Aquaculture nutrition, tập 18, số 4, 2012

Aquaculture Nutrition 2012 18; 349–368 doi: 10.1111/j.1365-2095.2011.00925.x 1 Department of Biology, American University of Beirut, Beirut, Lebanon; Auburn University, Auburn, AL, USA Department of Fisheries and Allied Aquaculture, KEY WORDS: Cherax quadricarinatus, crayfish, diet, feed, nutrition, redclaw Redclaw crayfish (Cherax quadricarinatus, von Martens 1868) is a freshwater decapod crustacean with a number of biological and commercial attributes that make it an excellent aquaculture species The redclaw aquaculture industry has been growing rapidly since the mid-1980s in tropical and subtropical regions of the world Redclaw aquaculture is mostly in extensive pond systems, but interest in developing more intensive systems is increasing To support continued intensification, development of high-quality practical diet formulations and information about redclaw nutrition requirements are necessary A number of studies have determined optimum dietary protein and lipid requirements for juvenile redclaw However, there is limited information on essential amino acid and fatty acid requirements Several studies report the presence of various digestive enzymes that have been linked to the ability of the species to digest a wide range of dietary components Furthermore, as in many other aquaculture species, there is a need to replace fishmeal with other protein sources A number of studies explored the possibility of replacing fish meal with various terrestrial plant sources of protein and lipids and showed that redclaw can be offered diets containing low-cost, plant-based ingredients without compromising survival, growth and, to a certain extent, reproduction Formulated diets containing less expensive, plant-based ingredients will contribute to a more profitable and environmentally sustainable redclaw aquaculture industry Finally, there is also a paucity of information on vitamin and mineral requirements of redclaw and little information on nutrient requirements of broodstock For the redclaw aquaculture industry to thrive, we need to have a better understanding of nutrient requirements at all life stages ª 2012 Blackwell Publishing Ltd Received 13 May 2011; accepted December 2011 Correspondence: I Patrick Saoud, Department of Biology, American University of Beirut, Beirut, Lebanon E-mail: is08@aub.edu.lb Aquaculture of the Australian redclaw crayfish Cherax quadricarinatus (von Martens 1868) is developing rapidly in tropical and some temperate regions of the world Webster et al (2002) stated that aquaculture of the species was mainly restricted to North-Eastern Australia, but redclaw aquaculture has expanded into South-East Asia and Central/South America and production is no longer restricted to Oceania The species grows well when offered diets developed for other crustaceans, but nutritional requirement data specific for redclaw have not been determined As culture methodology shifts from extensive and semi-intensive ponds into more intensive systems and as hatchery production becomes more common, we will need to develop species-specific feed formulations (Huner et al 1994; Medley et al 1994; Webster et al 1994, 2002; Curtis & Jones 1995) These diets should be less expensive than traditional shrimp feeds, offer a complete nutrient profile to the animal, be based on sustainable sources of raw ingredients and be available wherever the industry decides to grow The present manuscript reviews known nutritional requirements of redclaw crayfish based on existing literature and the experience of the authors In natural ecosystems, crayfish have polytrophic feeding habits and have been described as predators, omnivores and/or detritivores (Momot et al 1978; D’Abramo & Robinson 1989; Jones 1990; Brown 1995a; Momot 1995; Nystro¨m 2002; Garza de Yta et al 2011), consuming a variety of macrophytes, benthic invertebrates, algae and detritus (Brown 1995a; Nystro¨m 2002) Jones (1990) suggested that in general Cherax species are primarily detritivores, a statement supported by the findings of LoyaJavellana et al (1993) who reported that C quadricarinatus demonstrates an ontogenetic shift from non-selective feeding on decayed plant material or zooplankton to a selective feeding on decayed plant material Additionally, Jones (1995) observed that juvenile C quadricarinatus grow better when feeding on fresh zooplankton than when offered formulated flake diets (400 g kgÀ1 protein) but in both cases grew better when diets were supplemented with vegetal material The feeding behaviour (omnivorous/detritivorous) of redclaw appears to allow for the incorporation of a broad range of animal- and plant-based ingredients into formulations of practical diets for aquaculture (Jones 1990; Campan˜a-Torres et al 2005, 2006, 2008; Pavasovic et al 2007a) Loya-Javellana et al (1994) described the ontogeny of redclaw foregut from embryonic stage to adult, while the embryonic development of the digestive system of was described by Meng et al (2001) The digestive system of decapod crustaceans, including redclaw, can be divided into foregut, midgut and hindgut (Ceccaldi 1997; Meng et al 2001) The foregut comprises the mouth (with associated mandibles), oesophagus and a large part of the cardiac stomach where the masticating parts are located The oesophagus is a short, straight vertical structure that connects the mouth and the stomach The cardiac stomach, an oval like sac, is dorsal in the cephalothorax and leads into the pyloric stomach (elliptically shaped), situated in a ventro-posterior position in relation to the cardiac stomach The hepatopancreas (or midgut gland), a large, bilateral, multilobate diverticulum of the midgut with a basic unit called a blind tubule, occupies most of the cephalothoracic cavity The hepatopancreas has diverse functions including synthesis and secretion of digestive enzymes, nutrient absorption, storage of minerals, lipids and glycogen, and distribution of stored reserves during the intermoult period (Brown 1995a; Ceccaldi 1997; Verri et al 2001) In most crustaceans, the digestive epithelium of the hepatopancreas is comprised of at least four different cell types: E, R, F and B, and in some crustaceans, an M-cell is found (Jacobs 1928; Gibson & Barker 1979; Ceccaldi 1997; Verri et al 2001) E-cells (embryonic) arise by mitotic division at the distal tips of the each hepatopancreatic tubule and differentiate giving rise to R-cells and F-cells (Dall & Moriarty 1983; Ceccaldi 1997; Verri et al 2001) R-cells have microvilli and also contain lipid droplets and glycogen, and their primary role is storage (Dall & Moriarty 1983; Ceccaldi 1997) F-cells (fibrillar cells), similar to R-cells, have microvilli that might contribute to absorption These cells secrete and synthesize digestive enzymes and differentiate into B-cells (Dall & Moriarty 1983; Ceccaldi 1997) B-cells (blister cells) are associated with protein synthesis and enzyme secretion (Verri et al 2001) Another type of cells found in some crustaceans is the M-cells (midget cells) that might be involved in nutrient absorption and storage (Ceccaldi 1997; Guillaume & Choubert 2001) The midgut, not lined by chitin, begins at the posterior end of the stomach and extends throughout the abdomen terminating at the anus The hindgut is almost straight and impregnated with chitin, enlarging posteriorly into the rectum and terminates at the anus (see Ceccaldi 1997) Loya-Javellana et al (1995) measured the effect of animal size and feeding frequency on the foregut evacuation rates of redclaw Results indicated that evacuation rates did not differ significantly between size groups (medium, large) nor between feeding frequency groups (fed daily, fed every second day) However, the model specifications differed between feeding frequencies, i.e ingesta was evacuated linearly with time in the crayfish fed daily and according to a curvilinear pattern in those fed every second day, implying that crayfish are potentially capable of regulating their digestive processes according to food availability Moreover, the return of appetite in redclaw is rapid; the average return of appetite increased to >50% of the satiation meal at 5–10 h postfeeding, when the residuum of the previous meal was ca 60% or less The authors reported that based on these results, redclaw can resume feeding before a considerable proportion of an earlier meal is processed in the foregut, suggesting that the species is capable of optimizing the frequency of feeding during active foraging periods A variety of digestive enzymes including proteases, lipases and carbohydrases are found in the midgut gland (hepatopancreas) and gastric fluid of crayfish (Zwilling & Neurath 1981; Brown 1995a; Hammer et al 2000) including redclaw (Figueiredo et al 2001) Digestive enzymes are synthesized and secreted into the digestive tract by F- and B-cells in the midgut gland (Ceccaldi 1997; Verri et al 2001) The presence of a variety of enzymes in juvenile red- Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd claw has been linked to the ability of the species to digest a wide range of dietary components (Xue et al 1999; Figueiredo et al 2001; Lo´pez-Lo´pez et al 2003, 2005; Pavasovic et al 2007a) This complex digestive enzyme activity is affected by ontogeny (Figueiredo & Anderson 2003), moulting (Ferna´ndez et al 1997; Vega-Villasante et al 1999; Perera et al 2008), diet composition (Lo´pez-Lo´pez et al 2005; Pavasovic et al 2007a), circadian rhythms, photoperiod and quality of light, temperature, stage of larval development, changes during vitellogenesis (Ceccaldi 1997), feeding habits and even habitat (Figueiredo & Anderson 2009) Proteases, enzymes responsible for hydrolysis of peptide bonds in protein, are present in the gut of crustaceans in general They include trypsin or a trypsin-like serine protease, astacin, chymotrypsin and exopeptidases [e.g carboxypeptidases (A and B)] and aminopeptidases (New 1976; Vogt et al 1989; Brown 1995a; Ceccaldi 1997; Guillaume 1997; Navarrete del Toro et al 2006; Figueiredo & Anderson 2009) However, it is generally accepted that most crustaceans lack pepsin and stomach acid (see Brown 1995a; Guillaume 1997; Navarrete del Toro et al 2006) Total protease [two optimal pH peaks: 5.0 and 7.5 (gastric fluid) and 4.0 and 7.0 (midgut gland)], trypsin-like enzyme (EC 3.4.21.4), chymotrypsin-like enzyme (EC 3.4.21.1), carboxypeptidase A-like enzyme (EC 3.4.12.2), carboxypeptidase B-like enzyme (EC 3.4.12.3) and low levels of leucine aminopeptidase-like enzyme (EC 3.4.11.1) (Figueiredo et al 2001) are all found in the gut of crayfish but might change in activity and concentration depending on age and diet Ontogenetic changes in C quadricarinatus cause total proteases, trypsin, leucine aminopeptidase and carboxypeptidases A and B to exhibit high activity in juveniles and to decrease as the species grows (Figueiredo & Anderson 2003) Lipases are hydrolases that operate at the interface of emulsified lipid substances (Vogt 2002) They break down carboxyl ester bonds of triacylglycerols liberating carboxylic acids and glycerol Figueiredo et al (2001) reported lipase (EC 3.1.1.3) activity only in gastric fluid of adult C quadricarinatus, whereas Lo´pez-Lo´pez et al (2003) observed esterase–lipase activity in the hepatopancreas of juvenile redclaw Although aquatic animals in general are not efficient at utilizing carbohydrates as energy sources, some of the omnivorous crustaceans exhibit some carbohydrate digestion capabilities Thus, some of the major carbohydrases (amylases, laminarinases, chitinases) are found in the digestive system of many crustaceans (Dall & Moriarty 1983; Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd Ceccaldi 1997) The activity of some of these carbohydrases is age dependant (Figueiredo & Anderson 2003) and change with developmental stages of redclaw For example, amylase and laminarinase activities are significantly greater in large C quadricarinatus than at other stages, whereas protease activities decreased as the species grew The carbohydrases detected in the midgut gland and gastric fluid of adult C quadricarinatus also include a-amylase (EC 3.2.1.1), laminarinase (EC 3.2.1.6/EC 3.2.1.19), maltase (EC 3.2.1.20) and several para-nitrophenyl glycosidases (Figueiredo et al 2001) Xylanase activity was also reported in the digestive system of redclaw crayfish (Xue 1998; Crawford et al 2005) The presence of these carbohydrases would suggest that redclaw should be able to obtain a substantial amount of their metabolic energy needs from carbohydrates, yet research suggests that only a relatively