Diet

CHAPTER

Diet Douglas E Conklin Department of Animal Science, University of California, Davis, USA m m

Introduction Cultured fish are increasingly popular experimental animals (see Table 3.1). Part of the impetus behind this trend is the availability of a wide range of technological advances in fish husbandry arising from commercial aquaculture. Over the last decade, it has become more and more apparent that capture fisheries are nearing their productive limits. This has resulted in a growing shortfall of fisheries products to meet the demand of the burgeoning global population. The emerging field of aquaculture is seen as a means to increase supplies offish and shellfish even as natural harvests level off (New, 1997). In moving to produce ever-higher yields, aquaculturists have forged numerous improvements in fish culture technology including the development of effective formulated diets for several important food species. This chapter attempts to review what is known about fish nutrition as it applies to the development of formulated diets for the culture of fish specifically for laboratory research. Optimal nutrition is as important for sound research studies relating to the normal physiological and biochemical responses of fish as it would be for any other animal (Mehrle et al., 1977). While available information on nutritional needs for most species would not support the criterion of

Copyright 9 2000 AcademicPress

optimal nutrition, sufficient details are available to describe the general nutritional requirements of fish. Based on such information, diets can be formulated that support the culture of most species. Such diets may contain a number of nutrient excesses or produce less than optimal results but they support reasonable growth and survival and thus provide a starting point for refinements. As with all animals, the nutritional component of fish husbandry requires meeting at least two fundamental needs. Foremost is the requirement for a constant supply of energy. Energy liberated from the metabolism of organic compounds is essential to fuel the suite of biochemical processes maintaining biological organization and function. In addition, arrays of specific organic compounds as well as some inorganic elements are needed in the synthesis of the complex organic compounds. These compounds are used either for body maintenance or production of new tissue during growth and reproduction. A balance is required both in the array of required nutrients as well as the proportion of the total organic input devoted to energy use. Ideally this assortment of nutrients which must be moved through the aqueous environment should arrive to the fish relatively unchanged in composition and in a form that induces consumption. However, meeting this suite of objectives with formulated diets is quite difficult at present in that feed

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C o m m o n name

Genus and species

Common uses

Oncorhynchus spp.

Fisheries enhancement

Coldwater species Rainbow trout and Pacific salmon

Food production Atlantic salmon

Salmo salar

Food production

Warmwater species Channel catfish

Ictalurus punctatus

Food production

Tilapia

Tilapia spp. and Oreochromis spp.

Food production

Common carp

Cyprinus carpio

Food production Comparative biology

Gold fish

Carassius auratus

Neu roscience Ornamental culture

Killifish

Fundulus heteroclitus

Developmental biology

Fathead minnow

Pimephales promelas

Aquatic toxicology

Stickleback

Gasterosteus aculea

Aquatic toxicology

Guppy

Poecilia reticulata

Ornamental culture

Medaka

Oryzias latipes

Developmental biology Carcinogenicity assays

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Platyfish and swordtail

Carcinogenesis

Xiphophorus spp.

Ornamental culture

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Zebrafish

Danio rerio

Developmental biology

Electric eel and ray

Electrophorus electricus and Torpedo spp.

Neuroscience

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formulation technology is still being developed and data on nutrient needs of fishes are limited. In that sophisticated methods for accurately delivering nutrients to individual fish have yet to be developed, quantification of nutrient needs is quite coarse. The optimal dietary level of a nutrient is typically defined by the average growth of a group of fish on an 'as fed' basis. Under these conditions, nutrient losses during processing, storage, feeding and utilization are obscured. Another quandary is how to prevent intake of nutrients through routes other than the proffered ration. Feeding on available organisms from the aquatic environment or even direct uptake of nutrients, in the case of minerals, can also obscure actual requirements. Finally, in that the field of fish nutrition is relatively new, current information is not very robust. Often accepted insights into fish nutrition result from research with a single fish species or may sometimes be derived from nutritional studies with terrestrial animals and have yet to be actually tested in fish.

History Historically, fish were cultured using live or fresh food items. It was not until after the Second World War, that serious consideration was given to the production of formulated diets for use in various culture facilities, primarily trout hatcheries (Hardy, 1989). Trout were a fortunate species to start with because they hatched from large eggs and were ready to feed on prepared food immediately after consuming their supply of yolk. Ground meats were used for early trout diets with various nutritional supplements being added over time, as they were found necessary. Eventually, the ground meat portion was replaced with isolated protein sources and the supplemental meals were replaced with more refined feedstuffs. These improvements led to the advent of effective test diets to determine specific nutritional requirements of fish in the 1950s. A vitamin test diet used to quantify vitamin requirements for rainbow trout was first developed

