Aquaculture nutrition, tập 18, số 4, 2012

Growth wassignificantly greater in trials offered diets containing 365 g kg 1 protein with fish meal as protein source and 205 g kg 1crude protein entirely of non-animal material.The aut

1 2 1 1

Department of Biology, American University of Beirut, Beirut, Lebanon; 2 Department of Fisheries and Allied Aquaculture,Auburn University, Auburn, AL, USA

Redclaw crayfish (Cherax quadricarinatus, von Martens

1868) is a freshwater decapod crustacean with a number of

biological and commercial attributes that make it an

excel-lent 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

devel-oping 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

lim-ited 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

con-taining low-cost, plant-based ingredients without

compro-mising 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

Aquaculture of the Australian redclaw crayfish Cherax ricarinatus(von Martens 1868) is developing rapidly in tropi-cal and some temperate regions of the world Webster et al.(2002) stated that aquaculture of the species was mainlyrestricted to North-Eastern Australia, but redclaw aquacul-ture has expanded into South-East Asia and Central/SouthAmerica and production is no longer restricted to Oceania.The species grows well when offered diets developed forother crustaceans, but nutritional requirement data specificfor redclaw have not been determined As culture methodol-ogy shifts from extensive and semi-intensive ponds into moreintensive systems and as hatchery production becomes morecommon, we will need to develop species-specific feed formu-lations (Huner et al 1994; Medley et al 1994; Webster et al

quad-1994, 2002; Curtis & Jones 1995) These diets should be lessexpensive than traditional shrimp feeds, offer a completenutrient profile to the animal, be based on sustainablesources of raw ingredients and be available wherever theindustry decides to grow The present manuscript reviewsknown nutritional requirements of redclaw crayfish based onexisting literature and the experience of the authors

In natural ecosystems, crayfish have polytrophic feedinghabits 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)

sug-gested that in general Cherax species are primarily

detriti-vores, a statement supported by the findings of

Loya-Javellana et al (1993) who reported that C quadricarinatus

demonstrates an ontogenetic shift from non-selective

feed-ing 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

for-mulated flake diets (400 g kg 1 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

red-claw 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

con-nects 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

ven-tro-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 thedistal tips of the each hepatopancreatic tubule and differen-tiate giving rise to R-cells and F-cells (Dall & Moriarty1983; Ceccaldi 1997; Verri et al 2001) R-cells havemicrovilli and also contain lipid droplets and glycogen, andtheir primary role is storage (Dall & Moriarty 1983; Cec-caldi 1997) F-cells (fibrillar cells), similar to R-cells, havemicrovilli that might contribute to absorption These cellssecrete and synthesize digestive enzymes and differentiateinto B-cells (Dall & Moriarty 1983; Ceccaldi 1997) B-cells(blister cells) are associated with protein synthesis andenzyme secretion (Verri et al 2001) Another type of cellsfound in some crustaceans is the M-cells (midget cells) thatmight be involved in nutrient absorption and storage (Cec-caldi 1997; Guillaume & Choubert 2001) The midgut, notlined by chitin, begins at the posterior end of the stomachand extends throughout the abdomen terminating at theanus The hindgut is almost straight and impregnated withchitin, enlarging posteriorly into the rectum and terminates

at the anus (see Ceccaldi 1997)

Loya-Javellana et al (1995) measured the effect of mal size and feeding frequency on the foregut evacuationrates of redclaw Results indicated that evacuation ratesdid not differ significantly between size groups (medium,large) nor between feeding frequency groups (fed daily, fedevery second day) However, the model specifications dif-fered between feeding frequencies, i.e ingesta was evacu-ated linearly with time in the crayfish fed daily andaccording to a curvilinear pattern in those fed every secondday, implying that crayfish are potentially capable of regu-lating their digestive processes according to food availabil-ity Moreover, the return of appetite in redclaw is rapid;

ani-the average return of appetite increased to >50% of thesatiation meal at 5–10 h postfeeding, when the residuum ofthe previous meal was ca 60% or less The authorsreported that based on these results, redclaw can resumefeeding before a considerable proportion of an earlier meal

is processed in the foregut, suggesting that the species iscapable of optimizing the frequency of feeding duringactive foraging periods

A variety of digestive enzymes including proteases,

lipas-es and carbohydraslipas-es are found in the midgut gland topancreas) and gastric fluid of crayfish (Zwilling &

(hepa-Neurath 1981; Brown 1995a; Hammer et al 2000) ing redclaw (Figueiredo et al 2001) Digestive enzymes aresynthesized and secreted into the digestive tract by F- andB-cells in the midgut gland (Ceccaldi 1997; Verri et al

includ-2001) The presence of a variety of enzymes in juvenile

red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- red- 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;

Figuei-redo 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

lar-val 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

prote-ase, astacin, chymotrypsin and exopeptidases [e.g

carboxy-peptidases (A and B)] and aminocarboxy-peptidases (New 1976;

Vogt et al 1989; Brown 1995a; Ceccaldi 1997; Guillaume

1997; Navarrete del Toro et al 2006; Figueiredo &

Ander-son 2009) However, it is generally accepted that most

crus-taceans 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

(gas-tric 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

lev-els 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

juve-niles 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

carbox-ylic 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

diges-tion capabilities Thus, some of the major carbohydrases

(amylases, laminarinases, chitinases) are found in the

diges-tive system of many crustaceans (Dall & Moriarty 1983;

Ceccaldi 1997) The activity of some of these carbohydrases

is age dependant (Figueiredo & Anderson 2003) andchange with developmental stages of redclaw For example,amylase and laminarinase activities are significantly greater

in large C quadricarinatus than at other stages, whereasprotease activities decreased as the species grew The carbo-hydrases detected in the midgut gland and gastric fluid ofadult C quadricarinatus also include a-amylase (EC3.2.1.1), laminarinase (EC 3.2.1.6/EC 3.2.1.19), maltase(EC 3.2.1.20) and several para-nitrophenyl glycosidases (Fi-gueiredo 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 carbohydraseswould suggest that redclaw should be able to obtain a sub-stantial amount of their metabolic energy needs from car-bohydrates, yet research suggests that only a relativelysmall portion of their energetic needs are obtained fromcarbohydrates (see Pavasovic et al 2006; Garza de Yta

et al 2012) Additional work on carbohydrate digestibilityand assimilation by redclaw is warranted before definitivestatements can be made

Some crustaceans have been reported to possess

cellulas-es (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 cellulosenutritively Enzymatic hydrolysis of cellulose to glucosegenerally requires the synergistic action of three distinctclasses of cellulase enzymes: endoglucanases (endo-1,4-b-glucanases (EC 3.2.1.4) that cleave randomly internalb-1,4-glucosidic bonds; exoglucanases (exo-1,4-b-glucanases(EC 3.2.1.91) that cleave the disaccharide cellobiose fromthe non-reducing ends of the cellulose chains; and cellobias-

es (b-glucosidases, EC 3.2.1.21) that hydrolyse the ose to glucose (Wood 1985; Walker & Wilson 1991;Woodward 1991; Be´guin & Aubert 1994) Generally, higheranimals do not produce endogenous cellulases, but thepresence of symbiotic microorganisms in their alimentarytracts produces the necessary enzymes for cellulose diges-tion (Watanabe & Tokuda 2001)

cellobi-The occurrence of cellulase in the midgut gland and tric 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 interest-ing Cellulose, the principal constituent of most plant cellwalls, is known as the most abundant organic compoundand renewable energy source on earth (Aspinall 1980;BeMiller 2008) Although the idea of using an abundant

gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas- gas-.

Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd

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

popula-tions (up to 94%), suggesting that the activity is innate in

the crayfish and not in microbial symbionts Redclaw

cellu-lase 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

prefer-ence for soluble substrates (Xue et al 1999) The

occur-rence and activity of cellulase in C quadricarinatus is

consistent with the feeding behaviour of redclaw, which

consume significant amounts of plant materials and

decom-posing bacteria, fungi and animals (Byrne et al 1999; Xue

et al 1999) Byrne et al (1999) isolated an

endo-1,4-beta-glucanase cDNA sequence (termed CqEG) from the

hepa-topancreas 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

endog-enous multigene glycosyl hydrolase family 9 in redclaw

indicates that partial breakdown of plant cell

polysaccha-rides is a significant evolutionary strategy for the species

Results of their study suggested the presence of two

func-tional 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

cray-fish However, a study conducted by Pavasovic et al

(2006) indicated that the presence of cellulase (higher

activ-ity 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 do not increase

visco-sity of intestinal contents upon digestion, which in

turn would slow the passage of materials through the gut

(Pavasovic et al 2006) The authors concluded thatalthough cellulase activity is present in redclaw, there are

no detectable nutritive benefits of including insoluble lose (a-cellulose) in diet formulations of the species

cellu-In addition to proteases, lipases and carbohydrases,endonucleases probably also exist in redclaw Endonucleaseactivity has been reported in the digestive tract of variousother invertebrates including annelids, molluscs, echino-derms and arthropods (chelicerates, insects and crusta-ceans) (Yokoe & Yasumasu 1964; see also Watanabe &

Tokuda 2001 and references therein; Linton et al 2006)that are also probably members of the arsenal of digestiveenzymes in redclaw guts, but have yet to be isolated

Information derived from studies on biochemical sition and digestive enzyme activities on utilization of yolkduring embryonic development may provide some clues ofthe nutrient requirements for the embryos and thereforecan be used in understanding nutritional requirements ofbrood stock (Yao et al 2006; Luo et al 2008a) Luo et al

compo-(2008a) studied five digestive enzymes (trypsin, pepsin,lipase, amylase and cellulase) in embryonic redclaw, andall showed changes in enzymatic activity closely correlatedwith morphogenesis, hydrolysing the yolk and providingconstruction substances and energy resources for formation

of tissues, organs and various systems The activities of thedigestive enzymes were controlled by their genes andexpressed sequentially during development Specific activi-ties of pepsin and trypsin increased during early stages ofembryonic development, but pepsin activity decreased inlater stages (stage VI), while trypsin remained at high level

of activity (Luo et al 2008b) Furthermore, chymotrypsinactivity peaked in stage IV and then decreased significantlyduring the last stage of embryonic development Low levels

of lipase activity were also reported during embryonicdevelopment of redclaw (Luo et al 2008a) Specific activity

of amylase changed in a ‘V’ curve, increasing during laterstages (stage VI) Cellulase activity during embryonicdevelopment in redclaw was relatively low (Luo et al

2008a)

Research on the nutritional requirements and practical dietformulations for redclaw increased rapidly as the culture ofthe species became established with further advances occur-ring in the 21st century Dietary requirements of somenutrients have been determined for rapidly growing juve-niles only, with limited information for larger redclawapproaching 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

defi-ciencies in manufactured diets Currently, diets for the

commercial production of redclaw are based on

formula-tions 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;

Thomp-son 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

formu-lated diets with 200–300 g kg 1 crude protein and 50–

100 g kg 1lipids, 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

physio-logical stage and size, dietary characteristics of protein

quantity and quality (e.g digestibility), amount of

non-protein 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

dif-ferences in feeding habits between adult and juvenile

cray-fish have been attributed to slower growth of adult craycray-fish

and therefore lower protein requirements than in faster

growing juveniles (Lodge & Hill 1994)

Several studies have attempted to determine proteinrequirements of juvenile and preadult C quadricarinatusreared indoors or outdoors (see Table 1) Anson & Rouse(1996) evaluated growth response and survival of newlydetached (0.01 g) redclaw offered various commercial feeds(shrimp feed, catfish feed with or without Artemia naupliisupplement) ranging in protein content from a 320 g kg 1protein catfish diet to a 400 g kg 1 shrimp diet The

400 g kg 1shrimp diet resulted in best growth for the mals D’Agaro et al (2001) evaluated the dietary proteincontent (240 g kg 1 and 290 g kg 1; gross energy: 20.0–20.4 MJ kg 1) on growth performance of juvenile C quad-ricarinatus reared in a recirculating system No significantdifferences in growth were reported among treatments,probably because of protein-sparing effects from otherenergy sources Meade & Watts (1995) offered 0.01 g red-claw a number of commercially available formulated dietsand found that a 300 g kg 1crude protein, 100 g kg 1fatfeed provided best weight gain and survival as compared toall other treatments However, the authors note that suchfeeds do not provide complete nutritional needs of crayfish.Jones & Ruscoe (1996a) evaluated growth performance

ani-of juvenile redclaw in glass aquaria ani-offered five formulateddiets (four commercial formulations and one experimentalreference formulation) and one natural diet containingcrude protein ranging from 100–447 g kg 1 Growth wassignificantly greater in trials offered diets containing

365 g kg 1 protein (with fish meal as protein source) and

205 g kg 1crude 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 thatthey can be successfully cultured on a diet primarily com-posed of material of plant origin Similarly, Thompson

et al (2005) examined the growth performance of juvenileredclaw offered formulated practical diets containingincreasing percentages of dietary protein (300, 350 and

400 g kg 1) They found that juvenile redclaw can beoffered a 350 g kg 1 protein formulated practical diet with

a combination of plant-protein ingredients if fishmeal isexcluded

Natural food and forage can also supplement formulateddiets and spare proteins in the prepared feed Metts et al.(2007) reported that juvenile redclaw stocked semi-inten-sively and offered forage at a rate of 500 kg ha 1month 1may be able to utilize 130 g kg 1 protein diets Thompson

et al (2006) reported that juvenile redclaw offered dietscontaining 280 g kg 1 crude protein with or without fishmeal had significantly greater weight gain compared to red-claw offered 180 g kg 1crude protein with or without fish

.

Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd

meal (778% and 799%, respectively) They concluded that

pond-cultured redclaw performed well when offered diets

with 280 g kg 1 protein inclusion even if devoid of

fish-meal

Hernandez et al (2001) studied the effect of eighteen

iso-caloric (417.4–422.8 kcal 100 g 1

) diets containing six levels

of protein inclusion (250, 300, 350, 400, 450 and 500 g kg 1)

each at lipid levels of 40, 80 and 120 g kg 1, on growth and

survival of hatchling and juvenile redclaw reared under

con-trolled conditions The authors concluded that diets

contain-ing 300–350 g kg 1

protein (40–80 g kg 1

lipid) result inbest growth performance for both size classes Manomaitis

(2001) offered juvenile redclaw diets with various protein

inclusion levels (250–400%) for 7 weeks Final weight,

spe-cific 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 kg 1crude protein should be offered to newly released

redclaw, whereas for larger juveniles, a diet containing

300 g kg 1protein 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 kg 1) of

dietary protein and with 18.73–21.45 kJ g 1

gross energy(protein to energy ratio: 10.7–25.6 mg kJ 1

) Results showedthat highest mean weight (9.6 g) and SGR (3.64% day 1)

were achieved by offering a diet containing 310 g kg 1crude

protein The optimum dietary protein requirement,

calcu-lated from using a second-order polynomial (y= 1.142 +

0.484 0.0071x2, r2= 0.952), was 342 g kg 1

Similarresults were achieved by a later study conducted by Corte´s-

Jacinto et al (2005) determining the effect of various protein

(260, 310 and 360 g kg 1) and lipid (40, 80 and 120 g kg 1)

levels, with gross energy content of 17.5–19.4 kJ g 1

, ongrowth of juvenile C quadricarinatus Best growth was

observed when using dietary protein inclusion of 310 g kg 1

(80 g kg 1crude lipid) with gross dietary energy content of

19.69 kJ g 1 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

con-taining 310 g kg 1crude protein satisfy nutritional

require-ments 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 kg 1 protein diet stimulates

antioxidant response of superoxide dismutase (SOD) (SOD

is a cytosolic enzyme specific for scavenging superoxideradicals and is involved in protective mechanisms withininjured tissues following oxidative processes and phagocy-tosis) of juvenile redclaw

For earthen pond culture, it is not necessary to supplyhigh dietary protein because redclaw supposedly obtain asubstantial proportion of their nutrient requirements fromnatural food materials in the pond (Jones 1990; Jones &Ruscoe 1996b) Jones & Ruscoe (1996b) stocked juvenileredclaw in cages in a pond and offered diets containingcrude protein ranging from 100 to 447 g kg 1 Althoughcrayfish offered a reference crayfish diet (205 g kg 1 crudeprotein) grew better than crayfish offered all other diets,the authors suggested that the crayfish did not have adirect use of the feed offered but obtained the bulk of theirnutrition from natural productivity of the pond benthos In

a similar experiment, Thompson et al (2004) found that

220 g kg 1 dietary protein was sufficient for redclaw ture

cul-In other experiments, Pavasovic et al (2007b) reportedmaximum growth of subadult redclaw offered diets con-taining 250 g kg 1crude protein with a strong positive cor-relation between dietary protein and protein content in thetail However, other researchers did not observe a signifi-cant effect of dietary protein on percentage protein in red-claw 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 kg 1or greater protein inclusion are suitable for claw growout in ponds with natural productivity Dietswith 350 g kg 1 protein inclusion or greater are recom-mended for redclaw grown in closed recirculation systems.All diets should have a gross energy content of 18 kJ g 1,minimum These suggestions are supported by Corte´s-Jac-into et al (2004) who propose a minimum protein inclusion

red-in redclaw diets of 220 g kg 1with 15.21 kJ g 1 of ible energy

digest-No discussion of aquatic animal nutrition is completewithout mentioning broodstock diets Broodstock nutrition

is of high importance for successful reproduction and eggquality; adequate nutrients and energy in broodstock dietsare necessary for the onset of gonadal maturation, becausematernal nutrient intake during ovarian development iscritical and influences the composition of ovaries and thenutritional status of eggs Crustacean embryos rely exclu-sively on the nutrients and energy supplied by the egg(yolk) (Harrison 1997) In decapod crustaceans, protein is

a structural, functional and energy constituent of tissuesand plays an important role in spawning, fertilization and

.

Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd

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 kg 1 crude protein and 16.72 kJ g 1

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 kg 1 crude protein diet and

dietary protein levels of 320 g kg 1had 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

con-tent 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 kg 1

crude protein result in maturation offemale redclaw, but a dietary protein range from 284 to

355 g kg 1improved gonadal development and resulted in

more protein production in the hepatopancreas In

previ-ous 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

brood-stock females be offered diets with 350 g kg 1 protein and

a minimum of 18 kJ g 1 gross energy, a part of which

comes from fish oil to supply the necessary omega-3

HU-FAs

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

argi-nine, histidine, isoleucine, leucine, lysine, methioargi-nine,

phen-ylalanine, threonine, tryptophan and valine (D’Abramo &

Robinson 1989; Brown 1995b; Guillaume 1997) plus

aspar-agine 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 corre-lation between the dietary amino acid requirements of aspecies and the pattern of amino acids in whole body tissue(Cowey & Tacon 1983; Wilson & Poe 1985) Consequently,dietary amino acid requirements of growing animals areoften 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 requirementsmight first be deduced from the amino acid composition ofits tissues However, our experience suggests that whenusing body composition as reference of requirement, onewould overestimate dietary requirement of essential aminoacids and underestimate requirement of other protein com-ponents

Muzinic et al (2004) evaluated the amino acid tion of practical diets containing various levels of soybeanmeal (SBM) and brewer’s grains with yeast as replacementsfor fish meal, and results suggested that the amino acid levels

composi-in a 400 g kg 1crude protein diet were adequate for goodgrowth and survival of juvenile redclaw crayfish whicheverprotein source was used Similarly, Thompson et al (2005)noted that a complementary blend of SBM and other plant-protein sources used to replace FM in a 350 g kg 1proteindiet appeared to provide sufficient levels of essential aminoacids to meet requirements of redclaw In pond-cultured red-claw, diets containing 280 g kg 1 crude protein with orwithout fish meal may sufficiently satisfy the requirements ofessential amino acids of male and female redclaw (Thomp-son et al 2006) probably because natural productivity sup-plements the formulated feeds being offered Consequently,and based on their response to diets without fishmeal, onemay assume that methionine and lysine requirements of red-claw are relatively low Such assumptions are yet to beempirically tested

Knowledge of energetic utilization of farmed organisms isnecessary for the development of cost-effective diets

Energy from non-protein sources (lipids, carbohydrates)relative to protein levels must be supplied into diets in suf-ficient amounts to insure that protein is used for tissue syn-thesis as protein is considered the most expensive majorcomponent of crustacean diets (D’Abramo & Robinson1989; Cuzon & Guillaume et al 1997; Cho et al 2005) Ifthe non-protein energy to protein ratio is insufficient, die-tary protein may be catabolized and used as an energysource 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 kJ 1 and a crude protein content between 310 and

350 g kg 1 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 kcal 1 (240 and 294 g kg 1 protein,

respec-tively) 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± 2 mg kJ 1

(Rodrı´guez-Gonza´lez et al 2011) and 17.16 mg kJ 1

(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

nutri-tion as they provide energy and essential fatty acids

(EFAs), sterols, phospholipids and fat-soluble vitamins

nec-essary 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 kg 1 of feed by weight to ensure optimal

growth and survival (D’Abramo 1997) The lipid level

required for optimal growth is influenced by several factorsincluding quality and quantity of protein, availability,quantity and quality of other sources of energy and ade-quate provision of EFAs (D’Abramo 1997) as well as theability of the organism to digest carbohydrates and use glu-cose in its metabolism Lipids are often supplemented inexcess of minimal requirements to spare protein for somaticgrowth Such a protein-sparing effect of lipids was reported

in hatchling and juvenile redclaw offered diets containing

40–80 g kg 1 lipid (300–350 g kg 1 protein) (Hernandez

et al 2001), suggesting that this range of lipid inclusion toredclaw diets is suitable

A few studies investigated dietary lipid requirements ofredclaw under laboratory conditions (Hernandez et al 2001;Corte´s-Jacinto et al 2005; Zenteno-Savı´n et al 2008), andall seem to agree that a diet containing 80 g kg 1 dietarylipid with approximately 300 g kg 1 protein and grossenergy 17.5–19.1 kJ g 1

is suitable for good growth mance of juvenile C quadricarinatus while preventing diet-induced oxidative stress and protecting the integrity of theimmune function

perfor-Although lipids are necessary in redclaw diets, it appearsthat natural productivity can replace dietary lipids to a cer-tain extent Herna´ndez-Vergara et al (2003) evaluated theeffect of different dietary lipid levels (42, 82 and

123 g kg 1) on growth, survival and proximate tion of juvenile redclaw reared semi-intensively in outdoorstanks and observed no effect of treatment on the variousparameters Accordingly, it seems that in the presence ofsome natural productivity, a diet containing 42 g kg 1die-tary lipid (17.58 kJ g 1, 300 g kg 1crude protein) is suffi-cient for growth and survival of juvenile redclaw

composi-Differences in lipid metabolic routes between sexes wherefemales have higher carcass lipid content than males areoften reported (e.g Herna´ndez-Vergara et al 2003) This isgenerally attributed to storage of lipids for ova develop-ment or vitellogenesis Yet, studies on developing adequatediets for maturation of broodstock redclaw are rare (Rodrı´-guez-Gonza´lez et al 2006a,b, 2009a,b) Considering thatlipids are the main energy sources during ontogeny of crus-taceans and also structural components of cell membranes(Holland 1978; Harrison 1997), lack of research on the sub-ject seems surprising However, when one considers theease of collecting egg-bearing females from ponds, oneunderstands the lack of interest in broodstock maintenance.Nonetheless, as the industry grows and biosecurity issuesbecome more important and infectious diseases appear,indoor closed system hatcheries will become necessary andwith them special broodstock diets

.

Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd

According to Rodrı´guez-Gonza´lez et al (2006b), the

lipid requirements for the developing gonad of female

red-claw mainly originates from the diet Rodrı´guez-Gonza´lez

et al (2009b) studied the effect of dietary lipid levels (40,

80 and 120 g kg 1) 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 kg 1lipid diet In a

similar study on the effects of dietary lipids on female

red-claw reproduction, Li et al (2010) observed a significant

correlation between lipid transportation in the

hepatopan-creas 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

(lino-lenic (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

perfor-mance 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

suffi-cient 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 0 g kg 1

supplemental lecithin and/or cholesterol contained a

combi-nation 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

(lin-seed oil, canola oil, corn oil, beef tallow or menhaden oil)

that differ in fatty acid profile on growth response of

juve-nile 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 ofn-3 HUFA when offered to juvenile redclaw reared indoorsand lacking natural productivity The authors concludedthat redclaw can be fed diets containing plant-based oilswith high levels of 18-carbon unsaturated fatty acids Muzi-nic et al (2004) reported that practical diets containingvarious levels of SBM and brewer’s grains with yeast, asreplacements for fish meal, have both n-6 and n-3 highlyunsaturated fatty acids such as linoleic (18:2n-6), eicosapen-taenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids thatmay satisfy the EFA requirements of juvenile redclaw

The fatty acid profile during early embryonic ment of redclaw shows that the major fatty acids, oleic/

develop-vaccenic (18:1), palmitic (16:0), linoleic (18:2n-6) and mitoleic (16:1n-7) remain major during later developmentalstages and are required in larger quantities than other fattyacids (Alimon et al 2003) Monounsaturated fatty acidsconstituted the major moiety of the fatty acid profile, andthe PUFA were dominated by linoleic (n-6) series (low n-3

pal-to n-6 ratio) (Alimon et al 2003) Luo et al (2008a)reported that the predominant fatty acids of both neutraland polar lipids of redclaw during embryonic developmentwere C16:0, C18:0, C18:1n-9 and C18:3n-3

Saturated fatty acids (16:0 and 18:0) and monosaturatedfatty acids (16:1n-7 and 18:1n-9) are generally used forenergetic purposes, whereas PUFA (20:5n-3 and 22:6n-3)are important as structural components of cell membranesand in the development of the central nervous system (Luo

et al.2008a) However, even during vitellogenesis, there arehigh proportions of monounsaturated fatty acids in theovaries and hepatopancreas, suggesting their use as majorsources of energy (Li et al 2010) Such information wouldsuggest that broodstock diets could be formulated to con-tain more vegetable oils to be used for energy during vitel-logenesis without compromising development of eggs,which require some n-3 HUFAs found in expensive butnecessary fish oils

Phospholipids are added to the diet of crustaceans for ous reasons such as a source of energy; a major component

vari-of cell membranes; emulsification vari-of lipid aggregates duringdigestion and absorption; and because they play a majorrole in lipid transportation in the haemolymph (Coutteau

.

Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd

et al 1997; Teshima 1997) Thompson et al (2003a)

evalu-ated the effect of different levels of supplemental soybean

lecithin on growth and survival of juvenile redclaw crayfish

and found no effect The authors suggested that diets

con-taining enough lipids and 5 g kg 1 choline chloride by

weight may be sufficient for redclaw crayfish growth

Simi-lar observations were made by Thompson et al (2003b) in

a study examining growth performance of juvenile redclaw

reared over an 8-week period and offered diets with and

without supplemental lecithin and/or cholesterol

It is generally accepted that crustaceans are unable to

syn-thesize sterols de novo and require an exogenous dietary

source for growth, development and/or survival (see

Teshi-ma 1997; Kanazawa 2001) Cholesterol is the Teshi-major

essen-tial sterol in crustacean nutrition with an important role as

a cell constituent, metabolic precursor of steroid hormones

and moulting hormones (see Brown 1995b; Teshima 1997;

Shiau 1998) Dietary cholesterol requirement for optimal

growth performance in various crustacean species ranges

from 1.2 to 20 g kg 1of the dry weight of a diet (Teshima

1997; Kanazawa 2001)

Studies on cholesterol requirements of C quadricarinatus

are few Herna´ndez et al (2004) evaluated the effect of

die-tary cholesterol on growth and survival of redclaw and

observed no significant differences among treatments of

various cholesterol supplementations but noticed slight

growth increase in redclaw offered a diet with 5 g kg 1

cholesterol inclusion A growth study by Thompson et al

(2003b) whereby they offered C quadricarinatus juveniles

(0.2 g) practical diets with or without supplemental lecithin

and/or cholesterol showed no significant differences in

weight gain among treatments The authors interpreted

their results to suggest that redclaw could be farmed using

feeds less expensive than traditional marine shrimp feeds,

currently used in redclaw culture

Carotenoids, a family of over 600 natural lipid-soluble

pig-ments, are the most universally widespread (e.g bacteria,

algae, plants and animals) and structurally diverse

pigment-ing agents They are synthesized through the isoprenoid

pathway, which also produces diverse compounds such as

EFAs, steroids, sterols and vitamins A, D, E and K (see

Meyers & Latscha 1997; Lin˜a´n-Cabello et al 2002)

Crusta-ceans are unable to synthesize carotenoids de novo and must

obtain them from the diet (Meyers & Latscha 1997) Yet,most crustaceans contain and utilize carotenoid pigments;mainly in the carapace, eyes, blood eggs, midgut gland andovaries (Sagi et al 1995; Meyers & Latscha 1997) Func-tions of carotenoids include pigmentation, sources of provi-tamin A, antioxidants, positive effects on development,growth, maturation, reproduction and enhancement ofimmunity (see Meyers & Latscha 1997) The most commonpigments, derived from diets or from metabolic transforma-tion of precursor carotenoids, are astaxanthin, b-carotene,echinenone and canthaxanthin (Meyers & Latscha 1997).Astaxanthin has been described as the most frequent endproduct of carotenoid metabolism in crustacean (Meyers &Latscha 1997)

Rouse & Rash (1999) reported that astaxanthin added todiets offered to juvenile redclaw resulted in an increase insurvival and growth by 20% However, in a study by Har-paz et al (1998) where the effect of three carotenoidsources (dried alga cells prepared from Dunaliella salina(source of b-carotene), synthetic astaxanthin and alfalfameal) on growth and pigmentation of juvenile C quadrica-rinatuswas evaluated, no significant differences were found

on growth and survival Redclaws receiving enriched diets exhibited better pigmentation than thosereceiving carotenoid-free diets The authors suggested how-ever that adding alfalfa meal and artificial astaxanthin toredclaw diets produce desired body coloration

carotenoid-Carotenoids play an essential role before and after dal maturation processes (reviewed by Lin˜a´n-Cabello et al.2002) Sagi et al (1995) noted that target tissues for carot-enoid accumulation in C quadricarinatus are the ovary andcuticle In a study by Lin˜a´n-Cabello et al (2004), the effects

gona-of carotenoid (b-carotene and astaxanthin) and vitamin Ainjections were correlated with the ontogenic development

of oocytes in female redclaw Their results showed thatretinol palmitate had the greatest inductive effect on theprimary vitellogenic phase and on the indicators of onto-genic oocyte development Accordingly, we deduce thatcarotenoids and retinols are essential nutritive additivesduring gonadic maturation of redclaw and help givingadults a coloration that would help in marketing Addi-tional work should evaluate the effects of dietary carot-enoid supplementation on immune responses and generalphysiology of redclaw

Carbohydrates are generally an inexpensive source ofenergy for crustacean feeds (Shiau 1997) Although carbo-

.

Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd

hydrates are not considered essential nutrients, they are

incorporated into feeds to reduce costs, for their binding

activity during feed manufacturing and possibly for

protein-sparing effects (D’Abramo & Robinson 1989;

Guil-laume & Choubert 2001; Wouters et al 2001; Pillay &

Kutty 2005) Additionally, carbohydrates play several roles

in crustacean metabolism including glycogen storage, chitin

synthesis and the formation of steroids and fatty acids

(Parvathy 1971; Dall et al 1990; Ali 1993; Sa´nchez-Paz

et al.2006)

Crustaceans are able to utilize more complex

carbohy-drates (e.g starch, chitin) than simple sugars (e.g glucose)

(Shiau 1997), yet it is generally accepted that freshwater

crayfish have poor polysaccharide digestibility and are

unli-kely to obtain substantial nutrition from fibrous material

(Shiau 1997; Pavasovic et al 2006) However, dietary

fibres, water-soluble and water-insoluble polysaccharides

delay stomach emptying by increasing viscosity of the diet

(see Shiau 1997 and references therein), thus assisting in

digestion

Some polysaccharides such as carboxymethyl cellulose

cannot be digested even though some cellulase activity was

detected in the gut of redclaw Pavasovic et al (2006)

dem-onstrated cellulase activity in redclaw digestive systems, yet

observed no detectable nutritive benefits of including

insolu-ble cellulose (a-cellulose) in diet formulations for the species

Dietary inclusions of a-cellulose above 120 g kg 1

cantly reduced survival rate, feed conversion efficiencies and

signifi-general growth performance of redclaw Jones & Ruscoe

(1996c) assessed growth performance of redclaw maintained

in earthen ponds and offered five diets containing various

carbohydrate sources (maize, wheat, rice, sorghum, lupin

and barley) No significant differences in survival and growth

were observed among treatments This suggests that the

source of carbohydrate is not of particular importance in

redclaw nutrition In a recent experiment in Mexico, we

assessed the effect of stargrass hay supplementation to

for-mulated feed diets on redclaw growth and found no nutritive

value beyond what the animal obtains from formulated feed

Dietary carbohydrates also do not appear to affect or

improve gonadal maturation (Rodrı´guez-Gonza´lez et al

2006b) Nevertheless, carbohydrates will always constitute a

good portion of formulated redclaw diets because they are

an inexpensive source of energy and filler

Vitamins and minerals are essential micronutrients

neces-sary for normal life processes in crustaceans Deficiencies in

vitamins and/or minerals lead to slower growth, negativelyaffect reproduction and/or eventual mortality in crusta-ceans (Conklin 1997; Davis & Lawrance 1997) Informa-tion on specific vitamin and mineral requirements ofredclaw is scarce to non-existent It is assumed that vitaminand mineral requirements are similar to those of other crus-taceans (D’Abramo & Robinson 1989) However, gonadalmaturation was shown to be affected by vitamin levels

Lin˜a´n-Cabello et al (2004) reported that retinol has a nificant effect in oocyte maturation of C quadricarinatusand is an essential nutritive additive for gonadal matura-tion Luo et al (2004) found that excessive vitamin Eaffected reproduction of C quadricarinatus and that opti-mal content of vitamin E was 192 mg kg 1 Additionally,the authors speculated that vitamin E could protect C20:

sig-5n-3 and C22: 6n-3, necessary for the development of thenervous system, from oxidizing and improved the accumu-lation of important amino acids and fatty acids in fertilizedeggs

The nutritive value of a feed ingredient is based on itschemical composition and on an animal’s capacity todigest, absorb and utilize it Digestibility is the quantity ofthe nutrient or energy in the ingested feedstuff that is notexcreted in the faeces (NRC 1993; Lee & Lawrence 1997;

Guillaume & Choubert 2001) In general, freshwater ceans have higher apparent digestibility efficiency (ADE)and apparent crude protein digestibility (ACPD) values forhigh carbohydrate feeds than marine crustaceans, and bothhave high ACPD values for animal meals and purified pro-teins (Lee & Lawrence 1997)

crusta-Campan˜a-Torres et al (2005) evaluated dry matter andprotein digestibility of juvenile C quadricarinatus offereddiets supplemented with 150 g kg 1 of three plant-derived(soy paste, textured wheat and sorghum meal) and fouranimal-derived (two sardine meals, squid meal and red crabmeal) ingredients They found that plant-derived ingredi-ents and corresponding diets had better digestibility thananimal ingredients (see Table 2) In a subsequent study,Campan˜a-Torres et al (2006) reported that mean carbohy-drate and lipid digestibilities of vegetal ingredients and cor-responding diets were better than carbohydrate and lipiddigestibilities of animal ingredients although some of theanimal ingredients (e.g red crab) had high lipid digestibility(see Table 2) The authors concluded that redclaw are able

.

Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd

to efficiently consume diets containing a variety of

plant-and animal-derived ingredients, with better digestion

effi-ciency of plant-derived ingredients (see also

Campan˜a-Tor-res et al 2008) Similarly, Pavasovic et al (2007a)

evaluated the potential use of several ingredients (fish meal,

meat and bone meal, poultry meal, SBM, canola meal,

lupin meal and brewer’s yeast) in dietary formulations for

adult redclaw Results showed that apparent digestibility of

dry matter, crude protein and gross energy was better for

SBM diets than diets containing meat and bone meal

(Table 2) Accordingly, it seems that redclaw has the

capacity to utilize a broad range of dietary ingredients

including animal, single cell and plant matter in their diet

However, protein digestibility seems to be affected by other

dietary ingredients Supplementation of diets with either

300 g kg 1a-cellulose or Fuller’s earth significantly reduces

apparent dry matter digestibility (ADMD) and apparent

protein digestibility (APD) of redclaw (Pavasovic et al

2006)

Redclaw have the ability to modify their digestive

enzyme secretions in response to different ingredients in the

diet over time (Lo´pez-Lo´pez et al 2005; Pavasovic et al

2007a) Therefore, as they age and their food changes, they

adapt to the new dietary sources, particularly starches

(Lo´pez-Lo´pez et al 2005) However, digestibility of

nutri-ents is slightly better in juveniles than in preadults

(Cam-pan˜a-Torres 2001; Cam(Cam-pan˜a-Torres et al 2006, 2008) This

deterioration of digestibility with age might be because of

faster metabolism in early stages of development

(Guil-laume 1997)

Diets constitute a major expenditure in aquaculture Fishmeal and other marine meals (krill, shrimp, squid and scal-lop meals) are used as protein sources in many finfish andcrustacean diets, as they are considered excellent sources ofhigh-quality proteins, highly unsaturated fatty acids, vita-mins, minerals and attractants (Tacon & Akiyama 1997;Webster et al 2008) Fish oils have been used as a dietarylipid source in commercial aquafeeds because of their readyavailability, fair price and abundance of EFAs (Turchini

et al 2009) However, owing to aquaculture expansion,competition from other agricultural sectors, uncertainty inlong-term availability (e.g overfishing), yearly fluctuations

in supply, quality and potential price variation, there hasbeen considerable interest in partial or total replacement offish meal and other marine meals and fish oil with lessexpensive plant-protein meals and oils (see Naylor et al

2000, 2009; Venero et al 2008; Webster et al 2008)

Fishmeal replacement Few studies have evaluated thereplacement of fish meal in redclaw diets SBMreplacement

of fishmeal at various levels resulted in marginal growthdifferences among redclaw juveniles, but organisms offered

a fishmeal-based diet had better growth and more frequentmoulting than those offered graded levels of SBM (Garcı´a-Ulloa et al 2003; Muzinic et al 2004) Similarly, Gutierrez

& Rodrı´guez (2010) examined the effect of protein source(SBM) on growth of juvenile C quadricarinatus reared in

Table 2 Apparent dry matter digestibility (ADMD) coefficients for protein (APD), carbohydrates (ACD), lipids (ALD) and gross energy (AGED) of the various nutrient sources for redclaw (Cherax quadricarinatus)

.

Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd

individual containers Based on their results, best growth

was obtained with a mixture of 50% fish meal and 50%

SBM

Saoud et al (2008) evaluated the response of juvenile

redclaw offered six practical diets (260 g kg 1 crude

pro-tein, 70 g kg 1crude lipid) replacing fishmeal with poultry

by-product meal at various inclusion levels Yuniarti et al

(2011) performed a similar experiment but substituted

golden apple snail (Pomacea canaculata) meal for fishmeal

No significant differences in survival and growth were

detected among treatments in both experiments The

authors concluded that poultry by-product meal and

golden apple snail meal are potential candidates for fish

meal replacement in redclaw diets Similar results were

reported by Garza de Yta et al (2012) who evaluated

growth response of juvenile C quadricarinatus, reared in

tanks, offered soybean-based diets (350 g kg 1 crude

pro-tein, 71 g kg 1 lipids) containing either fish meal, poultry

by-product meal, ground pea meal or distillers’ dried grains

with solubles (DDGS) meal as protein source No

signifi-cant differences were found in survival, growth or feed

con-version ratio (FCR) of redclaw crayfish This might be

because natural productivity contributed to

supplementa-tion of minor deficiencies in essential amino acids

Thomp-son et al (2005) reported that juvenile redclaw reared in a

recirculating system can be offered practical diets

contain-ing 350 g kg 1crude protein with no fish meal if a

combi-nation of less expensive plant-protein ingredients such as

SBM, wheat, brewer’s grains with yeast is added to the

diet In pond culture of juvenile redclaw, practical diets

containing 280 g kg 1 crude protein with no fish meal but

containing a combination of plant-protein ingredients

(SBM, distillers’ DDGS and milo) was adequate for good

growth (Thompson et al 2006)

Forage (detrital) crops (e.g rice, hay) are often used in

freshwater crayfish cultivation (Ackefors 2000; Salame &

Rouse 2000; Jones et al 2002) for presumed benefits such

as supplementation of direct and indirect sources of food

and supplying protective cover for moulting crayfish as

they seek refuge from predators Fletcher & Warburton

(1997) tested fresh and decomposed duckweed (Spirodela

sp.) as feed for juvenile crayfish redclaw and found that

decomposed duckweed supported crayfish growth as well

as commercial pellets did The authors suggested that

prep-aration of diets using detrital aquatic plants may be a

cost-effective method of increasing redclaw production Salame

& Rouse (2000) evaluated forage-based feeding strategies

for redclaw reared in earthen ponds Juvenile redclaw

stocked at a density of 4 m 2 were offered two feeding

regimes: manufactured pellets and pellets+ forage grass (Cynodon plectostachyum) and janeiro grass (Erio-chloa polystachya)) at a rate of 100 kg ha 1month 1.Survival and yield were greater in ponds receiving pelletsand forage than survival and yield in ponds receiving pel-lets only However, Metts et al (2007) performed similarexperiments but found no benefit from forage supplementa-tion Generally, results of the majority of studies that havebeen performed do not support using forage in redclawaquaculture

(star-Fish oil replacement Fish oils rich in HUFA are ally used in aquatic animal feeds Aquaculturists would like

tradition-to replace them with terrestrial plant oils such as linseed oil,canola oil and corn oil, rich in linolenic acid (18:3n-3), lino-leic acid (18:2n-6) and oleic acid (18:1n-9) Thompson et al

(2010) evaluated growth response and fatty acid tion of juvenile redclaw crayfish offered diets containingvarious lipid sources such as linseed oil, canola oil, corn oil,beef tallow or menhaden oil Crayfish offered plant-derivedoils performed as well as those offered feeds based on men-haden oil containing high levels of n-3 HUFA The dietcontaining beef tallow had a higher percentage of saturatedfatty acids and resulted in poor growth The authors con-cluded that menhaden oil can be replaced by plant-basedoils with high levels of 18-carbon unsaturated fatty acids indiets of juvenile redclaw, thus reducing costs for producers

composi-We suggest further research before definitive conclusions aremade but believe that because redclaw are freshwater organ-isms, they probably can perform well without dietary EPAand DHA inclusion It is possible that they can elongateand desaturatea-linolenic acid

Crustaceans exhibit relatively slow and intermittent feedingactivity that has an impact on food acquisition and pro-cessing (Loya-Javellana et al 1995; Houser & Akiyama1997) These behavioural characteristics affect physicalproperties including water stability and durability of thepellets (Meyers & Zein-Eldin 1975; Lim & Cuzon 1994;

Houser & Akiyama 1997; Obaldo et al 2002) Pellets need

to be firmly bound to avoid breaking up into small cles that results in leaching of nutrients into water, reduc-tion in water quality, poor animal growth, inefficient feedconversion and low survival (Lim & Cuzon 1994; Houser

parti-& Akiyama 1997; Obaldo et al 2002) Binders affect pelletstability in three ways: they reduce void spaces resulting in

a more compact and durable pellet; act as adhesives

stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick- stick-.

Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd

ing particles together; and exert a chemical action on the

ingredients and alter the nature of the feed resulting in a

more durable pellet (Lim & Cuzon 1994; DeSilva &

Ander-son 1995) Presently, diets of redclaw consist of steam

pressed highly conglomerated pellets of about 5 mm in

diameter (see Ruscoe et al 2005) Redclaw can only ingest

large grain fragments; fine granules are propelled forward

and away from the mouth by currents induced by the

scaphognathites as water is passed through the gill chamber

(Ruscoe 2002) Moreover, redclaw move out of shelter

when food is offered, grasp large food items and return to

the relative safety of the artificial shelter where

consump-tion and digesconsump-tion can be completed without fear of

preda-tion (Loya-Javellana et al 1993) This means that many

pellets will lay on the pond bottom for a while before

redc-laws come searching for a second helping Such feeding

behaviour requires a very water durable pellet to ensure

optimal FCR and growth

Ruscoe et al (2005) assessed the water stability of

practi-cal crayfish research diets manufactured using various

bind-ers (agar, gelatin, carboxymethylcellulose and carrageenan)

They found that rate of dry matter loss decreased over time

and that carrageenan and carboxymethylcellulose are better

binders than agar and gelatin The 50 g kg 1 binder

con-centration slowed the decay rate by as much as 62% as

compared with 30 g kg 1 binder concentration

Addition-ally, 10% moisture alginate-bound pellets are more stable

than 50% binder-bound pellets Growth and survival are

not influenced by diet moisture although slightly better

growth was noted with moist diets offered to redclaw

cray-fish (Ruscoe et al 2000) According to Ruscoe et al

(2002), moist diets allow the manipulation and ingestion of

appropriately sized pieces as determined by the animal,

without the losses associated with abrasion, rasping and

subsequent fragmentation of hard-pellet diets This is in

agreement with the morphological evidence suggesting that

the mouthparts of juvenile redclaw are well suited to the

ingestion of soft, moist foods (Loya-Javellana & Fielder

1997), where pappo-serrate setae on the labrum allow for

gentle prey manipulation by pushing prey down in front of

the mouth opening (Garm 2004)

Currently, there are few if any commercial feeds specifically

formulated and manufactured for redclaw crayfish Progress

has been made over the past decade, but there are still

knowledge gaps in relation to nutrient requirements for

red-claw Some of the areas that require further research include

essential amino acid requirements, vitamin and mineralrequirements, pelleting technology to produce a dry but mal-leable pellet, an estimation of optimal feed regimens, brood-stock nutrient requirements and formulations of diets usingregionally available ingredients with least cost formulations.Presently, we recommend that semi-intensive farms use sink-ing diets containing 350 g kg 1crude protein, 60 g kg 1lip-ids, 18–20 MJ kg 1

digestible energy with crustaceanvitamin and mineral premix supplement and a water stability

of at least 30 min Broodstock diets should contain fish oiland carotenoid pigments We believe redclaw aquaculturehas reached critical mass and is growing With appropriatefeeds, nursery technology and growout protocols, produc-tion is set to increase in the near future

Abdu, U., Yehezkel, G & Sagi, A (2000) Oocyte development and polypeptide dynamics during ovarian maturation in the red-

Ackefors, H (2000) Freshwater crayfish farming technology in the

Ali, A (1993) Evaluation of different carbohydrates in the diet of

Alimon, A.R., Roustaian, P., Saad, C.R & Kamarudi, M.S (2003) Lipid content and fatty acid composition during early and late embryonic development of redclaw crayfish, Cherax

Aspinall, G.O (1980) Chemistry of cell wall polysaccharides In: The Biochemistry of Plants, A Comprehensive Treatise; (vol 3)

500 Academic Press, Inc., New York, NY.

Be´guin, P & Aubert, J.-P (1994) The biological degradation of

BeMiller, J.N (2008) Polysaccharides: occurrence, significance, and properties In: Glycoscience: Chemistry and Chemical Biology

Springer, Berlin, Heidelberg, New York.

Brown, P.B (1995a) Physiological adaptations in the

Brown, P.B (1995b) A review of nutritional research with crayfish.

Byrne, K.A., Lehnert, S.A., Johnson, S.E & Moore, S.S (1999) Isolation of a cDNA encoding a putative cellulase in the redclaw

Campan˜a-Torres, T.A (2001) Digestibility of vegetal and animal ingredients and diets for juvenile and pre-adult redclaw Cherax quadricarinatus Master of Science Thesis CIBNOR, S.C La Paz, B.C.S Me´xico.

.

Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd

Campan˜a-Torres, A., Martinez-Cordova, L.R.,

Villarreal-Colmen-ares, H & Civera-Cerecedo, R (2005) In vivo dry matter and

protein digestibility of three-plant derived and four

animal-derived feedstuffs and diets for juvenile Australian redclaw,

Campan˜a-Torres, A., Martinez-Cordova, L.R.,

Villarreal-Colmen-ares, H & Civera-Cerecedo, R (2006) Carbohydrate and lipid

digestibility of animal and vegetal ingredients and diets for

juvenile Australian redclaw crayfish, Cherax quadricarinatus.

Campan˜a-Torres, A., Martinez-Cordova, L.R.,

Villarreal-Colmen-ares, H & Civera-Cerecedo, R (2008) Carbohydrate and lipid

digestibility of animal and vegetal ingredients and diets for the

pre-adult redclaw crayfish, Cherax quadricarinatus (von

Ceccaldi, H.J (1997) Anatomy and physiology of the digestive

sys-tem In: Crustacean Nutrition, Vol 6 (D’Abramo, L.R.,

World Aquaculture, World Aquaculture Society, Baton Rouge,

LA.

Cho, C.Y & Bureau, D.P (2001) A review of diet formulation

strategies and feeding systems to reduce excretory and feed

Cho, S.H., Lee, S.M., Lee, S.M & Lee, J.H (2005) Effect of

die-tary protein and lipid levels on growth and body composition of

juvenile turbot (Scophthalmus maximus L) reared under optimum

240.

Conklin, D.E (1997) Vitamins In: Crustacean Nutrition, Vol 6

(D’Abramo, L.R., Conklin, D.E & Akiyama, D.M eds), pp.

Society, Baton Rouge, Louisiana, USA.

Corte´s-Jacinto, E., Villarreal-Colmenares, H., Civera-Cerecedo, R.

& Martı´nez-Co´rdova, L (2003) Effect of dietary protein level on

growth and survival of juvenile freshwater crayfish Cherax

Corte´s-Jacinto, E., Villarreal-Colmenares, H., Civera-Cerecedo, R.

& Naranjo-Pa´ramo, J (2004) Effect of dietary protein level on

the growth and survival of pre-adult freshwater crayfish Cherax

Corte´s-Jacinto, E., Villarreal-Colmenares, H., Cruz-Sua´rez, L.E.,

Civera-Cerecedo, R., Nolasco-Soria, H & Hernandez-Llamas,

A (2005) Effect of different dietary protein and lipid levels on

growth and survival of juvenile Australian redclaw crayfish,

Corte´s-Jacinto, E., Campa-Co´rdova, A´.I., Ascencio, F.,

Villarreal-Colmenares, H & Holguı´n-Pen˜a, R.J (2009) The effect of

pro-tein and energy levels in diet on the antioxidant activity of

juve-nile redclaw Cherax quadricarinatus (von Martens, 1868).

Coutteau, P., Geurden, I., Camara, M.R., Bergot, P & Sorgeloos,

P (1997) Review on the dietary effects of phospholipids in fish

invertebrates In: Proceedings 2nd Internat Conf on

Aquacul-ture Nutrition: Biochemical and Physiological Approaches to

30 Lousiana State University, Division of Continuing

Educa-tion, Baton Rouge, LA.

Crawford, A.C., Kricker, J.A., Anderson, A.J., Richardson, N.R.

& Mather, P.B (2004) Structure and function of a cellulase gene

Crawford, A.C., Richardson, N.R & Mather, P.B (2005) A parative study of cellulase and xylanase activity in freshwater

Curtis, M.C & Jones, C.M (1995) Observations of monosex ture of redclaw Cherax quadricarinatus von Martens (Decapoda:

159.

Cuzon, G & Guillaume, J (1997) Energy and protein: energy ratio In: Crustacean Nutrition, Vol 6 (D’Abramo, L., Conklin,

Aquacul-ture, World Aquaculture Society, Baton Rouge, Louisiana, USA.

D’Abramo, L.R (1997) Triacylglycerols and fatty acids In: tacean Nutrition, Vol 6 (D’Abramo, L.R., Conklin, D.E &

Aquacul-ture, World Aquaculture Society, Baton Rouge, Louisiana, USA.

D’Abramo, L.R & Robinson, E.H (1989) Nutrition of crayfish.

D’Abramo, L.R & Sheen, S.-S (1994) Nutritional requirements, feed formulation, and feeding practices for intensive culture of the freshwater prawn Macrobrachium rosenbergii Rev Fish Sci.,

2, 1–21.

D’Agaro, E., Lanari, D & Massimo, M (2001) Effects of stocking rate and dietary protein content on growth performance of red-

Dall, W & Moriarty, D.J.W (1983) Nutrition and digestion In:

261 Academic Press, New York, NY.

Dall, W., Hill, B.J., Rothlisberg, P.C & Staples, D.J (1990) The Biology of the Penaeidae Advances in Marine Biology pp 489.

Academic Press, London, UK 27.

Davis, D.A & Lawrance, A.I (1997) Minerals In: Crustacean Nutrition, Vol 6 (D’Abramo, L.R., Conklin, D.E & Akiyama,

Ferna´ndez, I., Oliva, M., Carrillo, O & Van Wormhoudt, A.

(1997) Digestive enzyme activities of Penaeus notialis during

Figueiredo, M.S.R.B & Anderson, A.J (2003) Ontogenetic changes

in digestive proteases and carbohydrases from the Australian freshwater crayfish, redclaw Cherax quadricarinatus (Crustacea,

Figueiredo, M.S.R.B & Anderson, A.J (2009) Digestive enzyme spectra in crustacean decapods (Paleomonidae, Portunidae and

Garcı´a-Guerrero, M., Racotta, I.S & Villarreal-Colmenares, H.

(2003) Variation in lipid, protein, and carbohydrate content

dur-ing the embryonic development of the crayfish Cherax

Garcı´a-Ulloa, G.M., Lo´pez-Chavarı´n, H.M., Rodrı´guez-Gonza´lez,

H & Villarreal-Colmenares, H (2003) Growth of redclaw

cray-fish Cherax quadricarinatus (Von Martens 1868) (Decapoda:

Parastacidae) juveniles fed isoproteic diets with partial or total

substitution of fish meal by soya bean meal: preliminary study.

Garm, A (2004) Mechanical functions of setae from the mouth

apparatus of seven species of decapod crustaceans J Morphol.,

260, 85–100.

Garza de Yta, A., Davis, D.A., Rouse, D.B., Saoud, I.P &

Gha-nawi, J (2011) Effects of stargrass hay supplementation on

488.

Garza de Yta, A., Davis, D.A., Rouse, D.B., Ghanawi, J & Saoud,

I.P (2012) Evaluation of practical diets containing various

terres-trial protein sources on survival and growth parameters of redclaw

Gibson, R & Barker, P.L (1979) The decapod hepatopancreas.

Guillaume, J & Choubert, G (2001) Digestive physiology and

nutri-ent digestibility in fishes In: Nutrition and Feeding of Fish and

Crustaceans (Guillaume, J., Kaushik, P., Bergot, P & Metailler,

Guillaume, J (1997) Protein and amino acids In: Crustacean

Nutri-tion, Vol 6 (D’Abramo, L.R., Conklin, D.E & Akiyama, D.M.

Gutierrez, M.L & Rodrı´guez, E.M (2010) Effect of protein source

on growth of early juvenile redclaw crayfish Cherax

Hammer, H.S., Bishop, C.D & Watts, S.A (2000) Activities of

three digestive enzymes during development in the crayfish

Harpaz, S., Rise, M., Arad, S & Gur, N (1998) The effect of

three carotenoid sources on growth and pigmentation of juvenile

Harrison, K.E (1990) The role of nutrition in maturation,

repro-duction, and embryonic development of decapods crustaceans: a

Harrison, K.E (1997) Broodstock nutrition and maturation diets.

In: Crustacean Nutrition, Vol 6 (D’Abramo, L.R., Conklin, D.

Soci-ety, Baton Rouge, LA.

Hernandez, M.P., Rouse, D.B & Olvera-Novoa, M.A (2001)

Australian crayfish (Cherax quadricarinatus) hatchlings and

Herna´ndez, P.V., Olvera-Novoa, M.A & Rouse, D.B (2004)

Effect of dietary cholesterol on growth and survival of juvenile

redclaw crayfish Cherax quadricarinatus under laboratory

Herna´ndez-Vergara, M.P., Rouse, D.B., Olvera-Novoa, M.A &

Davis, D.A (2003) Effects of dietary lipid level and source on

growth and proximate composition of juvenile redclaw (Cherax

quadricarinatus) reared under semi-intensive culture conditions.

Holland, D.L (1978) Lipid reserves and energy metabolism in the

larvae of benthic marine invertebrates In: Biochemical and

Bio-physical Perspectives in Marine Biology (Malins, D.C &

Houser, R.H & Akiyama, D.M (1997) Feed formulation ples In: Crustacean Nutrition, Vol 6 (D’Abramo, L.R., Conk-

Aquaculture Society, Baton Rouge, LA.

Huner, J.V., Moody, M.M & Thune, R.T (1994) Cultivation of freshwater crayfishes in North America In: Freshwater Crayfish Aquaculture in North America, Europe, and Australia Families

156 Food Products Press, New York, NY.

Jacobs, W (1928) Untersuchungen uber die cytology der dung in der mitteldarmdruse von Astacus leptodactylus Z Zel-

Jones, C.M (1990) The Biology and Aquaculture Potential of the Tropical Freshwater Crayfish Cherax quadricarinatus Queens- land Department of Primary Industries Information Series QI90028, 109 pp.

Jones, C.M (1995) Production of juvenile redclaw crayfish, Cherax

Jones, C.M & Ruscoe, I (1996a) Evaluation of six diets fed to redclaw, Charax quadricarinatus (von martens) (Decapoda: Parastacidae), under laboratory conditions In: Production Tech-

31 Final Report FRDC Project 92/119 Fisheries Research and Development, Canberra.

Jones, C.M & Ruscoe, I (1996b) Evaluation of six diets fed to redclaw, Cherax quadricarinatus (von Martens), (Decapoda: Parastacidae) held in pond enclosures In: Production Technol-

Final Report FRDC Project 92/119 Fisheries Research and Development, Canberra.

