Aquaculture research, tập 41, số 5, 2010

Aquaculture Research, 2010, 41, 611^612 doi:10.1111/j.1365-2109.2010.02510.x Editorial This issue is the result of the Special Session entitled ‘‘Basic And Applied Aspects Of Aquaculture Nutrition: Healthy Fish For Healthy Consumers’’ carried out on 17^18 September 2008 in Krakow, Poland as part of the Annual Meeting of the European Aquaculture Society This session was co-sponsored by the Organization for Economic Cooperation and Development (OECD) Co-operative Research Programme on Biological Resource Management for Sustainable Agriculture Systems,Trade and Agriculture Directorate In particular we appreciate help in the organization of the session by Dr Carl Christian Schmidt The European Aquaculture Society cosponsored the session and help from the Executive Director, Dr Alistair Lane, need to be recognized The Ohio State University, School of Environment and Natural Resources, contributed ¢nancially to the session One of the primary goals of ¢sh nutrition is to produce healthy food for human consumption This can only be achieved via a thorough understanding of ¢sh nutritional requirements and the appropriate choice of feed ingredients that will secure the economic e⁄ciency and sustainability of the industry, i.e decreased use of ¢shmeal and replacement with plant protein and lipid sources The conference intended to promote and focus the research on aquaculture nutrition by making a link to the human food chain The conference addressed the major areas of basic aspects of ¢sh nutrition by gathering experts in aquaculture nutrition that could outline the current state of knowledge in the ¢eld, and present coherent perspectives on the improvements needed in ¢sh culture to ful¢ll public expectations in terms of healthy food and its sustainable production Sustainable ¢sh production must concentrate on the use of plant protein and lipid sources in ¢sh diets to replace ¢shmeal resulting in healthy seafood from the human point of view Consequently, pollution originating from ¢sh and other aquatic animal farming must be decreased This will result in higher economic e⁄ciency of seafood production as the cost of ¢sh r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd food (diets) is the largest single cost (over 50%) of production From the very beginning of the session concept, results of the conference were to be transferred to professionals and therefore the subject of the regular peer-review process The intent was also to disseminate the results to the general public and the media in the form of a brochure that will summarize the major conclusions of the conference (abstracts) with the reference materials to each of the contributors, its ¢eld of expertise and current professional activity The session was also thought to contribute to public debate by focusing on the production of healthy human food at a time while the world ¢shery is shrinking and thus move from exploitation to sustainable production As outlined in the recent FAO ‘‘State of the World Aquaculture 2006’’document, the session intended to cross national and institutional boundaries and establish a framework for the large-scale development of aquaculture That was truly a forum for multidisciplinary interactions The programme areas that were covered by invited speakers can be grouped into the following major topics: (1) digestive tract morphology and regulation of nutrient uptake, (2) endocrine and neural regulation of food intake, (3) molecular biology tools to validate and compliment biological nutrition data, (4) major nutrient requirements, (5) replacement of ¢shmeal protein and lipids with plant ingredients, (6) speci¢c nutritional needs for di¡erent life stages of ¢sh, from larvae to broodstock nutrition and (7) management of aquaculture waste There are a number of challenges that must be overcome to maintain acceptable growth rates and feed e⁄ciency values at higher levels of substitution of ¢shmeal The ¢rst is cost of plant protein concentrates The second challenge facing the aquafeed industry as it moves to substitute higher amounts of ¢shmeal with plant proteins pertains to known nutritional limitations of plant proteins The third challenge to overcome to developing plant protein-based aquafeeds for intensively grown ¢sh species concerns unknown nutrients and biologically active 611 Editorial Aquaculture Research, 2010, 41, 611^612 materials in ¢shmeal that are not present in plant protein concentrates and that may be necessary dietary constituent for optimum growth and health of ¢sh These three applied aspects of ¢sh nutrition were reviewed but undoubtedly deserve special session in the near future as the logical consequence of the basic nutrition research These applied aspects will be 612 tackled with powerful new molecular biology tools, not only in a descriptive manner, but also by pathway-speci¢c metabolomic markers in ¢sh nutrition studies Konrad Dabrowski Ronald Hardy r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 611^612 Aquaculture Research, 2010, 41, 613^640 doi:10.1111/j.1365-2109.2009.02242.x REVIEW ARTICLE Live feeds for early stages of fish rearing Lu|¤ s E C Conceic°aìo1, Manuel Yu¤fera2, Pavlos Makridis3, So¢a Morais1 & Maria Teresa Dinis1 Center for Marine Sciences ^ CCMAR, University of Algarve, Faro, Portugal Instituto de Ciencias Marinas Andaluc|¤ a, CSIC, Apartado O¢cial, Puerto Real, Spain Institute of Aquaculture, Hellenic Center for Marine Research, Heraklion, Crete, Greece Correspondence: L E C Conceic°aìo, CCMAR, Universidade Algarve, Campus de Gambelas, 8005-139 Faro, Portugal E-mail: lconcei@ualg.pt Abstract Despite the recent progress in the production of inert diets for ¢sh larvae, feeding of most species of interest for aquaculture still relies on live feeds during the early life stages Independently of their nutritional value, live feeds are easily detected and captured, due to their swimming movements in the water column, and highly digestible, given their lower nutrient concentration (water content480%) The present paper reviews the main types of live feeds used in aquaculture, their advantages and pitfalls, with a special emphasis on their nutritional value and the extent to which this can be manipulated The most commonly used live feeds in aquaculture are rotifers (Brachionus sp.) and brine shrimp (Artemia sp.), due to the existence of standardized cost-e¡ective protocols for their mass production However, both rotifers and Artemia have nutritional de¢ciencies for marine species, particularly in essential n-3 highly unsaturated fatty acids (HUFA, e.g., docosahexaenoic acid and eicosapentaenoic acid) Enrichment of these live feeds with HUFA-rich lipid emulsions may lead to an excess dietary lipid and sub-optimal dietary protein content for ¢sh larvae In addition, rotifers and Artemia are likely to have sub-optimal dietary levels of some amino acids, vitamins and minerals, at least for some species Several species of microalgae are also used in larviculture These are used as feed for other live feeds, but mostly in the‘green water’technique in ¢sh larval rearing, with putative bene¢cial e¡ects on feeding behaviour, digestive function, nutritional value, water quality and micro£ora Copepods and other natural zooplankton organisms have also been used as live feeds, normally with considerably better r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd results in terms of larval survival rates, growth and quality, when compared with rotifers and Artemia Nonetheless, technical di⁄culties in mass-producing these organisms are still a constraint to their routine use Improvements in inert microdiets will likely lead to a progressive substitution of live feeds However, complete substitution is probably years away for most species, at least for the ¢rst days of feeding Keywords: microalgae, rotifers, Artemia, copepods, nutritional value, ¢sh larvae Introduction Live feeds are the main item in the diet of cultured ¢sh larvae and they are of particular importance when rearing marine ¢sh larvae of the altricial type Altricial larvae are those that remain in a relatively undeveloped state until the yolk sac is exhausted At ¢rst-feeding the digestive system is still rudimentary, lacking a stomach, and much of the protein digestion takes place in hindgut epithelial cells (Govoni, Boehlert & Watanabe 1986) Such a digestive system is in most cases incapable of processing formulated diets in a manner that allows survival and growth of the larvae comparable to those fed live feeds In fact, despite recent progress in the development of inert diets for ¢sh larvae (e.g., Lazo, Dinis, Holt, Faulk & Arnold 2000; Cahu & Infante 2001; Koven, Kolkovski, Hadas, Gamsiz & Tandler 2001), feeding of most species of interest for aquaculture still relies on live feeds during the early life stages Even the ‘Artemia replacement’ products increasingly used in commercial operations are normally used in co-feeding with live feeds (e.g., 613 Live feeds for ¢sh larvae L E C Conceic° aìo et al Curnow, King, Partridge & Kolkovski 2006; VegaOrellana, Fracalossi & Sugai 2006; Hamza, Mhetli & Kestemont 2007; Rosenlund & Halldo¤rsson 2007) However, the low digestive capacity of altricial larvae might not be the only aspect responsible for them normally requiring live feed Live preys are able to swim in the water column and are thus constantly available to the larvae Most formulated diets tend to aggregate on the water surface or, more commonly, sink within a few minutes to the bottom, and are thus normally less available to the larvae than live feeds In addition, since larvae are believed to be ‘visual feeders’, adapted to attack moving prey in nature, the movement of live feed in the water is likely to stimulate larval feeding responses Finally, live prey, with a thin exoskeleton and a high water content (normally 80%), have a lower nutrient concentration and may be more palatable to the larvae once taken into the mouth, compared with the hard, dry formulated diet This last point is rather critical as any feed item must enter the mouth whole, i.e., feed particles have to be smaller than the larva’s mouth gape, and are quickly accepted or rejected on the basis of palatability (FernaŁndez-D|¤ az, Pascual & Yu¤fera 1994; Bengtson 2003) The present paper aims to review the main types of live feeds used in aquaculture, their advantages and pitfalls, with a special emphasis on their nutritional value and the extent to which this may be manipulated It also reviews the main concerns and potential bene¢ts regarding the micro£ora composition of live feed when it is added to larval tanks Finally, it discusses the constraints of using live feeds to study ¢sh larvae nutritional requirements, the possibilities of using tracer studies to overcome such constraints and assessment of the future of live feeds in ¢sh larvae production Aquaculture Research, 2010, 41, 613^640 turn is used as food for the carnivorous larvae of many of the marine ¢sh and shrimp species presently farmed Finally, intensive rearing of bivalves has so far relied on the production of live microalgae, which comprises on average 30% of the operating costs in a bivalve hatchery For rearing marine ¢sh larvae according to the ‘green water technique’, algae are used directly in the larval tanks This technique is nowadays a normal procedure in marine larviculture, given that it has been widely reported to improve ¢sh larval growth, survival and feed ingestion (e.g., Òie, Makridis, Reitan & Olsen 1997; Reitan, Rainuzzo, Òie & Olsen 1997) The observed larval quality enhancement when using microalgae in the rearing water has been explained by di¡erent studies, which showed that microalgae seemed to provide nutrients directly to the larvae (Mo¡att 1981), to contribute to the preservation of live prey nutritional quality (Makridis & Olsen 1999), to promote changes in the visual contrast of the medium and in