in 1951 (Wolf, 1951). An improved vitamin test diet containing purified ingredients was published in 1953 (Halver, 1953) followed by an amino acid test diet towards the end of the decade (Halver, 1957). Trout not only readily accepted such test diets but as their husbandry requirements for cold well-oxygenated water could most easily be met by flow-through raceway culture systems, nutrient contamination from natural food sources was negligible. Following on the heels of the trout industry was the development of salmon culture (Pennell and Barton, 1996). Again, shaped by extensive research efforts to improve hatchery production, the farming of salmon in marine cages is now a highly developed aquaculture industry. Nutritional insights developed with various species of salmon have both extended and refined the earlier information gained with the related rainbow trout. In that trout and salmon have optimum temperature requirements in the range of 10-15~ this group is often referred to as 'coldwater' species. In contrast to coldwater species such as salmon, 'warmwater' fish are from the more temperate and tropical areas of the world. They are traditionally reared in freshwater ponds where temperatures in the range of 20-30~ promote not only rapid fish growth but also striking amounts of natural prey items. As a consequence, the need for complete nutritional information was not as critical for the aquaculture of warmwater species in that natural biota in the pond provided most or all of the food needed by the fish. Historically, methods of increasing production of warmwater pond species focused on enhancing the production of the total pond biota by using fertilizer to stimulate algal production at the base of the food chain. It was not until recently, as production density was increased beyond what could be supported by pond organisms, that the addition of supplementary feeds containing a broader and broader array of nutrients became obligatory (Hepher, 1988). As this trend continues and information on the nutritional needs of warmwater fish, such as catfish, carp, tilapia and others, continues to accumulate, formulation of pond feed is becoming less of an art and more of a science (see Wilson, 1991). For some species, such as the channel catfish grown in the southern United States, commercial formulated feeds come close to meeting all the nutritional needs of the species (Robinson, 1989). One other group of warmwater species receiving increasing industry attention is ornamental fish (Chapman et al., 1997). Currently the majority of

freshwater ornamental fish are produced in fertilized ponds. However, most marine ornamentals are collected from the wild. As with marine food fish, a combination of habitat destruction, pollution and overharvesting has led to declining natural populations of marine ornamentals. Development of appropriate culture techniques for marine ornamentals is of interest both to commercial aquaculturists and to conservation biologists (Andrews, 1990). As technologies are refined for culturing ornamental species, both freshwater and marine, development of suitable formulated diets will be a priority (Fernando et al., 1991; Earle, 1995; Kaiseretal., 1997). The fact that fishes are the most numerous ( ~ 21 500 species) of all vertebrate groups (almost half of all vertebrate species), and that detailed nutritional information is only available for a handful is not as daunting as it might seem at first sight (Figure 3.1). Unique feeding specializations are found among fishes, such as the species of the South American catfishes that feed on the gill filaments and blood of larger fish (Baskin et al., 1980). Fortunately, such peculiar adaptations are the exception so that the vast majority of fish can be adapted to feed on pellets or granules of an appropriate size. There also are fish species feeding at every trophic level - detritivore, herbivore, carnivore and omnivore - although most are either carnivorous or omnivorous. Fortunately for those wanting to culture the omnivores, they do not appear to have an absolute requirement of a varied input, in spite of being adapted to feed on an array of organisms in nature. Indeed they tend to thrive on similar formulations to that originally developed for the coldwater carnivorous species such as the rainbow trout even though it might not be theoretically optimum.

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Mammals 9%

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Birds 20%

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Repti 15 o, Amphibians 9% Figure 3,1 Fish as a percentage of total (~45 600) vertebrate species.

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Modified salmon diets have also been used for the culture of marine food fish, one of the newest areas of interest for commercial aquaculture. These species, such as yellowtail, red sea bream, turbot, etc., are typically reared at water temperatures above what is suitable for salmon and other coldwater species. Nevertheless, with some modifications primarily with respect to fatty acids, the older juveniles of these carnivorous species adapt quite well to these types of protein-rich diets. The biggest challenge has been in providing suitable feeds for the early stages of the marine species. The larval stages of many marine food species require restrictively fine particles at first feeding and thus these culture industries have been built around live food for the early stages of culture (Yoshimura et al., 1996). This is also true of many of the smaller warmwater species used as aquarium fish and more and more as laboratory animals (DeTolla et al., 1995; Westerfield, 1995; Kane et al., 1996; Rao et al., 1997). Early stages of these species do better on live feed organisms although once they have grown large enough to consume crumbles they can be switched to flake feeds. These commercial flake feeds intended for the home aquarist are formulated to be inclusive of any and all the nutrient needs identified for fish (Earle, 1995).