small portion of their energetic needs are obtained from carbohydrates (see Pavasovic et al 2006; Garza de Yta et al 2012) Additional work on carbohydrate digestibility and assimilation by redclaw is warranted before definitive statements can be made Some crustaceans have been reported to possess cellulases (EC 3.2.1.4) (Yokoe & Yasumasu 1964; Kristensen 1972; Brown 1995a; Xue et al 1999; Figueiredo & Anderson 2003, 2009) Cellulase activity is also present in all stages of growth in redclaw (Figueiredo & Anderson 2003), yet we have no definitive proof that redclaw can use cellulose nutritively Enzymatic hydrolysis of cellulose to glucose generally requires the synergistic action of three distinct classes of cellulase enzymes: endoglucanases (endo-1,4-bglucanases (EC 3.2.1.4) that cleave randomly internal b-1,4-glucosidic bonds; exoglucanases (exo-1,4-b-glucanases (EC 3.2.1.91) that cleave the disaccharide cellobiose from the non-reducing ends of the cellulose chains; and cellobiases (b-glucosidases, EC 3.2.1.21) that hydrolyse the cellobiose to glucose (Wood 1985; Walker & Wilson 1991; Woodward 1991; Be´guin & Aubert 1994) Generally, higher animals not produce endogenous cellulases, but the presence of symbiotic microorganisms in their alimentary tracts produces the necessary enzymes for cellulose digestion (Watanabe & Tokuda 2001) The occurrence of cellulase in the midgut gland and gastric fluid of redclaw (Byrne et al 1999; Xue et al 1999; Figueiredo et al 2001; Figueiredo & Anderson 2003; Crawford et al 2004; Pavasovic et al 2006) is very interesting Cellulose, the principal constituent of most plant cell walls, is known as the most abundant organic compound and renewable energy source on earth (Aspinall 1980; BeMiller 2008) Although the idea of using an abundant and low-cost ingredient in aquafeeds is exciting (Byrne et al 1999; Crawford et al 2004; Pavasovic et al 2007a), we believe that technological advances required to make cellulose a dietary energy source for aquatic organisms are yet to be described and might never be Regardless, we will review current literature on the subject The catalytic activity of cellulase in redclaw digestive tracts is not markedly inhibited by antibiotic treatment, despite a significant decrease in the gut bacterial populations (up to 94%), suggesting that the activity is innate in the crayfish and not in microbial symbionts Redclaw cellulase enzymes demonstrated broad substrate specificity, hydrolysing polysaccharides containing b-1,4 and mixed b-1,4 and b-1,3 glycosidic bonds but with a higher preference for soluble substrates (Xue et al 1999) The occurrence and activity of cellulase in C quadricarinatus is consistent with the feeding behaviour of redclaw, which consume significant amounts of plant materials and decomposing bacteria, fungi and animals (Byrne et al 1999; Xue et al 1999) Byrne et al (1999) isolated an endo-1,4-betaglucanase cDNA sequence (termed CqEG) from the hepatopancreas of redclaw, thus providing one of the first endogenous cellulase sequences in crustaceans Crawford et al (2004) complemented the study conducted by Byrne et al (1999) by presenting the genomic organization of CqEG According to the authors, the presence of an endogenous multigene glycosyl hydrolase family in redclaw indicates that partial breakdown of plant cell polysaccharides is a significant evolutionary strategy for the species Results of their study suggested the presence of two functional endoglucanase enzymes in redclaw that may be used to obtain energy (glucose) from soluble cellulose (see also Xue et al 1999), a tool to allow access to other nutrients within plant cells (Be´guin & Aubert 1994) or to reduce digestive viscosity of soluble polysaccharides leached from plant cell walls (Crawford et al 2004) Crawford et al (2005) reported that C quadricarinatus has the capacity to liberate glucose from carboxymethyl cellulose, indicating that cellulose substrates can be a source of energy for crayfish However, a study conducted by Pavasovic et al (2006) indicated that the presence of cellulase (higher activity in gastric fluid than midgut gland) in the gut of redclaw is unlikely to hydrolyse a-cellulose into glucose and thus would not allow for the supply of energy to the species Furthermore, the addition of a-cellulose to midgut gland extracts did not change solution viscosity, suggesting that insoluble non-starch polysaccharides not increase viscosity of intestinal contents upon digestion, which in turn would slow the passage of materials through the gut (Pavasovic et al 2006) The authors concluded that although cellulase activity is present in redclaw, there are no detectable nutritive benefits of including insoluble cellulose (a-cellulose) in diet formulations of the species In addition to proteases, lipases and carbohydrases, endonucleases probably also exist in redclaw Endonuclease activity has been reported in the digestive tract of various other invertebrates including annelids, molluscs, echinoderms and arthropods (chelicerates, insects and crustaceans) (Yokoe & Yasumasu 1964; see also Watanabe & Tokuda 2001 and references therein; Linton et al 2006) that are also probably members of the arsenal of digestive enzymes in redclaw guts, but have yet to be isolated Information derived from studies on biochemical composition and digestive enzyme activities on utilization of yolk during embryonic development may provide some clues of the nutrient requirements for the embryos and therefore can be used in understanding nutritional requirements of brood stock (Yao et al 2006; Luo et al 2008a) Luo et al (2008a) studied five digestive enzymes (trypsin, pepsin, lipase, amylase and cellulase) in embryonic redclaw, and all showed changes in enzymatic activity closely correlated with morphogenesis, hydrolysing the yolk and providing construction substances and energy resources for formation of tissues, organs and various systems The activities of the digestive enzymes were controlled by their genes and expressed sequentially during development Specific activities of pepsin and trypsin increased during early stages of embryonic development, but pepsin activity decreased in later stages (stage VI), while trypsin remained at high level of activity (Luo et al 2008b) Furthermore, chymotrypsin activity peaked in stage IV and then decreased significantly during the last stage of embryonic development Low levels of lipase activity were also reported during embryonic development of redclaw (Luo et al 2008a) Specific activity of amylase changed in a ‘V’ curve, increasing during later stages (stage VI) Cellulase activity during embryonic development in redclaw was relatively low (Luo et al 2008a) Research on the nutritional requirements and practical diet formulations for redclaw increased rapidly as the culture of the species became established with further advances occurring in the 21st century Dietary requirements of some nutrients have been determined for rapidly growing juveniles only, with limited information for larger redclaw approaching market weight or for broodstock This is Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd probably because most broodstock are collected from extensively stocked farm ponds where the animals have access to primary productivity to supplement possible deficiencies in manufactured diets Currently, diets for the commercial production of redclaw are based on formulations of other aquatic species, primarily penaeid shrimp feed but sometimes prawn and fish feed (Corte´s-Jacinto et al 2003, 2004, 2005; Garcı´ a-Ulloa et al 2003; Thompson et al 2003a,b) Redclaw have the capacity to adapt their digestive physiology in response to changes in their nutrient requirement or dietary profile (Pavasovic et al 2007b) and consequently have been reared on a wide range of feed formulations Redclaw diets could potentially be quite inexpensive to manufacture, considering that formulated diets with 200–300 g kgÀ1 crude protein and 50– 100 g kgÀ1 lipids, based primarily on vegetable rather than animal ingredients, allow for good survival and growth of the species (Corte´s-Jacinto et al 2004) Proteins and amino acids are essential nutrients required for maintenance, growth and reproduction in crustaceans as in other animals (Guillaume 1997) Protein requirements of crustaceans are affected by various factors including physiological stage and size, dietary characteristics of protein quantity and quality (e.g digestibility), amount of nonprotein energy in the feed, environmental factors (e.g temperature) and methodology used for dietary protein determination (D’Abramo & Robinson 1989; D’Abramo & Sheen 1994; Guillaume 1997; Thompson et al 2005, 2006; Rodrı´ guez-Gonza´lez et al 2006a) In general, a mixture of proteins of both animal and plant origin provide better growth than either alone because the mixture often contains a complementary blend of amino acids, which are more likely to meet or exceed the requirements (D’Abramo & Robinson 1989; Lovell 1998) Most crayfish exhibit an ontogenetic diet shift where adult crayfish incorporate greater levels of detritus and plants in their diet as compared to juvenile crayfish that feed mostly on invertebrates (Mason 1975; Loya-Javellana et al 1993; Lodge & Hill 1994; Momot 1995; Nystro¨m 2002) Such differences in feeding habits between adult and juvenile crayfish have been attributed to slower growth of adult crayfish and therefore lower protein requirements than in faster growing juveniles (Lodge & Hill 1994) Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd Several studies have attempted to determine protein requirements of juvenile and preadult C quadricarinatus reared indoors or outdoors (see Table 1) Anson & Rouse (1996) evaluated growth response and survival of newly detached (0.01 g) redclaw offered various commercial feeds (shrimp feed, catfish feed with or without Artemia nauplii supplement) ranging in protein content from a 320 g kgÀ1 protein catfish diet to a 400 g kgÀ1 shrimp diet The 400 g kgÀ1 shrimp diet resulted in best growth for the animals D’Agaro et al (2001) evaluated the dietary protein content (240 g kgÀ1 and 290 g kgÀ1; gross energy: 20.0– 20.4 MJ kgÀ1) on growth performance of juvenile C quadricarinatus reared in a recirculating system No significant differences in growth were reported among treatments, probably because of protein-sparing effects from other energy sources Meade & Watts (1995) offered 0.01 g redclaw a number of commercially available formulated diets and found that a 300 g kgÀ1 crude protein, 100 g kgÀ1 fat feed provided best weight gain and survival as compared to all other treatments However, the authors note that such feeds not provide complete nutritional needs of crayfish Jones & Ruscoe (1996a) evaluated growth performance of juvenile redclaw in glass aquaria offered five formulated diets (four commercial formulations and one experimental reference formulation) and one natural diet containing crude protein ranging from 100–447 g kgÀ1 Growth was significantly greater in trials offered diets containing 365 g kgÀ1 protein (with fish meal as protein source) and 205 g kgÀ1 crude protein (entirely of non-animal material) The authors concluded that redclaw does not seem to have a specific requirement for high levels of proteins and that they can be successfully cultured on a diet primarily composed of material of plant origin Similarly, Thompson et al (2005) examined the growth performance of juvenile redclaw offered formulated practical diets containing increasing percentages of dietary protein (300, 350 and 400 g kgÀ1) They found that juvenile redclaw can be offered a 350 g kgÀ1 protein formulated practical diet with a combination of plant-protein ingredients if fishmeal is excluded Natural food and forage can also supplement formulated diets and spare proteins in the prepared feed Metts et al (2007) reported that juvenile redclaw stocked semi-intensively and offered forage at a rate of 500 kg haÀ1 monthÀ1 may be able to utilize 130 g kgÀ1 protein diets Thompson et al (2006) reported that juvenile redclaw offered diets containing 280 g kgÀ1 crude protein with or without fish meal had significantly greater weight gain compared to redclaw offered 180 g kgÀ1 crude protein with or without fish Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd 0.022 0.02 9.7 0.2 and 8.52 0.07 3.22 1.08 Webster et al (1994) Keefe & Rouse (1999) D’Agaro et al (2001) Hernandez et al (2001) Manomaitis (2001) 1.04 Corte´s-Jacinto et al (2005) Corte´s-Jacinto et al (2009) 4.6 1.12 5.75 6.25 13.9 0.71 Thompson et al (2005) Thompson et al (2006) Metts et al (2007) Pavasovic et al (2007b) Zenteno-Savı´n et al (2008) 25.5 females Dı´az et al (2006) Rodrı´guez-Gonza´lez et al (2006a) Rodrı´guez-Gonza´lez et al (2009a) Thompson et al (2004) 1–2 23.0 21.8: females 23.1: males 0.71 Corte´s-Jacinto et al (2004) Corte´s-Jacinto et al (2003) Initial size (g) Reference 130–320 260–360 Same as Corte´s-Jacinto et al (2003) diets 130–280 180–280 300–400 220–420 220–450 320–350 220–370 280–400 260–360 220–450 240–294 250–500 250–400 250–460 200–550 230–430 230–550 Protein levels tested Mixture: Fish and SBMs and wheat middlings Anchovy fish meal Mixture: Menhaden fish meal (67%), SBM (46%), shrimp meal (44%) and wheat flour (11%) Mixture: Sardine, sorghum, soybean, red crab, squid meals, wheat meal and grenetine Mixture: Sardine, sorghum, soybean, red crab, squid meals, wheat meal and grenetine Mixture: Sardine, sorghum, soybean, squid meals, wheat meal and grenetine Mixture: Sardine, sorghum, soybean, red crab, squid meals, wheat meal and grenetine Rangen and Purina Mixture: Sardine, sorghum, soybean, and squid meals, wheat meal and grenetine Mixture: Sardine, sorghum, wheat, squid, red crab meals, soybean paste and grenetine Mixture: Menhaden fish meal (67%), SBM (50%), Brewer’s grains with yeast (35%), wheat gluten (41%) and wheat flour (14%) Mixture: Anchovy fish (65%) meal, SBM (48%), wheat flour (12.5%, milo (11.5%), BGY (35%) and wheat gluten (80%) Mixture: Menhaden fish meal (62%), SBM (48%), distillers’ grains with solubles (28%), milo (10%) and wheat gluten (72%) Mixture: Full-fat SBM, solvent-extracted SBM, wheat midds and whole wheat + alfalfa hay Fish meal, gelatin Mixture: Menhaden fish meal, soybean meal (SBM), shrimp head meal and ground corn Mixture: Corn, fish and SBMs Protein source Table Optimal dietary protein level for redclaw (Cherax quadricarinatus) 310 130 (with or without alfalfa hay) 250 280 (0 g kgÀ1 fish meal) 300 (150 g kgÀ1 fish meal) 350 (0 g kgÀ1 fish meal) 330 (recommended) 284–355 220 (recommended) 350 320 (calculated: 300) 350 430 280 (calculated) 240 (suggested) 300–350 400 300 (suggested) 310 342 (calculated) 220 256 (calculated) 310 330 (recommended) Optimal protein level (g kgÀ1 of diet) Individual cages within tanks, recirculating system Static experimental tanks Ponds Ponds Tanks, recirculating system Ponds Tanks, static system Recirculating system Tanks, static system Static experimental tanks Static experimental tanks Static experimental tanks Static experimental system Individual containers, recirculating system Tanks, recirculating system Tanks, recirculating system Semi-recirculating system Aquaria, recirculating system Culture type meal (778% and 799%, respectively) They concluded that pond-cultured redclaw performed well when offered diets with 280 g kgÀ1 protein inclusion even if devoid of fishmeal Hernandez et al (2001) studied the effect of eighteen isocaloric (417.4–422.8 kcal 100 gÀ1) diets containing six levels of protein inclusion (250, 300, 350, 400, 450 and 500 g kgÀ1) each at lipid levels of 40, 80 and 120 g kgÀ1, on growth and survival of hatchling and juvenile redclaw reared under controlled conditions The authors concluded that diets containing 300–350 g kgÀ1 protein (40–80 g kgÀ1 lipid) result in best growth performance for both size classes Manomaitis (2001) offered juvenile redclaw diets with various protein inclusion levels (250–400%) for weeks Final weight, specific growth rate (SGR) and percentage weight gain of the juveniles were positively correlated with increasing protein levels in the diet However, a second similar trial with larger juveniles resulted in no effect of dietary protein level on all test factors The author concluded that a diet of at least 400 g kgÀ1 crude protein should be offered to newly released redclaw, whereas for larger juveniles, a diet containing 300 g kgÀ1 protein is sufficient Corte´s-Jacinto et al (2003) evaluated the response of juvenile redclaw offered experimental diets containing seven levels (200, 250, 310, 370, 430, 490 and 550 g kgÀ1) of dietary protein and with 18.73–21.45 kJ gÀ1 gross energy (protein to energy ratio: 10.7–25.6 mg kJÀ1) Results showed that highest mean weight (9.6 g) and SGR (3.64% dayÀ1) were achieved by offering a diet containing 310 g kgÀ1 crude protein The optimum dietary protein requirement, calculated from using a second-order polynomial (y = 1.142 + 0.484 À 0.0071x2, r2 = 0.952), was 342 g kgÀ1 Similar results were achieved by a later study conducted by Corte´sJacinto et al (2005) determining the effect of various protein (260, 310 and 360 g kgÀ1) and lipid (40, 80 and 120 g kgÀ1) levels, with gross energy content of 17.5–19.4 kJ gÀ1, on growth of juvenile C quadricarinatus Best growth was observed when using dietary protein inclusion of 310 g kgÀ1 (80 g kgÀ1 crude lipid) with gross dietary energy content of 19.69 kJ gÀ1 Similar results were observed by Dı´ az et al (2006) Dietary protein also appears to have an effect on redclaw health Zenteno-Savı´ n et al (2008) reported that diets containing 310 g kgÀ1 crude protein satisfy nutritional requirements for optimal growth, while preventing diet-induced oxidative stress and protecting the integrity of the immune response in juvenile redclaw Similarly, Corte´s-Jacinto et al (2009) reported that a 350 g kgÀ1 protein diet stimulates antioxidant response of superoxide dismutase (SOD) (SOD Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd is a cytosolic enzyme specific for scavenging superoxide radicals and is involved in protective mechanisms within injured tissues following oxidative processes and phagocytosis) of juvenile redclaw For earthen pond culture, it is not necessary to supply high dietary protein because redclaw supposedly obtain a substantial proportion of their nutrient requirements from natural food materials in the pond (Jones 1990; Jones & Ruscoe 1996b) Jones & Ruscoe (1996b) stocked juvenile redclaw in cages in a pond and offered diets containing crude protein ranging from 100 to 447 g kgÀ1 Although crayfish offered a reference crayfish diet (205 g kgÀ1 crude protein) grew better than crayfish offered all other diets, the authors suggested that the crayfish did not have a direct use of the feed offered but obtained the bulk of their nutrition from natural productivity of the pond benthos In a similar experiment, Thompson et al (2004) found that 220 g kgÀ1 dietary protein was sufficient for redclaw culture In other experiments, Pavasovic et al (2007b) reported maximum growth of subadult redclaw offered diets containing 250 g kgÀ1 crude protein with a strong positive correlation between dietary protein and protein content in the tail However, other researchers did not observe a significant effect of dietary protein on percentage protein in redclaw tail muscle or even total body protein (Muzinic et al 2004; Thompson et al 2004) A summary of the literature thus suggests that diets with 250 g kgÀ1 or greater protein inclusion are suitable for redclaw growout in ponds with natural productivity Diets with 350 g kgÀ1 protein inclusion or greater are recommended for redclaw grown in closed recirculation systems All diets should have a gross energy content of 18 kJ gÀ1, minimum These suggestions are supported by Corte´s-Jacinto et al (2004) who propose a minimum protein inclusion in redclaw diets of 220 g kgÀ1 with 15.21 kJ gÀ1 of digestible energy No discussion of aquatic animal nutrition is complete without mentioning broodstock diets Broodstock nutrition is of high importance for successful reproduction and egg quality; adequate nutrients and energy in broodstock diets are necessary for the onset of gonadal maturation, because maternal nutrient intake during ovarian development is critical and influences the composition of ovaries and the nutritional status of eggs Crustacean embryos rely exclusively on the nutrients and energy supplied by the egg (yolk) (Harrison 1997) In decapod crustaceans, protein is a structural, functional and energy constituent of tissues and plays an important role in spawning, fertilization and normal development of embryos (Harrison 1990; Wouters et al 2001; Garcı´ a-Guerrero et al 2003; Rodrı´ guez-Gonza´lez et al 2006a) Asgari (2004) reported that based on spawning rate, fecundity, hatchability and egg size, a diet containing 400–450 g kgÀ1 crude protein and 16.72 kJ gÀ1 energy is optimal for redclaw crayfish broodstock, yet Rodrı´ guez-Gonza´lez et al (2006a) tested diets with lesser protein inclusion and found no differences in survival, final weight and fecundity of female broodstock However, regression analysis indicated that maximum spawning was from females offered a 300 g kgÀ1 crude protein diet and dietary protein levels of 320 g kgÀ1 had a significant effect on egg quality but not on biochemical composition of the eggs Such findings were recently corroborated by Li et al (2010) who found that using diets with higher protein content improves redclaw female spawning, especially when gonadosomatic index is >1.6 Rodrı´ guez-Gonza´lez et al (2009a) separated maturation from gonadal development as they relate to female broodstock diets and found that diets with 220–450 g kgÀ1 crude protein result in maturation of female redclaw, but a dietary protein range from 284 to 355 g kgÀ1 improved gonadal development and resulted in more protein production in the hepatopancreas In previous work, Rodrı´ guez-Gonza´lez et al (2006b) had observed that external sources of protein and energy were vital for nutrient accumulation in the gonad Additionally, protein contents in the gonad were correlated with gonadosomatic index; at mature stages, higher protein concentration was observed These gonadal proteins were a result of an active mobilization of energy reserves from exogenous sources, incorporated into the oocytes by endocytosis (Abdu et al 2000) Based on current knowledge, we suggest that broodstock females be offered diets with 350 g kgÀ1 protein and a minimum of 18 kJ gÀ1 gross energy, a part of which comes from fish oil to supply the necessary omega-3 HUFAs Determination of the exact amino acid requirements in crustaceans is difficult (Shiau 1998), and this is probably the reason for the paucity of reports on the specific amino acid requirements of redclaw In general, the essential amino acid requirements for most crustaceans include arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine (D’Abramo & Robinson 1989; Brown 1995b; Guillaume 1997) plus asparagine for crayfish (Brown 1995b) Tyrosine and cysteine are considered semi-essential in the diet as they potentially spare the requirement of phenylalanine and methionine, respectively (Guillaume 1997) There is a significant correlation between the dietary amino acid requirements of a species and the pattern of amino acids in whole body tissue (Cowey & Tacon 1983; Wilson & Poe 1985) Consequently, dietary amino acid requirements of growing animals are often assumed to be similar to the amino acid composition of the tissue proteins formed during growth Mitchell (1950) suggested that an animal’s amino acid requirements might first be deduced from the amino acid composition of its tissues However, our experience suggests that when using body composition as reference of requirement, one would overestimate dietary requirement of essential amino acids and underestimate requirement of other protein components Muzinic et al (2004) evaluated the amino acid composition of practical diets containing various levels of soybean meal (SBM) and brewer’s grains with yeast as replacements for fish meal, and results suggested that the amino acid levels in a 400 g kgÀ1 crude protein diet were adequate for good growth and survival of juvenile redclaw crayfish whichever protein source was used Similarly, Thompson et al (2005) noted that a complementary blend of SBM and other plantprotein sources used to replace FM in a 350 g kgÀ1 protein diet appeared to provide sufficient levels of essential amino acids to meet requirements of redclaw In pond-cultured redclaw, diets containing 280 g kgÀ1 crude protein with or without fish meal may sufficiently satisfy the requirements of essential amino acids of male and female redclaw (Thompson et al 2006) probably because natural productivity supplements the formulated feeds being offered Consequently, and based on their response to diets without fishmeal, one may assume that methionine and lysine requirements of redclaw are relatively low Such assumptions are yet to be empirically tested Knowledge of energetic utilization of farmed organisms is necessary for the development of cost-effective diets Energy from non-protein sources (lipids, carbohydrates) relative to protein levels must be supplied into diets in sufficient amounts to insure that protein is used for tissue synthesis as protein is considered the most expensive major component of crustacean diets (D’Abramo & Robinson 1989; Cuzon & Guillaume et al 1997; Cho et al 2005) If the non-protein energy to protein ratio is insufficient, dietary protein may be catabolized and used as an energy source to satisfy maintenance before somatic growth Con- Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd versely, if dietary energy to protein ratio is in excess, feed consumption may be reduced, resulting in a decrease in protein intake and other essential nutrients required for maximum growth Excessively high ratios of energy to nutrients can also lead to deposition of large amounts of body fat (Cuzon & Guillaume 1997) A review of various published results concerning dietary protein to energy requirements of redclaw suggests that optimal growth is obtained when the animals are offered feed with a protein to energy content between 16 and 20 mg kJÀ1 and a crude protein content between 310 and 350 g kgÀ1 of the diet by weight (D’Agaro et al 2001; Corte´s-Jacinto et al 2003, 2005, 2009) D’Agaro et al (2001) found no significant differences in growth of redclaw when offered diets containing protein to energy ratio of 50 and 60 mg kcalÀ1 (240 and 294 g kgÀ1 protein, respectively) and attributed their results to the protein-sparing capacity of the energy in the diets This protein-sparing effect was also observed by Hernandez et al (2001) Values for protein to energy ratio for optimal reproductive activity and gonadal development in redclaw were 18 ± mg kJÀ1 (Rodrı´ guez-Gonza´lez et al 2011) and 17.16 mg kJÀ1 (Rodrı´ guez-Gonza´lez et al 2006a), respectively, within the range observed as necessary for juvenile growth Dietary lipids play an important role in crustacean nutrition as they provide energy and essential fatty acids (EFAs), sterols, phospholipids and fat-soluble vitamins necessary for proper functioning of physiological processes and maintenance of biological structure and function of cell membranes (D’Abramo & Robinson 1989; Sargent et al 1989; D’Abramo 1997; Teshima 1997) Lipid used as energy source can also spare dietary proteins and reduce nitrogenous waste production (D’Abramo & Robinson 1989; Lim & Sessa 1995; Cho & Bureau 2001) However, high dietary lipid levels can cause significant reductions in growth rate, feed consumption and also might reduce the utilization of other nutrients resulting in reduced growth (D’Abramo 1997) Additionally, an increase in dietary lipid levels was linked to increases in the lipid content of midgut glands (hepatopancreas) (D’Abramo 1997) In general, nutritional studies with crustaceans indicate that lipid content of formulated diets should range between 50 and 80 g kgÀ1 of feed by weight to ensure optimal growth and survival (D’Abramo 1997) The lipid level Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd required for optimal growth is influenced by several factors including quality and quantity of protein, availability, quantity and quality of other sources of energy and adequate provision of EFAs (D’Abramo 1997) as well as the ability of the organism to digest carbohydrates and use glucose in its metabolism Lipids are often supplemented in excess of minimal requirements to spare protein for somatic growth Such a protein-sparing effect of lipids was reported in hatchling and juvenile redclaw offered diets containing 40–80 g kgÀ1 lipid (300–350 g kgÀ1 protein) (Hernandez et al 2001), suggesting that this range of lipid inclusion to redclaw diets is suitable A few studies investigated dietary lipid requirements of redclaw under laboratory conditions (Hernandez et al 2001; Corte´s-Jacinto et al 2005; Zenteno-Savı´ n et al 2008), and all seem to agree that a diet containing 80 g kgÀ1 dietary lipid with approximately 300 g kgÀ1 protein and gross energy 17.5–19.1 kJ gÀ1 is suitable for good growth performance of juvenile C quadricarinatus while preventing dietinduced oxidative stress and protecting the integrity of the immune function Although lipids are necessary in redclaw diets, it appears that natural productivity can replace dietary lipids to a certain extent Herna´ndez-Vergara et al (2003) evaluated the effect of different dietary lipid levels (42, 82 and 123 g kgÀ1) on growth, survival and proximate composition of juvenile redclaw reared semi-intensively in outdoors tanks and observed no effect of treatment on the various parameters Accordingly, it seems that in the presence of some natural productivity, a diet containing 42 g kgÀ1 dietary lipid (17.58 kJ gÀ1, 300 g kgÀ1 crude protein) is sufficient for growth and survival of juvenile redclaw Differences in lipid metabolic routes between sexes where females have higher carcass lipid content than males are often reported (e.g Herna´ndez-Vergara et al 2003) This is generally attributed to storage of lipids for ova development or vitellogenesis Yet, studies on developing adequate diets for maturation of broodstock redclaw are rare (Rodrı´ guez-Gonza´lez et al 2006a,b, 2009a,b) Considering that lipids are the main energy sources during ontogeny of crustaceans and also structural components of cell membranes (Holland 1978; Harrison 1997), lack of research on the subject seems surprising However, when one considers the ease of collecting egg-bearing females from ponds, one understands the lack of interest in broodstock maintenance Nonetheless, as the industry grows and biosecurity issues become more important and infectious diseases appear, indoor closed system hatcheries will become necessary and with them special broodstock diets According to Rodrı´ guez-Gonza´lez et al (2006b), the lipid requirements for the developing gonad of female redclaw mainly originates from the diet Rodrı´ guez-Gonza´lez et al (2009b) studied the effect of dietary lipid levels (40, 80 and 120 g kgÀ1) on female redclaw crayfish and their eggs Results indicated no significant differences in survival, final weight or fecundity However, dietary lipid content influenced size and weight of eggs, with greatest egg weight obtained from females offered the 87 g kgÀ1 lipid diet In a similar study on the effects of dietary lipids on female redclaw reproduction, Li et al (2010) observed a significant correlation between lipid transportation in the hepatopancreas and the ovaries, but it appeared that the lipid reserves in the hepatopancreas could not meet the requirements of ovaries The authors concluded that the lipid requirements of gonads come only partly from the diet Polyunsaturated fatty acids (PUFA) of the C18 series (linolenic (18:3n-3) and linoleic (18:2n-6) acids) and n-3 and n-6 highly unsaturated fatty acids [eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (ArA)] are considered essential in crustacean diets (see D’Abramo & Robinson 1989; D’Abramo 1997; Venero et al 2008) No studies on specific EFA requirements of redclaw were found; however, a few studies evaluated diets containing various levels of fatty acids on growth performance of the species Thompson et al (2003a) reported that a mixture of 5% cod liver oil and 1% corn oil added to the diets probably met the EFA requirements of juvenile redclaw The blend of these oils provides a mix of PUFA such as linoleic (18:2n-6), linolenic (18:3n-3), oleic (18:1n-9) acids and highly unsaturated fatty acids such as eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids, that is sufficient for redclaw survival and growth Similarly, Thompson et al (2003b) evaluated practical diets with and without supplemental lecithin and/or cholesterol offered to juvenile redclaw The authors indicated that diets with g kgÀ1 supplemental lecithin and/or cholesterol contained a combination of PUFA and HUFA in the diet, which satisfied the EFA requirements of juvenile redclaw Thompson et al (2010) examined the effect of different sources of lipids (linseed oil, canola oil, corn oil, beef tallow or menhaden oil) that differ in fatty acid profile on growth response of juvenile redclaws Results showed that whole-body fatty acid composition of redclaw differed among animals offered the various lipid sources, generally reflecting the fatty acid composition of the diets Plant oils rich in a-linolenic acid (18:3n-3), linoleic acid (18:2n-6) and oleic acid (18:1n-9) perform as well as menhaden oil containing high levels of n-3 HUFA when offered to juvenile redclaw reared indoors and lacking natural productivity The authors concluded that redclaw can be fed diets containing plant-based oils with high levels of 18-carbon unsaturated fatty acids Muzinic et al (2004) reported that practical diets containing various levels of SBM and brewer’s grains with yeast, as replacements for fish meal, have both n-6 and n-3 highly unsaturated fatty acids such as linoleic (18:2n-6), eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids that may satisfy the EFA requirements of juvenile redclaw The fatty acid profile during early embryonic development of redclaw shows that the major fatty acids, oleic/ vaccenic (18:1), palmitic (16:0), linoleic (18:2n-6) and palmitoleic (16:1n-7) remain major during later developmental stages and are required in larger quantities than other fatty acids (Alimon et al 2003) Monounsaturated fatty acids constituted the major moiety of the fatty acid profile, and the PUFA were dominated by linoleic (n-6) series (low n-3 to n-6 ratio) (Alimon et al 2003) Luo et al (2008a) reported that the predominant fatty acids of both neutral and polar lipids of redclaw during embryonic development were C16:0, C18:0, C18:1n-9 and C18:3n-3 Saturated fatty acids (16:0 and 18:0) and monosaturated fatty acids (16:1n-7 and 18:1n-9) are generally used for energetic purposes, whereas PUFA (20:5n-3 and 22:6n-3) are important as structural components of cell membranes and in the development of the central nervous system (Luo et al 2008a) However, even during vitellogenesis, there are high proportions of monounsaturated fatty acids in the ovaries and hepatopancreas, suggesting their use as major sources of energy (Li et al 2010) Such information would suggest that broodstock diets could be formulated to contain more vegetable oils to be used for energy during vitellogenesis without compromising development of eggs, which require some n-3 HUFAs found in expensive but necessary fish oils Phospholipids are added to the diet of crustaceans for various reasons such as a source of energy; a major component of cell membranes; emulsification of lipid aggregates during digestion and absorption; and because they play a major role in lipid transportation in the haemolymph (Coutteau Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd Experimental treatments1 FO/FO 14:0 14:1n-5 16:0 16:1n-7 18:0 18:1n-9 18:1n-7 18:2n-6 18:3n-6 18:3n-3 18:4n-3 20:0 20:1n-11 20:1n-9 20:2n-6 20:3n-6 20:4n-6 20:3n-3 20:4n-3 20:5n-3 22:0 22:1n-11 22:1n-9 22:2n-6 22:4n-6 22:5n-3 24:0 22:6n-3 24:1n-9 SFA2 MUFA PUFA n-6 C18 PUFA n-3 C18 PUFA n-6 LC-PUFA n-3 LC-PUFA 33.9 0.6 151.4 62.0 37.9 266.3 1.5 82.5 0.7 18.3 5.1 1.4 3.6 32.8 7.1 2.2 5.6 2.4 7.8 31.7 0.4 15.7 4.8 0.5 1.5 18.0 0.6 117.4 1.4 225.6 388.7 300.8 83.2 23.4 16.9 177.2 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± VO/FO 0.95b 0.04b 0.28b 1.42b 1.30a 3.54b 0.13b 2.97a 0.27a 0.64a 0.40 0.06 0.05b 0.07c 0.18a 0.07a 0.16b 0.07a 0.32b 1.53b 0.03 0.65b 0.12b 0.10a 0.09b 0.99b 0.08a 5.46b 0.74 1.67b 2.62b 12.62a 3.19a 1.01a 0.56a 7.95b 18.7 0.3 129.5 34.5 43.3 245.8 0.9 212.5 2.8 91.3 3.1 1.2 1.4 17.9 12.6 4.6 4.2 5.6 6.0 19.5 0.3 5.9 2.4 1.1 0.6 10.2 0.6 70.4 1.3 193.4 310.4 444.5 215.3 94.4 23.1 111.6 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± T-test (VO/FO versus UF/FO) UF/FO 0.61a 0.01a 1.34a 0.15a 0.53b 2.89a 0.16a 5.83b 0.27b 2.27b 1.60 0.19 0.09a 0.81b 0.39b 0.35b 0.06a 0.19b 0.27a 0.61a 0.02 0.33a 0.09a 0.03b 0.09a 0.13a 0.11a 1.26a 0.09 0.68a 4.27a 8.88b 6.09b 1.23b 0.88b 1.06a 16.4 0.4 133.3 31.2 42.3 252.0 0.7 208.5 2.6 89.4 3.9 1.3 1.1 15.0 13.0 5.1 4.0 5.8 5.9 18.2 0.4 4.5 2.0 1.1 0.8 9.0 0.9 74.0 1.2 194.6 308.3 441.4 211.1 93.3 24.0 112.9 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.47a 0.04a 5.19a 2.76a 1.24b 3.91a 0.10a 8.40b 0.06b 4.77b 0.82 0.04 0.17a 1.10a 0.52b 0.18b 0.15a 0.30b 0.41a 0.25a 0.17 0.80a 0.21a 0.04b 0.02a 0.18a 0.02b 1.00a 0.08 7.69a 5.72a 15.16b 8.35b 5.58b 0.49b 1.39a ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ** * ns ns ns ns ns ns ns ns ns Table Fatty acid composition (mg g)1 of lipid) of rainbow trout fillets under different treatments at the