its chemical composition (Naas, Naess & Harboe 1992; Naas, Huse & Iglesias 1996) and to play an important role in the micro£ora diversi¢cation of both the tank and the larval gut (Nicolas, Robic & Ansquer 1989; Reitan et al 1997; Skjermo & Vadstein 1999; Olsen, Olsen, Attramadal, Christie, Birkbeck, Skjermo & Vadstein 2000) More recently, Rocha, Ribeiro, Costa and Dinis (2008) showed that ¢sh larvae feeding ability is also in£uenced by the presence of microalgae in the tank However, this e¡ect is not the same among species and has been shown to be more pronounced with gilthead seabream than with Senegalese sole larvae (Rocha et al 2008) Main species of microalgae Microalgae Main utilizations of microalgae Microalgae constitute the ¢rst link in the oceanic food chain, i.e., the primary producer, due to its ability to synthesize organic molecules using solar energy In aquaculture, microalgae are produced as a direct food source for various ¢lter-feeding larval stages of organisms such as bivalve molluscs (clams, oysters and scallops), the larval stages of some marine gastropods (abalone) and early stages of penaeid shrimp larvae (Yu¤fera & LubiaŁn 1990) They are also used as an indirect food source, in the production of zooplankton (e.g., rotifers and Artemia), which in 614 The ¢rst microalgae species produced for aquaculture were selected from those produced naturally in pioneering ¢sh farms and were probably the easiest ones to cultivate (Muller-Fuega, Moal & Kaasa 2004) However, other species were later investigated based on their biological characteristics and performance under laboratory culture conditions, as well as on their nutritional and energetic properties Among the most important selection criteria for microalgae, the following can be highlighted: cell size appropriate to the demands of the consumer organisms; adequate nutritional value; high digestibility; r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 613^640 Aquaculture Research, 2010, 41, 613^640 ease of culture at high densities; short life cycle, reproducible in captivity; and tolerance to environmental variations Using these selection criteria, 16 genera of microalgae are generally produced nowadays Still, some species dominate and it is possible to relate such species to their corresponding utilization Class Baccilariophyceae (Diatoms) are usually given to bivalve molluscs and crustacean larvae as they are rich in silicates, which constitute their cell walls (frustules) and are necessary for bivalves and crustaceans for the formation of rigid structures Classes Prasinophyceae (e.g., Tetraselmis suecica, Tetraselmis Chuii) and Chrophyceae (e.g., Dunaliella tertioleta, Chrorella minutissima) are ideal food for crustacean larvae, when complemented by Baccilariophyceae for silicate supply Class Prymnesiophyceae (e.g., Isochrysis galbana) is widely used to culture marine ¢sh larvae (Brown 1991), while class Thraustochytriidae (e.g., Schizochtrium sp.), which consists of heterotrophic chromists, has mostly been used as feed for live prey species (Brachionus sp and Artemia sp.) Microalgae product types A general feature of marine microalgae is their high polyunsaturated fatty acid (PUFA) content However, the availability of microalgae as an a¡ordable PUFA source is limited At present, many ¢sh farms have their own facilities to produce microalgae for use during the ¢rst feeding of marine ¢sh and crustacean larvae The investment in such facilities is high and can represent 30% of a hatchery operating cost (Coutteau & Sorgeloos 1992) In addition, productivity may be variable depending on the season New products and methodologies with better coste¡ectiveness have been investigated and developed This includes microcapsules, dried microalgae, yeasts or yeast-based diets, bacteria, thraustochytrids (Knauer & Southgate 1999; Langdon & Únal 1999) and algal pastes (Heasman, Diemar, O’Connor, Sushames & Foulkes 2000) A fast-growing range of such commercial products is available, including live microalgae concentrates, and frozen and freeze-dried microalgae Results using these products are generally good For instance, centrifuged concentrates of Pavlova lutheri in combination with Chaetoceros calcitrans or Skeletonema costatum yielded 85^90% of the growth of a mixed diet of live microalgae for oyster larvae Saccostrea glomerata (Heasman et al 2000) Hatcheries that already have the infrastructure for Live feeds for ¢sh larvae L E C Conceic° aìo et al algal mass production may also prepare their own concentrates on-site, and thus limit their algal production to less busy periods of the season, better manage their requirements for microalgae and also reduce costs due to over-production (Knuckey, Brown, Robert & Frampton 2006) Techniques for growing microalgae Various techniques have been developed to grow microalgae on a large scale, ranging from less controlled extensive to monospeci¢c intensive cultures However, the controlled production of microalgae is still a complex and expensive procedure Culture of microalgae for aquaculture purposes (rearing of mollusc, shrimp and ¢sh larvae) takes place mostly onsite, i.e., in the ¢sh farms where they are utilized, although a new industry is emerging for the production of microalgae and delivery in lyophilized, frozen or other form to the farms (Navarro & Sarasquete 1998; Muller-Fuega et al 2004) There are various methods for the culture of microalgae, but the induction of ‘blooms’ by fertilization (addition of nutrients to the culture medium) is the most usual In ¢sh farms, stock cultures are kept under controlled conditions and protected from contamination by other microalgae, ciliates and potentially harmful bacteria Up-scaling of the microalgae cultures takes place in several steps, and the ¢nal large-scale cultures are often located outdoors under natural light conditions Cultures may be monospeci¢c or polyspeci¢c Polyspeci¢c cultures are normally carried out in open air tanks, or ponds, with water volumes exceeding 100 m3, while monospeci¢c cultures are carried out in medium (o100 m3) or small (o5 m3) containers, in which environmental variables are controlled Microalgae cultures may be divided according to the technology applied in the following types, listed according to the ascending intensity of the culture and average cell density: earth ponds, raceways, plastic polyethylene bags (100^500 L), open cylindrical tanks constructed of polymer ¢breglass and tubular and £at-plate photo-bioreactors (Zmora & Richmond 2004) Earth ponds and raceways are more open systems, exposed to weather conditions, and where there is a higher risk of contamination In addition, the cell density remains at relatively low levels in comparison with the other culture systems Bags and cylindrical tanks are quite common approaches for the production of microalgae in ¢sh farms in the Mediterranean region r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 613^640 615 Live feeds for ¢sh larvae L E C Conceic° aìo et al Photo-bioreactors are a relatively recent advance for the production of microalgae in aquaculture farms They enable the production of large biomasses of microalgae in high-cell-density cultures, in a small area and with a low input of labour Tubular (serpentine and manifold) and £at-plate bioreactors are commonly used for this purpose Small volumes of high-density microalgae are easier to handle by ¢sh farmers but present some problems as well, as they require the use of experienced personnel and all microalgae production is dependent on a few cultures Such devices may need a cooling system, as heat is accumulated in the system, and automated addition of carbon dioxide In relation to the timing of harvest of microalgae during the culture period, culture strategies can be divided into: (i) batch cultures, where no growth medium is added after initial inoculation; and semicontinuous cultures, where a part of the culture is harvested and new growth medium is added subsequently, several times during the culture period (Ii, Hirata, Matsuo, Nishikawa & Tase 1997) According to Fogg (1975), the limiting factors for microalgae production by photoautotrophy, resulting from the production method, are the exhaustion of nutrients, reduction in illumination due to the increase in cell density (shadowing) and the inhibition of cell division due to the accumulation of catabolites However, the various factors may be interdependent and a parameter that is optimal for one set of conditions is not necessarily optimal for another Algal cultures need to be enriched with nutrients in order to overcome the nutrient de¢ciencies of seawater This includes the macronutrients nitrate and phosphate (in an approximate ratio of 6:1), silicate (if growing diatoms) and micronutrients, comprising various trace metals and the vitamins thiamin (B1), cyanocobalamin (B12) and sometimes biotin The Walne medium and the F/2 medium are the two most extensively used enrichment media and are suitable for the growth of most algae There are also commercially available nutrient solutions that are suitable for mass production of microalgae in large-scale extensive systems These solutions contain only the most important nutrients and are made of agriculturegrade rather than laboratory-grade fertilizers Heterotrophic culture may provide a cost-e¡ective, large-scale alternative method of cultivation for some microalgae that utilize organic carbon substances as their sole carbon and energy source This mode of growth eliminates the requirement for light and, therefore, o¡ers the possibility of considerably in- 616 Aquaculture Research, 2010, 41, 613^640 creasing the microalgal cell concentration and, hence, volumetric productivity in batch systems In the last decade, knowledge of the cultivation of heterotrophic marine algae that accumulate PUFAs has increased However, knowledge and production of such products are limited and restricted only to very few companies (Muller-Fuega et al 2004) Furthermore, as heterotrophic algae are not used directly as feed in the aquaculture industry, their performance is not known and is yet to be determined Two heterotrophic species that are commonly used in live food enrichment emulsions or in ¢sh diets, due to their high levels of docosahexaenoic acid (22:6n-3; DHA), are the dino£agellate Crypthecodinium cohnii and the fungal thraustochytrid, Schizochytrium sp (De Swaarf, Pronk & Sijtsma 2003) Nutritional value of microalgae Whenever microalgae are used as a direct food source or as an indirect food source, in the production of rotifers, Artemia or copepods, growth of the animals is usually superior when a mixture of several microalgal species is used (Becker 2004) This probably occurs as di¡erent species compensate one another for eventual de¢ciencies in given nutrients Special care is needed when selecting microalgae for ongrowing live feeds for marine ¢sh larvae, in order to avoid the nutritional de¢ciencies of the latter, in particular in terms of n-3 highly unsaturated fatty acids (HUFA) The dry matter composition of microalgae is highly variable, even within a given species, with protein contents ranging from 12% to 35%, lipid from 7.2% to 23% and carbohydrates from 4.6% to 23% (Becker 2004) The protein content of microalgae is a major factor in determining its nutritional value and may change considerably with the composition of the growing medium (Becker 2004) Nitrogen concentration seems to be particularly important in this respect De¢ciencies in the n-3 HUFA contents of microalgae may cause severe mortalities and/or quality problems in shrimp, mollusc and marine ¢sh larvae In addition, such de¢ciencies may also cause reduced fecundity of rotifer and copepod cultures Signi¢cant concentrations of eicosapentaenoic acid (EPA; 20:5n-3) are normally present in diatom species (C calcitrans, Chaetoceros gracilis, S costatum andThalassiosira pseudonana), Nannochloropsis sp., T