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Live foods for larvae and fry As indicated above, many small aquarium species as well as the larval stage of many larger marine fish species are either unable or reluctant to feed on prepared feeds. This may be from a combination of factors relating either directly to size of the particle, subtle organoleptic factors or the lack of characteristic prey movement (Kanazawa, 1995). For these species, a wide variety of live feeds have been examined for their usefulness. While live feeds generally give excellent results, in most cases, provision of live foods means the investigator has to become involved in the culture of the prey item in addition to the fish. This added effort and cost is the primary disadvantage of live feeds. There are also other potential disadvantages such as the possibility of variable nutritional planes and the possibility of introducing disease. Because their unique characteristics tend to ameliorate these

disadvantages, two particular live food items, brine shrimp nauplii and rotifers, tend to be preferred by culturists.

Brine shrimp One live food organism requiring minimal culture effort is the freshly hatched nauplii of brine shrimp Artemia spp. In nature, brine shrimp produce a cystencapsulated egg that is resistant to complete desiccation and death of the organism. These cysts are harvested, primarily from the Great Salt Lake in Utah, externally dried and packaged. The cysts will remain viable for a period of years but hatch readily within a day upon water immersion (Sorgeloos et al., 1977). Disease concerns can be minimized by first removing the bacterial contaminated cyst wall with chlorine bleach and then inducing hatching (Campton and Busack, 1989). Brine shrimp nauplii are less than a half a millimeter in size, strongly pigmented, and have a swimming movement that attracts first-feeding fish. They are used widely both in aquaculture and in the rearing of laboratory species (Sorgeloos, 1980). Often they are used in combination with a prepared feed of unknown efficacy to enhance the overall nutrition of the fish (Abi-Ayad and Kestemont, 1994). While attractive because of their 'off the shelf' attributes, nutritionally they can be lacking and, more importantly, for some fish larvae they are too large.

Rotifers Both a freshwater species (Lim and Wong, 1997) and several brackish-water species of rotifer are commonly available within the aquaculture industry (Hagiwara et al., 1997). Many fish and fish fry with a small mouth gape and which were previously fed on various protozoans do particularly well on rotifers, roughly half the size of brine shrimp nauplii. The euryhaline rotifer Brachionus plicatilis has proven to be essential for the effective commercial culture of marine fish larvae and is now cultured in large-scale intensive systems under carefully controlled conditions (Yoshimura et al., 1996; Lubzens et al., 1997). Related freshwater species also appears to be useful for the smaller freshwater aquarium species (Lim and Wong 1997; Chapman et al., 1998) and can be used for first feeding of zebrafish in lieu of protozoan prey (Westerfield, 1995). A disadvantage is that cysts are

not readily available and rotifer use entails the additional burden of culturing either algae or yeast in order to maintain the rotifer cultures (Fulks and Main, 1991).

Enrichment of zooplankton Experience has shown that freshly hatched brine shrimp nauplii and rotifers can be deficient in essential fatty acids particularly required by marine fish larvae (Rainuzzo et al., 1997). As a consequence, a great deal of effort has been made to develop methods to enhance the fatty acid profile of live foods for some of the long-chain unsaturated fatty acids. Feeding lipidenriched diets (Nanton and Castell, 1998) can significantly enhance fatty acid composition of zooplankton. For example, since Euglena gracilis is high in these essential fatty acids, it has been found to be a good supplement to feed to rotifers and A r t e m i a (Hayashi et al., 1993). The most widely used enrichment technique has been to first grow yeast in a medium containing fish oil in order to increase the percentage of desirable fatty acids in the yeast. Subsequent feeding of rotifers and brine shrimp nauplii on the enriched yeast results in a more nutritionally desirable prey item for larval fish (Furuita et al., 1996). Yeast is relatively easy to culture and the enrichment technique is now well established. Direct enrichment is also possible by manipulating the system so that the zooplankton organisms have lipid-filled guts when they are consumed by the fish larvae. Typically, emulsified fish oil droplets are added to the rotifer or brine shrimp culture medium and the zooplankton allowed to gorge on the droplets. The various techniques and strategies for presenting dietary polyunsaturated fatty acids to larval fish either through single cell organisms or through purified oils have recently been reviewed by several authors (Sargent et al., 1997; Coutteau and Sorgeloos, 1998). In addition to fatty acids, live feeds can be used to deliver other nutrients such as vitamins (Coutteau and Sorgeloos, 1998) or mendicants to larval fish (Robles et al., 1998). Another method of introducing dietary elements which are either limited or lacking all together in live prey is to practice co-feeding of larvae with a mixture of live and inert diets. This approach appears to be a promising mechanism to reduce the amount of the live diet needed (Canavate and Fernandez-Diaz 1999) as long as the fish larvae have not been habituated to only live feed (Dutton, 1992; Fernandez-Diaz et al., 1994).