Jones, C.M & Ruscoe, I (1996c) Assessment of carbohydrate source in five diets fed to redclaw, Cherax quadricarinatus (von Martens) (Decapoda:Parastacidae), under earthen pond condi- tions In: Production Technology for Redclaw Crayfish (Cherax

Fisheries Research and Development, Canberra.

Jones, P.L., Chavez, J.R & Mitchell, B.D (2002) Production of Australian freshwater crayfish in earthen-based systems using pel-

Kanazawa, A (2001) Sterols in marine invertebrates (review

Keefe, A & Rouse, D (1999) Protein requirements for juvenile Australian redclaw crayfish, Cherax quadricarinatus Freshwater

Kristensen, J.H (1972) Carbohydrases of some marine brates with notes on their food and on the natural occurrence of

Lee, P.G & Lawrence, A.L (1997) Digestibility In: Crustacean Nutrition, Vol 6 (D’Abramo, L.R., Conklin, D.E & Akiyama,

World Aquaculture Society, Baton Rouge, LA.

Li, J., Guo, Z., Gan, X., Wang, Q & Zhao, Y (2010) cal changes during vitellogenesis in the red claw crayfish,

e455.

Lim, C & Cuzon, G (1994) Water stability of shrimp pellet: a

Lim, C & Sessa, D.J (1995) Nutrition and Utilization Technology

in Aquaculture AOCS Press, Champaign, IL.

.

Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd

Lin˜a´n-Cabello, M.A., Paniagua-Michel, J.J & Hopkins, P.M.

(2002) Bioactive roles of carotenoids and retinoids in

Lin˜a´n-Cabello, M.A., Medina-Zendejas, R., Sa´nchez-Barajas, M.

& Herrera, A.M (2004) Effects of carotenoids and retinol in

oocyte maturation of crayfish Cherax quadrucarinatus Aquacult.

Linton, S.M., Greenaway, P & Towle, D.W (2006) Endogenous

Lodge, D.M & Hill, A.M (1994) Factors governing species

com-position, population size, and productivity of cool-water

Lo´pez-Lo´pez, S., Nolasco, H & Vega-Villasante, F (2003)

redclaw crayfish Cherax quadricarinatus Comp Biochem

Lo´pez-Lo´pez, S., Nolasco, H., Villarreal-Colmenares, H &

Civera-Cerecedo, R (2005) Digestive enzyme response to supplemental

ingredients in practical diets for juvenile freshwater crayfish

Lovell, R.T (1998) Nutrition and Feeding of Fish, 2nd edn

Klu-wer Academic Publishers, Boston, MA.

Loya-Javellana, G.N & Fielder, D.R (1997) Development trends

in the mouthparts during growth from juvenile to adult of the

tropical freshwater crayfish, Cherax quadricarinatus von

Mar-tens, 1868, (Decapoda: Parastacidae) Invertebr Reprod Dev.,

32, 167–175.

Loya-Javellana, G.N., Fielder, D.R & Thorne, M.J (1993) Food

choice by free-living stages of the tropical freshwater crayfish,

118, 299–308.

Loya-Javellana, G.N., Fielder, D.R & Thorne, M.J (1994)

Ontog-eny of foregut in the tropical freshwater crayfish Cherax

Loya-Javellana, G.N., Fielder, D.R & Thorne, M.J (1995)

Fore-gut evacuation, return of appetite and gastric fluid secretion in

the tropical freshwater crayfish, Cherax quadricarinatus

Luo, W., Wang, Q., Zhao, Y.-L., Gu, Z.-M., Mi, G.-Q., Huang,

X.-M & Liu, Q.-W (2004) Effects of Dietary Vitamin E on

Reproduction of Redclaw Crayfish Cherax quadricarinatus.

Luo, W., Zhao, Y.-L & Yao, J.-J (2008a) Biochemical composition

and digestive enzyme activities during embryonic development

Luo, W., Zhao, Y., Zhou, Z., An, C & Ma, Q (2008b) Digestive

enzyme activity and mRNA level of trypsin in embryonic

red-claw crayfish, Cherax quadricarinatus Chin J Oceanol Limnol.,

26, 62–68.

Manomaitis, L (2001) Assessment of the crude protein

require-ment of juvenile redclaw crayfish (Cherax quadricarinatus) MSc.

Thesis, Auburn University, Auburn AL.

Mason, J.C (1975) Crayfish production in a small woodland

Meade, M.E & Watts, S.A (1995) Weight gain and survival of

juvenile crayfish Cherax quadricarinatus fed formulated feeds.

Medley, P.B.C., Jones, C.M & Avault, J.W Jr (1994) A global

perspective on the culture of Australian redclaw crayfish, Cherax

quadricarinatus: production, economics and marketing World

Meng, F.-L., Zhao, Y.-L., Chen, L.-Q., Gu, Z.-M., Xu, G.-X &

Liu, Q.-W (2001) Embryonic development of redclaw crayfish

Metts, L.S., Thompson, K.R., Xiong, Y.L., Kong, B., Webster, C.

D & Brady, Y.J (2007) Use of alfalfa hay, compared to feeding practical diets containing two protein levels, on growth, survival, body composition, and processing traits of Australian redclaw crayfish, Cherax quadricarinatus, grown in ponds J World

Meyers, S.P & Latscha, T (1997) Carotenoids In: Crustacean Nutrition, Vol 6 (D’Abramo, L.R., Conklin, D.E & Akiyama,

Aquaculture Society, Baton Rouge, LA.

Meyers, S.P & Zein-Eldin, Z.P (1975) Binders and pellet stability

in development of crustacean diets Proc World Maricult Soc.,

3, 351–364.

Mitchell, H.H (1950) Some species and age differences in amino acid requirements In: Protein and Amino Acid Requirements

New York, NY.

Momot, W.T (1995) Redefining the role of crayfish in aquatic

Momot, W., Gowing, H & Jones, P.D (1978) The dynamics of

Muzinic, L.A., Thompson, K.R., Morris, A., Webster, C.D., Rouse, D.B & Manomaitis, L (2004) Partial and total replace- ment of fish meal with soybean meal and brewer’s grains with yeast in practical diets for Australian redclaw crayfish Cherax

Navarrete del Toro, M.A., Garcı´a-Carren˜o, F.L., Dı´az, L.M., is-Guerrero, L & Saborowski, R (2006) Aspartic proteinases in the digestive tract of marine decapod crustaceans J Exp Zool., 305A, 645–654.

Cel-Naylor, R.L., Goldburg, R.J., Primavera, J.H., Kautsky, N., ridge, M.C.M., Clay, J., Folke, C., Lubchenco, J., Mooney, H.

Beve-& Troell, M (2000) Effect of aquaculture on world fish supplies.

Nutri-Nystro¨m, P (2002) Ecology In: Biology of Freshwater Crayfish

Obaldo, L.G., Divakaran, S & Tacon, A.G (2002) Method for determining the physical stability of shrimp feeds in water Aqua-

Parvathy, K (1971) Glycogen storage in relation to the moult cycle in two crustaceans Emerita asiatica and Ligia exotica Mar.

Pavasovic, A., Richardson, N.A., Mather, P.B & Anderson, A.J.

(2006) Influence of insoluble dietary cellulose on digestive enzyme activity, feed digestibility and survival in the redclaw crayfish, Cherax quadricarinatus (von Martens) Aquacult Res.,

37, 25–32.

.

Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd

Pavasovic, A., Anderson, A.J., Mather, P.B & Richarson, N.A.

(2007a) Effect of a variety of animal, plant and cell-based feed

ingredients on diet digestibility and digestive enzyme activity in

redclaw crayfish, Cherax quadricarinatus (Von Martens 1868).

Pavasovic, A., Anderson, A.J., Mather, P.B & Richardson, N.A.

(2007b) Influence of dietary protein on digestive enzyme activity,

growth and tail muscle composition in redclaw crayfish, Cherax

Perera, E., Moyano, F.J., Dı´az, M., Perdomo-Morales, R.,

Mon-tero-Alejo, V., Rodriguez-Viera, L., Alonso, E., Carrillo, O &

Galich, G.S (2008) Changes in digestive enzymes through

developmental and molt stages in the spiny lobster, Panulirus

Pillay, T.V.R & Kutty, M.N (2005) Aquaculture, Principles and

Practices, 2nd edn pp 630 Blackwell Publishing Ltd, Oxford,

UK.

Rodrı´guez-Gonza´lez, H., Garcı´a-Ulloa, M., Herna´ndez-Llamas, A.

& Villarreal, H (2006a) Effect of dietary protein level on

spawn-ing and egg quality of redclaw crayfish, Cherax quadricarinatus.

Rodrı´guez-Gonza´lez, H., Herna´ndez-Llamas, A., Villarreal, H.,

Saucedo, P.E., Garcı´a-Ulloa, M & Rodrı´guez-Jaramillo, C.

(2006b) Gonadal development and biochemical composition of

female crayfish Cherax quadricarinatus (Decapoda: Parastacidae)

in relation to the Gonadosomatic Index at first maturation.

Rodrı´guez-Gonza´lez, H., Villarreal, H., Garcı´a-Ulloa, M &

Her-na´ndez-Llamas, A (2009a) Evaluation of practical diets

contain-ing different protein levels on gonad development of female

–355.

Rodrı´guez-Gonza´lez, H., Villarreal, H., Garcı´a-Ulloa, M &

Her-na´ndez-Llamas, A (2009b) Dietary lipid requirements for

opti-mal egg quality of redclaw crayfish, Cherax quadricarinatus.

Rodrı´guez-Gonza´lez, H., Villarreal, H., Garcı´a-Ulloa, M.,

Herna´n-dez-Llamas, A., Garcı´a-Ulloa, M., Va´zquez-Boucard, C &

Ser-rano-Pinto, V (2011) Effect of dietary protein-to-energy ratio on

reproduction in female redclaw crayfish Cherax quadricarinatus.

Rouse, D & Rash, J (1999) Influence of astaxanthin in the diets

of juvenile and adult redclaw crayfish Cherax quadricarinatus.

Ruscoe, I.M (2002) Improving ingestion of artificial diets by

Che-rax quadricarinatus MSc Thesis Deakin University,

Warrnam-bool, Vic., Australia.

Ruscoe, I.M., Jones, P.L & Jones, C.M (2000) Effects of feeding

moist and dry diets to redclaw crayfish Cherax quadricarinatus in

Ruscoe, I.M., Jones, P.L & Jones, C.M (2002) A comparison of

moist and dry diets fed to redclaw crayfish Cherax

Ruscoe, I.M., Jones, C.M., Jones, P.L & Caley, P (2005) The

effects of various binders and moisture content on pellet stability

93.

Sagi, A., Rise, M., Isam, K & Arad, S (1995) Carotenoid and

their derivatives in organs of the maturating female crayfish

Salame, M.J & Rouse, D.B (2000) Forage-based feeding in

Sa´nchez-Paz, A., Garcia-Carren˜o, F.L., Muhlia-Almaza´n, A., Peregrino-Uriarte, A.B., Herna´ndez-Lo´pez, J & Yepiz-Plascen- cia, G (2006) Usage of energy reserves in crustaceans during starvation: status and future directions Insect Biochem Mol.

Saoud, I.P., Rodgers, L.P., Davis, D.A & Rouse, D.B (2008) Replacement of fish meal with poultry by-product meal in prac- tical diets for redclaw crayfish (Cherax quadricarinatus) Aqua-

Sargent, J., Henderson, R.J & Tocher, D.R (1989) The lipids In:

New York.

Shiau, S.Y (1997) Carbohydrates and fiber In: Crustacean tion, Vol 6 (D’Abramo, L.R., Conklin, D.E & Akiyama, D.M.

Aquaculture Society, Baton Rouge, LA.

Shiau, S.-Y (1998) Nutrient requirements of penaeid shrimps.

Tacon, A.G.J & Akiyama, D.M (1997) Feed ingredients In: Crustacean Nutrition, Vol 6 (D’Abramo, L.R., Conklin, D.E &

Aquacul-ture, World Aquaculture Society, Baton Rouge, LA.

Teshima, S (1997) Phospholipids and sterols In: Crustacean Nutrition, Vol 6 (D’Abramo, L.R., Conklin, D.E & Akiyama,

Aquaculture Society, Baton Rouge, LA.

Thompson, K.R., Muzinic, L.A., Christian, T.D., Webster, C.D., Manomaitis, L & Rouse, D.B (2003a) Lecithin requirements of juvenile Australian redclaw crayfish Cherax quadricarinatus.

Thompson, K.R., Muzinic, L.A., Christian, T.D., Webster, C.D., Manomaitis, L & Rouse, D.B (2003b) Effect on growth, sur- vival, and fatty acid composition of Australian redclaw crayfish

Thompson, K.R., Muzinic, L.A., Yancey, D.H., Webster, C.D., Rouse, D.B & Xiaon, Y (2004) Effects of feeding practical diets containing various protein levels on growth, survival, body com- position and processing traits of Australian redclaw crayfish,

Thompson, K.R., Muzinic, L.A., Engler, L.S & Webster, C.D (2005) Evaluation of practical diets containing different protein levels, with or without fish meal, for juvenile on Australian red-

Thompson, K.R., Metts, L.S., Muzinic, L.A., Dasgupta, S & Webster, C.D (2006) Evaluation of practical diets containing different protein levels, with or without fish meal, on growth, survival, body composition and processing traits of male and female Australian redclaw crayfish (Cherax quadricarinatus)

Thompson, K.R., Bailey, T.J., Metts, L.S., Brady, Y.J & Webster, C.D (2010) Growth response and fatty acid composition of juvenile redclaw crayfish (Cherax quadricarinatus) fed different

Turchini, G.M., Torstensen, B.E & Ng, W.-K (2009) Fish oil

Vega-Villasante, F., Fernandez, I., Preciado, R.M., Oliva, M., Tovar, D & Nolasco, H (1999) The activity of digestive enzymes during the molting stages of the arched swimming Calli-

.

Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd

Venero, J.A., Davis, D.A & Lim, C.E (2008) Use of plant protein

sources in crustacean diets In: Alternative Protein Sources in

Aquaculture Diets (Lim, C.E., Webster, C.D & Lee, C.S eds),

Verri, T., Mandal, A., Zilli, L., Bossa, D., Mandal, P.K., Ingrosso,

L., Zonno, V., Vilella, S., Ahearn, G.A & Storelli, C (2001)

D-Glucose transport in decapod crustacean hepatopancreas.

Vogt, G (2002) Functional anatomy In: Biology of Freshwater

NewYork, NY.

Vogt, G., Stocker, W., Starch, V & Zwilling, R (1989)

Biosynthe-sis of Astacus protease, a digestive enzyme from crayfish

Walker, L.P & Wilson, D.B (1991) Enzymatic hydrolysis of

Watanabe, H & Tokuda, G (2001) Animal cellulases Cell Mol.

Webster, C.D., Goodgame-Tiu, L.S., Tidwell, J.H & Rouse, D.B.

(1994) Evaluation of practical feed formulations with different

protein levels for juvenile redclaw crayfish (Cherax

Webster, C.D., Thompson, K.R., Muzinic, L.A., Rouse, D.B &

Manomaitis, L (2002) Culture and nutrition of redclaw crayfish,

Webster, C.D., Thompson, K.R., Metts, L.S & Muzinic, L.A.

(2008) Use of distillers grains with solubles and brewery

by-products in fish and crustacean diets In: Alternative Protein

Sources in Aquaculture Diets (Lim, C.E., Webster, C.D & Lee,

Wilson, R.P & Poe, W.E (1985) Relationship of whole body and

egg amino acid patterns to amino acid patterns in channel

388.

Wood, T.M (1985) Properties of cellulolytic enzyme systems

Woodward, J (1991) Synergism in cellulase systems Bioresour.

Xue, X.M., Anderson, A.J., Richardson, N.A., Anderson, A.J., Xue, G.P & Mather, P.B (1999) Characterisation of cellulase activity in the digestive system of the redclaw crayfish (Cherax

Yao, J.-J., Zhao, Y.-L., Wang, Q., Zhou, Z.-L., Hu, X.-C., Duan, X.-W & An, C.-G (2006) Biochemical compositions and diges- tive enzyme activities during the embryonic development of

Yokoe, Y & Yasumasu, I (1964) The distribution of cellulase in

Yuniarti, A., Aan, K & Hariarti, A.M (2011) Utilization of Golden Apple Snail (Pomacea canaculata) Meat Meal in the Diet

of Redclaw Crayfish (Cherax quadricarinatus) Asian-Pacific Aquaculture and Giant Prawn Conference 2011, India, Abstract,

619 pp.

Zenteno-Savı´n, T., Corte´s-Jacinto, E., Va´zquez-Medina, J.P & larreal-Colmenares, H (2008) Oxidative damage in tissues of juvenile crayfish (Cherax quadricarinatus von Martens, 1868) fed

154.

Zwilling, R.H & Neurath, H (1981) Invertebrate proteases In:

Methods in Enzymology, Vol 80 (Wood, W.A & Kellogg, S.T.

.

Aquaculture Nutrition 18; 349–368 ª 2012 Blackwell Publishing Ltd

DE LAGroupe de rechereche en recyclage biologique et aquiculture, De´partement des sciences animales, Universite´ Laval, Que´bec,Que´bec, Canada

Factors influencing apparent digestibility coefficient (ADC) of

nutrients from a plant protein–based diet supplemented with

microbial phytase were investigated in a series of experiments

with rainbow trout (Oncorhynchus mykiss) The influence of

phytase level, water temperature, feed particle size and

addi-tion of a protease/non-starch polysaccharidase (PNSP)

enzyme cocktail were tested in a phytase-supplemented

(2000 FTU kg)1) diet Finally, the influence of Ca/P ratio,

addition of 1,25-hydroxycholeocalciferol, or inclusion of lactic

acid (LA) in diets with and without phytase was evaluated

Addition of microbial phytase improved ADC of dry matter

(DM), protein, ash and minerals (P, Ca, Mg, Fe and Zn)

(P < 0.05) Reducing feed particle size potentiated the effect of

phytase on P and ash ADC, as did the addition of a PNSP

enzyme cocktail; the latter also significantly improved DM

ADC in both control and phytase-supplemented diets

Increasing the Ca/P ratio reduced the effect of phytase on

P and ash ADC Addition of 1,25-dihydroxycholecalciferol

and LA had no effect on DM, P and ash ADC in control diets

and tended to reduce the phytase-induced increase in P ADC

key words: digestibility, fish, mineral, phosphorus, phytase

Received 18 September 2009, accepted 26 June 2011

Correspondence: Grant Vandenberg, De´partement des sciences animales,

Pavillon Paul Comtois, Universite´ Laval, Que´bec, Que´bec G1V 0A6,

Canada E-mail: grant.vandenberg@fsaa.ulaval.ca

*Present address: Brandon Research Centre, Agriculture and Agri-Food

Canada, P.O Box 1000A, RR3, Brandon, Manitoba R7A 5Y3, Canada.