suecica, Tetraselmis chuii, D tertioleta and C minutissima (Brown 1991; Becker 2004) High concentrations of r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 613^640 Aquaculture Research, 2010, 41, 613^640 DHA are found in I galbana and P lutheri (Brown 1991; Becker 2004) and particularly in Thraustochytriidae (e.g., Schizochtrium sp.), which can contain over 70% of its weight as lipids and have a DHA content up to 35% of their total fatty acids (Sijtsma & de Swaarf 2004) Microbial aspects in phytoplankton cultures During the culture of microalgae, a high organic load is progressively accumulated, which becomes the substrate for the proliferation of bacteria Bacterial cells may attach to microalgae cells or may proliferate in the water Bacteria associated with microalgae may reach very high values of culturable bacteria, such as 108 mL À or 103 cell À (Salvesen, Reitan, Skjermo & Òie 2000) In batch cultures, the level of bacteria is lower during the exponential phase and reaches a maximum during the stationary phase (Salvesen et al 2000; Makridis, Alves Costa & Dinis 2006) In semi-continuous cultures, where there is a periodic replacement of part of the culture by new growth medium, numbers of bacteria tend to stabilize at a value lower than that observed in similar batch cultures Unsuccessful culture of microalgae, characterized by low growth rates and an extended lag phase of the cultures, may result in a high bacterial load (Nicolas et al 1989; Salvesen et al 2000) The cultured microalgae cells and the bacterial communities associated with the microalgae cultures are in constant interaction, resulting either in the suppression of growth of speci¢c groups of bacteria or in decreased growth of the cultured microalgae (Munro, McLean, Barbour & Birkbeck1995; Fukami, Nishijima & Ishida 1997; Suminto & Hirayama 1997; Kokou, Ferreira, Tsigenopoulos, Makridis, Kotoulas, Magoulas & Divanach 2007) The outcome of these interactions may depend on the method of microalgae production, the microalgae species grown, the growth media used, the quality of seawater and the growth phase of the culture (Salvesen et al 2000; Makridis et al 2006) Antimicrobial activity has been detected in extracts of microalgae (Du¡ & Bruce 1966; Austin & Day 1990; Austin, Baudet & Stobie 1992; Tendencia & dela Penìa 2003) and in bacteria isolated from microalgae (Makridis et al 2006) This antimicrobial activity can be caused by (i) associated microbiota (Makridis et al 2006); (ii) antimicrobial proteins or fatty acids produced by the microalgae cells (Kokou et al 2007); or Live feeds for ¢sh larvae L E C Conceic° aìo et al (iii) free oxygen radicals produced due to the photosynthetic activity of the microalgae cells (Marshall, de Salas, Oda & Hallegraef 2005) Examination of the bacterial populations present in microalgae cultures using molecular approaches able to detect non-culturable and culturable bacterial strains revealed a di¡erent picture than studies of the culturable microbiota Cultures of P lutheri, I galbana, C calcitrans, S costatum, C gracilis and Chaetoceros muelleri harboured a broad spectrum of species belonging to the groups of a-Proteobacteria, b-Proteobacteria, g-Proteobacteria, Cytophaga^ Flavobacterium^Bacteroides (CFB) bacteria group, Actinobacteria and Bacillus Members of the Roseobacter clade and the CFB group were dominant in the microalgae cultures In microalgae cultures, culturable members of the Vibrio group were absent or present in very low numbers (Salvesen et al 2000; Tendencia & dela Penìa 2003; Sainz-Hernandez & Maeda-Martinez 2005; Makridis et al 2006) Addition of microalgae to the rearing tanks of marine ¢sh larvae has a positive e¡ect on the growth and survival of the larvae It has been suggested that this positive e¡ect may be due to the bacteria associated with the microalgae cultures (Reitan, Rainuzzo, Òie & Olsen 1993) However, evidence for this is still weak and further studies are needed Rotifers Main utilization of rotifers Since the 1970s, the rotifers and more speci¢cally Brachionus plicatilis constitute an essential part of the feeding during the larval stages of marine ¢sh and crustaceans (Yu¤fera 2001; Lubzens & Zmora 2003) Its body size (between 70 and 350 mm depending on the strain and age) makes this organism an appropriate prey to start feeding after the resorption of vitelline reserves of many species In fact, Brachionus is widely used as ¢rst food during a period of days or weeks depending on the reared species, being replaced afterwards by a larger prey species, usually Artemia nauplii (Yu¤fera, Rodr|¤ guez & LubiaŁn 1984; Polo, Yu¤fera & Pascual 1992; Olsen et al 2000) Obviously, ¢sh species having a wider mouth gape at the onset of feeding may start directly on Artemia nauplii Besides the above-mentioned body size feature, the main advantages of this organism to be used as live prey in hatchery large-scale production are the following: (i) a high population growth rate, (ii) feeding by ¢ltration of particles in suspension, being able to r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 613^640 617 Live feeds for ¢sh larvae L E C Conceic° aìo et al ingest microalgae, yeast, bacteria and organic particles, (iii) a good tolerance to culture conditions and handling, something common in euryhaline animals, and (iv) to exhibit an appropriate energy content and reasonable nutritional value that, in addition, is relatively modi¢able by dietary manipulation during routine feeding and/or by means of speci¢c post-culture enrichment Another advantage of this prey is that it can be made permanently available Rotifers are a renewable resource and the production chain can be completely established in the hatchery, avoiding the dependence on external supplies The major inconveniences arise from the occurrence of episodic collapses as well as from the e¡ort required for maintaining the whole plankton chain Raising rotifers for larviculture requires the production of large amounts of organisms with appropriate size and nutritional quality In order to meet these objectives, many di¡erent strains have been isolated from nature and acclimated to the laboratory conditions In addition, di¡erent mass culture techniques have been developed, and a variety of microalgae species, yeast and commercial products have been tested and used as food during the routine culture and the following enrichment period Main species and strains of rotifers Variations in female’s body size were already observed in the late 1970s, but were noted mainly during the 1980s when di¡erent morphotypes and strains were described according to body size and spine shape The di¡erent strains were grouped into large (L-type), medium (SM-type) and small (S-type) Brachionus plicatilis (Yu¤fera 1982; Fukusho & Okaushi 1983; Snell & Carrillo1984; Fu, Hirayama & Natsukari 1991; Go¤mez & Serra 1995) Currently, this group is considered to be a multi-species complex of 9^15 di¡erent species and biotypes This complex includes species formally described as Brachionus plicatilis sensu stricto, Brachionus rotundiformis, Brachionus ibericus and Brachionus manjavacas, together with a series of lineages discernible by molecular techniques (Segers 1997; Ciros-Pe¤rez, Go¤mez & Serra 2001; Go¤mez, Serra, Carvalho & Lunt 2002; Suatoni,Vicario, Rice, Snell & Caccone 2006; Fontaneto, Giordani & Serra 2007; Mills, Lunt & Go¤mez 2007) Besides the formal species, at least the lineages Brachionus sp Nevada, Brachionus sp Cayman and Brachionus sp Austria have been identi¢ed as common in hatcheries (Papakostas, Dooms, Triantafyllidis, Deloof, Kappas, Dierckens, 618 Aquaculture Research, 2010, 41, 613^640 De Wolf, Bossier, Vadstein, Kui, Sorgeloos & Abatzopoulos 2006; Dooms, Papakostas, Ho¡man, Delbare, Dierkens, Triantafyllidis, De Wolf, Vadstein, Abatzopoulos, Sorgeloos & Bossier 2007; Kostopoulou & Vadstein 2007; Baer, Langdon, Mills, Schulz & Hamre 2008) The clari¢cation of the taxonomical situation will continue in the coming years From a practical point of view, as a prey for larviculture, it is still useful to use the identi¢cation of large (L), medium (SM) and small (S and SS) morphotypes referring to the relative body size (Table 1) Rearing techniques for rotifers Since the establishment of the basis for the culture of Brachionus (Ito 1960), di¡erent techniques of mass culture have been developed in order to obtain high and constant productions (see for instance: Hirata & Mori 1967; Theilacker & McMaster 1971; Hirata 1974; Yu¤fera & Pascual 1980; Gatesoupe & Robin 1981; Yoshimura, Hagiwara, Yoshimatsu & Kitajima 1996; Suantika, Dhert, Nurhudah & Sorgeloos 2000; Dhert, Rombaut, Suantika & Sorgeloos 2001; Park, Lee, Cho, Kim, Jung & Kim 2001; Lubzens & Zmora 2003; Olsen 2004) The Brachionus plicatilis complex of species and biotypes reproduce mostly by parthenogenesis, although a sexual phase may occur under speci¢c environmental conditions The most relevant aspect is its high fecundity, which allows a population duplication time of 24^48 h (Hirayama & Kusano 1972; Hirayama, Watanabe & Kusano 1973; Yu¤fera, LubiaŁn & Pascual 1983; Korstad, Olsen & Vadstein 1989) Provided that the appropriate abiotic and feeding conditions are supplied, a single parthenogenetic female Table Accepted species (in bold) and other biotypes belonging to Brachionus plicatilis species complex grouped according to former size-related classi¢cation (Go¤mez et al., 2002; Baer et al., 2008) L -biotypes 220^340 lm SM -biotypes 150^220 lm S and SS biotypes 100^150 lm B plicatilis B manjavacas B ‘Nevada’ B ‘Austria’ B ibericus B ‘coyrecupiensis’ B ‘almenara’ B ‘tiscar’ B ‘cayman’ B ‘towerinniensis’ B rotundiformis B ‘lost’ L, large; SM, medium; S and SS, small and super small The numbers indicate the approximate range of adult’s body length in each group r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 613^640 Aquaculture Research, 2010, 41, 613^640 may generate a large o¡spring in a few days The rearing techniques developed for rotifers take advantage of this characteristic of their reproductive biology The Brachionus population exhibits an exponential growth while the favourable conditions persist, followed by a decrease and cessation in growth when the food is exhausted or the chemical and microbiological conditions fall out beyond tolerance ranges Therefore, all the di¡erent culture systems that have been developed attempt to maintain the exponential growth by supplying high food levels and by preventing excessive accumulation of nitrogenous waste The best descriptor of the health and growth status of the cultured population is the egg ratio This index is a direct indicator of fecundity, and therefore of the potential for growth in the following hours (Snell & Carrillo1984;Yu¤fera et al.1984) The best growth rates occur approximately at a temperature between 20 and 35 1C, a salinity of 10^35 and a pH of 7.0^9.0 (Hirayama & Kusano 1972; Pascual & Yu¤fera 1983; Miracle & Serra 1989; Yu¤fera & Navarro 1995) The renovation pattern of the culture media (water plus algal cells) determines the basic methods, from batch culture (no renovation) to semi-continuous culture (partial renovation) and continuous culture (permanent renovation) In addition, independent of the culture system, to establish a complete production chain, three basic volume scales have to be considered: the stock culture (50^500 mL) aiming to maintain the genetic strain under optimal conditions; the starter culture (from to 50 L) proceeding directly from the genetic strain and used to inoculate large volume cultures; and the mass culture (from 50 L to several m3) Obviously, this basic chain may change according to the operating conditions of each hatchery In the batch culture system, an inoculum with a relatively low individual density is seeded in a dense microalgae suspension and the rotifer population grows for several days until the exhaustion of the algal cells The