Other live feeds While widely used, brine shrimp nauplii and rotifers are not a universal solution indicating there is still much to learn regarding live feeds. For example, for some marine fish species investigators have had better success with copepods than with brine shrimp (Shansudin et al., 1997). While it is still not completely clear why some species of fish larvae appear to do better on copepods than brine shrimp, it appears again to be associated with lipid requirements rather than size (Ronnestad et al., 1998). Copepod culture techniques are somewhat cumbersome (Uhlig, 1984; Stottrup and Norsker, 1997). In addition, many copepods have the further behavioral disadvantage of tending to stay on the culture vessel walls and thus not be available to the fish larvae. There are also fish larvae that strongly benefit from having algae in the culture media. Some species require algae as a direct food source for the first few days of endogenous feeding (Rodrigues and Hirayama, 1997) but for others the advantage conferred by the algal cells is not clear (Reitan et al., 1997). While such alternatives as copepods and necessary supplements as algae are of interest, it is likely that as the specific nutrient advantage of each is elucidated there will be an attempt to incorporate the factor into either brine shrimp nauplii or rotifers.

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Manufactured feeds

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While live feeds have proved necessary, ideally fish culturists would prefer manufactured feeds because of the greater control over nutrient quality and reduced labor costs. Leaching and consequent dissolution of vital nutrients into the aqueous environment has made this difficult. This not only results in nutrient deficiencies but also pollutes the enveloping aqueous environment. In many respects, the field is just beginning to deal with the problem of delivering quantifiable amounts of nutrients to animals. Typically, significant but unknown amounts of nutrients are lost to the aqueous environment before the targeted fish consumes the feed. Even under conditions where uneaten feed is negligible, leaching of soluble ingredients from pellets can be consequential (Goldblatt et al., 1979). Loss of water-soluble vitamins can be over 50% before trout,

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a fish that feeds relatively rapidly, snaps up a pellet (Slinger et al., 1979). Loss of soluble amino acids from diets makes it difficult to meet essential amino acid requirements of larval fish (Lopez-Alvarado and Kanazawa, 1994; Lopez-Alvarado et al., 1994). As the accurate assessment of the amount of food actually consumed by fish is a daunting task, most fish are fed 'to excess'. Uneaten residue then serves as substrate for a variety of microbial organisms. Small fish can gain appreciable nutrition from organic detritus (Lemke and Bowen, 1998).

Microencapsulated diets

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As feed particle size is reduced, the problem of leaching is greatly exacerbated. Potentially one could avoid the problem of leaching if the fine food particles could be encased with an impenetrable film. Unfortunately, conventional microencapsulation technology tends to use protein films, which do not prevent the movement of small water-soluble molecules. Consequently, most researchers feel that manufactured diets replacing live feeds should have a complex wall structure made up of several layers. A layer of lipid is probably a necessary component of the microcapsule wall in order to prevent leaching of water-soluble components (Lopez-Alvarado et al., 1994; Langdon and Buchal, 1998). This is a complicated research task involving defining nutrient requirements while trying at the same time to determine appropriate delivery systems. It is likely that the goal of developing effective artificial micro-feeds meeting the needs of fish larvae presently dependent on live food diets will remain elusive (Watanabe and Kiron 1994; Southgate and Partridge 1998).

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Purified and semipurifed feeds

type of diet used in nutritional studies with fish. While the ingredients are refined, complex organics rather than individual nutrients are used. For example, the amino acid in a semipurifed diet would be supplied through isolated proteins, such as casein and gelatin. Other typical ingredients would be corn starch as a carbohydrate source, corn or fish oil as a source of fatty acids along with some sort of binder such as gluten or agar. Vitamin and mineral mixtures are used, as is the case with purified diets. As indicated earlier, there are a number of such diets in the literature, which have been developed to study the nutritional requirements of various aquaculture species such as trout and catfish (National Research Council, 1993). Similar semipurifed diets can be made for the smaller laboratory fish species such as medaka (DeKoven et al., 1992) using the general insights gained with the well-known aquaculture species.

Practical and commercial feeds Practical feeds are manufactured from readily available ingredients that have a minimal amount of processing apart from drying and grinding. Commercial feeds for aquaculture which are also sometimes used in laboratory settings are of this type. Common feedstuffs making up practical diets might be fish and soybean meals, fish oils, ground wheat and corn. As with the other types of diets, mixtures of known vitamins and minerals are also added. Due to the limited definition of their contents, nutrient input when using these feeds is somewhat uncertain. On the other hand, commercial feeds typically are designed to have a margin of safety above what is minimally needed by the target fish species and are thus suitable for a number of uses in the laboratory (Ako, 1999).