The rapid expansion of the aquaculture industry has led to

concerns about the environmental impact of intensive

production, particularly the excessive loading of phosphorus

in freshwater ecosystems (Davenport et al 2003), as well asabout the sustainability of certain industry sectors related tothe demand on fish meal supplies (Tacon & Metian 2008) Inparticular, the production of carnivorous fish species has comeunder scrutiny, as it is a net consumer of fisheries resourcesbecause of the need for high-quality fish meal to meet theirnutritional requirements (Naylor et al 2009) For example, in

2006, the global production of salmon and trout, which resented 11% of the total global aquafeed production, con-sumed 27% of the fish meal used in commercial aquaculturefeeds (Tacon & Metian 2009) Pressures to reduce fish mealconsumption, combined with the increasing price forfish meal as a result of increasing global demand, have resulted

rep-in research aimed at findrep-ing fish meal replacements

A variety of plant protein sources, including oilseed meals(Sanz et al 1994; Riche & Brown 1999; Carter 2000; Thiessen

et al 2004), legume species (Hughes 1991; Gouveia et al.1993; Drew et al 2005) and processing by-products (Sugiura

et al 1998b; Regost et al 1999; Lee et al 2002), has beenstudied as potential fish meal replacements They have shownpromise because of their adequate palatability and nutri-tional profile; however, a number suffer from the presence ofspecific antinutritional factors (Tacon 1997; Bureau et al.1998; Glencross et al 2003) One of these is phytate (myo-inositol 1, 2, 3, 4, 5, 6-hexakis dihydrogen phosphate), which

is the major storage form of phosphorus (P) in plant seeds(Graf 1986) and which represents 50–90% of the P found inplant protein sources The phytate molecule, being negativelycharged, forms complexes with a variety of divalent cations(Davies & Nightingale 1975), starch (Maga 1982) and pro-teins (Knuckles et al 1985), thus reducing the availability of

a variety of nutrients Although endogenous phytase activityderived from intestinal microorganisms has been demon-strated in selected fish species (Shi et al 2008), phytate-

bound nutrients are largely unavailable to monogastric

animals, which lack sufficient activity of endogenous phytase

to liberate phytate-P (Pointillart et al 1984)

Since the work of Nelson et al (1968) revealed the utility

of exogenous phytase addition on P utilization in chicks, a

large body of work has shown the ability of supplemented

phytase to increase P utilization in a range of monogastric

species, including humans (Sandberg & Anderson 1988),

rodents (Williams & Taylor 1985), poultry (Nelson et al

1971) and swine (Moser et al 1982) More recently, the

commercial availability of microbial phytase for the animal

feed industry has prompted increased research investigating

the supplementation of domestic animal diets In these

spe-cies, the efficacy of phytase is affected by a number of dietary

factors, including the Ca/P ratio (Lei et al 1994; Qian et al

1996), the co-addition of vitamin D or vitamin D metabolites

(Edwards 1993), or a number of organic acids (Li et al 1998;

Kemme et al 1999b) Understanding the influence of these

factors on enzyme function is critical to ensure optimal

response to supplementary phytase

A number of studies have shown the utility of phytase

supplementation for a variety of fish species fed plant–based

diets Fish species that consume high levels of plant–based

dietary proteins, including carp (Schaefer et al 1995), catfish

(Jackson et al 1996; Eya & Lovell 1997; Li & Robinson 1997)

and tilapia (Gur 1998), respond well to supplemented

phy-tase A number of reports have also demonstrated the

effec-tiveness of microbial phytase supplementation in increasing P

digestibility in salmonids diets having a significant proportion

of the dietary fish meal replaced by plant protein–based

ingredients (Brown 1993; Rodehutscord & Pfeffer 1995;

Lanari et al 1998; Vielma et al 1998; Forster et al 1999)

A recent review of work describing the application of phytase

in fish species concludes that, to date, there has been a lack of

information relating to dietary and environmental factors

affecting the ability of supplementary phytase to affect

nutrient digestibility in fish (Cao et al 2007)

The objective of the current study was to characterize the

influence of phytase addition on nutrient digestibility from a

plant protein–based diet and to identify dietary and

envi-ronmental factors that affect the response to phytase

sup-plementation

Rainbow trout (initial mass 51.1 ± 3.4 g) were obtained

from a certified disease-free hatchery (Pisciculture des

Alleghanys, St-Phile´mon, QC, Canada) Fish (n = 150) wererandomly distributed into 80-l cylindroconical tanks and fed

a commercial diet (Martin Feed Mills, Elmira, ON, Canada)for 7 days prior to the start of the experiment All tanks weresupplied with water from a recirculation system receivingapproximately 25% daily make-up water at a flow rate of

30 L h)1 Suspended solids were removed using a sand filter,effluent ammonia removed using a trickling biofilter;

ammonia and nitrite concentrations were monitored twiceweekly to assess biofilter performance Water temperatureand dissolved oxygen were continuously monitored bycomputer and automatically maintained at 15.0 ± 0.5Cand 10.3 ± 0.5 mg L)1, respectively Water pH was mea-sured daily and maintained by the addition of calcium car-bonate to the sand filter and sodium carbonate to the effluentwater The experimental animals were kept in accordancewith the guidelines of the Canadian Council of Animal Careand the Comite´ de protection des animaux, Universite´ Laval

A plant protein–based diet (Table 1) was modelled afterAdelizi et al (1998) and formulated to meet nutrientrequirements (NRC, 1993) Ingredients were obtained from acommercial feed mill (Martin Feed Mills) The levels ofavailable methionine and lysine were estimated using proteinavailability values, and crystalline forms were supplemented

to ensure an adequate amino acid balance Vitamin andmineral supplements were added to recommended levels(Ontario Ministry of Natural Resources Formulation VIT-

9608 and MIN-9504, respectively) and supplied by BASFCorp (Georgetown, ON, Canada) Sipernat 50 [source ofacid insoluble ash (AIA)] was obtained from Degussa-Hu¨lsCanada Inc (Brampton, ON, Canada) Microingredientswere first mixed and then slowly added to the macroingre-dients to ensure a homogenous mixture The mash was steamconditioned, cooled, and a liquid suspension of microbialphytase (Natuphos 5000L; BASF Corporation, Mississauga,

ON, Canada) added to the basal diets and mixed for 15 minusing a Hobart Legacy 30 quart mixer (Hobart Corporation,Troy, OH, USA) to provide a homogenous mixture Dietswere cold-pelleted using a laboratory mill (California PelletMill, Crawfordsville, IN, USA) equipped with a 3.5-mm die;

there was no significant subsequent heating of the mashresulting from the pelleting process The resulting pellets weredried in a forced air oven (30C for 24 h), sieved and stored

in airtight containers at 5C until required

Experimental diets were fed to triplicate tanks (80 L tanksholding 50 g body weight fish stocked at 30 kg m)3)

.

Aquaculture Nutrition 18; 369–379  2011 Blackwell Publishing Ltd

Tanks were individually equipped with automatic faeces

settling columns as previously described (Vandenberg & de la

Nou¨e 2001) Diets were fed for 7 days to acclimate fish to the

new diet, which was followed by a 5-day faeces collection

period Diets were fed to apparent satiation once daily

(08:00 h), following the recovery of collected faeces For all

treatment groups, daily faecal collections were pooled and

stored at)80 C until required for further processing

A series of three trials were carried out with rainbow troutusing the system described earlier to characterize the influ-ence of dietary phytase supplementation on digestibility ofnutrients from a plant protein–based diet In Trial 1, theeffect of graded increases in phytase activity (0, 2000, 4000and 6000 FTU kg)1; where 1 FTU liberates 1 lmol inor-ganic phosphorus per minute from 5.1· 10)3M sodiumphytate at 37C at pH 5.5) was examined; these levels werechosen based on preliminary studies indicating a requirementfor higher levels of phytase activity versus warm-bloodedspecies (results not shown) Because temperature significantlyaffects phytase activity (Keis 1999b), the impact of varyingwater temperatures across a range that would be encounteredwhen raising rainbow trout (5, 10, 15 and 20C) was eval-uated for diets supplemented with 2000 and 4000 FTU kg)1

in Trial 2 Because the results of Trial 1 showed that nofurther improvements in apparent digestibility coefficient(ADC) were obtained at levels of phytase inclusion exceeding

4000 FTU phytase kg)1, the remainder of the trials describedlater used diets supplemented with 0 and 2000 FTU phy-tase kg)1 In Trial 3, a series of dietary manipulations wasundertaken: The influence of feed particle size (1 versus0.25 mm screen using a Wiley Laboratory Mill, Model 4;Thomas Scientific, Swedesboro, NJ, USA) was examinedbecause of possible impact of rate and extent of digestion onphytase activity The interaction between phytase and theaddition of a protease/non-starch polysaccharidase (PNSP;

5 g kg)1Avizyme; Finnfeeds International, Wiltshire, UK)enzyme cocktail was examined The PNSP has the potential

to digest the protein polysaccharide network of the plantproteins thus altering the exposure of phyate-P to the phytaseenzyme The ratio of Ca/P was altered (0.75 : 1, 1 : 1 and

2 : 1) because this ratio has been shown to impact thedigestion of these two minerals in terrestrial animals Theimpact of supplementing 1, 25-dihydroxycholecalciferol(10 lg kg)1; Sigma Chemical Co., St Louis, MO, USA), aform of vitamin D involved in Ca and P absorption, wasadded to the basal diet Finally, the basal diet was supple-mented with the organic acid, LA (30 g kg)1; Sigma Chem-ical Co.), which can impact pH in the digestive tract andtherefore mineral digestion All of the aforementionedtreatments in trial 3 were tested against a control basal diet intriplicate tanks in a completely randomized design

Frozen faeces samples were freeze-dried, ground using a1-mm screen and stored at)30 C until required for analysis.Using standard methods (AOAC, 1990), feed and faeces were

#VIT-9608 Provides per kg diet (DM basis): vitamin A (as retinyl acetate):

3750 IU; vitamin D3 (as cholecalciferol): 3000 IU; vitamin E (as

dl-a-tocopheryl-acetate): 75 mg; vitamin K (as menadione

acid (as ascorbyl polyphosphate): 75 mg; d-biotin: 0.21 mg; choline

(as chloride): 1500 mg; folic acid: 1.5 mg; niacin (nicotinic acid):

15 mg; pantothenic acid: 30 mg; pyridoxine: 7.5 mg; riboflavin:

9 mg; thiamin: 1.5 mg.

#MIN-9504 Provides per kg diet (DM basis): sodium chloride (39%,

analysed for dry matter (DM) (drying in a vacuum oven at

65C for 18 h) and ash (incineration at 550 C for 18 h)

Crude protein (N· 6.25) was quantified using the

semi-automatic Kjedahl method (AOAC method 7.B01-7.B04;

Føss Electric, Hillerød, Denmark), lipid in the feed via ethyl

ether extraction (Soxtec System HT12; Foss Tecator AB,

Hoganas, Sweden) and energy using an adiabatic bomb

calorimeter (Parr Instrument Co., Moline, IL, USA)

Phosphorus was determined following ash digestion in 10%

nitric acid using a Technicon AutoAnalyzer apparatus

(Technicon Corporation, TarryTown, NY, USA) employing

the vanadate/molybdate method of analysis (Varley 1966)

Other minerals (Ca, Mg, Mn, Cu, Zn and Fe) were analysed

following ash digestion in 2 N hydrochloric acid using

atomic absorption spectrophotometer employing standard

methods (AOAC, 1990) Phytase activity and phytate were

measured according to (Radecki 1999) and (Radecki & Chen

1999), respectively Acid insoluble ash was measured

according to Atkinson et al (1984) with particular care taken

to thoroughly rinse the filtrate with boiling, de-mineralized

water ADC was calculated as described by Cho & Slinger

(1979): ADC = 1) (F/D · Di/Fi), where F = % nutrient

of feces, D = % nutrient of diet, Di= % indigestibile

marker (AIA) of diet, Fi= % indigestible marker (AIA) of

feces

All presented data are mean values of duplicate analyses from

three replicate tanks Apparent digestibility values were

subjected to analysis of variance using treatment, tank

rep-licate and their interactions with the statistical model as

appropriate ADC values in Trial 2 were analysed as a 2· 4

factorial design (phytase level· temperature) The effects of

Ca/P with and without phytase was analysed as a 3· 2

fac-torial design The effects of various dietary factors [particle

size, PNSP, 1,25-dihydroxycholecalciferol (HCC) and LA

supplementation] were analysed as a 2· 2 factorial design

Means were compared using the Duncans multiple range post

hoctest and were considered significant at P < 0.05

No mortalities were observed during the digestibility trials

The total phosphorus and phytate-P fractions on the

ingre-dients used to formulate the experimental diet are given in

Table 2 Phytate-P levels ranged from 51% to 88% of the

total P in the individual ingredients; in the experimental diet,

65% of the total P was in the form of phytate-P The results

of Trial 1 examining the influence of microbial phytaseaddition to the plant protein–based diet on nutrient ADC aregiven in Table 3 The addition of phytase significantlyincreased (P < 0.05) the ADC of a variety of mineralsincluding P, Ca, Mg, Fe, Zn and to a lesser degree Cu and

Mn Furthermore, ash, DM and protein ADCs were alsosignificantly higher (P < 0.05) as a result of phytase supple-mentation Phosphorus, Ca, ash and protein ADCs weremaximized with 4000 FTU kg)1 phytase, whereas theADCs of the other parameters were maximized with 2000FTU kg)1

In Trial 2, water temperature significantly affected(P < 0.05) the influence of phytase on P and ash (repre-senting an indicator of total mineral availability) ADCs(Fig 1) The effect of water temperature on the phytase-in-duced increase in nutrient ADC is more pronounced at the

4000 FTU kg)1level versus 2000 FTU kg)1as temperatureincreases; however at 5C, there was no increase in P or ash

Table 3 Nutrient ADC and influence of level of microbial phytase supplementation in rainbow trout fed a plant protein–based diet

Pooled SEM

activity): (0) 196, (2000) 1875, (4000) 3694 and (6000) 5636.

a,b,c

Within individual nutrients, means having different scripts are significantly different (P < 0.05).

and percentage phytate-phosphorus of the macro ingredients used in the dietary formulation

Analysed according to Radecki (1999).

Values based on three duplicate samples.

.

Aquaculture Nutrition 18; 369–379  2011 Blackwell Publishing Ltd

ADC when phytase activity was increased from 2000 to

4000 FTU kg)1 Whereas P and ash ADCs increased

significantly when water temperature was increased from 10

to 15C when 2000 FTU kg)1phytase was added to the diet,

P and ash ADCs were maximized between 5 and 10C with

4000 FTU phytase kg)1(Fig 1)

Trial 3 showed that decreasing feed particle size did not

influence DM, ash or P digestibility in control diets

(P > 0.05); however, a synergistic effect of decreasing

parti-cle size was noted for ash and P when 2000 FTU

phy-tase kg)1 was added (Table 4) The addition of a PNSP

enzyme cocktail in Trial 4 significantly increased DM ADC

(P < 0.05) but had no influence on P or ash ADC in control

diets The effects of phytase on DM, ash and P ADC were

amplified when phytase and the PNSP enzyme cocktail were

added simultaneously (Table 5)

The influence of changing the ratio of Ca/P on the control

and phytase-supplemented diets is shown in Fig 2 For both

control- and phytase-added diets, ash and P ADCs did not

follow consistent patterns For example, in the

phytase-sup-plemented diets, ash ADC was similar for the 0.75 : 1 and the

2 : 1 groups, but lower in the 1 : 1 group The ADC for P

was highest in the 0.75 : 1 group, and significantly lower

(P < 0.05) in both the 1 : 1 and 2 : 1 groups One trend,

however, can be observed: as the Ca/P ratio increases, theresponse of the ADCs for both ash and P to added phytasetends to decrease

The addition of 1,25-dihydroxycholecalciferol (HCC;Table 6) or lactic acid (LA; Table 7) to the experimentaldiet with and without phytase did not significantly alterADC for DM and ash; however, the increase in P ADCfollowing phytase addition was significantly lower(P < 0.05) when either HCC or LA were combined withphytase

10 C

15 C

20 C

A,X A,X

A,X A,X

B,X

A,X

B,Y

B,Y C,X

A-DWithin individual variables, different values are significantly different (P < 0.05).

XYWithin individual temerpatures, different values are significantly different (P < 0.05).

Figure 1 Phosphorus and ash apparent digestibility coefficient from

temperatures.

Table 4 Influence of microbial phytase supplementation and feed particle size reduction (PSR) on apparent nutrient digestibility coefficients in rainbow trout fed a plant protein–based diet

Dietary

Phytase activity

basal diets were ground using a 1-mm screen.

2

Table 5 Influence of microbial phytase supplementation and tion of a protease/non-starch polysaccharidase (PNSP) on apparent nutrient digestibility coefficients in rainbow trout fed a plant protein– based diet

addi-Dietary

Phytase activity

The present study clearly demonstrates that the addition of

microbial phytase to a plant protein–based diet fed to

rain-bow trout significantly increases the ADC of a number of

nutrients and identifies a variety of factors that affects the

response to phytase The dietary formulation employed was

adapted from previous work, which showed that a similar

diet provided adequate growth rates versus a commercial

control diet (Adelizi et al 1998) Work in our laboratory hasshown that the formulation used in this study results in sig-nificantly lower growth rate versus a fish meal–based controldiet (G.W Vandenberg, unpublished data) However, thecurrent formulation does permit the evaluation of thepotential of microbial phytase addition to diets containingelevated levels of phytate-P and will be increasingly appli-cable as superior plant protein sources are identified ascandidates for fish meal replacement in salmonid diets(Hughes 1991; Gomes et al 1995; Carter 2000)

The phytase enzyme employed in the present study is anacid phosphatase that catalyses the hydrolysis of phosphateesters from phytate (Shute et al 1988) Given that the mo-nogastrics do not possess significant endogenous phytaseactivity (Pointillart et al 1984), the availability of phytate-P

is considered negligible in these species (Maga 1982) and isconsidered completely unavailable when formulating prac-tical rations The addition of microbial phytase significantlyincreases P digestibility in domestic monogastrics; Jongbloed

et al (1992) reported that the addition of 1500 FTU tase kg)1 increased P digestibility of a practical swine dietfrom 16% to 46%, with phytate-P digestibility increasingfrom )1.4% to 74% Optimal phytase inclusion levels forswine have been reported in the range of 800–1200 FTU kg)1,resulting in a 40% increase in overall P retention (Mroz et al

phy-1994) Similar increases in P availability following phytasesupplementation have been reported in a variety of mono-gastric species (Broz et al 1994; Denbow et al 1995; Punna &

Roland 1999; Atia et al 2000), resulting in consistent, andsignificant increases in P digestibility The effect of phytase on

P availability has been evaluated in a number of fish species,

Table 6 Influence of microbial phytase supplementation and

1,25-dihydroxycholecalciferol (HCC) addition apparent nutrient

digest-ibility coefficients in rainbow trout fed a plant protein–based diet

Table 7 Influence of microbial phytase supplementation and lactic acid addition (LA) on apparent nutrient digestibility coefficients in rainbow trout fed a plant protein–based diet

Dietary

Phytase activity

Figure 2 The influence of Ca/P ratio on P and ash apparent

.