growth pattern follows a sigmoid curve and the maximum rotifer density attained increases with an increase in the initial food concentration following a saturation response The whole production is harvested at the end of the exponential phase and used Commonly, a small part of this production is used as a starter in the next production cycle two or three times The quality of this starter (inoculum) is decisive for the success of the rotifer culture The periodic use of the starter coming directly from the stock culture guarantees the health of the cultured population and the stability of the production This is a very common system in hatcheries, and many exam- Live feeds for ¢sh larvae L E C Conceic° aìo et al ples of its application may be found in the literature (Lubzens 1987; Dhert et al 2001) In the semi-continuous system, harvesting and media renovation are frequent, and account for a notable part of the total volume Harvested volume ranges from 10% to 50% and the harvesting frequency ranges from to days The culture may last several weeks When the renovation frequency is every 24 h or less and the renovated volume is constant, the culture can be associated with a continuous culture (Boraas 1983; Schluter, Soeder & Growneweg 1987) Like the batch system, the semicontinuous culture is widely used in larviculture and is usually combined with batch culture (Hirayama & Nakamura1976; Suantika et al 2000; Lubzens & Zmora 2003; Olsen 2004) The continuous rotifer culture systems are based on the chemostat methodology (Droop & Scott 1978; James & Abu Rezeq 1989) In chemostat-like systems, the daily renovation rate is constant After the initial growth, the population reaches a steady state, maintaining an almost constant rotifer density during weeks During the steady state, the growth rate is equal to the renovation rate The female density attained during the steady state, and consequently the production, depends on the food level (Boraas 1983; Schluter et al.1987;Walz1993; Navarro & Yu¤fera1998) The use of a concentrate microalgae paste, freezedried microalgae or commercial feeds allows higher levels of cell and particle concentration to be attained in the culture media than that normally obtained with microalgae suspension cultures (Hirayama & Nakamura 1976; Yu¤fera & Navarro 1995) This has been the basis for the development of super-intensive culture techniques in which the rotifer density and production are notably higher than that obtained with traditional methods (Yoshimura et al 1996; Fu, Ada, Yamashita, Yashida & Hino 1997; Suantika et al 2000; Park et al 2001; Suantika, Dhert, Rombaut, Vandenberghe, De Wolf & Sorgeloos 2001;Yoshimura, Tanaka & Yoshimatsu 2001) This kind of mass culture is associated with continuous or similar systems, with permanent addition of a high-quality diet that ensures rapid rotifer growth Rotifers may invest a large amount of energy in relation to their body biomass in reproduction Therefore, failure in food supply following a period of large investment in egg production may result in collapse of the rotifer culture Another important issue in relation to highdensity production of rotifers is the removal of ammonia Several techniques have been suggested, such as lowering of pH, ion exchanger, membrane ¢lter r 2009 The Authors Journal Compilation r 2009 Blackwell Publishing Ltd, Aquaculture Research, 41, 613^640 619 Nutritional management of waste outputs in aquaculture D P Bureau & K Hua lenges related to potential environmental impacts of wastes of nutritional or biological origins, and recent progresses relative to nutritional strategies aimed at reducing or managing better the release of wastes by aquaculture operations Aquaculture wastes and potential environmental impacts Rearing ¢sh in an intensive manner involves the transformation of dietary inputs into ¢sh biomass This process generates wastes, which are, in many cases, di⁄cult to contain and recover The release of wastes into aquatic ecosystems by aquaculture operations may result in nutrient enrichment of these ecosystems These wastes can, in turn, potentially lead to environmental changes The type and magnitude of these changes will be highly dependent on the biological, chemical and physical characteristics of the receiving ecosystem Di¡erent water bodies will react di¡erently to in£ux of the same amount of certain wastes Close or highly sheltered sites with poor £ushing rates may also respond very di¡erently from wide-open sites, with high currents and £ushing actions (Reid, Liutkus, Robinson, Chopin, Blair, Lander, Mullen, Page & Moccia 2008) The contribution of other sources of wastes, whether from natural (riparian vegetation, leaf litter, alluvial sediments) or anthropogenic (agricultural, municipal, household, etc.) origins can be very signi¢cant, and therefore must also be taken into consideration (Camargo & Alonso 2006) Besides biological, chemical and physical factors, the perception of these changes will also be dependent on numerous socio-economical factors For example, a slight increase in primary productivity (e.g microalgae) or secondary productivity (pelagic zooplankton, macrozoobenthos) may be perceived negatively in areas with signi¢cant touristic and recreational activities Increases in primary and secondary productivities could potentially be perceived as neutral, or even bene¢cial, in areas where environmental degradation has already occurred, or in regions where natural productivity of water bodies is limited and wild ¢sh and invertebrates harvests play important roles in the regional economy, or where socio-economical conditions are such that the environmental, recreational and aesthetical attributes of the ecosystem are not highly valued The expectations and value systems of the various local stakeholders play a great role in the de¢nition of ‘assimilative capa- 778 Aquaculture Research, 2010, 41, 777–792 city’ and ‘environmental impacts’ These parameters are not as objective as they are often assumed to be The ‘release of wastes’cannot be systematically equated to ‘deleterious environmental changes’as it is frequently assumed in much of the literature or popular press The assimilative capacity of the environment and the potential environmental impacts of aquaculture wastes may be de¢ned, assessed and perceived very di¡erently, depending on a multitude of environmental, economical and societal factors Nonetheless, there is a growing consensus around the world that aquaculture operations, notably those operating in sensitive areas, need to act in increasingly environmental and socio-economically sustainable manners Signi¢cant e¡orts should be invested to try to minimize the release of wastes and/or the environmental impacts of aquaculture operations In this context, it is important to understand that di¡erent types of wastes will be of concern to di¡erent types of aquaculture operations and different strategies may be relevant for di¡erent production scenarios or local conditions It is, consequently, wiser to ¢rst develop a basic understanding of some of the major issues associated with the di¡erent types of wastes for di¡erent types of aquaculture operations in di¡erent environments SW Solid wastes generally represent a very high proportion of the ‘mass’ of wastes released by most ¢sh culture operations These wastes are generally of faecal or feed (feed wastage) origin and consist either of relatively fast-sinking particles (settleable solids) or particles that remains in suspension in the water column for extended periods of time (suspended solids) On land-based ¢sh culture operations, rapidly settling SW particles can be recovered through various technological approaches (e.g waste traps, ¢lter, settling ponds) Consequently, suspended solids arguably represent the main issue from a waste output and environmental impact perspective Suspended solids can a¡ect the e⁄ciency of water usage on the farm by decreasing the e¡ectiveness of the treatment systems The release of suspended solids in the environment can have direct negative impacts on health of vertebrates and invertebrates and indirect e¡ects on water quality parameters by favouring thermal strati¢cation of water bodies, which, in turn, interferes with mixing and dispersion of oxygen (O2) and nutrients to deeper layers Suspended solids may also r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 777^792 Aquaculture Research, 2010, 41, 777^792 Nutritional management of waste outputs in aquaculture D P Bureau & K Hua negatively a¡ect drinking water treatment, industrial uses and recreational and aesthetic attributes of water bodies Siltation or the sedimentation of SW (notably solid organic matter) is, arguably, the main concern for cage culture operations Degradation of settled organic matter by benthic biota (bacteria and other organisms) leads to consumption of O2 and the production of carbon dioxide (CO2) and ammonia (NH3) Excessive localized sedimentation of organic matter may result in signi¢cant reduction in dissolved oxygen (DO) levels, notably in an environment with poor mixing of the water layers during period of warm water temperatures (Magni, Rajagopal, van der Velde, Fenzi, Kassenberg,Vizzini, Mazzola & Giordani 2008) The decline in DO can also promote the formation of reduced compounds, such as toxic hydrogen sulphide (H2S), which can have direct adverse e¡ects on aquatic animals The resulting low-DO and elevated NH3 and H2S concentrations can be especially damaging to the benthic biota (Magni et al 2008) Decomposition of the SW will also result in the mineralization/dissolution of various elements, notably nitrogen (N) and P, which can then become available to primary producers and can contribute further to eutrophication of the water body Eutrophication (also called hypernutri¢cation) refers to the enrichment of aquatic ecosystem by nutrients, causing accelerated algal growth and associated undesirable disturbance to the water ecosystem ecosystems limited in N (Dugdale 1967) It has been shown that in many freshwater ecosystems, the concentration and availability of P has a direct e¡ect on the biomass of algae (Gibson 1997) Bostr˛m, Persson and Broberg (1988) de¢ned bioavailable P as the sum of immediately available P and those forms that can yield the available P under conditions of physical, chemical and biological processes The most available P forms are orthophosphates (Pi; H2PO4À , HPO24 À and PO34 À ), although organic compounds containing P can be taken up in variable amount by algae (Wetzel 2001) However, the uptake of organic P has to be preceded by external phosphatase hydrolysis to release orthophosphates (Jansson 1988) Therefore, the input of Pi into aquatic systems is an important factor that determines the primary productivity Anthropogenic activities are the major sources of P input into the aquatic system This includes urban, industrial and agricultural activities (Carpenter, Caraco, Correll, Howarth, Sharpley & Smith 1998) In intensive animal production systems, a great amount of dietary P supplied to farmed animals is released to the environment In addition, a proportion of the phosphate fertilizers applied to the soil unutilized by crops, can become ¢xed in the soil in an unavailable form to plant Accumulated soil P can be released to runo¡ and end up in water bodies as either the dissolved form or particulate form (Sharpley & Rekolainen 1997) Nitrogenous wastes P wastes Phosphorus is the most limiting factor for algae growth and eutrophication phenomenon in many freshwater ecosystems Nutrient enrichment can cause important ecological e¡ects on aquatic communities, as the overproduction of organic matter by primary producers and its subsequent decomposition usually lead to reduced DO concentrations in the bottom water strata and sediments of aquatic ecosystems with low water turnover rates, as described above Red¢eld (1958) identi¢ed that the composition of aquatic