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Ideally, in order to determine the exact nutrient requirements of animals, a purified diet would offer a researcher maximal control over the nutritional aspect of husbandry. Purified diets are made up of individual amino acids, simple sugars, individual fatty acids, and mixtures of required vitamins and minerals alongwith some sort of filler such as pure cellulose. In general, however, purified diets are not effective with fish and performance has been problematic. Both rapid leaching and poor palatability are probably to blame. Somewhat more effective for fish are semipurified diets.A semipurified diet is the most common

Energy- immediate nutritional needs As poikilothermic (cold-blooded) animals, the temperature of the culture water primarily determines dietary energy requirements for fish. Under laboratory conditions of constant temperature and only nominal activity, energy requirements of fish for

maintenance are much less that for terrestrial vertebrates because of the buoyancy of water. Key factors in determining the energy needs of fish are size and physiological state. These differences may not be noticed in laboratory culture where fish are typically fed to excess. Metabolism is higher in smaller fish than in larger ones. This is true both within a species, where the size difference is typically the result of age difference, and between different species with differing sizes at maturity. Energy is also required in processing food as well as in the synthesis of tissues. These are important elements for aquaculture where the goal is to achieve the maximum amount of growth with as little feed as possible. The details of energy utilization with regard to laboratory husbandry, however, are less important because while rapid growth may be desirable, efficient food conversion is less critical.

Tissue synthesis In nature, much of the energy used by fish comes from protein. As protein-rich feedstuffs tend to be more expensive, aquaculturists attempt to formulate diets so that energy for metabolism comes from lipid or carbohydrate. Conserving protein for use in growth is referred to as protein sparing and is important to the economic success of commercial aquaculture. As protein is economically important to commercial aquacuhurists, a great deal of effort has been devoted to defining the protein requirements of fish, such information being of interest to researchers involved with laboratory culture of fish. Efficient utilization of dietary protein will reduce ammonia production. This being the breakdown product of protein catabolism in fish. As ammonia and other nitrogenous metabolites arising from it are toxic to fish, these products have to be removed by dilution or biological filtration. Under these conditions, catabolism of excess protein will result in increased system maintenance requirements.

Protein Fish require the same array of essential amino acids common to other vertebrate species (Table 3.2). Quantitative requirements have been successfully based on mimicking the amino acid profile of fish muscle. As muscle makes up around 80% of the tissue

TABLE 3.2: Qualitative and quantitative amino acid requirements of fish Essential amino acid requirement

Non-essential

(g/lO0 g protein)

amino acids

Arginine 3.3-5.9

Alanine

Histidine I[3-2.1

Aspartic acid

Isoleucine 2.0-4.0

Cystine

Leucine 2.8-5.3

Glutamic acid

Lysine 4.1-6.1

Glycine

Methionine 2.2-6.5

Proline

Phenylalanine 5.0-6.5

Serine

Threonine 2.0-4.0

Tyrosine

Tryptophan 0.3-I .4 Valine 2.3-4.0

mass, the amino acid profile of fish muscle tissue provides a reasonable estimate of the ratio of essential amino acids that will be required for growth (van der Meer and Verdegem, 1996). There are however apparently some differences in other uses of essential amino acids. Thus, while these muscle amino acid patterns are quite similar among fish, comparisons of amino acid requirements based on growth tests suggest there are differences between salmon and carp (Akiyama et al., 1997). It is not yet known if these differences can be expanded to embrace the warmwater versus coldwater groupings, The protein requirement of fish is a reflection of the quantitative requirement for the 10 essential amino acids. The closer the balance of essential amino acids in the protein is to the balance required by the animal, the more efficient protein utilization, thus lowering the apparent requirement for protein, The apparent requirement for protein can also be influenced by other factors such as digestibility and palatability. Rapidly growing early juvenile fish do better on high protein (~ 50% of the diet) rations with the optimum level of protein declining as they grow. In general, coldwater species like salmon have higher requirements for protein ( ~ 40O/oof the diet) than warmwater species (~30% of the diet) (Lochmann and Phillips, 1994; Wilson, 1994a). Matching the necessary balance of essential amino acids for fish, in particular arginine, lysine and methionine, generally requires the use of animal protein. Typically this is fishmeal in practical diets and casein for purified diets, including those for the smaller ornamental species (Shim and Ng, 1988; DeKoven et al., 1992). While neutralized free amino acids can

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be used to examine requirements on an experimental basis (Rodehutscord et al., 1997; Twibell and Brown, 1997) supplementary amino acids are not used in practical diets. This is because protein-bound amino acids are utilized more efficiently than free amino acids (Ng et al., 1996). The better utilization of proteinbound amino acids is true even if leaching is taken into account (Zarate and Lovell, 1997).