Aquaculture Nutrition 18; 369–379  2011 Blackwell Publishing Ltd

either by pretreatment of ingredients with phytase (Cain &

Garling 1995; Ramseyer et al 1999; Van Weerd et al 1999)

or direct addition to the diet (Brown 1993; Rodehutscord &

Pfeffer 1995; Schaefer et al 1995; Jackson et al 1996; Hughes

& Soares 1998; Oliva-Teles et al 1998; Vielma et al 1998;

Nwanna et al 2006; Baruah et al 2007) Although the

addition of phytase to diets of non-carnivorous fish species is

immediately applicable, a number of reports have shown that

microbial phytase can increase nutrient availability from

salmonid diets having a significant proportion of the fish meal

replaced by plant-based proteins Studies in salmonids have

used a wide range of phytase levels (500–4500 FTU kg)1);

however, there is a lack of information regarding optimal

phytase inclusion levels, particularly in practical diets

The present study reveals that P ADC is optimized at

4000 FTU kg)1, with no further increase in at 6000 FTU kg)1

(Table 3) Forster et al (1999) reported a linear increase in P

and phytate digestibility following addition of graded levels of

phytase (500–4500 FTU kg)1) in rainbow trout diets

con-taining high levels of canola protein concentrate In a soybean

meal–based diet fed to rainbow trout, Sugiura et al (2001)

reported that the absorption of P increased from 26.6% (no

phytase added) to 90.1% (4000 FTU phytase added per kg

dry diet); the relationship was not linear, and the efficacy of

supplemental phytase decreased as the inclusion level

in-creased In this study, at the highest level of phytase inclusion,

phytate ADC was 45%, suggesting that higher levels of

phy-tase addition may have further improved its ADC A number

of studies in other monogastrics have shown that a variety of

dietary factors contribute to the efficacy of microbial phytase,

including the level of available and total P and phytate P (Yi

et al.1996b; Kemme et al 1997); therefore, slight differences

in these parameters between studies make difficult the

deter-mination of an optimal phytase level

The addition of phytase also improved the ADC of a

number of other minerals including Ca, Mg, Fe and Zn

(Table 2) This effect has been widely reported in a number of

species (Adeola et al 1995; Kemme et al 1999b), and

al-though not directly studied to a large extent in fish, there

exists evidence that dietary phytate significantly reduces

growth and feed efficiency (Richardson et al 1985) as well as

the availability of Zn, Fe and Cu to salmonids (Spinelli et al

1983) The interaction between phytate and zinc was

con-firmed in catfish, as increasing levels of dietary phytate

reduced vertebral zinc deposition (Satoh et al 1989), and

increased the dietary requirement of zinc by a factor of 10

(Gatlin & Phillips 1989) More recently, the influence of

dietary phytase on trace mineral ADC has confirmed the

ability of phytase to improve the ADC of a number of

minerals in rainbow trout (Sugiura 1998; Vielma et al 1998;Cheng & Hardy 2003) and striped bass (Papatryphon et al.1999) In the current study, the response of Cu and Mn tosupplemental phytase was not consistent and may be due

to the fact that macro and trace minerals were supplemented

to NRC levels, which may have masked some effects, as hasbeen previously suggested in other species (Adeola 1995).The addition of microbial phytase significantly increasedthe ADC of protein (Table 3), an effect that is related to thesecondary effects of phytate binding to proteins Under cer-tain conditions, phytate has the ability to bind to dietaryproteins (Knuckles et al 1985), thus reducing their avail-ability, as well as a variety of proteolytic enzymes (Caldwell1992) Supplementation with microbial phytase has beenreported to increase protein ADC in a swine (Kemme et al.1999a) and poultry (Ravindran et al 1999a), and althoughthe magnitude of response of protein ADC is generally lower

to that of P, dietary phytase leads to an overall increase in Nretention (Yi et al 1996a) The effect of phytase supple-mentation on protein digestibility in diets for fish is less clear.Although several reports have shown a response of proteindigestibility to phytase supplementation (Vielma et al 2004),

a number of studies have not (Cao et al 2007) The nisms related to phytase-induced alteration of proteindigestibility remain largely uncharacterized; further research

mecha-is required to better understand thmecha-is aspect Interestingly, theADC of both P and protein was significantly lower at thehighest level of phytase inclusion; a tendency for lowerADCS of a number of other components was also noted(Table 1) Few other studies have supplemented phytase atlevels above 4000 FTU kg)1in diets for salmonids; therefore,there is little evidence in the literature for this observation

In warm water fish species including Rohu, (Labeo rohi)(Baruah et al 2007) and African catfish (Clarias gariepinus)(Nwanna et al 2006), this observation has been reported for

P and protein ADC, albeit at lower ranges of phytaseinclusion Reduced nutrient digestibility at higher levels ofphytase inclusion may be related to product inhibition of thephytase reaction as a result of enhanced enzyme activity,although direct evidence in the current study is not available.Figure 1 confirms the importance of temperature on theability of phytase to affect P and ash digestibility Phytaseactivity is maximized at 55C, below which the activitydecreases linearly to approximately 10% relative activity at

5C (Keis 1999a); the activity of phytase at 15 C is reported

to be 25% that of its activity at 37C (Sugiura 1998) Resultsfrom the current study suggest that phytase may have limitedutility in water temperatures in the range of 5–10C, irre-spective of level of phytase supplementation

.

Aquaculture Nutrition 18; 369–379  2011 Blackwell Publishing Ltd

Feed particle size is observed to affect the ability of phytase

to increase nutrient ADC (Table 4) The phytate in oilseeds is

localized within globoids throughout the seed, whereas in

cereal crops, phytate is isolated within the aleurone layer

(Ravindran et al 1994), which is particularly resistant to

digestion Thus, if not adequately disrupted, the phytase

enzyme may not have full access to the phytate

Further-more, a smaller particle size and thus, larger effective surface

area, will permit increased phytate diffusion out of the feed

particles (Bedford & Schulze 1998) In a similar manner, the

co-administration of phytase and a commercial cocktail of

PNSPs likely chemically degraded the

protein–polysaccha-ride network around the phytate, thus allowing greater

availability for phytate–phytase interaction (Ravindran et al

1999b) A better understanding of particle size and impact of

NSP enzymes (and any potential interactions) is warranted

given the present findings Salmonid fish do seem to be

negatively affected by very fine grinding of feed ingredients as

is the case in terrestrial monogastrics (Wondra et al 1995);

thus, the potential benefits of fine grinding and/or NSP

enzyme supplementation regarding response to phytase

supplementation should be more thoroughly studied

In the current study, the addition of 1,

25-dihydroxyc-holeocalciferol had no effect on the availability of nutrients

from control or phytase-supplemented diets (Table 6) This is

in contrast with a number of studies reporting the direct

effect of vitamin D analogs on P availability, and the

syn-ergistic effects related to P ADC in poultry following phytase

supplementation in combination with vitamin D or

meta-bolic analogs (Edwards 1993; Mitchell & Edwards 1996;

Qian et al 1997) Vitamin D is essential for normal growth of

terrestrial animals as well as rainbow trout (Barnett et al

1979), although the metabolic roles of vitamin D and its

analogs in fish are not well characterized A number of

studies have shown that injection of vitamin D analogs

induces alteration of P homoeostasis in a variety of fish

species (Swarup & Srivastav 1982; Fenwick et al 1984;

Fenwick & Vermette 1989; Swarup et al 1991), although

Vielma et al (1999) reported that orally applied vitamin D3

in P replete and deficient diets had no influence on P

metabolism Our findings are in agreement with those of

Vielma et al (1998), who reported a lack of response of P

metabolism in rainbow trout fed graded levels of

cholecal-ciferol with and without microbial phytase

The supplementation of postweaning swine diets with

citric, lactic, propionic and formic and fumaric acids or their

salts has been suggested to improve growth through

improved digestion, absorption and retention of a variety of

nutrients during critical production phases (Partanen &

Mroz 1999) More recently, a synergistic effect on P ADCfollowing phytase addition has been observed for a number

of organic acids in swine (Li et al 1998; Kemme et al

1999b) This effect is likely related the reduction in gastric

pH, thus promoting phytase activity, as well as the chelatingproperties of certain acids, which may reduce specific mineralinteractions that reduce their availability (Partanen & Mroz1999) Sugiura et al (1998a) reported that supplementation

of fish meal–based diets with 50 g kg)1 citric acid cantly increased P ADC; this group has also demonstratedthe additive effects of citric acid to plant protein–based dietsupplemented with phytase, which was not observed when asoybean–fish meal diet was tested (Sugiura 1998) Theseresults suggest complex interactions between different forms

signifi-of dietary P, citric acid and phytase The lack signifi-of a synergisticresponse between LA and phytase in the present study maysimply be related to the level of LA employed (30 g kg)1w/w),which may not be sufficient to affect an adequate reduction indietary pH

Increasing the Ca/P ration has been previously reported

to reduce the effectiveness of phytase in both swine andpoultry diets (Lei et al 1994; Qian et al 1996; Li et al 1999)

This effect is likely due to the formation of insolubleCa-phytate complexes, thus rendering dietary phytateinsensitive to phytase supplementation (Qian et al 1996) Inthe present study, there was no consistent influence on ADCfor ash or P resulting from increasing the Ca/P ratio in thecontrol diets (Fig 2) In phytase-supplemented diets,however, increasing Ca/P ratio reduced the phytase-relatedincrease in P and ash ADC, suggesting a Ca-induced inter-ference with phytase activity Although not directly tested, it

is interesting to speculate on the possibility that the calciumcarbonate used as a source of supplemental Ca may also act

as a dietary buffer, which may have also contributed to thereduction in response to supplemented phytase

This work was supported by the Natural Sciences andEngineering Research Council (NSERC Strategic ProjectsProgram) and BASF Canada Inc GWV was the recipient of

an NSERC Postgraduate Scholarship The authors aregrateful to Martin Feed Mills who generously supplied thedietary ingredients, Richard Prince and Pierre Castonguayfor their help with the feed formulation, Jean Bricault,Francine Gigue`re and Andre´ Roy for their skilled analyticalassistance and the staff of the Laboratoire re´gional dessciences aquatiques for their assistance with the feedingexperiments

.

Aquaculture Nutrition 18; 369–379  2011 Blackwell Publishing Ltd

Adelizi, P.D., Rosati, R.R., Warner, K., Wu, Y.V., Muench, T.R.,

White, M.R & Brown, P.B (1998) Evaluation of fish-meal free

diets for rainbow trout, Oncorhynchus mykiss Aquac Nutr., 4,

255–262.

Adeola, O (1995) Digestive utilization of minerals by weanling pigs

fed copper- and phytase-supplemented diets Can J Anim Sci.,

75, 603–610.

Adeola, O., Lawrence, B.V., Sutton, A.L & Cline, T.R (1995)

Phytase-induced changes in mineral utilization in

zinc-supple-mented diets for pigs J Anim Sci., 73, 3384–3391.

AOAC (1990) Official Methods of Analysis, 15th edn Association of

Official Analytical Chemists Inc., Arlington, VA.

Atia, F.A., Waibel, P.E., Hermes, I., Carlson, C.W & Walser, M.M.

(2000) Effect of dietary phosphorus, calcium, and phytase on

performance of growing turkeys Poult Sci., 79, 231–239.

Atkinson, J.L., Hilton, J.W & Slinger, S.J (1984) Evaluation of acid

insoluble ash as an indicator of feed digestibility in rainbow trout

(Salmo gairdneri) Can J Fish Aquat Sci., 41, 1384–1386.

Barnett, B.J., Cho, C.Y & Slinger, S.J (1979) The essentially of

cholecalciferol in the diets of rainbow trout Comp Biochem.

Physiol., 63A, 291–297.

Baruah, K., Pal, A.K., Sahu, N.P., Debnath, D., Nourozitallab, P &

Sorgeloos, P (2007) Microbial phytase supplementation in rohu,

labeo rohita, diets enhances growth performance and nutrient

digestibility J World Aquac Soc., 38, 129–137.

Bedford, M.R & Schulze, H (1998) Exogenous enzymes for pigs

and poultry Nutr Res Rev., 11, 91–114.

Brown, P.B (1993) Comparison of fecal collection methods for

443–447 Institut national de la recherche agronomique, Paris,

France.

Broz, J., Oldale, P., Perrin, V.A.H., Rychen, G., Schulze, J & Nunes,

C.S (1994) Effects of supplemental phytase on performance and

phosphorus utilization in broiler chickens fed a low phosphorus

diet without addition of inorganic phosphates Br Poult Sci., 35,

273–280.

Bureau, D.P., Harris, A.M & Y., C.C (1998) The effects of purified

alcohol extracts from soy products on feed intake and growth of

chinook salmon (Oncorhynchus tshawytscha) and rainbow trout

(Oncorhynchus mykiss) Aquaculture, 161, 27–43.

Cain, K.D & Garling, D.L (1995) Pretreatment of soybean meal

with phytase for salmonid diets to reduce phosphorus

concentra-tions in hatchery effluents Prog Fish Cult., 57, 114–119.

Caldwell, R.A (1992) Effect of calcium and phytic acid on the

activation of trypsinogen and the stability of trypsin J Agric.

Food Chem., 40, 43–46.

Cao, L., Wang, W., Yang, C., Yang, Y., Diana, J., Yakupitiyage, A.,

Luo, Z & Li, D (2007) Application of microbial phytase in

fish feed Enzyme Microb Tech., 40, 497–507.

Carter, C.G (2000) Fish meal replacement by plant meals in

ex-truded feeds for Atlantic salmon, Salmo salar L Aquaculture, 185,

299–311.

Cheng, Z.J & Hardy, R.W (2003) Effects of extrusion and expelling

processing, and microbial phytase supplementation on apparent

digestibility coefficients of nutrients in full-fat soybeans for

rain-bow trout (Oncorhynchus mykiss) Aquaculture, 218, 501–514.

Cho, C.Y & Slinger, S.J (1979) Apparent digestibility measurement

in feedstuffs for rainbow trout In: Finfish Nutrition and Fishfeed

Technology, Vol II (Halver, J.E & Tiews, K eds), pp 239–247.

Heenemann Verlagsgesellschaft, Berlin, Germany.

Davenport, J., Black, K., Burnell, G., Cross, T., Culloty, S., aratne, S., Furness, B., Mulcahy, M & Thetmeyer, H (2003) Aquaculture: The Ecological Issues Blackwell Science Ltd, Malde, Oxford, Melbourne, Berlin.

Ek-Davies, N.T & Nightingale, R (1975) The effects of phytate on intestinal absorption of zinc, and secretion of zinc, and whole body retention of zinc, copper, iron and manganese in rats Br J Nutr.,

34, 243–250.

Denbow, D.M., Ravindran, V., Kornegay, E.T., Yi, Z & Hulet, R.M (1995) Improving phosphorus availability in soybean meal for broilers by supplemental phytase Poult Sci., 74, 1831– 1842.

Drew, M.D., Racz, V.J., Gauthier, R & Thiessen, D.L (2005) Effect

of adding protease to coextruded flax:pea or canola:pea products

on nutrient digestibility and growth performance of rainbow trout (Oncorhynchus mykiss) Anim Feed Sci Technol., 119, 117–128 Edwards, H.E.J (1993) Dietary 1,25-dihydroxycholecalciferol sup- plementation increases natural phytate phosphorus utilization in chickens J Nutr., 123, 567–577.

Eya, J.C & Lovell, R.T (1997) Net absorption of dietary phorus from various inorganic sources and effect of fungal phytase

phos-on net absorptiphos-on of plant phosphorus by channel catfish Ictalurus punctatus J World Aquac Soc., 28, 386–391.

handling of phosphate in American eels Fish Physiol Biochem., 7, 351–358.

Fenwick, J.C., Smith, K., Smith, J & Flik, G (1984) Effect of ious vitamin D analogs on plasma calcium and phosphorus and intestinal calcium absorption in fed and unfed American eels, Anguilla rostrata Gen Comp Endocrinol., 55, 398–404.

var-Forster, I., Higgs, D.A., Dosanjh, B.S., Rowshandeli, M & Parr, J (1999) Potential for dietary phytase to improve the nutritive value

of canola protein concentrate and decrease phosphorus output in rainbow trout (Oncorhynchus mykiss) held in 11 degree C fresh water Aquaculture, 179, 109–125.

Gatlin, D.M III & Phillips, H.F (1989) Dietary calcium, phytate and zinc interactions in channel catfish Aquaculture, 79, 259–266 Glencross, B.D., Boujard, T & Kaushik, S.J (2003) Influence of oligosaccharides on the digestibility of lupin meals when fed to rainbow trout, Oncorhynchus mykiss Aquaculture, 219, 703–713 Gomes, E.F., Rema, P & Kaushik, S.J (1995) Replacement of fish meal by plant proteins in the diet of rainbow trout (Oncorhynchus mykiss): digestibility and growth performance Aquaculture, 130, 177–186.

Gouveia, A., Olivia Teles, A., Gomes, E & Rema, P (1993) Effect of cooking/expansion of three legume seeds on growth and food utilization by rainbow trout In: Fish Nutrition in Practice (Kanshik, S.J & Luquet, P eds) Institut national de la recherche agronomique, Paris, France pp 933–938.

Graf, E (1986) Phytic Acid: Chemistry and Applications Pilatus Press, Minneapolis, MN.

Gur, N (1998) Enzymatic supplements in aquafeeds Fish Isr., 31, 78–86.

Fishbreed-Hughes, S.G (1991) Use of lupin flour as a replacement for full-fat soy in diets for rainbow trout (Oncorhynchus mykiss) Aquaculture,

93, 57–62.

Hughes, K.P & Soares, J.H.J (1998) Efficacy of phytase on phorus utilization in practical diets fed to striped bass Morone saxatilis Aquac Nutr., 4, 133–140.

phos-Jackson, L.S., Li, M.H & Robinson, E.H (1996) Use of microbial phytase in channel catfish Ictalurus punctatus diets to improve utilization of phytate phosphorus J World Aquac Soc., 27, 309– 313.

.

Aquaculture Nutrition 18; 369–379  2011 Blackwell Publishing Ltd

Jongbloed, A.W., Mroz, Z & Kemme, P.A (1992) The effect of

supplementary Aspergillus niger phytase in diets for pigs on

con-centration and apparent digestibility of dry matter, total

phos-phorus, and phytic acid in different sections of the alimentary

tract J Anim Sci., 70, 1159–1168.

Keis, A.K (1999a) Phytase: mode of action In: Phytase in Animal

Nutrition and Waste Management: a BASF Reference Manual

(Coelho, M.B & Kornegay, E.T eds), pp 205–212 BASF

Cor-poration, Mount Olive, NJ.

Keis, K (1999b) Phytase- mode of action In: Phytase in Animal

E.T eds), pp 205–212 BASF Corp, Mount Olive, NJ.

Kemme, P.A., Radcliffe, J.S., Jongbloed, A.W & Mroz, Z (1997)

Factors affecting phosphorus and calcium digestibility in diets for

growing-finishing pigs J Anim Sci., 75, 2139–2146.

Kemme, P.A., Jongbloed, A.W., Mroz, Z., Kogut, J & Beynen, A.C.

(1999a) Digestibility of nutrients in growing-finishing pigs is

affected by Aspergillus niger phytase, phytate and lactic acid levels.

1 Apparent ileal digestibility of amino acids Livest Prod Sci., 58,

107–117.

Kemme, P.A., Jongbloed, A.W., Mroz, Z., Kogut, J & Beynen, A.C.

(1999b) Digestibility of nutrients in growing-finishing pigs is

affected by Aspergillus niger phytase, phytate and lactic acid levels.

2 Apparent total tract digestibility of phosphorus, calcium and

magnesium and ileal degradation of phytic acid Livest Prod Sci.,

58, 119–127.

Knuckles, B.E., Kuzmicky, D.D & Betschart, A.A (1985) Effect of

phytate and partially hydrolyzed phytate on in vitro protein

digestibility J Food Sci., 50, 1080–1082.

Lanari, D., DAgaro, E & Turri, C (1998) Use of nonlinear

regression to evaluate the effects of phytase enzyme treatment of

plant protein diets for rainbow trout (Oncorhynchus mykiss).