biomass in terms of carbon, N and P is relatively constant, with the relationship of C:N:P being 105:15:1 It is generally assumed that that any element below the Red¢eld ratio in the ecosystem is likely to be a limiting factor for the aquatic biota production On the basis of this ratio, freshwater ecosystems are generally considered to be limited in P and marine In marine ecosytems, N inputs generally control primary production and enhance eutrophication phenomena (Camargo & Alonso 2006) While P is often the foremost limiting nutrient for algal growth in freshwater ecosystems, N inputs can also play an important role in net primary production in lakes and streams with low N:P loading ratios (Camargo & Alonso 2006) À Ionized ammonia (NH1 ), nitrite (NO2 ) and nitrate À (NO3 ) are the most common forms of dissolved inorganic N in aquatic ecosystems These ions can be present naturally as a result of atmospheric deposition, surface and groundwater runo¡, point and non-point sources derived from human activities, dissolution of N-rich geological deposits, N2 ¢xation by certain prokaryotes and biological degradation of proteinaceous material (Camargo & Alonso 2006) These nitrogenous wastes can directly result in deleterious e¡ects Unionized NH3 is very toxic to aquatic animals, r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 777^792 779 Nutritional management of waste outputs in aquaculture D P Bureau & K Hua particularly to ¢sh, whereas NH1 is much less toxic Concentrations between 0.01 and 0.02 mg NH3N L À have been proposed to protect sensitive aquatic animals (Camargo & Alonso 2006) Nitrite can have a toxic e¡ect on aquatic animals as it can interfere with the O2-carrying capacity of haemoglobin (or haemocyanin in crustaceans) Water quality criteria within the range 0.08^0.35 mg NO2-N L À have been proposed to protect sensitive aquatic animals, at least during short-term exposures (Camargo & Alonso 2006) Nitrate has a relatively low toxicity and a maximum level of mg NO3-N L À has been proposed to protect sensitive aquatic animals (Camargo & Alonso 2006) As NH3 is toxic at low concentration and it is a determinant factor of wastewater treatment and exchange requirement of land-based aquaculture operations, various approaches have been adopted to reduce or remove nitrogenous wastes High water exchange limits the e¡ectiveness (or coste¡ectiveness) of various technological approaches aimed at converting nitrogenous wastes (bio¢lters) or reducing SW outputs (e.g ¢lters, settling ponds, etc.) À Elevated concentrations of N ions (NH1 , NO2 and À NO3 ) stimulate or enhance the development, maintenance and proliferation of primary producers (phytoplankton, benthic algae, macrophytes), contributing to the eutrophication of aquatic ecosystems as described above In some extreme cases, the production of certain toxic algae (cyanobacteria, dino£agellates and diatoms) may be stimulated by elevated N ions levels The toxins produced by algae can remain inside algal cells or they may be released into the surrounding water (extracellular toxins) Animals may be directly exposed to toxins by absorbing toxins from water, drinking water with toxins, or ingesting algal cells via feeding activity The algal toxins can be also bioaccumulated and biomagni¢ed through food chains and food webs (Camargo & Alonso 2006) and reach toxic levels in some organisms (molluscs, ¢sh) destined for human consumption Nutritional strategies for reducing waste output The nutritional origins of wastes Fish ingest various compounds as part of their diets These ingested compounds (or sub-components thereof) are digested, absorbed, metabolized, partly retained in the body and partly excreted, as faecal 780 Aquaculture Research, 2010, 41, 777–792 material or through the gills or the urine by the animal This is achieved through a large number of complex chemical reactions In practice, however, simple principles of nutrition can be used to describe or predict the excretion of wastes by ¢sh and, by extension, ¢sh culture operations (Cho, Hynes, Wood & Yoshida 1994) Figure presents a simple nutrient or elemental partitioning scheme (budget) for ¢sh that can be used for this purpose SW Dietary components ingested by ¢sh are subject to digestion and the digestion end products are absorbed Indigested, indigestible or unabsorbed compounds, as well as a small amount of endogenous material (cell sloughed-o¡, digestive enzymes, intestinal micro-organisms and their by-products) are egested as faecal material by the animal This material is largely insoluble (i.e solid) and egested as more or less cohesive faecal particles by ¢sh These faecal wastes form the bulk of the SWoutput of most ¢sh culture operations (Reid, McMillan & Moccia 2006) Feed wastage is generally well managed on ‘modern’ commercial ¢sh culture operations and recent estimates for salmonid cage culture operations suggest that this has been reduced to o1% (C L Podemski, per comm., March 2008) Consequently, a very large proportion (495%) of SW released by ¢sh culture operations should be of faecal origin Solid wastes can be estimated as follows: SW ¼ feed consumed  ð1 À ADCÞ þ feed wasted where feed consumed and feed wastes are expressed on a dry matter basis and ADCDM is the apparent digestibility coe⁄cient of dry matter of the feed Solid P wastes (SWP) and solid N wastes (SWN) can be estimated as follows: SWP ¼feed consumed  P content of feed  ð1 À ADCP Þ þ feed wasted  P content of feed SWN ¼feed consumed  N content of feed  ð1 À ADCcrude protein Þ þ feed wasted  N content of feed The amount, composition and physicochemical characteristics of the faecal material egested by ¢sh, and the SW output of aquaculture operations are, therefore, mainly a function of the amount (concentration) and digestibility of various components (proteins, lipids, starch, NSP, minerals, etc.) of the diet consumed by the animal (Cho & Bureau 1997), r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 777^792 Aquaculture Research, 2010, 41, 777^792 Nutritional management of waste outputs in aquaculture D P Bureau & K Hua Feed wastes Feed Intake Feces Undigested Solid wastes Digested Urinary and branchial excretion Dissolved wastes Retained Fish biomass Figure Simple nutrient partitioning scheme that can be used for predicting waste outputs from ¢sh although various biological and environmental parameters can also play a role (Reid et al 2008; Brinker 2009) Faecal material egested by salmonid ¢sh fed high-quality commercial-extruded diets generally consists of a relatively equal ‘mix’ of minerals (20^ 60%), carbohydrates (10^30%) protein (10^30%) and lipids (2^25%) Protein and lipids are generally more digestible than minerals and total carbohydrates However, because salmonid ¢sh feeds generally contain much higher levels of protein and lipids than minerals and carbohydrates, signi¢cant levels of these relatively highly digestible components are present in the manure Dissolved wastes (DW) Dissolved wastes output of ¢sh populations is a direct function of the consumption, metabolism and retention of nutrients by the animals Digested dietary compounds (nutrients) are absorbed and can potentially be metabolized by the animal to support various processes and functions (Fig 1) A signi¢cant proportion of the digested/absorbed nutrients is generally retained in the form of body tissue components (body proteins, phospholipids, triglycerides, etc.) It can be estimated that for most commercial-intensive ¢sh culture operations around the world, between 25% and 65% of the mass of digestible nutrients consumed by the animal is retained in the biomass produced This proportion is highly dependent on the ¢sh species and composition of feed used but numerous other factors, such as life stage, production/ rearing system, environmental conditions and husbandry practices, also have a determinant e¡ect The absorbed nutrients that are not retained are metabolized, and end-products of the catabolism of these nutrients can be eliminated by the animal through branchial or urinary excretion Amino acids need to be deaminated to allow their carbon backbone to be used in various reactions Consequently, amino acids catabolism also results in the release of NH3, which the ¢sh and crustaceans mainly excreted through their gills Ammonia represents approximately 80^ 90% of nitrogenous metabolic wastes of ¢sh and crustacean The catabolism of purine bases (from nucleic acids) and arginine results in the production of urea, which is mainly excreted in the urine Urea generally only represents about 10% of dissolved N waste (DWN) outputs of ¢sh (Kaushik & Cowey1991) There are many di¡erent kinds of DW excreted by ¢sh but NH3, orthophosphates and CO2 are arguably the most signi¢cant, quantitatively and environmentally speaking The environmental impacts of the release of the dissolved forms of N and P have been discussed above Carbon dioxide is not considered to be an environmental pollutant but it can be a signi¢cant concern for aquaculture operations operating recirculation systems and because it tends to accumulate in the water and needs to be ‘stripped o¡’ to maintain water quality and prevent hypercapnia in the animals Dissolved P wastes (DWP) and DWN can be estimated as follows: DWP ¼digestible P intake À retained P DWN ¼digestible N intake À retained N As the various wastes produced by ¢sh ¢nd their origins in the intake, digestion and metabolism of r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 777^792 781 Nutritional management of waste outputs in aquaculture D P Bureau & K Hua dietary compounds, nutritional strategies o¡er a direct way of managing the release of wastes of ¢sh culture operations The modi¢cation of feed composition has, indeed, been demonstrated as a very e¡ective way of reducing waste outputs by ¢sh culture operations (Cho et al 1994; Cho & Bureau 1997) Endogenous factors, such as ¢sh species and size/age, may also have very signi¢cant impacts (Azevedo, Cho, Leeson & Bureau 2004; Azevedo, Leeson, Cho & Bureau 2004) It is necessary to improve our understanding of the basis and relative contribution of these various determinants in order to develop nutritional and breeding strategies aimed at minimizing waste outputs from ¢sh culture operations Nutritional management of waste outputs: progresses and challenges Numerous stakeholders of the aquaculture industry have been proactive in the evaluation of nutritional strategies to reduce or manage waste outputs of commercial ¢sh operations The invested e¡orts have resulted in very signi¢cant reduction of waste outputs (per unit of ¢sh produced) by ¢sh culture operations over the past four decades In the 1970s and early 1980s, for example, commercial steam-pelleted troutfeed formulae were relatively low in protein (approximately 36% protein), fat (8^12% fat) and digestible energy (DE) (o14 MJ) but rich in starch and ¢bre (35^40%) as well as being high in P (42%) (Table 1) Progress in feed formulation, together with the introduction of modern feed production technology (extrusion, etc.), has resulted in the production of feed with higher digestible (or useful) nutrient densities, which enabled signi¢cant reduction in the amount of feed required to produce one unit of biomass In the 1970s and early 1980s, feed conversion ratio (FCR, feed:gain) of 1.5:2.5 were common for marketsize rainbow trout (1kg) fed the commercial feeds available at that period in North America Today, the use of higher digestible nutrient-density-extruded feeds [e.g 440% digestible protein (DP), 425% fat, 419 MJ DE] allows FCR of about (ranging from 0.9 to 1.2) for these ¢sh This signi¢cant decrease in FCR was also accompanied by very signi¢cant decrease in total SW, solid and dissolved P wastes (Table 1) The production of 1tonne of ¢sh biomass nowadays probably results in less than half of the amount of SW (manure) than what was the case 30 years ago Optimization of