end of the second phases (day 35) of the experimental finishing period Data represent means ± standard error (n = 3; N = 9) Values in the same row with different superscripts are significantly different (P < 0.05; ANOVA and Student–Newman–Keuls post hoc test) The results of the independent T-test between VO/FO and UF/FO are also reported as: *P < 0Æ05, **P < 0Æ01, ***P < 0Æ001; ns = not significant See Table for experimental treatments abbreviations and Table for diets abbreviations See Table for fatty acid class abbreviations Growth performance during the experimental finishing trial was consistent with what was expected, with unfed fish (UF/FO) showing a growth reduction during the weeks of food deprivation, followed by a phase of accelerated growth (Table 3), commonly referred to as compensatory growth, as previously documented for rainbow trout (Dobson & Holmes 1984) The VO/FO treatment showed a slightly reduced growth performance compared to FO/FO fish, without any indication of lipo-compensatory growth, as previously reported for other finfish species when shifted from a FO-deprived diet to a FO-based diet (Turchini et al 2007) However, it is important to underline that the present trial was not specifically designed to assess growth performance, as a relatively limited number of fish were used (although individually tagged) which were then sampled at regular time intervals Thus, whilst the sampling was implemented randomly, it could have been responsible for reducing the overall reliability of these results Therefore, the growth and feed utilization performance needs to be considered as indicative only However, it Aquaculture Nutrition 18; 441–456 Ó 2012 Blackwell Publishing Ltd Table Fatty acid composition (mg g)1 of lipid) of rainbow trout fillets under different treatments at the end of the third phases (day 56) of the experimental finishing period Experimental treatments1 FO/FO 14:0 14:1n-5 16:0 16:1n-7 18:0 18:1n-9 18:1n-7 18:2n-6 18:3n-6 18:3n-3 18:4n-3 20:0 20:1n-11 20:1n-9 20:2n-6 20:3n-6 20:4n-6 20:3n-3 20:4n-3 20:5n-3 22:0 22:1n-11 22:1n-9 22:2n-6 22:4n-6 22:5n-3 24:0 22:6n-3 24:1n-9 SFA2 MUFA PUFA n-6 C18 PUFA n-3 C18 PUFA n-6 LC-PUFA n-3 LC-PUFA 34.2 0.6 157.8 64.5 41.6 280.8 2.7 82.6 1.1 18.6 5.4 1.3 3.8 34.6 7.3 2.4 5.9 2.5 7.8 33.2 0.5 16.6 5.1 0.6 1.0 19.1 0.8 114.7 0.9 236.2 409.6 302.2 83.7 24.0 17.3 177.2 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± VO/FO 0.50b 0.11 0.93b 1.54b 0.79 3.76b 0.20b 1.86a 0.30a 0.16a 0.18 0.23 0.10b 0.28b 0.16a 0.20a 0.16b 0.05a 0.08b 1.33b 0.02 0.08b 0.05b 0.06a 0.20 0.42b 0.04 3.01c 0.22 1.24b 4.69b 6.70a 1.84a 0.29a 0.61a 4.71c 19.6 0.4 131.7 36.5 43.7 247.0 1.8 197.0 2.5 82.6 2.9 1.4 1.7 19.7 12.8 4.7 4.5 5.6 6.4 19.4 0.7 6.7 2.6 1.0 0.9 11.5 0.8 76.9 1.6 198.0 318.0 428.9 199.5 85.5 24.0 119.8 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± T-test (VO/FO versus UF/FO) UF/FO 1.51a 0.04 5.77a 3.26a 0.56 1.26a 0.47ab 13.95b 0.16b 6.95b 1.65 0.04 0.22a 1.85a 0.75b 0.03b 0.18a 0.34b 0.34a 0.84a 0.29 0.88a 0.26a 0.06b 0.05 0.78a 0.03 2.96a 0.08 7.81a 7.80a 15.15b 13.91b 5.32b 0.80b 4.78a 19.8 0.3 129.2 34.2 43.3 244.4 1.1 202.8 2.6 85.3 5.0 1.2 1.7 19.0 12.8 4.5 4.7 5.6 6.1 22.8 0.7 7.0 2.7 1.2 1.0 11.0 0.7 91.7 1.4 194.8 311.8 457.2 205.4 90.4 24.1 137.3 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.02a 0.06 2.90a 0.36a 1.78 2.77a 0.30a 3.70b 0.31b 2.50b 0.70 0.21 0.11a 0.46a 0.08b 0.10b 0.26a 0.02b 0.28a 0.71a 0.14 0.60a 0.16a 0.06b 0.13 0.71a 0.13 0.50b 0.26 4.74a 4.39a 5.76b 3.73b 2.30b 0.55b 1.29b ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns * ns ns ns ns ns ns ns ** ns ns ns ns ns ns ns * Data represent means ± standard error (n = 3; N = 9) Values in the same row with different superscripts are significantly different (P < 0.05; ANOVA and Student–Newman–Keuls post hoc test) The results of the independent T-test between VO/FO and UF/FO are also reported as: *P < 0Æ05, **P < 0Æ01, ***P < 0Æ001; ns = not significant See Table for experimental treatments abbreviations and Table for diets abbreviations See Table for fatty acid class abbreviations is interesting to report that whilst not statistically significant, UF/FO fish recorded a 7.2% reduction in final weight compared to VO/FO fish, but, on the other hand, significantly less FO-based feed was utilized by UF/FO fish compared to VO/FO fish (29.5% reduction) Thus, from an economical and environmental point of view, this can be viewed as a positive result for such a strategy, as significantly less FO was used to obtain fish with an even higher n-3 LC-PUFA content, yet to the detrimental of loss of potential weight gain Aquaculture Nutrition 18; 441–456 Ó 2012 Blackwell Publishing Ltd The primary purpose of the present study was to test the hypothesis previously proposed by Palmeri et al (2009) This study proposed that by reducing the total fat content of fillets of fish previously fed a FO-deprived diet before the dietary shift to a FO-based finishing diet, the restoration of an overall FO-like fatty acid profile would be more efficient and therefore require less FO As such, a 2-week period of food deprivation was imposed on fish (previously fed for 18 weeks a VO-based diet; VO; 50% linseed oil and 50% sunflower oil), prior the finishing period to reduce the initial total 26.0 ** Coefficient of distance (D) 24.0 UF/FO VO/FO 22.0 20.0 18.0 16.0 14.0 12.0 Week Figure Variation in the coefficient of distance (D) for fillet fatty acid composition in VO/FO and UF/FO treatments in comparison with the control treatment (FO/FO) during the different phases of the experimental finishing period for 56 days Each dot represents means ± standard error (n = 3; N = 6), and dots at the same time with the different superscripts are significantly different (**P < 0.01, independent T-test) content of fat in the fish body In the previous study by Palmeri et al (2009), implemented on the freshwater Murray cod, fish did not show any reduction in the lipid content, either in the fillet or the whole body during the period of food deprivation, and thus, it was not possible to accept or reject the proposed hypothesis In the present trial, rainbow trout unfed for the 2-week period showed a significant reduction in body weight and fillet fat content and therefore, the required assumption to validate the proposed hypothesis was achieved Lipid mobilization during the period of food deprivation in rainbow trout has been formerly studied by Jezierska et al (1982), who reported that upon a period of food deprivation, visceral and muscle lipid reserves contributed most to energy metabolism Similarly, other studies on various salmonid species have reported that food deprivation is responsible for the overall reduction of total fillet fat content as a result of reduced fillet lipid content and reduced body weight (Einen et al 1998; Cook et al 2000; Regost et al 2001; Turchini et al 2004; Morkore et al 2008) What is particularly interesting is that whilst there is evidence that food deprivation in rainbow trout plays a major role in affecting the transcription rate of several genes (Salem et al 2007) and also affects a large number of plasma, liver and muscle metabolites (Kullgren et al 2010), the actual modification of the fatty acid composition during food deprivation is, thus 14:0 n-3 LC-PUFA 120% 16:0 110% n-3 C18 PUFA SFA 100% 90% 80% 22:6n-3 18:1n-9 70% 60% 22:5n-3 MUFA 20:5n-3 18:2n-6 18:3n-3 20:4n-6 n-6 LC-PUFA Line of Equality (Day 0) 1st phase (week 2, day 14) 2nd phase (week 5, day 35) 3rd phase (week 8, day 56) n-6 C18 PUFA Figure Fatty acid composition of trout fillet for the two experimental treatment groups (VO/FO and UF/FO) expressed as a per cent fraction of fatty acid composition observed in the UF/ FO group relative to the VO/FO group Values were computed from fatty acid (FA) composition (% w/w) as FA(UF/FO group) Ä FA(VO/FO group) · 100 Based on this calculation, a value of 100% represents equality between tissue profiles, as it was at time zero, the commencement of the experimental finishing period See Table for fatty acid class abbreviations Aquaculture Nutrition 18; 441–456 Ó 2012 Blackwell Publishing Ltd Table The fillet fatty acid composition (% w/w; selected fatty acid and fatty acid classes only) observed in treatments UF/FO and VO/FO at day 35, and computed by the dilution model using data relative the 2nd phase of the experimental finishing (day 14–35) The variation between observed and computed values (= observed Ä computed) is also reported and analysed UF/FO 14:0 16:0 18:1n-9 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:5n-3 22:6n-3 SFA MUFA PUFA n-6 C18 PUFA n-3 C18 PUFA n-6 LC-PUFA n-3 LC-PUFA VO/FO Variation Computed Observed T-test Computed Observed T-test UF/VO 2.31 13.98 25.97 18.85 7.75 0.52 2.27 1.27 9.43 20.91 35.18 43.91 19.06 8.18 2.46 14.21 1.73 14.12 26.69 22.08 9.47 0.42 1.93 0.95 7.84 20.61 32.65 46.73 22.35 9.88 2.54 11.96 * ns ns * * ** *** *** *** ns * ns * * ns *** 2.14 14.03 26.53 20.60 8.81 0.46 2.03 1.10 8.05 21.06 34.14 44.79 20.85 8.98 2.52 12.44 1.97 13.65 25.93 22.41 9.63 0.44 2.06 1.07 7.42 20.40 32.74 46.86 22.70 9.96 2.43 11.77 ns ns ns * * ns ns ns ** ns ns ns * ** ns ** 0.75 1.01 1.03 1.17 1.22 0.82 0.85 0.75 0.83 0.99 0.93 1.06 1.17 1.21 1.03 0.84 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.01 0.02 0.24 0.18 0.07 0.01 0.03 0.00 0.14 0.01 0.32 0.32 0.19 0.06 0.01 0.15 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.16 0.59 0.28 0.81 0.47 0.02 0.02 0.02 0.13 0.87 0.62 1.46 0.80 0.55 0.05 0.19 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.02 0.28 0.14 0.33 0.16 0.01 0.04 0.01 0.05 0.39 0.20 0.47 0.33 0.16 0.01 0.03 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.07 0.14 0.38 0.46 0.17 0.01 0.07 0.02 0.12 0.19 0.55 0.63 0.48 0.09 0.09 0.06 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± T-test VO/FO 0.07 0.04 0.00 0.04 0.07 0.04 0.01 0.02 0.00 0.04 0.01 0.03 0.04 0.08 0.02 0.00 0.92 0.97 0.98 1.09 1.09 0.97 1.01 0.98 0.92 0.97 0.96 1.05 1.09 1.11 0.97 0.95 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.02 0.03 0.02 0.03 0.04 0.02 0.05 0.03 0.02 0.02 0.02 0.02 0.03 0.02 0.03 0.01 ns ns * ns ns * * ** * ns ns ns ns ns ns *** Data represent means ± standard error (n = 3; N = 6) Within each couple of data set, the results of the independent T-test are reported as: *P < 0Æ05, **P < 0Æ01, ***P < 0Æ001; ns = not significant Table The fillet fatty acid composition (% w/w; selected fatty acid and fatty acid classes only) observed in treatments UF/FO and VO/FO at day 56, and computed by the dilution model using data relative the entire experimental finishing period (day 0–56) The variation between observed and computed values (= observed Ä computed) is also reported and analysed UF/FO 14:0 16:0 18:1n-9 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:5n-3 22:6n-3 SFA MUFA PUFA n-6 C18 PUFA n-3 C18 PUFA n-6 LC-PUFA n-3 LC-PUFA VO/FO Variation Computed Observed T-test Computed Observed T-test UF/VO 1.76 13.23 24.98 22.32 9.92 0.47 1.84 0.99 7.43 19.93 32.69 47.38 22.60 10.56 2.66 11.56 2.05 13.41 25.35 21.04 8.86 0.48 2.36 1.14 9.52 20.21 32.35 47.44 21.32 9.38 2.50 14.24 * ns ns ns * ns ** ns ** ns ns ns ns * ns ** 2.01 13.69 25.61 20.48 8.84 0.49 2.06 1.13 8.06 20.61 34.11 45.28 20.74 9.47 2.55 12.53 2.08 13.94 26.14 20.85 8.74 0.48 2.06 1.22 8.14 20.96 33.65 45.39 21.12 9.05 2.54 12.68 ns ns * ns ns ns ns ns ns ns ns ns ns ns ns ns 1.17 1.01 1.01 0.94 0.89 1.03 1.28 1.15 1.28 1.01 0.99 1.00 0.94 0.89 0.94 1.23 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.03 0.10 0.11 0.27 0.11 0.01 0.04 0.03 0.26 0.12 0.10 0.16 0.28 0.13 0.10 0.38 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.10 0.28 0.21 0.43 0.29 0.03 0.08 0.07 0.08 0.45 0.36 0.71 0.43 0.27 0.05 0.15 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.03 0.09 0.10 0.23 0.09 0.01 0.04 0.02 0.23 0.11 0.09 0.14 0.24 0.11 0.09 0.33 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.16 0.60 0.12 1.50 0.74 0.02 0.08 0.08 0.30 0.83 0.79 1.61 1.49 0.57 0.07 0.48 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± T-test VO/FO 0.08 0.02 0.01 0.03 0.04 0.04 0.03 0.06 0.04 0.03 0.01 0.02 0.03 0.03 0.05 0.03 1.04 1.02 1.02 1.02 0.99 0.97 1.00 1.07 1.01 1.02 0.99 1.00 1.02 0.96 1.00 1.01 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.09 0.05 0.00 0.08 0.09 0.04 0.04 0.07 0.04 0.05 0.02 0.04 0.07 0.06 0.01 0.04 ns ns ns ns ns ns ** ns ** ns ns ns ns ns ns ** Data represent means ± standard error (n = 3; N = 6) Within each couple of data set, the results of the independent T-test are reported as: *P < 0Æ05, **P < 0Æ01, ***P < 0Æ001; ns = not significant far, a topic