Aquaculture, 161, 343–354.

Lee, K.J., Dabrowski, K., Blom, J.H., Bai, S.C & Stromberg, P.C.

(2002) A mixture of cottonseed meal, soybean meal and animal

byproduct mixture as a fish meal substitute: growth and tissue

gossypol enantiomer in juvenile rainbow trout (Oncorhynchus

mykiss) J Anim Physiol Anim Nutr., 86, 201–213.

Lei, X.G., Ku, P.K., Miller, E.R., Yokoyama, M.T & Ullrey, D.E.

(1994) Calcium level affects the efficacy of supplemental microbial

phytase in corn-soybean meal diets of weanling pigs J Anim Sci.,

72, 139–143.

Li, M.H & Robinson, E.H (1997) Microbial phytase can replace

inorganic phosphorus supplements in channel catfish Ictalurus

Li, D., Che, X., Wang, Y., Hong, C & Thacker, P.A (1998) Effect of

perfor-mance and phosphorus, nitrogen and calcium digestibility in

growing swine Anim Feed Sci Technol., 73, 173–186.

Li, D., Che, X.R., Wang, Y.Q., Qiao, S.Y., Cao, H., Johnson, W &

Thacker, P (1999) The effect of calcium level on microbial phytase

activity and nutrient balance in swine Asian-Australas J Anim.

Sci., 12, 197–202.

Maga, J.A (1982) Phytate: its chemistry, occurence, food

interac-tions, nutritional significance and methods of analysis J Agric.

Food Chem., 30, 1–8.

Mitchell, R.D & Edwards, H.M Jr (1996) Effects of phytase and

1,25-dihydroxycholecalciferol on phytate utilization and the

quantitative requirement for calcium and phosphorus in young

broiler chickens Poult Sci., 75, 95–110.

Moser, R.L., Poe, E.R., Moser, B.D & Lewis, A.J (1982) Effect of

grain source and dietary levels of oat hulls on phosphorus

and calcium utilization in the growing pig J Anim Sci., 54, 800–

Nelson, T.S., Shieh, T.R., Woodzinski, R.J & Ware, J.H (1968) The availability of phytate phosphorus in soya bean meal before and after treatment with mold phytase Poult Sci., 47, 1842–1848.

Nelson, T.S., Shieh, T.R., Wodzinski, R.J & Ware, J.H (1971) Effect of supplemental phytase on the utilization of phytate phosphorus by chicks J Nutr., 101, 1289–1292.

NRC (1993) Nutrient Requirements of Fish National Academy Press, Washington, D.C.

Nwanna, L.C., Ajani, E.K & Bamidele, F (2006) Determination of optimum dietary phytase level for the growth and mineral deposition in African catfish (Clarias gariepinus) Aquat Sci., 54, 75–82.

Oliva-Teles, A., Pereira, J.P., Gounveia, A & Gomes, E (1998) Utilisation of diets supplemented with microbial phytase by sea- bass (Dicentrachus labrax) juveniles Aquat Living Resour., 11, 255–259.

Papatryphon, E., Howell, R.A & Soares, J.H Jr (1999) Growth and mineral absorption by striped bass Morone saxatilis fed a plant feedstuff based diet supplemented with phytase J World Aquac.

Phy-Punna, S & Roland, D.A Sr (1999) Influence of supplemental microbial phytase on first cycle laying hens fed phosphorus- deficient diets from day one of age Poult Sci., 78, 1407–1411.

Qian, H., Kornegay, E.T & Conner, D.E Jr (1996) Adverse effects

of wide calcium:phosphorus ratios on supplemental phytase cacy for weanling pigs fed two dietary phosphorus levels J Anim.

effi-Sci., 74, 1288–1297.

Qian, H., Kornegay, E.T & Denbow, D.M (1997) Utilization of phytate phosphorus and calcium as influenced by microbial phy- tase, cholecalciferol, and the calcium:total phosphorus ratio in broiler diets Poult Sci., 76, 37–46.

Radecki, B (1999) Determination of phytic acid in feed by HPLC.

In: Phytase in Animal Nutrition and Waste Management: A BASF

Corporation, Mount Olive, NJ pp 751–752.

Radecki, B & Chen, J (1999) Determination of phytase activity in straight products premixes and feeds In: Phytase in Animal Nutrition and Waste Management: A BASF Reference Manual (Coelho, M.B & Kornegay, E.T eds), pp 743–750 BASF Corporation, Mount Olive, NJ.

Ramseyer, L., Garling, D.J., Hill, G & Link, J (1999) Effect of dietary zinc supplementation and phytase pre-treatment of soy- bean meal or corn gluten meal on growth, zinc status and zinc- related metabolism in rainbow trout, Oncorhynchus mykiss Fish Physiol Biochem., 20, 251–261.

Ravindran, V., Ravindran, G & Sivalogan, S (1994) Total and phytate phosphorus contents of various foods and feedstuffs of plant origin Food Chem., 50, 133–136.

.

Aquaculture Nutrition 18; 369–379  2011 Blackwell Publishing Ltd

Ravindran, V., Cabahug, S., Ravindran, G & Bryden, W.L (1999a)

Influence of microbial phytase on apparent ileal amino acid

digestibility of feedstuffs for broilers Poult Sci., 78, 699–706.

Ravindran, V., Selle, P.H & Bryden, W.L (1999b) Effects of phytase

supplementation, individually and in combination, with glycanase,

on the nutritive value of wheat and barley Poult Sci., 78, 1588–

1595.

Regost, C., Arzel, J & Kaushik, S (1999) Partial or total

replace-ment of fish meal by corn gluten meal in diet for turbot (Psetta

maxima) Aquaculture, 180, 99–117.

Richardson, N.L., Higgs, D.A., Beames, R.M & McBride, J.R.

(1985) Influence of dietary calcium, phosphorus, zinc and sodium

phytate level on cataract incidence, growth and histopathology in

juvenile chinook salmon (Oncorhynchus tshawytscha) J Nutr.,

115, 553–567.

Riche, M & Brown, P (1999) Incorporation of plant protein

feed-stuffs into fish meal diets for rainbow trout increases phosphorus

availability Aquac Nutr., 5, 101–105.

Rodehutscord, M & Pfeffer, E (1995) Effects of supplemental

microbial phytase on phosphorus digestibility and utilization in

rainbow trout (Oncorhynchus mykiss) Water Sci Technol., 31,

143–147.

Sandberg, A.S & Anderson, H (1988) Effect of dietary phytase on

the digestion of phytate in the stomach and small intestine of

humans J Nutr., 118, 469–473.

Sanz, A., Morales, A.E., de la Higuera, M & Cardenete, G (1994)

Sunflower meal compared with soybean meal as partial substitutes

for fish meal in rainbow trout (Oncorhynchus mykiss) diets: protein

and energy utilization Aquaculture, 128, 287–300.

Satoh, S., Poe, W.E & Wilson, R.P (1989) Effect of supplemental

phytate and/or tricalcium phosphate on weight gain, feed efficiency

and zinc content in vertebrae of channel catfish Aquaculture, 80,

155–161.

Schaefer, A., Koppe, W.M., Meyer-Burgdorff, K.H & Guenther,

K.D (1995) Effects of a microbial phytase on the utilization of

native phosphorus by carp in a diet based on soybean meal Water

Sci Technol., 31, 149–155.

Shi, P., Huang, H., Wang, Y., Luo, H., Wu, B., Meng, K., Yang, P.

& Yao, B (2008) A novel phytase gene appA from Buttiauxella sp.

GC21 isolated from grass carp intestine Aquaculture, 275, 70–75.

Shute, J.K., Baker, R., Billington, D.C & Gani, D (1988)

Mecha-nism of the myo-inositol phosphatase reaction J Chem Soc.

Chem Commun., 31, 422–423.

Spinelli, J., Houle, C.R & Wekell, J.C (1983) The effect of phytates

on the growth of rainbow trout (Salmo gairdneri) purified diets

containing varying quantities of calcium and magnesium

Aqua-culture, 30, 71–84.

Sugiura, S.H (1998) Development of low-pollution feeds for

sustain-able aquaculture, Vol Ph.D University of Washington, Seattle,

WA.

Sugiura, S.H., Dong, F.M & Hardy, R.W (1998a) Effects of dietary

supplements on the availability of minerals in fish meal:

pre-liminary observations Aquaculture, 160, 283–303.

Sugiura, S.H., Dong, F.M., Rathbone, C.K & Hardy, R.W (1998b)

Apparent protein digestibility and mineral availabilities in

various feed ingredients for salmonid feeds Aquaculture, 159, 177–

202.

Sugiura, S.H., Gabaudan, J., Dong, F.M & Hardy, R.W (2001)

Dietary microbial phytase supplementation and the utilization

of phosphorus, trace minerals and protein by rainbow trout

[Oncorhynchus mykiss (Walbaum)] fed soybean meal-based diets.

Aquacult Res., 32, 583–592.

hyper-calcaemia in male catfish Clarias batrachus Gen Comp nol., 46, 271–274.

Endocri-Swarup, K., Das, V.K & Norman, A.W (1991) Dose-dependent

and hyperphosphatemia in male cyprinoid Cyprinus carpio Comp Biochem Physiol A Mol Integr Physiol., 100, 445–447 Tacon, A.G.J (1997) Fishmeal replacers: review of antinutrients within oilseeds and pulses A limiting factor for the aquafeed green revolution? In: Feeding Tomorrows Fish (Tacon, A.G.J & Bas- urco, B eds), pp 153–182 Centre international de hautes e´tudes agronomiques me´diterrane´ennes, Zaragoza, Spain.

Tacon, A.G.J & Metian, M (2008) Global overview on the use of fish meal and fish oil in industrially compounded aquafeeds: trends and future prospects Aquaculture, 285, 146–158.

Tacon, A.G.J & Metian, M (2009) Fishing for aquaculture: food use of small pelagic forage fish – a global perspective Rev Fish Sci., 17, 305–317.

non-Thiessen, D., Maenz, D., Newkirk, R., Classen, H & Drew, M (2004) Replacement of fishmeal by canola protein concentrate in diets fed to rainbow trout (Oncorhynchus mykiss) Aquac Nutr.,

10, 379–388.

Van Weerd, J.H., Khalaf, K.A., Aartsen, F.J & Tijssen, P.A.T (1999) Balance trials with African catfish Clarias gariepinus fed phytase-treated soybean meal-based diets Aquac Nutr., 5, 135– 142.

Vandenberg, G & de la Nou¨e, J (2001) Apparent digestibility comparison in rainbow trout (Oncorhynchus mykiss) assessed using three methods of faeces collection and three digestibility markers Aquac Nutr., 7, 237–245.

Varley, J.A (1966) Automatic method for determination of nitrogen, phosphorus and potassium in plant material Analyst, 91, 452– 455.

Vielma, J., Lall, S.P., Koskela, J., Schoner, F.J & Mattila, P (1998) Effects of dietary phytase and cholecalciferol on phosphorus bio- availability in rainbow trout (Oncorhynchus mykiss) Aquaculture,

163, 307–321.

Vielma, J., Lall, S.P., Koskela, J & Mattila, P (1999) Influence of low dietary cholecalciferol intake on phosphorus and trace element metabolism by rainbow trout (Oncorhynchus mykiss, Walbaum) Comp Biochem Physiol A Mol Integr Physiol., 122, 117–125 Vielma, J., Ruohonen, K., Gabaudan, J & Vogel, K (2004) Top- spraying soybean meal-based diets with phytase improves protein and mineral digestibilities but not lysine utilization in rainbow trout,

Williams, P.J & Taylor, T.J (1985) A comparative study of phytate hydrolysis in the gastrointestinal tract of the golden hamster (Mesocricetus auratus) and the laboratory rat Br J Nutr., 54, 429–435.

Wondra, K.J., Hancock, J.D., Behnke, K.C., Hines, R.H & Stark, C.R (1995) Effects of particle size and pelleting on growth per- formance, nutrient digestibility, and stomach morphology in finishing pigs J Anim Sci., 73, 757–763.

Yi, Z., Kornegay, E.T & Denbow, D.M (1996a) Effect of microbial phytase on nitrogen and amino acid digestibility and nitrogen retention of turkey poults fed corn-soybean meal diets Poult Sci.,

75, 979–990.

Yi, Z., Kornegay, E.T., Ravindran, V., Lindemann, M.D & Wilson,

bioavailabilities of phosphorus and other nutrients in soybean meal-based semipurified diets for young pigs J Anim Sci., 74, 1601–1611.

.

Aquaculture Nutrition 18; 369–379  2011 Blackwell Publishing Ltd

Nutrition Laboratory, Institute of Aquatic Economic Animals, School of Life Science, Sun Yat-Sen University, Guangzhou,

China

A growth trial was conducted to estimate the optimum

requirement of dietary zinc (Zn) for grass carp

(Ctenophar-yngodon idella) Triplicate groups of grass carp (3.97 ±

0.05 g) were fed diets containing graded levels (13, 25, 34,

53, 89 and 135 mg kg 1) of Zn for 8 weeks Grass carp fed

with dietary Zn levels higher than 34 mg kg 1significantly

increased final body weight, weight gain and specific

growth rate (P< 0.05) For body composition, fish fed

with dietary Zn levels higher than 53 mg kg 1significantly

decreased the moisture contents but increased the lipid

con-tents of whole body and liver Whole body, scales,

verte-brae and liver mineralization were all affected significantly

(P < 0.05) by dietary Zn levels Zn contents in whole body,

scales, vertebrae and plasma were linearly increased up to

the 53 mg kg 1 dietary Zn and then remained stable

beyond this level Grass carp fed with dietary Zn levels

higher than 53 mg kg 1 significantly increased

triacyglyce-ride and total cholesterol contents and plasma alkaline

phosphatase activity in plasma (P< 0.05) Broken-line

analysis indicated that 55.1 mg kg 1 dietary Zn was

required for maximal tissue storage and mineralization as

well as optimal growth of grass carp

KEY WORDS: body composition, diet, grass carp

(Ctenophar-yngodon idella), growth performance, mineralization, zinc

requirement

Received 24 March 2011; accepted 28 July 2011

Correspondence: Hui-Jun Yang, Institute of Aquatic Economic Animals,

School of Life Science, Sun Yat-sen University, 135 Xin’gang Xi Road,

Guangzhou 510275, China E-mail: edls@mail.sysu.edu.cn

Zinc (Zn) is an essential micronutrient required for normalgrowth and metabolic functions by vertebrates and is essen-tial for fish (NRC 1993) It is an essential trace metalrequired in more than 1000 structural, regulatory and cata-lytic proteins necessary for normal physiology, growth anddevelopment in all animals (Eide 2006; Maret & Kr El2007) Bone growth retardation is a common finding invarious conditions associated with Zn deficiency, suggesting

a physiological role of Zn in the growth and mineralization

of bone tissue (Yamaguchi 1998)

Zn deficiency signs in fish include impaired growth,increased mortality, eye cataracts, short body dwarfism andlow tissue Zn (Lall 1989) Dietary Zn levels were found toaffect not only the appetite, growth rate and mortality ofthe carp, but also the levels of Zn, Fe and Cu in the tissues(Ogino & Yang 1979) Channel catfish-fed Zn-deficientdiets show reduced growth rate, anorexia, reduced serum

Zn and reduced bone Zn and Ca deposition (NRC 1993)

Fish can obtain Zn directly from the water or via their diet(Handy 1996) However, normal Zn levels in freshwater(Gatlin & Phillips 1989) and in seawater (Willis & Sunda1984) are known to be suboptimal to meet metabolicrequirements of rapidly growing fish species In addition,because the presence of excessive levels of dietary Zn candisrupt transport and absorption systems, thus affectingthe nutritional status of other metals including Fe, Cu and

Cd, it is desirable to avoid unnecessarily high tion of Zn which also may limit the loading of minerals inthe aquatic environment (Buentello et al 2009) Therefore,the diets offered to aquatic animals have to contain theoptimal level of Zn to maintain health and high growthperformance Quantitative dietary Zn requirements for sev-eral species of fresh fish have been determined and rangefrom 15 to 80 mg kg 1 (Ogino & Yang 1978, 1979; Gatlin

supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa- supplementa-.

ª 2012 Blackwell Publishing Ltd

doi: 10.1111/j.1365-2095.2011.00935.x .2012 18; 380–387

Aquaculture Nutrition

& Wilson 1983; 1984; Gatlin et al 1991; Eid 1994; Lin

2008; Buentello et al 2009)

Production of grass carp (Ctenopharyngodon idella)

con-stitutes the largest aquaculture industry of finfish in China

In 2008, the production of grass carp reached 3.71 million

tons, which was 18% of freshwater aquaculture production

in China (Ministry of Agriculture 2009) Basing on the

pre-vious Zn studies of other fresh fishes, it must be necessary

to supplement Zn in the diet of grass carp However, the

information on Zn requirement for this fish is very poor

The present study was designed to determine the

require-ment of dietary Zn of grass carp and effects of Zn levels

on growth performance and tissue mineral content of this

fish

The basal diet formulation and proximate analysis are

given in Table 1 Casein (Hulunbeier Sanyuan Milk Co.,

Ltd, Inner Mongolia, China) and gelatin (Rousselot

Gela-tin Co., Ltd, Guangdong, China) were used as the protein

source Fish oil (Gaolong Industrial Company Ltd, Fujian,

China) and corn oil (Defeng Starch Sugar Company,

Guangdong, China) were used as lipid sources, and

corn-starch (Langfang Starch Factory, Hebei, China) was used

as a carbohydrate source Six semi-purified experimental

diets were formulated to contain graded levels of Zn by

supplementing the basal diet with 0, 10, 20, 40, 80 and

120 mg kg 1Zn in the form of Zn sulphate (ZnSO4·7H2O)

Element analysis (Zn, Fe, Ca, P, Mg and K) of the

experi-mental diets is shown in Table 2

Diet ingredients were ground through a 60-mm mesh All

the dry ingredients were weighed and mixed for 15 min, and

then fish oil and corn oil were added and mixed for 15 min

Distilled water was added to the premixed dry ingredients

and thoroughly mixed until homogenous in a Hobart-type

mixer The pellets were obtained (1.5 mm in diameter) using

a pelletizer (Institute of Chemical Engineering, South ChinaUniversity of Technology, Guangzhou, China) and air dried

to a moisture content of<100 g kg 1

The noodle-like dietswere ground, sieved and stored in plastic bags at 20 °Cuntil used

Juvenile grass carp were obtained from a local hatchery.Before the experiment, the fish were acclimated to a basaldiet (diet 1) for 2 weeks After the acclimatization, the fish

Table 1 Composition of the basal diet

vita-min A, 25 000 IU; vitavita-min E, 400; vitavita-min D3, 24 000 IU; one, 40; pyridoxine HCl, 40; cyanocobalamin, 0.1; biotin, 6; calcium pantothenate, 100; folic acid, 15; niacin, 200; inositol, 2000; and cellulose was used as a carrier.

menadi-2

sul-phate, 14.56; potassium chloride, 5.3; ferrous sulsul-phate, 0.9; ferric citrate, 3.1; magnesium sulphate, 6.93; manganese sulphate, 0.05; cupric sulphate, 0.03; cobalt chloride, 0.1; potassium iodide, 0.0035; sodium selenite, 0.0015; aluminium chloride, 0.025; and cellulose was used as a carrier.