feed composition and manufacturing techniques, therefore, proved to be a very e¡ective 782 Aquaculture Research, 2010, 41, 777–792 Table Theoretical estimates of waste outputs of rainbow trout fed di¡erent feed formulae Feed Parameters Chemical composition Crude protein (%) Nitrogen (N) (%) Lipid (fat) (%) Digestible energy (MJ kg À 1) Phosphorus (P) (%) Apparent digestibility coefficient (%)à Dry matter (DM) Crude protein (CP) Gross energy (GE) Phosphorus (P) Theoretical feed conversion ratiow (feed:gain) Solid wastesz Total solid wastes kg tonne À of feed fed kg tonne À of fish produced Solid nitrogen wastes kg tonne À fish produced Solid phosphorus wastes kg tonne À fish produced Dissolved wastesz Dissolved nitrogen wastes kg tonne À fish produced Dissolved phosphorus wastes kg tonne À fish produced 1980s 2000s 36 5.8 10 14 2.5 44 7.0 24 19 1.1 65 85 70 50 1.5 78 88 80 60 1.1 350 540 220 250 13 19 48 43 16 ÃEstimated values wEstimated based on an estimated digestible energy requirement of 21.5 MJ to grow a rainbow trout from 10 to 1000 g zEstimated using the biological method of Cho et al (1994) means of reducing wastes outputs It is worth noting that these changes in feed composition and manufacturing practices were not driven by a need to reduce waste outputs but rather to improve the coste¡ectiveness of feeds and reduce the production cost of ¢sh With these major gains behind us, much more limited reductions in waste outputs are expected, at least for salmonid ¢sh culture operations Non-negligible reductions in waste outputs by ¢sh culture operations could still be achieved in a cost-e¡ective fashion through careful selection and quality control of feed ingredients, ¢ne-tuning of feed formulations, use of feed additives and processing of certain ingredients Major reductions in waste outputs could, nonetheless, be achieved for other major commercial ¢sh species cultured around the world, notably ¢sh species fed lower quality, lower nutrient density feeds (carp, tilapia, etc.) or for artisanal production systems, r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 777^792 Aquaculture Research, 2010, 41, 777^792 Nutritional management of waste outputs in aquaculture D P Bureau & K Hua where improperly balanced diets are used For example, the simple use of nutritionally adequate dry feeds instead of trash ¢sh has been shown to reduce N waste outputs of cuneate drum reared in marine net pens by 450% while sustaining the same growth rates (Wang, Guo, Li & Bureau 2006) Slim pro¢t margins, due to increasing competition, stagnant or decreasing product prices and rising production costs, may limit the ability of many ¢sh culture operations to focus on reduction of waste outputs and also, in some cases, even lead to the undoing of some of the progresses achieved However, as highlighted above, reduction of waste outputs of ¢sh and improvement of cost-e¡ectiveness of the feed are not incompatible objectives Factors that a¡ect the availability of nutrients also are those that a¡ect waste outputs In some cases, handling of wastes can also be very costly and limit the productivity of the infrastructure For example, nutritional management of wastes may be especially cost-e¡ective for land-based aquaculture facilities operating recirculation aquatic systems as solid organic matter, NH3 and CO2 are waste products that impose a signi¢cant burden on the water treatment infrastructure For aquaculture facilities operating in the public domain (e.g net pens in lakes, rivers and oceans) and facing increasing public scrutiny about their potential (or perceived) environmental impacts, devoting meaningful e¡orts to the management of waste outputs may be the key to insuring long-term sustainability and acceptability of these enterprises Strategies for the management of SW outputs As indicated above, very signi¢cant reduction of SW outputs by ¢sh culture operations has been achieved through improvement of the feed formulations and feed manufacturing techniques Non-negligible reduction of SW outputs by ¢sh culture operations could still be achieved with the selection of more highly digestible ingredients and exclusion of poorly digested ingredients from the feed formulation Quality control of the ingredients can be an e¡ective approach for controlling SW outputs It may, however, be di⁄cult to economically reduce ‘indigestible matter’content in feeds to levels below 10% (i.e economically produce feed with dry matter digestibility 490%) Consequently, improvement of feed e⁄ciency (reduction of the amount of feed required to produce 1unit of biomass), perhaps, remains the most promising avenue to allow further reduction of SW by ¢sh culture operations using high-quality feeds Improving feed e⁄ciency can be achieved by further increasing the digestible nutrients density and ‘nutrient balance’ of feeds beyond what is currently achieved A better understanding of nutrient requirements and utilizations of ¢sh and of the composition and bioavailability of nutrients in feed ingredients is essential in this context In the current context of high feed ingredient commodity prices, notably high ¢sh meal and ¢sh oil prices, feed manufacturers have been forced to significantly modify their feed formulations Increased reliance on more economical feed ingredients of plant or animal origins, with high ¢bre (NSP) or mineral contents, and lower dry matter or organic matter digestibility, may result in increases in SW outputs of some ¢sh culture operations Simple processing of common ingredients, such as thermal treatment, air classi¢cation or elutriation of ¢sh meal, meat and bone meal, poultry by-products meal or plant products (pea, canola meal, soybean meal, sun£ower meal) to reduce ash, starch and NSP contents of these ingredients, have been shown to be an e¡ective and relatively economical approach to producing feeds resulting in lower waste outputs More advanced processing of plant proteins to produce highly digestible, low-phytate P, plant protein concentrates is also a very e¡ective method in ultimately reducing solid, N and P wastes outputs of ¢sh culture operations Very signi¢cant improvement in the dry matter digestibility of pea products produced using di¡erent technological approaches provides a nice example of the potential reduction in SW outputs that can be achieved through ingredient processing (Table 2) Pea may be separated into their components (protein, starch and ¢bre) by dry or wet processing techniques (Thiessen, Campbell & Adelizi 2003; Thiessen 2004) Dry milling of dehulled peas produces pea £our with particles of various sizes and densities Subsequent air classi¢cation separates the less dense protein fraction from the coarser starch fraction to produce a concentrated protein fraction Wet milling involves milling of peas followed by solubilization of the protein in water or acid washings (Thiessen 2004) This method results in a more pure protein fraction, producing highly digestible pea protein concentrates (Table 2) More highly processed ingredients are generally more digestible and result in much lower amounts of SW However, they are relatively expensive at this point in time to use at high levels in feed formulations More work is required to improve the cost-e¡ectiveness of various processing methods r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 777^792 783 Nutritional management of waste outputs in aquaculture D P Bureau & K Hua Aquaculture Research, 2010, 41, 777–792 Table Digestibility of ¢eld pea and of increasingly processed derivatives for rainbow trout Digestibility Ingredients Dry matter (%) Protein (%) Starch (%) Energy (%) Whole, raw Dehulled, raw Dehulled, extruded Protein flour, 50% crude protein (CP) Protein concentrate, 77% CP Protein concentrate, 85% CP 42 47 74 84 94 98 91 91 94 95 97 97 14 25 100 66 - 55 57 78 87 96 98 Source: Thiessen (2004) Manipulation of the physico-chemical characteristics of SW Advances in nutritional strategies to reduce P waste outputs The biophysical characteristic of faecal wastes is an issue that has received very little attention in the past However, the properties of faecal waste (e.g particle size, density, stability, settling velocity, etc.) have signi¢cant implications for the deposition and dispersal of SW and, consequently, for waste management and environmental impacts This issue has been recently reviewed by Reid et al (2008) At present, our understanding of the factors that a¡ect the biophysical properties of ¢sh faeces is still limited and more research is needed (Reid et al 2008) It is known that diet composition, or speci¢c chemical components, can signi¢cantly a¡ect the physico-chemical properties of faecal material Low dietary incorporation levels (1^3 kg tonne À of feed) of guar gum, for example, greatly improved the stability and settling characteristics of faecal material egested by rainbow trout, thereby facilitating the recovery of SW from land-based farm e¥uents (Brinker, Koppe & R˛sch 2005a, b) Improvements imparted by guar gum reduced post-¢ltration e¥uent load to about 20^35% in rainbow trout, Oncoryhnchus mykiss (Walbaum) Faecal leaching decreased signi¢cantly with increasing stability (Brinker 2008) This bene¢cial e¡ect of guar gum to faecal stability is dose responsive and high-viscosity guar gum performed signi¢cantly better than mid-viscosity guar gum (Brinker 2007) Ogunkoya, Page, Adewolu and Bureau (2006) observed that the combination of soybean meal and an enzyme cocktail signi¢cantly reduced the stability (cohesiveness) of faecal material produced by rainbow trout More easily breakable and dispersible faecal wastes could potentially minimize localized impacts of some cage culture operations Phosphorus utilization by ¢sh has been the topic of numerous research projects over the past 20 years and is now fairly well understood Both digestibility and quantity will determine the fate of P fed to ¢sh The undigested fraction of the P of the diet is excreted in the faeces by ¢sh The digestible fraction of P that exceeds ¢sh requirement is excreted through urine as orthophosphates Both digestibility and quantity will determine the fate of P fed to ¢sh Experimental evidences suggest that P requirement is around 0.4^0.6% of digestible P (0.2^0.3 g MJ À DE) for most ¢sh species Fish receiving only the digestible P amount required to meet the requirement for growth, excrete only minute amounts of metabolic P (ca.5 mg P kg À BWday À 1) indicating that digestible P intake of the ¢sh is directed almost completely towards deposition (Rodehutscord 1996; Bureau & Cho 1999) A threshold of plasma phosphate concentration exists below which minimal renal P excretion occurs (Bureau & Cho 1999) Therefore, nutritional management of P output of ¢sh culture operations should be based on the reduction of total P level of feeds, improvement of dietary P digestibility and formulation to digestible P level meeting but not greatly exceeding P requirements The form of P excreted may also have a signi¢cant e¡ect on potential environmental impacts Not all forms of P excreted by ¢sh are equal to stimulate primary productivity In order to be utilized by algae and other plants, P must be soluble Therefore, P excreted in the urine of ¢sh is highly available to plants The potential of other forms of P excreted by ¢sh (P excreted in the faeces) is determined by their chemical nature Faecal phytate-P and other organic forms of P can be solubilized by bacteria and other organisms in the aquatic environment Mineral phosphates may 784 r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 777^792 Aquaculture Research, 2010, 41, 777^792 Nutritional management of waste outputs in aquaculture D P Bureau & K Hua also be mineralized through the action of bacteria and other living organisms or through simple chemical equilibrium process, determined by the dissociation constant (pK) of the chemical forms present Bone-P or hydroxyapatite, is