that has received relatively little attention in salmonid fish Jezierska et al (1982) reported that in unfed rainbow trout, the modification of fatty acid composition was considerably diverse in different body departments Specifically, in fish muscle, the authors reported an increase in SFA, a decrease in MUFA and a relatively constant Aquaculture Nutrition 18; 441–456 Ó 2012 Blackwell Publishing Ltd PUFA content as the result of a 27-day period of food deprivation However, within the relatively constant PUFA class, the authors recorded a decline in 18:2n-6 and an increase in 20:4n-6, 20:5n-3 and 22:6n-3 In Atlantic salmon (Salmo salar) unfed for up to 86 days, an increase in total n-3 PUFA content, and a simultaneous decrease in SFA content, was recorded in the belly flap, whilst in the leaner central part of the fillet MUFA, 20:5n-3 and n-6 PUFA increased and 16:0 decreased upon food deprivation (Einen et al 1998) Clearly, whilst food deprivation seems to be responsible for specific trends and modifications of fatty acid composition of different fish tissues, the fatty acid composition of fish tissues before the food deprivation period could have a major role in determining such modifications In the present study, unfed fish were previously fed a VO diet, which was extremely rich in 18:2n-6 and 18:3n-3, and consequently, these fatty acids were highly abundant in initial fish samples before food deprivation, whilst n-3 LC-PUFA were relatively limited In the present study, by comparing the fillet fatty acid composition (as % w/w) of initial fish and after weeks of food deprivation in the UF/FO treatment (specific data not reported but easily evincible from Tables and 5), it was shown that food deprivation was responsible for the reduction of 20:5n-3, 14:0 and 16:0 with an actual decrease of 4.8%, 4.3% and 3.1% of their initial values, respectively On the other hand, 20:4n-6 and 22:6n-3 values increased by 4.3% and 10.4%, respectively However, the most interesting results were relative to the variation in the percentage content of 18:2n-6 and 18:3n-3, which upon the period of food deprivation increased by 1.7% and 2.6%, respectively Thus, whilst the period of food deprivation was effective in reducing the total fat content of the fillet (Fig 1), interestingly and unexpectedly, it was also responsible for a further divergence from the FO-like fatty acid profile of the fillet, as evidenced from the recorded coefficient of distance (D) (Fig 2) The reduction of total fat content of the fillet in unfed fish (UF/FO) was then responsible for a much faster recovery towards a FO-like fatty acid profile during the following phases in which fish were fed with the FO diet (Fig 2), validating the proposed hypothesis However, the modification of the fatty acid composition of fish fillets during food deprivation, coupled with the postponement of administering of the finishing diet, and therefore the postponing of the washing-out effect, resulted in no real beneficial effect of the tested hypothesis on the actual efficiency of the finishing strategy In fact, as seen in Fig and Table 4, the 1st phase of food deprivation significantly impacted on the fillet fatty acid profile, making the fillet composition of UF/FO fish very different from that of VO/FO, which were already moving towards a FO-like fatty acid make-up These differences were levelled during the subsequent feeding period, attributable to the faster recovery of the FO-like fatty acid make-up in UF/FO fish However, an interesting aspect was that the final content of 20:5n-3, 22:6n-3 and, in general, n-3 LC-PUFA in the fillets of UF/FO fish were significantly higher than VO/FO fed fish (Table and Fig 3), clearly suggesting that the period of food deprivation was positively impacting on the deposition/retention of these fatty acids The dilution model proposed by Robin et al (2003), and subsequently largely validated (Jobling 2003, 2004a,b; Turchini et al 2006), was efficiently capable of predicting the fatty acid composition of trout fillets during the different phases of this trial (Tables and 9) In fact, in only a few exceptions, the comparison of observed and computed fatty acid values in the VO/FO treatment resulted in statistically significant differences Thus, considering the model as an accurate tool to describe fatty acid modification after a dietary shift, it is possible to use the results of such a model to gain a better understanding of how the modification of the fatty acid profile was affected by the period of food deprivation in UF/FO fish In the period immediately following the weeks of food deprivation (Table 8), the observed values for 18:2n-6 and 18:3n-3 (and in general C18 PUFA) were significantly higher than those predicted by the model, indicating that the previous period of food deprivation was clearly increasing the retention of these two fatty acids Interestingly, C18 PUFAs were the main fatty acids characteristic of the previous VO diet and therefore abundantly present in the fillet of initial fish On the contrary, 14:0, 20:4n-6, 20:5n-3, 22:5n-3, 22:6n-3 and n-3 LC-PUFA (all fatty acids typical of the FO diet used during the finishing period) recorded significantly lower values compared to the values predicted by the model This is therefore suggesting that the previous period of food deprivation was decreasing the efficiency of deposition of these fatty acids However, when the entire experimental finishing period is considered (Table 9), the results are almost the opposite: the unfed group recorded lower values for C18 PUFA and higher values for 20:5n-3 and 22:6n-3, compared to values predicted by the model This discrepancy can be explained by the fact that by considering the entire finishing period as a whole, there is a combination of effects acting on the modification of the fatty acid profile, such as the effects of the period of food deprivation, the initial reduction and subsequent rapid regain of fillet fat content, and the postponement of the FO finishing diet Therefore, observing the variation between observed and computed values, it is manifested that overall, the period of food deprivation was responsible for a higher deposition/ retention of 20:5n-3, 22:6n-3 and in general n-3 LC-PUFA compared to fish under the normal finishing period However, the overall modification of other fatty acids, and specifically the washing-out of C18 PUFA, was basically unaffected by the previous short-term period of food deprivation Aquaculture Nutrition 18; 441–456 Ó 2012 Blackwell Publishing Ltd In summary, the suggested strategy resulted in mixed results: (i) significantly less FO was used by the UF/FO treatment compared to the VO/FO treatment (56.7 g versus 80.4 g of FO per fish, respectively) to achieve a final product characterized by an almost identical total n-3 LC-PUFA content, when expressed as mg per 100 g of edible product (1671 mg per 100 g in VO/FO and 1672 mg per 100 g in UF/FO); (ii) the UF/FO treatment was more efficient in utilizing and depositing dietary n-3 LC-PUFA (the total n-3 LC-PUFA administered was 20.2 and 14.3 g per fish; total n-3 LC-PUFA harvested in total edible product was 8.5 and 7.7 g per fish, and the resulting n-3 LC-PUFA deposition/ retention efficiency was 42.2% and 54.3% for VO/FO and UF/FO, respectively); and (iii) the fish under the UF/FO treatment showed a considerable loss of potential weight gain Therefore, as a conclusive remark, innovative strategies that can reduce the fillet lipid content before the implementation of a finishing period, but at the same minimise weight loss, should be investigated and pursued This research was supported under the Australian Research CouncilÕs Discovery Projects funding scheme (project DP1093570) The views expressed herein are those of the authors and are not necessarily those of the Australian Research Council The first author, Thanuthong, T is the recipient of a Thai Government scholarship funded by Thai Government, and this support is gratefully acknowledged The authors also express their gratitude to Bob Collins for his general and technical support throughout the research project and to Dr Richard Smullen (RidleyAquafeed, Ridley AgriProducts, Narangba, QLD, Australia) for kindly providing some of the raw materials used in the experimental diets Bell, J.G., Henderson, R.J., Tocher, D.R & Sargent, J.R (2004) 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Aquaculture, 300, 148–155 Cook, J.T., Sutterlin, A.M & McNiven, M.A (2000) Effect of food deprivation on oxygen consumption and body composition of growth-enhanced transgenic Atlantic salmon (Salmo salar) Aquaculture, 188, 47–63 Dobson, S.H & Holmes, R.M (1984) Compensatory growth in the rainbow-trout, Salmo-Gairdneri Richardson J Fish Biol., 25, 649–656 Aquaculture Nutrition 18; 441–456 Ó 2012 Blackwell Publishing Ltd Einen, O., Waagan, B & Thomassen, M.S (1998) Starvation prior to slaughter in Atlantic salmon (Salmo salar) – I Effects on weight loss, body shape, slaughter- and fillet-yield, proximate and fatty acid composition Aquaculture, 166, 85–104 Francis, D.S., Turchini, G.M., Jones, P.L & De Silva, S.S (2007) Growth performance, feed efficiency and fatty acid composition of juvenile Murray cod, Maccullochella peelii peelii, fed graded levels of canola and linseed oil Aquacult Nutr., 13, 335–350 Glencross, B.D & Turchini, G.M (2010) Fish oil replacement in starter, grow-out and finishing feeds for farmed aquatic animals In: Fish Oil Replacement and Alternative Lipid Sources in Aquaculture Feeds (Turchini, G.M et al eds), pp 373–404 CRC Press, Taylor & Francis group, Boca Raton, FL, USA Jezierska, B., Hazel, J.R & Gerking, S.D (1982) Lipid mobilization during starvation in the rainbow trout, Salmo gairdneri Richardson, with attention to fatty-acids J Fish Biol., 21, 681–692 Jobling, M (2003) Do changes in Atlantic salmon, Salmo salar L., fillet fatty acids following a dietary switch represent wash-out or dilution? 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(Salmo salar) fed vegetable oils Food Chem., 87, 567–580 Torstensen, B.E., Bell, J.G., Rosenlund, G., Henderson, R.J., Graff, I.E., Tocher, D.R., Lie, O & Sargent, J.R (2005) Tailoring of Atlantic salmon (Salmo salar L.) flesh lipid composition and sensory quality by replacing fish oil with a vegetable oil blend J Agric Food Chem., 53, 10166–10178 Turchini, G.M., Mentasti, T., Crocco, C., Sala, T., Puzzi, C., Moretti, V.M & Valfre, F (2004) Effects of the extensive culture system as finishing production strategy on biometric and chemical parameters in rainbow trout Aquacult Res., 35, 378– 384 Turchini, G.M., Francis, D.S & De Silva, S.S (2006) Modification of tissue fatty acid composition in Murray cod (Maccullochella peelii peelii, Mitchell) resulting from a shift from vegetable oil diets to a fish oil diet Aquacult Res., 37, 570–585 Turchini, G.M., Francis, D.S & De Silva, S.S (2007) Finishing diets stimulate compensatory growth: results of a study on Murray cod, Maccullochella peelii peelii Aquacult Nutr., 13, 351–360 Turchini, G.M., Torstensen, B.E & Ng, W.K (2009) Fish oil replacement in finfish nutrition Rev Aquacult., 1, 10–57 Aquaculture Nutrition 18; 441–456 Ó 2012 Blackwell Publishing Ltd Aquaculture Nutrition 2012 18; 457–464 1 2,3 doi: 10.1111/j.1365-2095.2011.00915.x 1 Department of Aquaculture, The Stanisaw Sakowicz Inland Fisheries Institute, Oczapowskiego, Olsztyn, Poland; Department of Fish Pathology and Immunology, The Stanisaw Sakowicz Inland Fisheries Institute, Oczapowskiego, Olsztyn, Poland; Department of Microbiology and Clinical Immunology, Faculty of Veterinary Medicine, University of Warmia and Mazury, Oczapowskiego, Olsztyn, Poland; Department of Ichthyology, Faculty of Environmental Sciences and Fisheries, University of Warmia and Mazury, Oczapowskiego, Olsztyn, Poland Juvenile European pikeperch, Sander lucioperca, were fed commercial feed (group C) or experimental feed supplemented with NuProÒ nucleotide-rich Saccharomyces cerevisiae yeast protein (extract obtained through a cell wall removal process) in doses of 20, 40 or 60 g kg)1 feed (groups N2, N4, N6) for weeks Growth, non-specific immunity parameters, histological structure of the liver and intestine, proximate whole-body composition and blood biochemical parameters were assayed It was noted that brewerÕs yeast extract has immunomodulatory proprieties NuProÒ in doses of 40 and 60 g kg)1 feed strongly stimulated non-specific (innate) cellular and humoral immunity in pikeperch The experimental feed did not have a significant impact on pikeperch growth (P > 0.05) The proximate composition of the