The technology has been successfully applied commercially in Belize by Belize aquaculture (McIntosh, 2000a, b & c, 2001). It also has been applied with success in shrimp farming in Indonesia, Malaysia (Nyan Taw 2004, 2005, 2008, 2010 &2011). The combination of two technologies, partial harvesting and biofloc, has been studied in northern Sumatra, Indonesia (Nyan Taw 2008 et. al). Presently, a number of studies by major universities and private companies are using biofloc as a single cell protein source in aquafeeds. Biofloc, harvested from fish or shrimp ponds could in near future possibly replace the expensive protein rich fish meal. In any aquaculture business as defined by economics - savings are also considered as profit. Savings such as from feed, time, energy, stability and sustainability can be calculated as profit. With emerging viral problems and rising costs for energy, biosecure with biofloc technology appears to be an answer for sustainable production.

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