Lipid and carbohydrate requirements

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Ideally carbohydrates and lipids serve as the primary energy sources in the diet offish in order to spare protein for growth. In contrast to mammals and birds, carbohydrates are not particularly useful as an energy source in fish. Lipids are ideal for fish in that stores can serve both as an energy reserve and as a buoyancy factor. The buoyant eggs of numerous marine fish species contain triglyceride-rich oil droplets in their eggs as the primary energy source (Heming and Buddington, 1988). Species of marine fish utilizing oil droplets are characterized by rapid development fueled by these triglycerides of the oil (Mourente and Vazquez, 1996). While carbohydrates are not essential for fish, they are a common constituent of the diet. Cheaper than proteins and fats, carbohydrates such as cornstarch are used in fish diets as a source of energy. Fish generally utilize complex carbohydrates such as starch more effectively than simple sugars but starches must be cooked to promote digestion. Coldwater and marine fish species are able to utilize up to around 20% of the diet as carbohydrates while warmwater pond species tend to be able to use 30-40% in the diet (Wilson, 1994b). Excessive amounts of digestible carbohydrates in the diet lead to an increase in mesentery fat deposits. Fish can effectively convert excess energy into storage lipids. Lipids, fatty acids and phospholipids are also vital elements of biological membranes. Fish are unable to synthesize the two specific fatty acids linoleic acid (18:2 n-6) 1 and linolenic acid (18:3 n-3) and thus require dietary sources (Kanazawa et al., 1Fattyacid nomenclature- shorthandconvention,in the example 18:2 n-6; the numberbeforethe colon indicatesthe numberof carbon atoms in the chain (18),the number followingthe colon indicates the number of double bonds (2), and the number following the 'n' indicates the position of the first double bond from the methylend (betweenthe sixth and seventhcarbon atoms).

1979). Each of these parent fatty acids, through elongation and desaturation, gives rise to some important fatty acids of each series. Linoleic acid gives rise to arachidonic acid (20:4 n-6) and linolenic acid give rise to eicosapentaenoic acid (20:5 n-3) and docosahexaenoic acid (22:6 n-3). Physiologically more significant than the respective parent fatty acid, these long-chain polyunsaturated fatty acids (PUFA) play a key role in maintaining the fluidity, flexibility and permeability of cellular membranes. They are also the precursors of the hormone-like compounds, eicosanoids. 3 Specific requirements for individual fatty acids is related to the ability of the species to desaturate and elongate them as well as the need to meet the challenge of such environmental variables as temperature and salinity (Sargent et al., 1989). All fish appear to require n-3 PUFAs of C18 chain length or greater. These fatty acids need to be provided in the diet at around 1-2%. Marine fish appear to lack the ability to elongate C18 fatty acids efficiently and thus growth and survival is enhanced with the addition of eicosapentaenoic acid (20:5 n-3) and/or docosahexaenoic acid (22:6 n-3). The requirement for dietary sources of these longer chain fatty acids is also seen in marine fish larvae (Sargent et al., 1997). Freshwater species do not seem to have this requirement for the C20 and C22 PUFA but may have additional requirements for n-6 PUFAs. In analyzing commonly used live or fresh foods used for maturation in freshwater ornamental fish Tamaru et al. (1997) suggested arachidonic acid (20:4 n-6) might be important in reproduction. The ability to modify the carcass composition of fish to enhance the content of PUFAs has been appealing because of their purported health effects in humans (Steffens, 1997). Bell and co-workers (1995) found that the carcass composition of PUFA could easily be enriched in turbot by feeding high levels of these essential fatty acids. It should be noted however that some peroxidation in these oils is difficult to prevent even with added antioxidants (Gonzalez et al., 1992) and thus some caution in use of high levels of PUFA is appropriate. Increased lipid levels in diets with the increased oxidative challenge often requires the addition of more vitamin E (Baker and Dvies, 1996). One effective way of adding fatty acids to the diet is through the addition of phospholipids. Dabrowski (1986) points out that fish eggs contain large amounts of phospholipids and thus it is possible that larval fish may have a requirement for phospholipids. A number of experiments have shown enhanced growth and survival when lecithin, a convenient phospholipid

source, is added to the diet of numerous fish particularly marine species (Kanazawa, 1993). Dietary phospholipids are beneficial in the diet of fish larvae in terms of survival, growth and resistance to stress (Coutteau et al., 1997; Kanazawa, 1997). The addition of lecithin was even found beneficial in a practical diet for juvenile goldfish (Lochmann and Brown, 1997).

Vitamins and minerals Most people are familiar with the dramatic impact that a lack of vitamins, and to some extent minerals, can have on animals. Determining the impact on fish has been arduous because of the aqueous environment and the permeability of the fish gill to ions. On the one hand, water-soluble vitamins in the feed can be lost before the fish engulfs it. On the other hand, fish can gain vitamins by feeding on organisms in the water or through intestinal synthesis. Minerals can also be gained from the water by uptake of ions across the gill membranes.