fish bodies and the hepatosomatic and viscerosomatic (VSI) indices were also not affected, which indicated that the tested diets had no negative impact on the metabolism or deposition of nutrients in fish tissues The lower levels of transaminases AST and ALT, which were noted in the groups with the two highest doses of NuProÒ (P < 0.05), might indicate improved liver function It was also demonstrated that the brewerÕs yeast extract stimulates the absorption activity of intestinal epithelial cells key words: brewerÕs yeast, growth, Immune response, Nucleotide, Saccharomyces cerevisiae, Sander lucioperca Received 18 January 2011, accepted 21 September 2011 Correspondence: Sylwia Jarmołowicz, Department of Aquaculture, The Stanisław Sakowicz Inland Fisheries Institute, Oczapowskiego 10, 10-719 Olsztyn, Poland E-mail: jarmolowicz@infish.com.pl Ó 2012 Blackwell Publishing Ltd The application of brewerÕs yeast, mainly the Saccharomyces cerevisiae strain, as a source of nutrients and bioactive compounds in aquaculture started as early as the beginning of the 1990s (Ferreira et al 2010) Commercial brewerÕs yeast is an inactive yeast (dead yeast cells) by-product of the brewing process The cell wall, which can comprise 20–25% of the dry weight of the cell, consists of about 85–90% polysaccharide The polysaccharide component consists of a mixture of mannan, glucan and small amounts of chitin (Nguyen et al 1998) Numerous experimental studies on fish have indicated that compounds such as mannan oligosaccharides, glucans and chitin strongly stimulate fish immune systems (i.e Esteban et al 2001; Couso et al 2003; Torrecillas et al 2007) Complete removal of the cell wall yields yeast extract It is mainly the product of enzymatic digestion of the yeast cell constituents by endogenous (in an autolysis process) and exogenous (in a hydrolysis process) yeast enzymes (Bekatorou et al 2006; Ferreira et al 2010) The disrupted yeast cells are significantly more highly digestible and have a higher protein content than intact brewerÕs yeast (Rumsey et al 1991) Thus, S cerevisiae yeast protein has been used successfully as an alternative protein source with a few fish species (Oliva-Teles & Goncalves 2001; Li & Gatlin 2003; Craig & McLean 2005) and can replace 50% of fish meal protein with no negative effects on fish performance Yeast extract is also an important source of amino acids, vitamins and nucleotides (in the form of nucleic acids; Ferreira et al 2010) These are present mostly as ribonucleic acid (RNA) and represent approximately 13–23% of the total nitrogen (Anupama & Ravindra 2000) Although the role of nucleotides in fish diets has been studied for over 30 years, the results of experiments performed in the last decade are especially promising It has been demonstrated that nucleotides added to basal diets can positively affect fish growth, innate and adaptive immune responses, disease resistance, lipid metabolism and intestinal structure (Li et al 2005; reviewed by Li & Gatlin 2006) In the light of these, the present experiment was designed to study the effect of the commercial product NuProÒ (nucleotide-rich S cerevisiae yeast protein extract obtained through the cell wall removal process; Alltech Inc., Nicholasville, KY, USA) added to practical diets on the growth and health of juvenile pikeperch, Sander lucioperca Thus, the rearing indexes, non-specific cellular and humoral immunity parameters, histological structure of the liver and intestine and proximate whole-body composition were investigated Additionally, blood biochemical parameters such as aspartate (AST) and alanine (ALT) aminotransferase, alkaline phosphatase (ALP), ceruloplasmin and bilirubin were analysed as markers of intestinal epithelium, kidney and liver damage resulting from inappropriately composed diets for fish (Dunier et al 1995; Velisek et al 2010) The influence of pikeperch of exogenous nucleotides was investigated for the first time In comparison with other percids, the pikeperch holds a significant position in European aquaculture, and this species is in high consumer demand (Zake˛s´ 2003; Schulz et al 2005) The pikeperch were reared initially at the Department of Sturgeon Breeding in Pieczarki, Inland Fisheries Institute (IFI) in Olsztyn, Poland In the early stages of rearing, the larvae were fed brine shrimp (Artemia sp.) and formulated feed (commercial trout starter) Formulated feed was fed exclusively from day 14 of rearing (larval age 18 days posthatch) (Szkudlarek & Zake˛s´ 2007) After weeks of rearing, 420 pikeperch juveniles were transported in plastic bags containing oxygen to the Department of Aquaculture, IFI in Olsztyn, where the experiment properly took place When the fish had achieved a mean body weight of about 37 g, they were placed in 12 tanks with volumes of 0.2 m3 each The mean initial stocking density was 2.3 kg m)3 (35 individuals per tank) The physicochemical parameters of the water measured at the rearing tank outflows during the experiment were as follows: temperature – 21.9 °C (±0.3); oxygen content – 5.07 mg O2 L)1 (±0.62); and total ammonium nitro- gen (TAN = NHþ -N + NH3-N) and nitrite (NO2-N) – 0.177 ± 0.098 mg TAN L)1 and 0.015 ± 0.004 NO2-N mg L)1, respectively Water pH in outflows ranged from 7.6 to 7.8 The light intensity at the water surface of the tanks was 50–60 lx The base feed used was commercial trout feed with a proximate composition of protein – 500 g kg)1, lipids – 180 g kg)1, carbohydrates – 150 g kg)1 (manufacturer data, Nutreco, Boxmeer, Netherlands) and with a granulation size of 1.9 mm supplemented with NuProÒ extract (Alltech Inc.) in dosages of 20, 40 and 60 g kg)1 feed The quantities of extract were mixed with mL of distilled water and added to 100 g of base feed and then sealed in an AGA Labor vacuum pump (Lublin, Poland) The control group comprised the base feed without extract supplementation The proximate compositions of the feeds tested are presented in Table Four feed treatment groups of fish (fish fed commercial feed, group C; fish fed feed supplemented with three elevated dosages of brewerÕs yeast extract – 20 g kg)1 feed – group N2; 40 g kg)1 feed, group N4; and 60 g kg)1 feed, group N6), each in three replicates (n = 3), were reared for 56 days The feed was delivered with automatic band feeders (4305 FIAP; Fish Technic Gmbh, Ursensollen, Germany) for 18 h per day (10:00–04:00) The daily feed ration was from 1.3% of the stocked biomass (first weeks of rearing) to 1% of the stock biomass (last weeks of rearing) Table Proximate composition (g kg)1 of dry weight) of experimental diets Diets Crude protein Crude lipids NFE2 Crude fibre Crude ash Gross energy (MJ kg)1 feed)3 Total nucleic acids4 C N2 N4 N6 512.5 174.4 201.0 19.0 93.1 22.5 0.0 512.7 169.9 202.8 19.1 95.5 22.4 1.1 507.4 165.2 214.5 18.7 94.2 22.3 2.2 497.9 159.9 232.2 18.2 91.8 22.1 3.3 In NuProâ the crude protein content is around 470 g kg)1 (manufacturer data) Nitrogen free extracts = 100 ) (crude protein + crude lipid + crude fibre + crude ash) (Shearer 1994) Gross energy calculated from the chemical composition using the following energy conversion factors: 24 kJ g)1 proteins, 39 kJ g)1 lipids and 17 kJ g)1 NFE (Jobling 1994) Nitrogen in NuProâ yeast protein is present in nucleic acids (manufacturer data) Aquaculture Nutrition 18; 457–464 Ó 2012 Blackwell Publishing Ltd Body weight (W ± 0.01 g) and total length (TL ± 0.1 cm) measurements were taken on all fish at the beginning and end of the experiment (day 56) Rearing indices were calculated with the following formulas: daily growth rate (DGR, g day)1) = (Wf – Wi) · T)1; specific growth rate (SGR, % day)1) = 100 · [(lnW2 – lnW1) · t)1]; condition factor (CF) = (W · 100) · TL)3; feed conversion ratio (FCR) = TFI · (FB – IB))1; protein efficiency ratio (PER) = (FB – IB) · TFP)1 (n = 3; 35 fish tank)1) At the end of the experiment, 15 fish from each replicate were randomly selected (n = 15; fish tank)1), and their viscera (±0.01 g) and liver (±0.001 g) were weighed to determine the hepatosomatic index (HSI, %) = 100 · (LW · W)1) and the viscerosomatic index (VSI, %) = 100 · (VW · W)1), where Wi – initial mean body weight (g); Wf – final mean body weight (g); T – rearing time (day); W – body weight (g); TL – total length (cm); FB – final stock biomass (g); IB – initial stock biomass (g); TFI – total feed ingested (g); TFP – total feed protein (g); LW – liver weight (g); and VW – viscera weight (g) On day 56 of the experiment, the livers and digestive tract mid-sections were excised from seven randomly selected individuals from each dietary treatment (n = 7; 2–3 fish tank)1) and subjected to histological analyses The tissues were fixed with BouinÕs solution, cleared in xylene and embedded in paraffin blocks, which were then sliced into 5-lm sections with a rotation microtome (Leica, Bensheim, Germany) The preparations were stained with haematoxylin and eosin (H&E) and analysed under a light microscope (Nikon E600, Tokyo, Japan) MultiScanBase v 8.08 (Computer Scanning System Ltd., Warsaw, Poland) was used for viewing the preparations and measuring the structures The histological measurements were analysed (10 consecutive, adjacent, non-overlapping fields per individual sample) according to the procedures described in previous studies (Kowalska et al 2010) At the conclusion of the experiment, blood was drawn from the caudal veins of ten randomly selected individuals from each dietary treatment variant (n = 10; 3–4 fish per tank) After centrifuging (5000 g, 10 min) the samples with the Aquaculture Nutrition 18; 457–464 Ó 2012 Blackwell Publishing Ltd kinetic method (IFCC; PTH Hydrex, Warsaw, Poland), the blood plasma biochemical parameters were determined: aspartate aminotransferase activity AST (IU); ALT (IU); and ALP (IU) Bilirubin levels (mM) and the activity of ceruloplasmin (IU) were determined with spectrophotometry (Rice et al 1986) Biochemical tests carried out with colorimetric methods were performed using an EPOL spectrophotometer (Poll-Ltd., Warsaw, Poland) (Svobodova et al 1986; Siwicki et al 2003) Samples of blood and pronephros were taken (56 day) from ten individuals from each dietary treatment with the aim of isolating leucocytes Blood samples were diluted : in RPMI 1640 medium (Sigma-Aldrich, St Louis, MO, USA) without Ca2+/Mg2+(Biomed, Warszawa, Poland) The pronephros was removed aseptically and pushed through a 60-lm nylon mesh with RPMI 1640 medium (Sigma) with L-glutamine and heparin (Biomed) Leucocytes were isolated by density gradient according to Rowley (1990) Briefly, separating mixture was prepared by building up : of Gradisol L (1.077 mg L)1; Aqua-Medica, Ło´dz´, Poland) and Gradisol G (1.115 mg L)1; Aqua-Medica) After centrifugation (1000 g, 40 min, 10 °C), two bands were formed, the first one rich in lymphocytes and the second one rich in neutrophils and macrophages/monocytes The cells from each band were collected and washed twice in culture medium to attain maximum purity and then diluted to a concentration of · 106 cells mL)1 in RPMI-1640 (Sigma) with the addition of hepes, L-glutamine and 10% foetal serum (FCSGibco, Berlin, Germany) Cell counts and viability testing by the dye exclusion method (0.1% trypan blue) were performed in a Bu¨rker chamber The metabolic activity of phagocytes was determined with respiratory burst activity (RBA) after stimulating the cells with phorbol myristate acetate (PMA; Sigma) according to the method described by Secombes (1990) The phagocytic activity of polymorphonuclear and mononuclear cells was determined with spectrophotometry using the potential killing activity (PKA) test described by Rook et al (1985) The proliferative response of lymphocytes stimulated with concanavalin A (ConA; Sigma) and lipopolysaccharides (LPS; Sigma) mitogens was determined using spectrophotometry with the MTT test described by Mosmann (1983) Extinction was read at a wavelength of 620 nm The content of total protein and total immunoglobulin (Ig) levels in serum was determined with the spectrophotometric method according to the method described by Siwicki et al (1994) Lysozyme activity in serum was did not have an impact on fish growth, final condition factor (CFf), FCR or the PER (P > 0.05; Table 2) The values of the hepatosomatic (HSI) and viscerosomatic (VSI) indexes were also similar in all of the groups analysed (P > 0.05; Table 2) determined with the turbidimetric method (Parry et al 1965) with modifications in fish species by Studnicka & Siwicki (1986) The proximate composition of the fish was determined at the conclusion of the experiment in five individuals from two