only soluble at very low pH (e.g pH o3^4) and, in practice, can be considered inert and it is doubtful if any of it is potentially plant available Estimates of P waste outputs for cage farms should ideally take into account the di¡erent forms of P excreted, not just the total P waste output Hua, de Lange, Niimi, Cole, Moccia, Fan and Bureau (2008) integrated the available information on P nutrition of ¢sh and constructed a factorial model to estimate P digestibility, retention and waste outputs of salmonid ¢sh species This model operates within the framework of the FISH-PRFEQ bioenergetics model (Cho & Bureau 1998; Bureau, Gunther & Cho 2003) has been shown to generate relatively highly accurate estimates of waste outputs of ¢sh culture operations and allow theoretical estimation of the di¡erent types of P wastes (dissolved vs solid; inorganic vs organic solid P wastes) such as phosphorylated protein, creatine, phospholipids and nucleic acids, are apparently highly digestible to ¢sh (490% digestible) Phytate-P, another form of organic P, however, is not digestible to ¢sh The digestibility of mineral phosphates, such as dicalcium phosphate and rock phosphate, varies with their degree of solubility but is generally high (60^95% digestible) Digestibility of bone-P is variable between ¢sh species and depends mostly on gastric acid secretion by the animal For rainbow trout, a ¢sh with a true acid stomach, digestibility of bone-P is between 40% and 60% Through a detailed meta-analysis of the published data, Hua and Bureau (2006) developed a model to estimate digestible P content of salmonid ¢sh feeds Digestible P content of salmonid ¢sh feeds can be estimated as follows (Fig 2): Digestible P ¼ 0:68 bone-P þ phytate-P þ 0:84 organic P þ 0:89 Ca monobasic=Na=K Pi supplement þ 0:64 Ca dibasic Pi supplement þ 0:51 phytase=phytate Formulating on a digestible P basis Formulating feeds to a precise digestible P content can be a di⁄cult task because both P content and estimates of the digestibility of P common feed ingredients in the literature are highly variable (Table 3) Phosphorus content of feed ingredients is highly variable and P is found under di¡erent chemical forms in di¡erent ingredients (Hua & Bureau 2006) These forms can broadly be classi¢ed in four groups: organic P, phytate-P, mineral phosphates and bone-P (hydroxyapatite) Digestibility of these di¡erent forms of P di¡ers widely for ¢sh Organic P compounds, Table Apparent digestibility coe⁄cients (ADC) of phosphorus in common ingredients in salmonid diets Ingredient ADC (%) of phosphorus Fish meal Meat and bone meal Poultry by-product meal Blood meal Feather meal Corn gluten meal Soybean meal NaH2PO4 Ca(H2PO4)2 CaHPO4 Ca10(OH)2(PO4)6 or Ca3(PO4)2 17–81 22–67 38–66 70–104 68–82 o10 27–46 95–98 93–94 54–77 37–64 À 0:02 ðphytase=phytateÞ2 À 0:03 ðbone-PÞ2 À 0:14 bone-P  Ca monobasic=Na=K Pi supplement  ðP < 0:0001; R2 ¼ 0:96Þ A digestibility trial with rainbow trout fed diets formulated with di¡erent practical ingredients demonstrated that the model of Hua and Bureau (2006) was highly reliable and practical Judicious use of feed additives to further reduce P wastes Exogenous phytase (microbial or fungal phytase) can be supplemented in diets to improve the P digestibility of plant ingredients Phytate present in plant ingredients has to be dephosphorylated, primarily by phytase, to be available for intestinal absorption Phytase (myo-inositol hexaphosphate phosphohydrolase) catalyses the stepwise removal of Pi from phytic acid (Ravindran, Bryden & Kornegay 1995) Phytase is di¡erentiated into 3-phytase and 6-phytase based on their initial dephosphorylation position of the ester bond of the phytate In general, microbial phytase is 3-phytase, whereas phytase in plants and fungi is 6-phytase (Ravindran et al 1995) Certain plant ingredients have high endogenous phytase activities, such as rye, wheat and barley (Weremko, Fandrejewski, Zebrowska, Han, Kim & Cho 1997) In monogastric mammals, such as pig and poultry, en- r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 777^792 785 Nutritional management of waste outputs in aquaculture D P Bureau & K Hua Aquaculture Research, 2010, 41, 777–792 Dietary P Bone-P 68% Bone-P2 –3% Organic P 84% Phytate-P 0% Phytase 51% Phytase2 –2% Ca Mono/ Na/K Pi 89% Ca-Di Pi 64% Bone-P*Mono-Pi –14% Figure Classi¢cation scheme for phosphorus (P) compounds in ingredients and feeds and a model to estimate digestible P content of salmonid ¢sh feeds (source: Hua & Bureau 2006) dogenous phytases present in plant ingredients contribute to the hydrolysis of phytate However, phytase is heat labile, and a temperature of 70^80 1C can cause partial or total inactivation of endogenous phytase (Ravindran et al.1995) Extrusion, the major processing method for ¢sh diets, is likely to destroy endogenous phytase activity (Hughes & Soares 1998; Forster, Higgs, Dosanjh, Rowshandeli & Parr 1999; Vandenberg 2001) Therefore, research on the e¡ect of phytase on ¢sh diets has focused on exogenous phytase The incorporation of exogenous phytase in diets has been shown to improve the digestibility of P in salmonid (Rodehutscord & Pfe¡er 1995; Lanari, D’Agaro & Turri 1998; Vielma, Lall, Koskela, Schoner & Mattila 1998; Sugiura, Gabaudan, Dong & Hardy 2001; Vandenberg 2001) and other ¢sh species, such as carp, Cyprinus carpio L (SchÌfer, Koppe, MeyerBurgdor¡ & Gunther 1995), channel cat¢sh, Ictalurus punctatus (Ra¢nesque) (Jackson, Li & Robinson 1996; Yan, Reigh & Xu 2002), African cat¢sh, Clarias Gariepinus (Burchell) (Van Weerd, Khalaf, Aartsen & Tijssen 1999), striped bass, Morone saxatilis (Walbaum) (Hughes & Soares 1998), Japanese £ounder, Paralichthys olivaceus (Temminck and Schlegel) (Masumoto, Tamura & Shimeno 2001) and seabass, Dicentrachus labrax (Linnaeus) (Oliva-Teles, Pereira, Gounveia & Gomes 1998) There is some indication that the e⁄cacy of microbial phytase is more prominent in warm water species (Forster et al 1999) The use of phytase probably only makes sense for diets with digestible P contents below the requirement of the ¢sh and containing signi¢cant levels of plant ingredients, i.e in which indigestible P is mostly phytate-P Pre-treatment of feed ingredients with phytase is a promising alternative It has been observed that pretreatment of plant ingredients can also improve P di- 786 gestibility and utilization Dephytinization of plant ingredient improved P utilization in Atlantic salmon, Salmo salar L (Storebakken, Shearer & Roem 1998) and rainbow trout (Vielma, Ruohonen & Peisker 2002) Low-phytate cultivar grains, in which single gene mutation resulting in blockage of phytic acid accumulation, have been developed to improve phytate P digestibility Phosphorus digestibility in low-phytate barley and corn and barley was signi¢cantly higher than regular grains for rainbow trout (Sugiura, Raboy, Young, Dong & Hardy 1999; Overturf, Raboy, Cheng & Hardy 2003) Organic acids have been used in swine and poultry diets to improve P utilization The early study of Pileggi, De Luca, Cramer and Steenbock (1956) showed that citric acid improved the P utilization in phytate-containing diets in rats Citric acid also increased phytate-P utilization in chicks and pigs (Boling,Webel, Mavromichalis, Parsons & Baker 2000) Organic acids have been shown to be e¡ective to improve P (and other minerals) utilization in ¢sh Dietary supplementation of citric acid, Na citrate and EDTAwas able to improve P digestibility of ¢sh meal to rainbow trout (Sugiura, Dong & Hardy 1998) Formic acid at supplementation at and 10 mL kg À diet signi¢cantly improves P digestibility and retention in rainbow trout (Vielma & Lall 1997) Supplementing citric acid at levels ranging from to 30 g kg À diet to diets containing ¢sh or ¢sh bone meals improved P utilization by rainbow trout and red sea bream, Pagrus major (Temminck and Schlegel) (Vielma, Ruohonen & Lall 1999; Sarker, Satoh & Kiron 2005) Pandey and Satoh (2008) observed that1% citric acid supplementation signi¢cantly improved P retention in rainbow trout fed low ¢sh meal-based diet Supplementing organic acids have also been obeserved to improve P digestibility of the diets of agastric ¢sh species (Leng, Lun, Li,Wang & Xu 2006) The positive e¡ect r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 777^792 Aquaculture Research, 2010, 41, 777^792 Nutritional management of waste outputs in aquaculture D P Bureau & K Hua of organic acid is probably due to the solubilization of bone minerals in ¢sh meal, as well as a chelating e¡ect that reduces the antagonistic interaction between Ca and P that could precipitate Ca and P at the intestinal brush border (Sugiura et al 1998; Sarker et al 2005) Baruah, Pal, Sahu, Jain, Mukherjee and Debnath (2005) observed that supplementation (30 g kg À diet) of citric acid signi¢cantly increased bone P content in Indian carp (Rohu), Labeo rohita (Hamilton), fed a soybean meal-based diet These authors also observed signi¢cant interaction between citric acid and phytase enzyme on bone P content, indicating a synergetic e¡ect of these two feed additives High level of free-form organic acids in feeds can result in reduced feed intake, stomachal and duodenal mucosa damage, bone demineralization and metabolic acidosis (Partanen & Mroz 1999; Gauthier 2002, 2005), which could ultimately negatively a¡ect the growth performance of the animal Sugiura et al (1998) observed that citric acid levels up to 5% (50 g kg À diet) did not a¡ect the feed intake of rainbow trout but resulted in marked reduction of feed intake in gold¢sh, Carassius auratus (Linnaeus) Organic acids can possibly disturb the acid^base balance and mineral homoeostasis More research is warranted in this aspect Strategies for reducing N waste outputs Manipulation of diet composition The main factors a¡ecting N metabolic waste outputs are those that in£uence the catabolism and deposition (retention) of amino acids (protein) by the ¢sh Amino acid composition of the diet is consequently a factor that has a signi¢cant e¡ect on the amount of NH3 produced by ¢sh Feeding amino acids in excess of the requirement will result in the catabolism of the amino acid with an associated excretion of NH3 and loss of energy Diet formulated with protein sources of poorer amino acid pro¢le will result in lower digestible N retention e⁄ciency and greater NH3 excretion Optimizing amino acid composition of ¢sh feeds remains a challenge as there is very signi¢cant variability in estimates of essential amino acid requirements and disagreement about the mode of determination of these requirements of ¢sh The balance between DP and DE of the diet (DP/DE ratio) is another key factor There is no doubt that non-protein energy sources can spare dietary amino acids from being utilized as energy sources, thereby improving e⁄ciency of protein utilization for protein deposition in both rainbow trout (Cho & Woodward 1989; Ruohonen, Vielma & Grove 1998; Ste¡ens, Rennert, Wirth & Krˇger 1999) and Atlantic salmon (e.g Einen & Roem 1997; Grisdale-Helland & Helland 1997; Helland & Grisdale-Helland 1998; Hillestad, Johnsen, Austreng & —sgÔrd 1998) This is commonly referred to as ‘protein sparing’ Protein sparing by dietary lipids has been shown to occur in most ¢sh species Azevedo et al (2004b) observed that digestible N retention e⁄ciency (N retained/digestible N intake) linearly increased from 28%, 32%, 34% to 36% for large rainbow trout as the dietary protein/lipid ratios decreased from 57/20, 51/22, 46/24 to 43/26 respectively The increase in N retention e⁄ciency results in a decrease in DWN outputs of the ¢sh Protein sparing by digestible carbohydrate, such as gelatinized starch, has also been demonstrated but may be limited especially when the diet already contains a high level of lipids or a relatively low DP/DE ratio Overall, experimental data suggest that DP/DE ratio of about18 g MJ À e¡ectively reduces amino acid catabolism (and consequently