Vitamins Qualitative vitamin requirements for several fish species are known and mirror those seen in various warm-blooded vertebrates. Undoubtedly, this reflects the similarity of biochemical processes used by all vertebrates for intermediary metabolism and for the synthesis of specific compounds in the maintenance and production of tissue. While there are some differences, these typically relate to the ability or lack thereof to synthesize particular compounds rather than differences in the synthetic pathways themselves. Quantitative vitamin requirements are less well known and estimates can vary by an order of magnitude or more depending on the source of information. For example, contrast the two left-hand columns of Table 3.3, both of which relate to estimates of the vitamin needs of trout and the related species of salmon. Some of the reasons for these large differences most certainly relate to the relative newness of the field compounded by the inherent difficulty of measuring actual intake of water-soluble nutrients presented in an aqueous environment. But there are also a number of other factors involved, an understanding of which is useful to those considering developing a diet or selecting an existing formulation.

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Thiamin (mg kg -1)

10

1

1

0.5

Riboflavin (mg kg -1)

20

4

9

4

Niacin (mg kg -1)

150

10

14

28

Vitamin B6 (mg kg -I) Pantothenate (mg kg -I)

10 40

2 20

3 10-15

5-6 30-50

Biotin (rag kg -I)

I

0.1

R

I

Folacin (mg kg -I)

5

I

1.5

NR

> z

Vitamin B12 (rag kg -I) Ascorbic acid (mg kg i)

0.02 100

0.01 10

R 3

NR 5-6

oo

Choline (mg kg -I)

3000

500-4000

400

1500

Inositol (mg kg -~)

400

250

NR

440

Vitamin A (IU kg -I)

2500 IU

2500-5000 IU

1000-2000 IU

4000-20 000 IU

Vitamin E (IU or mgkg ~)

30 IU

28 IU

25mg

100mg

Vitamin D 3 (IU kg -~) Vitamin K (mg kg -I)

2400 IU 10

2400 IU 0.5

500-1000 IU R

not determined not determined

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As indicated above, there is clearly an element of refinement with time that is leading to better and better estimates of the actual requirements. Often early research studies covered relatively wide increments in order to make certain the experimental treatments adequately covered the potential range of deficiency to surplus. As noted, the species most often used in the United States during the early phase of research on fish nutrition were salmonids, either Pacific salmon or rainbow trout. Hatching from a relatively large egg, these species would accept formulated diets from first feeding onwards. Also, these fish quickly engulfed proffered feed enabling progress to be made in defining nutrient requirements even with diets that were not particularly water stable. A summary of this early work taken from the National Research Council's (1981) report on the nutrient requirements of salmonids is listed in the left-hand column of Table 3.3. In that these results were to provide general guidance with respect to fish nutrition, requirements were defined with caution so as to err on the side of excess. Such conservative estimates of need would provide a large margin of safety regardless of application. On the other hand, based on what was known from terrestrial vertebrates with regard to deleterious impacts with an excess of the fat-soluble vitamins A, D and E, estimates of the requirements for these vitamins were kept at a minimal level. In contrast to the first report (1981), a subsequent report (National Research Council, 1983) on the nutrition of warmwater species such as catfish and carp enumerated minimal requirements. These differences in approach have given rise to the belief that trout and salmon and by inference other carnivorous species have higher vitamin requirements than warmwater omnivorous fish species. Woodward (1994), however, has argued that there is little difference if a range of experimental evidence is examined and consideration is given to the poor water satiability of the classic purified experimental diet. The conclusions drawn by Woodward, based on his examination of the literature as to minimal dietary requirements, are shown in the second column of Table 3.3 and are quite close to those found for catfish and carp. This suggests that the dietary requirement for vitamins such as thiamin, riboflavin, niacin, vitamin 136,pantothenate, biotin, folate, and vitamin B12 are likely to be similar for most fish species. It is thought that the lack of response to removal of folic acid and vitamin B12 from the diet of carp reflects microbial synthesis in the gut rather than an actual species difference (Kashiwadaetal., 1970, 1971).

It should be noted, however, that many of the vitamin requirements were established using practical diets in which the feedstuffs contain additional sources of vitamins. Consequently, when making up semipurified diets Kaushik and co-authors (1998) found a modest increase (20-30%) was necessary for salmon and sea bass. Researchers wanting to incorporate vitamin mixtures into research diets to rear fish for laboratory experimentation should also use a similar safety margin.

Vitamin C While the majority of vitamins can be added in the form of a premix in order to meet established requirements, the case for vitamin C is a little more complicated. It appears that not all fish require vitamin C. A number of fish have the enzymatic capability to synthesize ascorbic acid (Yamamoto et al., 1978; Touhata et al., 1995). Both sturgeon (Moreau et al., 1996) and carp (Sato et al., 1978) apparently synthesize ascorbic acid at a rate sufficient to meet their nutritional needs. Thus it would appear that each species must be investigated as to its need for dietary ascorbic acid. There also may be a change with age. Contrary to the earlier findings for juvenile carp, GouillouCoustans and colleagues (1998) found dietary ascorbate was beneficial for carp larvae. As ascorbic acid is prone to rapid oxidation, most feed manufacturers now use one of the stabilized forms, either a phosphate or a sulfate derivative. Again there appear to be some important differences in the ability of various species to use both of these derivatives. While some species like tilapia (Abdelghany, 1996) can use either, others like the sea bass (Amerio et al., 1998) do best on the phosphate derivatives. Species such as trout can readily utilize the phosphate derivatives, converting ascorbyl-2-phosphate to ascorbic acid in rainbow trout (Miyasaki et al., 1992). Such species may also synthesize the phosphate form for tissue storage (Miyas aki et al., 1991 ).