tanks of each dietary treatment (n = 2) Whole fish from one tank were combined and analysed together The proximate composition of the feed was also determined Protein content was determined with the Kjeldahl method using a nitrogen to protein conversion factor of 6.25 Lipid was determined with the Soxhlet method, and ash content was identified by burning samples at a temperature of 550 °C [Association of Official Analytical Chemists (AOAC) 1975] The content of raw fibre was also determined (Tecator Fibertec System M 1020, Ho¨gana¨s, Sweden) The mean size of the hepatocytes and hepatocyte nuclei of the fish from the four dietary treatments was similar (P > 0.05, Table 3) No significant differences among groups were noted in mean intestinal muscularis thickness, mucosal fold height, height of the intestinal epithelial cells or their nuclei (P > 0.05, Table 3) However, the height of the supranuclear zone in the intestinal epithelial cells was significantly higher in groups N4 and N6 in comparison with the control group (P < 0.05, Table 3) The results of the study were analysed statistically using the GraphPad Prism program (GraphPad Software Inc., La Jolla, CA, USA) Differences between groups were tested with single-factor analysis of variance ANOVA When statistically significant differences were noted (P £ 0.05), TukeyÕs post hoc test was applied Supplementing feed with yeast extract did not have a significant impact on the final contents of water, lipid, protein or ash of the fish (P > 0.05, Table 4) The fish that received the two highest extract concentrations in their diets (N4 and N6) exhibited significantly lower AST and ALT activity in comparison with the control group (P < 0.05, Table 5) The levels of alkaline ALP, ceruloplasmin and bilirubin activity were similar in all of the dietary treatment groups (P > 0.05, Table 5) No deaths were noted in any of the dietary groups during the rearing period The feed supplemented with yeast extract Dietary treatments C Initial body weight (g) Final body weight (g) Initial total length (cm) Final total length (cm) Daily growth rate (g day)1) Specific growth rate (% day)1) Initial condition factor Final condition factor Feed conversion ratio Protein efficiency ratio HSI (%) VSI (%) 37.5 74.9 16.6 20.4 0.67 1.24 0.81 0.86 0.93 2.09 0.86 8.65 N2 ± ± ± ± ± ± ± ± ± ± ± ± 3.94 6.77 0.48 0.52 0.05 0.05 0.03 0.02 0.04 0.17 0.25 1.21 34.7 69.8 16.1 20.1 0.63 1.25 0.82 0.85 0.92 2.11 0.82 7.86 N4 ± ± ± ± ± ± ± ± ± ± ± ± 1.59 3.62 0.33 0.40 0.04 0.03 0.04 0.05 0.02 0.13 0.21 0.89 N6 38.0 73.6 16.5 20.3 0.64 1.18 0.84 0.87 0.99 2.00 0.76 7.89 ± ± ± ± ± ± ± ± ± ± ± ± 2.43 3.86 0.37 0.34 0.03 0.03 0.01 0.02 0.03 0.08 0.13 1.20 36.9 70.1 16.4 20.2 0.59 1.14 0.82 0.84 1.02 1.97 0.83 7.73 ± ± ± ± ± ± ± ± ± ± ± ± 1.18 1.62 0.10 0.11 0.02 0.03 0.01 0.02 0.03 0.06 0.23 1.30 Table Rearing parameters of pikeperch fed experimental diets for 56 days (mean ± SD, n = and n = 15 for VSI and HSI) HSI, hepatosomatic index; VSI, viscerosomatic index There were no significant differences between experimental groups for any parameter (P > 0.05) Aquaculture Nutrition 18; 457–464 Ó 2012 Blackwell Publishing Ltd Table Histological morphometrics of liver and gut samples of pikeperch fed experimental diets (mean ± SD, n = 7) Dietary treatments Morphometric data C N2 Size of hepatocyte (lm) Size of nuclei (lm) Hepatonuclei index Muscularis thickness (lm) Height of mucosal fold (lm) Height of enterocytes (lm) Height of supranuclear zone (lm) Size of nuclei (lm) 13.7 4.5 0.3 143.0 474.0 29.3 11.3 4.8 ± ± ± ± ± ± ± ± 0.8 0.2 0.0 39.5 39.5 4.7 2.2 0.2 N4 13.8 4.7 0.3 152.5 491.8 28.6 12.6 4.9 ± ± ± ± ± ± ± ± 1.0 0.1 0.0 38.9 61.4 3.0 1.1 0.1 N6 13.3 4.5 0.3 147.6 500.2 29.8 14.3 4.8 ± ± ± ± ± ± ± ± 0.9 0.2 0.0 25.3 62.1 2.7 0.8* 0.2 13.2 4.5 0.4 145.1 505.9 31.0 13.3 4.8 ± ± ± ± ± ± ± ± 1.1 0.2 0.1 19.4 31.1 1.4 0.8* 0.2 *Significant differences between groups (P < 0.05) Table Proximate composition (g kg)1 of dry weight) of whole bodies of juvenile pikeperch fed experimental diets (mean ± SD, n = 2) Dietary treatments C Dry matter Protein Lipids Ash 298.7 177.1 92.5 34.3 N2 ± ± ± ± 1.8 0.1 1.3 0.2 286.3 176.1 79.6 34.3 N4 ± ± ± ± 0.6 0.1 0.4 0.0 276.5 171.8 76.0 31.7 N6 ± ± ± ± 0.0 0.1 0.4 0.1 285.3 174.7 81.4 31.8 ± ± ± ± 0.1 0.1 0.4 0.1 non-specific humoral defence mechanisms presented by lysozyme, total protein and total immunoglobulin (Ig) levels in serum are presented in Table Significantly, higher lysozyme and Ig activity in the blood serum was confirmed in the experimental groups of fish fed diets supplemented with 40 and 60 g of NuProÒ kg)1 feed (P < 0.05) Significant differences were not noted among the control and experimental groups only with regard to protein levels (P > 0.05) There were no significant differences between experimental groups for any parameter (P > 0.05) The influence of brewerÕs yeast extract on the non-specific cellular defence mechanisms in healthy pikeperch is presented in Table The analyses of the results showed that NuProÒ administered in doses of 40 and 60 g kg)1 feed statistically significantly activated the metabolic activity (RBA) of blood phagocytes and pronephric macrophages (P < 0.05) Increases were also noted in the intracellular PKA of the blood and pronephric phagocytes in groups N4 and N6 (P < 0.05; Table 6) In the two highest doses, NuProÒ statistically significantly stimulated blood and pronephric lymphocyte T stimulated by ConA and lymphocyte B stimulated by LPS as compared with the control group (P < 0.05; Table 6) The Supplementing diets with yeast extract did not significantly impact the growth rates of juvenile pikeperch Morphometric analysis of the intestines indicated a significant increase in the supranuclear zone of enterocytes in fish fed diets supplemented with 40 and 60 g of NuProÒ kg)1 feed (equivalent to 2.2 and 3.3 g nucleotides kg)1 feed) Such changes in intestinal morphology result in greater cell absorption activity and better digestion of nutrients in the intestine, which usually leads to improved growth performance and feed utilization Presumably, in the present study, the growth-enhancing effect might be masked, because the pikeperch were fed a high-quality, high-protein diet throughout the feeding trial Generally, the high demand for nucleotides occurs during periods of rapid growth (Carver 1994); thus, the growthpromoting effect after nucleotide supplementation on most Table Biochemical parameters of the blood of juvenile pikeperch fed experimental diets (mean ± SD, n = 10) Dietary treatments C Aspartate aminotransferase (IU) Alanine aminotransferase (IU) Alkaline phosphatase (IU) Ceruloplasmin (IU) Bilirubin (mM) 12.1 7.2 2.6 28.9 5.1 N2 ± ± ± ± ± 1.3 1.5 0.5 2.2 0.8 *Significant differences between groups (P < 0.05) Aquaculture Nutrition 18; 457–464 Ó 2012 Blackwell Publishing Ltd 10.8 6.0 2.5 31.4 5.5 N4 ± ± ± ± ± 1.8 1.4 0.4 2.4 0.6 9.8 5.1 2.6 27.4 5.0 N6 ± ± ± ± ± 1.2* 0.9* 0.6 2.0 0.7 10.0 5.4 2.9 29.1 5.2 ± ± ± ± ± 1.1* 0.8* 0.5 2.3 0.5 Table Influence of experimental diet on the non-specific cellular and humoral defence mechanisms in pikeperch (mean ± SD, n = 10) Dietary treatments C Non-specific cellular immunity Metabolic activity of blood phagocytes (RBA; OD 620 nm) Metabolic activity of pronephric macrophages (RBA; OD 620 nm) Potential killing activity of blood phagocytes (PKA; OD 620 nm) Potential killing activity of pronephric phagocytes (PKA; OD 620 nm) Proliferative response of blood lymphocytes stimulated by ConA Proliferative response of pronephric lymphocytes stimulated by ConA Proliferative response of blood lymphocytes stimulated by LPS Proliferative response of pronephric lymphocytes stimulated by LPS Non-specific humoral immunity Lysozyme activity (mg L)1) Total protein level (g L)1) Total Immunoglobulin (Ig) level (g L)1) 0.34 0.45 0.31 0.35 0.40 0.43 0.32 0.36 N2 ± ± ± ± ± ± ± ± 0.03 0.04 0.03 0.04 0.05 0.04 0.03 0.05 41.0 ± 3.1 63.5 ± 5.5 14.5 ± 1.0 0.35 0.47 0.33 0.38 0.43 0.46 0.35 0.39 N4 ± ± ± ± ± ± ± ± 0.04 0.03 0.04 0.05 0.03 0.04 0.05 0.04 41.5 ± 3.0 62.8 ± 5.8 14.9 ± 0.9 0.44 0.52 0.40 0.43 0.49 0.53 0.38 0.46 N6 ± ± ± ± ± ± ± ± 0.05* 0.04* 0.03* 0.03* 0.04* 0.04* 0.03* 0.04* 45.3 ± 1.8* 63.5 ± 4.3 16.1 ± 0.8* 0.43 0.50 0.39 0.41 0.46 0.52 0.37 0.44 ± ± ± ± ± ± ± ± 0.03* 0.03* 0.03* 0.04* 0.03* 0.03* 0.03* 0.03* 44.2 ± 1.4* 63.5 ± 4.9 15.7 ± 0.9* LPS, lipopolysaccharides; RBA, respiratory burst activity *Significant differences between groups (P < 0.05) juvenile or sub-adult fish does not occur (Li et al 2004) It is plausible that using brewerÕs yeast extract during the larval stage might improve pikeperch growth indexes The analysis of the proximate composition of pikeperch did not show any significant changes when the feed was supplemented with NuProÒ The lack of differences in wholebody composition of fish concurs with observations of other authors who analysed brewerÕs yeast protein or nucleic acid supplementation (Takii et al 1999; Peres & Oliva-Teles 2003) The VSI, HSI and the sizes of hepatocytes and their nuclei were also unaffected BrewerÕs yeast extract supplementation to the base feed did not interfere with the metabolism or the deposition of nutrients in pikeperch tissues Additionally, the biochemical assays of the blood of pikeperch indicated that none of the yeast extract dosages applied in the current study compromised liver, gut or kidney function The level of bilirubin, ceruloplasmin and ALP remained at similar levels in all dietary treatment groups The lower levels of the transaminases AST and ALT were noted in groups with the two highest doses of NuProÒ, which may indicate improved liver function (Metwally 2009) The beneficial effects of dietary nucleotides on hepatocytes in mammals have been more widely investigated, and nucleotides were found to improve hepatic function and promote earlier restoration of nitrogen balance following liver injury (reviewed by Sauer et al 2009) According to the authorsÕ knowledge, this paper is the first report of the positive effect of dietary nucleotides on serum enzymes in fish NuProÒ in doses of 40 and 60 g kg)1 feed strongly stimulated all of the analysed parameters of non-specific (innate) cellular immunity in pikeperch Both doses increased the metabolic and potential killing activities of the phagocytes and the proliferative responses of T and B lymphocytes Increases in the values of two indicators of non-specific humoral immunity, that is, lysozyme activity and overall Ig levels, were also noted in these groups Other research also has shown that exogenous nucleotides can influence both humoral and cellular components of the innate immune system of common carp (Sakai et al 2001) and hybrid striped bass (Li et al 2004) The innate immune system of fish provides the first line of immune defence When fish encounter pathogenic micro-organisms, the non-specific defence mechanisms are initially more important than the specific (adaptive) immune response, because the latter requires a longer time for antibody build-up and specific cellular activation (Anderson 1992) The stimulatory mechanism of nucleotide activity on fish immunity has yet to be fully explained; however, it appears likely it is linked to the great demand of intensively proliferating immune system cells for nucleotides (Gil 2002) It has been demonstrated that, like in the majority of tissues, immune cells are incapable of synthesizing nucleotides de novo and are dependent on assimilating exogenous nucleotides from the diet (Quan 1992) The current study indicated that brewerÕs yeast extract has strong immunostimulant properties and can be used as a source of nucleotides The optimal dose determined for pikeperch is 40 g of NuProÒ kg)1 feed (containing approximately 2.2 g exogenic nucleotides kg)1 feed) Additionally, NuProÒ can be added in this dosage to practical fish diets (with high-protein content) without adverse effects on fish metabolism or tissues Future studies will focus on determining the optimal dose for stimulating the antibacterial and Aquaculture Nutrition 18; 457–464 Ó 2012 Blackwell Publishing Ltd antiviral protection of pikeperch in intensive systems of culture to reduce mortality caused by infectious diseases The authors would like to extend their sincere thanks to the Alltech Inc for supplying NuProÒ yeast extract and funding biochemical analysis of proximate composition The authors would also like to thank John Sweetman for his interest and support of this work and El_zbieta Ziomek for her technical assistance with the histological studies The study was 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