DWN) without a¡ecting growth rate and feed e⁄ciency of salmonid ¢sh species Values reported in the literature suggest that smaller ¢sh and/or juvenile stages of ¢sh generally have higher feed e⁄ciency and N retention e⁄ciency than larger and/or post-juvenile ¢sh, and their optimum dietary DP/DE appears to be di¡erent (Ronsholdt 1995; Einen & Roem 1997) Digestible N retention e⁄ciency, nonetheless, rarely exceeds 50% in rainbow trout and 60% in Atlantic salmon fed diets with low DP/DE (16^18 g DP MJ À DE) It is not clear to what extent this signi¢cant catabolism of amino acids, despite ample supply of non-protein energy (indicated by high lipid deposition), is related to the inevitable losses of amino acids or catabolism of amino acids that are in excess of requirement Increasing DE, achieved through an increase in the lipid content of the diet, decreased catabolism of lysine in rainbow trout even when lysine is the ¢rst limiting nutrient and dietary lysine level is highly de¢cient (Encarnac°aìo, de Lange, Rodehutscord, Hoehler, Bureau & Bureau 2004) Inevitable catabolism could be a rather elastic concept for ¢sh The lysine-sparing e¡ect exerted by lipids was not observed with other energyyielding nutrients The e¡ect of di¡erent nutrients on the e⁄ciency of amino acid utilization cannot be explained solely on the basis of their energy contribution [even when expressed on a net energy (NE) basis] Leucine, an EAA with the same entry point into the TCA cycle as are fatty acids and lysine, was shown to r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 777^792 787 Nutritional management of waste outputs in aquaculture D P Bureau & K Hua be an e¡ective NE source but it had no e¡ect on the ef¢ciency of lysine utilization (Encarnac°aìo, de Lange & Bureau 2006) DE supplied as starch, even at low intake levels, was clearly less e¡ective than lipids at ‘sparing’ amino acid from catabolism (Tapia-Salazar M., Hua K., Bureau D P unpubl obs.) Impacts of endogenous and exogenous factors An increasing number of studies also indicate that catabolism of amino acids is very sensitive to a number of biological factors Studies have highlighted clear di¡erences between species and ¢sh at di¡erent life stages in terms of e⁄ciency of utilization of dietary amino acids (Berg & Bremset 1998; Rasmussen & Ostenfeld 2000; Refstie, Korsoen, Storebakken, Baeverfjord, Lein & Roem 2000; Azevedo, Cho, et al 2004; Azevedo, Leeson, et al 2004) There is a clear association between protein deposition and weight gain and between e⁄ciency of amino acid utilization and feed e⁄ciency Studies (Azevedo, Cho, et al 2004; Azevedo, Leeson, et al 2004; Dumas, de Lange, France & Bureau 2007) indicate that starting at a weight of about 400 g, EAA catabolism and the ratio of lipid to protein deposited (LD/PD) increases dramatically with an increase in body weight in rainbow trout This shift is not seen in Atlantic salmon of similar weights, reared under the same conditions and fed the same diets These observations point to the need to investigate the endogenous determinants of amino acid utilization in ¢sh This could then be translated into the development of approach for genetic selection or metabolic modulation Water temperature is frequently assumed to have a signi¢cant impact of metabolic N waste excretion by ¢sh However, studies with rainbow trout reared at di¡erent temperature showed that water temperature from to 15 1C had no e¡ect on digestible N retention e⁄ciency (Azevedo, Cho & Bureau 1998) Increasing water temperature results in increasing feed intake, growth and N waste outputs per ¢sh per unit of time but not appear to have any e¡ect on the ratio of N waste produced to DP or N consumed Accurately estimating waste outputs The ¢rst step towards an objective assessment of environmental impact and sustainability of aquaculture operations is having access to objective estimates of the amount of waste associated with production (actual or planned) Various attempts to directly monitor waste outputs from ¢sh populations 788 Aquaculture Research, 2010, 41, 777–792 or land-based ¢sh culture facilities has been shown to be a high-cost yet a highly inaccurate process (Cho, Hynes, Wood & Yoshida 1991; Cho et al 1994) There have been few attempts to estimate waste outputs from salmonid cage culture operations (Merican & Phillips 1985; Kelly, Stellwagen & Bergheim 1996) where direct monitoring and estimation of waste outputs is even more di⁄cult, costly and likely inaccurate than it is for land-based operations It has been demonstrated that estimation of waste outputs could be accurately and economically carried out with great £exibility based on feed inputs and feed component utilization by ¢sh (Cho et al 1991, 1994) Several studies have used nutritional mass balance models to estimate the nutrient loading from landbased and cage culture operations (e.g., Cho et al 1994; McDonald, Tikkanen, Axler, Larsen & Host 1996; Cho & Bureau 1998; Stigebrandt 1999; Davies 2000; Bureau et al 2003; Papatryphon, Petit,Werf, Sadasivam & Claver 2005) and the good concurrence between measured nutrient concentrations in farm e¥uent water not only allowed validation of the nutrient waste model estimations but also supported the robust nature of these nutritional models for estimating nutrient loading from land-based systems (e.g., Cho et al.1991,1994; Kelly 1995; Kelly et al.1996; McDonald et al 1996; Green, Hardy & Brannon 2002; Papatryphon et al 2005; d’Orbcastel, Blancheton, Boujard, Aubin, Moutounet, Przybyla & Belaud 2008) Comparison of waste output estimate generation with the FISHPRFEQ bioenergetics model (Cho & Bureau 1998; Bureau et al 2003) with more direct measures of waste outputs of rainbow trout over ¢ve production cycles at a model freshwater rainbow trout cage farm indicated that the model generates a reliable estimate of waste outputs for open water net pen operations The use of a simple nutritional model by ¢sh culture operations could be a very e¡ective tool for monitoring and setting goals for the reduction of waste outputs Conclusion Wastes produced by ¢sh ¢nd their origins in the intake, digestion and metabolism of dietary compounds Nutritional strategies o¡er a simple yet e¡ective way to manage the release of wastes of ¢sh culture operations Di¡erent forms and types of wastes will be of concern to di¡erent types of aquaculture operations, and consequently di¡erent strategies may be suited to di¡erent production scenarios or local conditions Signi¢cant reduction of waste r 2010 The Authors Journal Compilation r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41, 777^792 Aquaculture Research, 2010, 41, 777^792 Nutritional management of waste outputs in aquaculture D P Bureau & K Hua outputs by ¢sh culture operation can be e¡ectively achieved through improvements of feed formulation aimed at improving the e⁄ciency of digestion and retention of nutrients by ¢sh Reduction of waste outputs could also be achieve through the use of dietary additives, such as enzymes or organic acids, and relatively simple processing techniques, to improve digestibility and availability of various ingredients, especially the non-conventional ones Reduction of waste outputs of ¢sh can often go hand in hand with improvement of cost e¡ectiveness of the feed and contributes to maintaining environmental and economical sustainability of aquaculture operations References Azevedo P.A., Cho C.Y & Bureau D.P (1998) E¡ects of feeding level and water temperature on growth, nutrient and energy utilization and waste outputs of rainbow trout (Oncorhynchus mykiss) Aquatic Living Resources 11, 227^238 Azevedo P.A., Cho C.Y., Leeson S & Bureau D.P (2004) Growth, nitrogen, and energy utilization by four juvenile salmonid species: diet, species, and size e¡ects Aquaculture 234, 393^314 Azevedo P.A., Leeson S., Cho C.Y & Bureau D.P (2004) Growth and feed utilization of large size rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) reared in freshwater: diet and species e¡ects, and responses over time Aquaculture Nutrition 10, 401^411 Baruah K., Pal A.K., Sahu N.P., Jain K.K., Mukherjee S.C & Debnath D (2005) Dietary protein level, microbial phytase, citric acid and their interactions on bone mineralization of Labeo rohita (Hamilton) juveniles Aquaculture Research 36, 803^812 Berg O.K & Bremset G (1998) Seasonal changes in the body composition of young riverine Atlantic salmon and brown trout Journal of Fish Biology 52, 1272^1288 Boling S.D., Webel D.M., Mavromichalis I., Parsons C.M & Baker D.H (2000) The e¡ects of citric acid on phytatephosphorus utilization in young chicks and pigs Journal of Animal Science 78, 682^689 Bostr˛m B., Persson G & Broberg B (1988) Bioavailability of di¡erent phosphorus forms in freshwater systems Hydrobiologia 170, 133^155 Brinker A (2007) Guar gum in rainbow trout (Oncorhynchus mykiss) feed: the in£uence of quality and dose on stabilisation of faecal solids Aquaculture 267, 315^327 Brinker A (2008) Improving the mechanical characteristics of faecal waste in rainbow trout: the in£uence of ¢sh size and treatment with a non-starch polysaccharide (guar gum) Aquaculture Nutrition 15, 229^240 Brinker A (2009) Improving the mechanical characteristics of faecal waste in rainbow trout: the in£uence of ¢sh size and treatment with a non-starch polysaccharide (guar gum) Aquaculture Nutrition 15, 229^240 Brinker A., Koppe W & R˛sch R (2005a) Optimised e¥uent treatment by stabilised trout faeces Aquaculture 249, 125^144 Brinker A., Koppe W & R˛sch R (2005b) Optimizing trout farm e¥uent treatment by stabilizing trout feces: a ¢eld trial North American Journal of Aquaculture 67, 244^258 Bureau D.P & Cho C.Y (1999) Phosphorus utilization by rainbow trout (Oncorhynchus mykiss): estimation of dissolved phosphorus waste output Aquaculture 179, 127^140 Bureau D.P., Gunther S & Cho C.Y (2003) Chemical composition and preliminary theoretical estimates of waste outputs of rainbow trout reared on commercial cage culture operations in Ontario North American Journal of Aquaculture 65, 33^38 Camargo J.A & Alonso A (2006) Ecological and toxicological e¡ects of inorganic nitrogen pollution in aquatic ecosystems: a global assessment Environment International 32, 831^849 Carpenter S.R., Caraco N.F., Correll D.L., Howarth R.W., Sharpley A.N & Smith V.H (1998) Nonpoint pollution of surface waters with phosphorus and nitrogen Ecological Applications 8, 559^568 Cho C.Y & Bureau D.P (1997) Reduction of waste output from salmonid aquaculture through feeds and feeding The Progressive Fish-Culturist 59,155^160 Cho C.Y & Bureau D.P (1998) Development of bioenergetic models and the Fish-PrFEQ Aquatic Living Resources 11, 199^210 Cho C.Y & Woodward W.D (1989) Studies on the protein to energy ratio in diets for rainbow trout (Salmo gairdneri) In: Energy Metabolism of Farm Animals Proc 11th Symposium, EAPP Publication No 43 (ed byY van der Honing & W.H Close), pp 37^48 Pudoc, Wageningen, the Netherlands Cho C.Y., Hynes J.D.,Wood K.R & Yoshida H.K (1991) Quantitation of ¢sh culture wastes by biological (nutritional) and chemical (limnological) methods; the development of high nutrient dense (HND) diets In: Nutritional Strategies and Aquaculture Waste Proceedings of the 1st International Symposium on Nutritional Strategies in Management of AquacultureWaste (ed by C.B Cowey & C.Y Cho), pp.37^ 50 University of Guelph, Ontario, Canada Cho C.Y., Hynes J.D.,Wood K.R & Yoshida H.K (1994) Development of high nutrient-dense, low pollution diets and prediction of aquaculture wastes using biological approaches Aquaculture 124, 293^305 Davies I.M (2000) Waste production by farmed Atlantic salmon (Salmo salar) in Scotland International Council for the Exploration of the Sea, Annual Science Conference, CM 2000/O:01,12pp d’Orbcastel E.R., Blancheton J.P., Boujard T., Aubin J., Moutounet Y., Przybyla C & Belaud A 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