Other vitamin-like factors L-Carnitine promotes the utilization of long chain fatty acids (Chatzifotis et al., 1995). Dietary L-carnitine heightened membrane impermeability to fluorescein in guppies (Schreiber et al., 1997). The possible dietary requirement for carnitine may be a result of other nutritional disorders. In that vitamin C is

involved in its synthesis, one might look first at defining the requirement for this known factor. A number of investigators have suggested a possible vitamin role for carotenoids in fish (Tacon, 1981; Torrissen and Christiansen, 1995), however, demonstrating an unambiguous effect has been more difficult. While some experiments have shown improved growth and survival in groups fed diets supplemented with carotenoids such as astaxanthin no convincing metabolic explanation has been established. Particularly when purified diets are being used, even the possibility that astaxanthin could act as a feeding attractant cannot be ruled out (Christiansen et al., 1994). While large amounts of pigments are deposited in the eggs of some fish such as salmon, Choubert et al. (1998) were unable to show any evidence of an effect of feeding carotenoid-supplemented diets to the female parent.

Minerals As fish can take up minerals from the water via the gills, it is difficult to show dietary requirements for fish - particularly with practical diets. An exception is phosphorus in that levels in waters tend to be low. Asgard and Shearer (1997) found that ~ 10 g phosphorus per kilogram of diet was required by juvenile Atlantic salmon Salmo salar. Dietary deficiency of phosphorus initially resulted in a reduction of calcium and phosphorus tissue levels particularly in the mineral-rich bones and scales (Baeverfjord et al., 1998). Development of abnormally soft bones resulted in scoliosis of the spine and a wrinkly appearance of the ribs. Eventually, growth was severely impaired and mortality rates elevated. Interestingly, as opposed to terrestrial animals, it appears that a high calcium to phosphorus ratio in the diet does not interfere with dietary phosphorus utilization (Vielma and Lall, 1998). It also appears that vitamin D has more minor effects on mineralization in fish than it does in terrestrial animals (O'Connell and Gatlin, 1994). Trace mineral requirements have been identified for iron, copper, manganese, zinc and selenium (Watanabe et al., 1997). Marine species can absorb most of their requirement for these trace minerals from ingested sea water. Even for freshwater fish, it is likely that under most conditions and certainly with practical diets supplementation with these elements is unnecessary although they are usually added for

assurance. In that in terrestrial animals a number of additional mineral requirements have been identified, it is likely that similar requirements for minerals exist for fish. These unidentified requirements are presently being supplied either through uptake from the culture water or by accidental dietary inclusion as a component of other feedstuffs. Mineral requirements cannot be discounted until studies have been carried out where both of these possible sources are controlled. The recent studies showing that boron in the water plays a role in stimulating embryonic growth of trout (Eckhert, 1998) and zebrafish (Rowe et al., 1998) are telling examples.

Summary Information on the nutrition of a few well-studied aquaculture species provides appropriate information on which to base diet formulation for other species of fish that are being used in the laboratory but there are several caveats. While studies are still quite limited, a number of differences as to requirements for specific nutrients in various species of fish have been established. This would suggest that further differences will be discovered as the informational base on fish nutrition is expanded. Also it should be remembered that the culture of food fish is typically carried under strikingly different husbandry conditions from those fish being reared in the laboratory. The impact of husbandry conditions on the nutritional plane needed for optimal husbandry has yet to be seriously investigated.

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References Abdelghany, A.E. (1996).J. WorldAquacult. $oc. 27, 449455. Abi-Ayad, A. and Kestemont, P. (1994). Aquaculture 128, 163-176. Akiyama, T., Oohara, I. and Yamamoto, T. (1997). Fish. Sci. 63,963-970. Ako, H. (1999). Internat. Aquafeed 8, 30-36. Amerio, M., Ruggi, C., Rovelli, R.M. and Volker, L. (1998).Aquaculture 159, 233-237. Andrews, C. (1990).J. FishBiol. 37 (Supplement A), 53-59. Asgard, T. and Shearer, K.D. (1997). Aquacult. Nutr. 3, 17-23.

Baeverfjord, G., Asgard, T. and Shearer, K.D. (1998). Aquacult. Nutr. 4, 1-11.

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