Table 2 Zn supplement and analysed

mineral content of experimental diets

with similar body weight were selected and randomly

dis-tributed to 18 experimental fibreglass tanks (98 L9

48 W9 42 H cm, water volume of 200 L, 30 fish per

tank), which were connected to a recirculation system The

initial body weight averaged 3.97 g (SEM= 0.05 g with

n= 18) Each diet was assigned to triplicate tanks The

feeding trial lasted for 8 weeks The fish were fed with a

daily ration of 30–40 g kg 1

of body weight divided intotwo meals per day The fish were weighed every 2 weeks

for daily ratio estimated During the trial period, the

photoperiodic cycle was 12-h light/12-h dark One-third of

the rearing water would be drained weekly We determined

water quality parameters before we drained the water and

after we replenished the water, respectively Water quality

parameters were as follows (mean± SEM): temperature,

26.4± 2.3 °C; dissolved oxygen, 7.4 ± 0.35 mg L 1

; totalammonia-nitrogen, 0.089± 0.005 mg L 1; pH, 7.3± 0.4,

respectively Zn concentrations of the rearing water were

<0.02 mg L 1

during the trial period

At the beginning of the feeding trial, 20 fish were randomly

sampled for the analyses of whole body composition At

the end of the 8-week feeding trial, 24 h fasted fish in each

tank were weighed and sampled for tissue analysis Nine

fish from each tank were randomly collected, in which

three was used for the analysis of whole body composition

and six were anaesthetized with tricaine methane

sulpho-nate (MS222) (50 mg L 1) for blood and scales collection

and organ dissection White muscle from both sides of the

fillets without skin was dissected The plasma was

sepa-rated by centrifugation (1790 g for 10 min) and stored at

20°C until analysed Fish carcasses and another six fish

were cooked in a microwave oven for 5 min, and the

sur-rounding tissues were removed from the vertebrae

Vertebrae and scales were rinsed with distilled water,

dried and ground for mineral analyses To determine the

mineral contents on a fat-free dry basis, ground vertebrae

were extracted twice with 50-mL chloroform and methanol

(1 : 1, v/v), firstly air dried at room temperature and then

oven-dried at 105°C for 24 h Approximately, 0.10–0.15 g

dried and finely ground samples(the feed, whole body,

ver-tebrae, scales and liver) were digested with 15 mL 65–68%

nitric acid and 2 mL 72% perchloric acid using Kjeldahl

flasks After digestion, samples were diluted with deionized

water to 50 mL and determined for mineral contents (Zn,

Fe, Ca, P, Mg and K) by inductively coupled plasma

atomic emission spectrophotometer (ICP; model: IRIS

Advantage (HR), Thermo JarrelAsh Corporation, Boston,USA)

Diets and fish samples (including white muscle and liver)were analysed in duplicate for proximate composition

Moisture, crude protein, crude lipid and ash were mined using standard methods (AOAC 1995) Moisturewas determined by drying in an oven at 105°C for 24 h;

deter-crude protein (N9 6.25) was analysed by the Kjeldahlmethod after acid digestion (1030-Auto-analyzer, Tecator,Ho¨gana¨s, Sweden); crude lipid was determined by theether-extraction method by Soxtec System HT (Soxtec Sys-tem HT6; Tecator); crude ash by incineration in a mufflefurnace at 550°C for 5 h Activities of plasma alkalinephosphatase (ALP), along with the concentrations ofplasma triacyglyceride (TG), total cholesterol (T-CHO) and

Zn, were assayed within 3 days by enzymatic procedureusing automatic biochemical analyser and attached kit(Hitachi 7170; DAICHI, Tokyo, Japan)

All data are presented as means± SEM and subjected toone-way analysis of variance to determine whether signifi-cant differences occurred among treatments If a significantdifference was identified, differences among means werecompared by Duncan’s multiple range test (Duncan 1955)

by P< 0.05 All statistical analysis was performed using aSPSS 13.0 software package (SPSS 13.0 for Windows; SPSSInc., Chicago, IL, USA) Broken-line model (Robbins et al

1979) was used to estimate the requirement of dietary Zn

The equation used in the model is Y = L U(R XLR),

in which Y is the parameter (such as whole-body Zn tent) chosen to estimate the requirement, L is the ordinate,and R is the abscissa of the breakpoint R is taken as theestimated requirement XLRmeans X less than R, and U isthe slope of the line for XLR By definition, R XLR= 0when X> R

con-At the end of 8-week trial, only eight fish died during theexperiment and mortality was unrelated to treatments Noabnormal behaviours or body shapes of fish were observed

in any treatment group As showed in Table 3, grass carpfed with dietary Zn levels higher than 34 mg kg 1 signifi-cantly increased final body weight (FBW), weight gain(WG) and specific growth rate (SGR), while dietary Zn

.

Aquaculture Nutrition 18; 380–387 ª 2012 Blackwell Publishing Ltd

levels higher than 53 mg kg 1 significantly increased feed

efficiency (FE), feed intake (FI) and protein efficiency ratio

(PER) (P < 0.05) Apparent retention of Zn decreased

sig-nificantly with increasing dietary Zn level, except that

apparent retention of Zn of diet 4 was higher than diet 2

and 3 (P< 0.05)

For body composition (Table 4), grass carp fed with dietary

Zn levels higher than 53 mg kg 1 significantly decreased

the moisture contents but increased the lipid contents of

whole body and liver Dietary Zn levels had no significanteffect on condition factor (CF), viscerosomatic index (VSI)

or hepatosomatic index (HSI) However, grass carp fedwith dietary Zn levels higher than 34 mg kg 1 significantlyincreased intraperitoneal fat ratio (IPF)

Dietary Zn levels higher than 53 mg kg 1 significantlydecreased the ash contents of whole body, scales and verte-brae (Table 5) Whole body, scales, vertebrae and liver

Table 3 Growth performance and feed utilization of grass carp-fed experimental diets at the end of the growth trial

Diet

Zn)

Final body weight (g)

Protein efficiency

Apparent retention

Table 4 Body composition and morphometric indices of grass carp-fed experimental diets at the end of the growth trial

.

Aquaculture Nutrition 18; 380–387 ª 2012 Blackwell Publishing Ltd

mineralization were all affected significantly (P < 0.05) by

dietary Zn levels (Table 6) Zn contents in whole body,

scales and vertebrae were linearly increased up to the

53 mg kg 1 dietary Zn and then remained stable beyond

this level, while dietary Zn levels had no effect on Zn

con-tent in the liver (P< 0.05) Contrarily, grass carp fed with

dietary Zn levels higher than 53 mg kg 1 significantly

decreased the Ca, P and Mg contents in whole body, scales,

vertebrae and liver Similar trend was found in Fe contents,but there was no significant effect on Fe content in scales

Plasma Zn content was significantly increased with theincrease of dietary Zn concentration to 53 mg kg 1and thenmaintained constant beyond this level (P< 0.05) Grass carpfed with dietary Zn levels higher than 53 mg kg 1 signifi-cantly increased TG and T-CHO contents and ALP activity

in plasma (P< 0.05)

Based on WG, the requirement of dietary Zn for the mal growth of juvenile grass carp was 53 mg kg 1(Table 3) Broken-line analysis showed that, based on the

opti-Zn content in whole body, scales and vertebrae, the ments of dietary Zn for maintaining maximum Zn storages

require-in juvenile grass carp were 53.8, 55.1 and 51.8 mg kg 1(Figs 1–3)

Table 5 Ash concentration in whole body, scales and vertebrae of

grass carp-fed experimental diets at the end of the growth trial

Mean values within a row with unlike superscript letters were

.

Aquaculture Nutrition 18; 380–387 ª 2012 Blackwell Publishing Ltd

During the trial period, Zn concentration of the rearing

water was <0.02 mg L 1

This level of Zn present in freshwater was apparently insufficient to meet the metabolic

requirements of grass carp Signs of Zn deficiency in this

experiment were characterized by poor growth and feedutilization (Table 3) and reduced tissue Zn content(Table 5, 6)

Weight gain and SGR results in the present work gested that fish fed the diet with Zn<53 mg kg 1were Zn-deficient, which was in accordance with the results of otherstudies For dietary Zn levels were found to affect theappetite, growth rate and mortality of the carp (Ogino &Yang 1979)

sug-The dietary Zn levels influenced the digestibility of tein and carbohydrate, especially that of protein (Ogino &Yang 1978) In the present trial, the decrease in FE andPER for fish fed with Zn-deficient diet indicated that theprotein of the diet was being poorly digested In addition,dietary Zn levels also affected the appetite in the presenttrial, and similar results were observed in the carp (Ogino

pro-& Yang 1979) and Nile tilapia (Eid 1994) Fish fed the deficient diet developed anorexia that might also due to thepoor digestibility of protein

Zn-Zn homoeostasis is maintained in fish by regulating theexcretory mechanisms and controlling gastrointestinaluptake (Watanabe et al 1997) In the present study,although Zn contents were higher in fish fed the Zn-supple-mented diets, Zn deposition in fish body was lower than Znintake from the diets, so Zn retention decreased with increas-ing dietary Zn levels, particularly for dietary levels above

53 mg kg 1(Table 3) At the same time, there was no moreeffect neither in growth performance nor in body Zn contentafter the Zn was met at 53 mg kg 1, so the excess Zn justexcreted into water

In the present trial, dietary Zn levels higher than

53 mg kg 1 significantly increased the body lipid content(Tables 4 & 7) Ogino & Yang (1978) also found that the

Figure 1 Effect of dietary Zn on the whole-body Zn content of

juvenile grass carp-fed experimental diets for 8 weeks The

Figure 2 Effect of dietary Zn on the Scale Zn content of juvenile

grass carp-fed experimental diets for 8 weeks The breakpoint of

Figure 3 Effect of dietary Zn on the Vertebrae Zn content of

juve-nile grass carp-fed experimental diets for 8 weeks The breakpoint

Table 7 Biochemical compositions of plasma from grass carp-fed

Mean values within a row with unlike superscript letters were

rainbow trout fed the low-Zn diet was lower in crude

pro-tein and lipid contents However, the information on

die-tary Zn effect on lipid metabolism is very poor, so it needs

more trial to confirm the reason of the increased body lipid

content of grass carp

In the present trial, body Zn contents increased

signifi-cantly to increasing dietary levels of Zn until a plateau was

reached (Tables 6 & 7) Similar responses have also been

reported for tissues Zn of the carp (Ogino & Yang 1979),

scale and bone Zn of blue tilapia and red drum (McClain

& Gatlin 1988; Gatlin et al 1991), whole body and plasma

Zn of Nile tilapia (Eid 1994) and so on

Opposite to Zn concentration, ash, Ca, P, Mg and Fe

con-centrations in whole body, vertebrae, scales and liver seem to

be inversely related to dietary Zn levels (Table 5 & 6)

There may be a competition between Zn and Fe for

binding sites of the divalent cations transporter 1 (Cousins

& McMahon 2000) Ogino & Yang (1978) found that the

Fe contents changed inversely proportional to the dietary

Zn levels in rainbow trout Dietary zinc levels were also

found to affect the levels of the tissues Fe and Cu of the

carp (Ogino & Yang 1979), liver Fe of channel catfish

(Gatlin & Phillips 1989) and whole body Fe of Nile tilapia

(Eid 1994) In the present study, the same trend was seen

both in whole body, vertebrae and liver Fe levels

Slaunwhite (1988) suggested that Zn partially substitutes

for Ca in bone composition because of similarities in

physi-cal–chemical properties between these two minerals

Chan-nel catfish fed the basal diet had decreased whole-body Zn

levels and increased whole-body Ca levels (Scarpa & Gatlin

1992) In the present study, the same trend was seen both

in whole body, scales, vertebrae and liver Ca levels

Other-wise, the values of Ca-to-P ratios in the whole body for

dif-ferent fish species are from 0.7 to 1.6 described by Lall

(2002) Fishes have to hold the Ca-to-P ratios steady, so P

followed the similar trend as Ca in this study It seems that

the Ca and P concentrations were the major reason for

higher ash content of fish-fed Zn-deficient diet

In our early trial about Mg requirement of grass carp

(unpublished), Zn contents in whole body and vertebrae of

fish fed high level of Mg decreased significantly, while in

the present trial, the body Mg contents changed inversely

to the dietary Zn levels So, this might also be caused by

competitive inhibition of these two cations during intestinal

absorption

Plasma alkaline phosphatase activity is considered a

sen-sible Zn status indicator in animals (Swinkels et al 1996)

Thus, in the present study, the significantly lower ALP

activity in plasma observed in fish fed the Zn-deficient diet

This response was also observed in channel catfish (Gatlin

& Wilson 1983) and Nile tilapia (Do Carmo E Sa´ 2004)

Results of this study indicated that a proper level of dietary

Zn was essential for normal physiology, growth, efficient feedutilization and bone mineralization of juvenile grass carp

Excess Zn not only caused excessive excretion of Zn but alsohad a negative effect on bone mineralization According tothese results, 55.1 mg kg 1dietary Zn was set as the require-ment of grass carp juvenile for maximal tissue storage, miner-alization and optimal growth This value is within the ranges

of the Zn requirements reported for other fish species

This work was funded by Team Project of Natural ScienceFoundation of Guangdong Province (10351064001000000)

AOAC (1995) Official Methods of Analysis Association of Official Analytical Chemists, Arlington, 1141 pp.

Buentello, J.A., Goff, J.B & Gatlin, D.M III (2009) Dietary zinc

Cousins, R.J & McMahon, R.J (2000) Integrative aspects of zinc

Do Carmo E Sa´, M (2004) Optimum zinc supplementation level

in Nile tilapia Oreochromis niloticus juveniles diets Aquaculture,

Eide, D.J (2006) Zinc transporters and the cellular trafficking of

Gatlin, D.M III & Phillips, H.F (1989) Dietary calcium, phytate

Gatlin, D.M III & Wilson, R.P (1983) Dietary zinc requirement

Gatlin, D.M III & Wilson, R.P (1984) Zinc supplementation of

Gatlin, D.M III, O’Connell, J.P & Scarpa, J (1991) Dietary zinc requirement of the red drum, Sciaenops ocellatus Aquaculture,

92, 259–265.

Handy, R.D (1996) Dietary exposure to toxic metals in fish In:

Toxicology of Aquatic Pollution: Physiological, Cellular and

Cam-bridge University Press, CamCam-bridge, UK.

Lall, S.P (1989) The minerals In: Fish Nutrition, 2nd edn, Vol 1

Inc., San Diego, CA USA.

Lall, S.P (2002) The minerals In: Fish Nutrition, 3rd edn (Halver,

York.

Lin, Y (2008) Dietary Zinc Requirements of Juvenile Hybrid

.

Aquaculture Nutrition 18; 380–387 ª 2012 Blackwell Publishing Ltd

Maret, W & Kr El, A (2007) Cellular zinc and redox buffering

capacity of metallothionein/thionein in health and disease Mol.

McClain, W & Gatlin, D.M III (1988) Dietary zinc requirement

of Oreochromis aureus and effects of dietary calcium and phytate

Ministry of Agriculture (2009) China Fisheries Yearbook China

Agricultural Press, Beijing, China, pp 220 (in Chinese).

NRC (National Research Council) (1993) Nutrient Requirements

of Fish National Academy Press, Washington, DC.

Ogino, O & Yang, G.Y (1978) Requirement of rainbow trout for

Ogino, C & Yang, G.Y (1979) Requirement of Carp for Dietary

Robbins, K.R., Norton, H.W & Baker, D.H (1979) Estimation of

Scarpa, J & Gatlin, D.M (1992) Dietary zinc requirements of channel catfish, Ictalurus punctatus, swim-up fry in soft and hard

Watanabe, T., Kiron, V & Satoh, S (1997) Trace minerals in fish

Willis, J.N & Sunda, W.G (1984) Relative contributions of food and water in the accumulation of zinc by two species of marine

Yamaguchi, M (1998) Role of zinc in bone formation and bone

.

Aquaculture Nutrition 18; 380–387 ª 2012 Blackwell Publishing Ltd

1,2 1,2 1,2 1,2 1,2 1,2

1

CSIRO Food Futures Flagship, Cleveland, QLD, Australia;2 CSIRO Marine and Atmospheric Research, Cleveland, QLD,

Australia;3 Ridley Aquafeed, Narangba, QLD, Australia

This study examined the digestibilities of whole diets and

ingredients of a range of starch/cereal grains when fed to

barramundi (Lates calcarifer) The ingredients included wheat,

oats, sorghum, triticale, barley, among others Twelve diets

were prepared using a twin-screw extruder to mimic modern

aquafeed-manufacturing processes The diets were then fed to

juvenile barramundi and the faeces collected using stripping

methods Significant variability in the digestible energy and

starch value was seen among the test ingredients and diets Diet

starch digestibility ranged from 49.1% to 93.9% Ingredient

starch digestibility ranged from 18.0% to 96.5% Among the

different starch ingredients, a strong negative correlation

between the ingredient amylopectin content and starch

digestibility of that ingredient was observed This relationship

was also mirrored by a similar relationship, although not as

strong, between total starch and starch digestibility This study

demonstrates that barramundi have limited ability to digest

starch and that there is an effect of inclusion level on the ability

of the animal to digest this nutrient But it also demonstrates

that there is a wide range of cereal sources that can be

effec-tively used as starch sources in extruded diets for this species

key words: Asian seabass, barley, digestibility, extrusion,

grain, wheat

Received 2 February 2011, accepted 7 August 2011

Correspondence: Brett Glencross, CSIRO Food Futures Flagship, PO Box

120, Cleveland, QLD 4163, Australia E-mail: brett.glencross@csiro.au

The use of cereal grains such as wheat, barley, oats and corn

in carnivorous fish diets is a routine practice throughout

the world (Gatlin et al 2007; Hardy 2010) Their inclusion isprimarily to provide adequate starch to assist the extrusionprocess with pellet binding and expansion (Hilton et al 1981;

Jeong et al 1991) Despite this, there is actually limitedinformation on the nutritional and functional value of manycereal grain varieties when fed such fish species (Krogdahl

et al 2005; Gatlin et al 2007) Each of the different cerealgrains differs subtly in their chemical composition (Table 1)

These differences offer potential advantages and tages with their inclusion, provided the implications of theirinclusion are understood (Glencross et al 2007) While mostcereal grains are low in antinutritional factors (Francis et al

disadvan-2001), their inclusion does introduce a significant level ofstarch which may be of limited nutritional value to manycarnivorous species (Stone 2003; Krogdahl et al 2005; Hua

& Bureau 2009) They also introduce other carbohydrates ofnon-nutritive value, in the form of non-starch polysaccha-rides (NSP), which have been shown to have variable effects

on the nutritive value of diets to fish (Hansen & Storebakken2007; Glencross et al 2008)

The digestion of starch (as either amylose or amylopectin)has been shown to vary considerably as a function of inclu-sion level and starch state (Bergot & Breque 1983; Hua &

Bureau 2009) Studies examining the digestibility of waxy ornormal maize starch found little difference in digestibility(Enes et al 2008) It was reported by Stone (2003) thatincreasing the level of native (ungelatinized) wheat starchresulted in a reduction in energy digestibility in barramundi(Lates calcarifer) Similar effects were also observed in othercarnivorous species like Cod (Gadus morhua), rainbow trout(Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)(Stone 2003; Krogdahl et al 2005) Although an increase incarbohydrate digestion has been seen with increasing starchcontent in species like carp (Cyprius carpio), in Atlantic sal-mon (S salar) no effect on digestibility has been noted with