Aquaculture nutrition, tập 17, số 6, 2011

GR activity of fish fed diets containing MHA from Hepatopan-creas CAT, GST and GR activities were quadratic responses MDA, protein carbonyl content, anti-superoxide anion and anti-hydroxy

1 2 2 1

(NIFES), Bergen, Norway

Vitamin K belongs to the lipid soluble vitamins, and occurs

source in animal feed Menadione is unstable during feed

processing and storage and the dietary content may reach

critically low levels Recent publications also question the

availability of menadione in feed for salmonids Vitamin K

plays vital roles in blood coagulation and bone

mineraliza-tion in fish, but the suggested minimum requirement varies

considerably depending on the vitamin K source used

Vitamin K deficiency is characterized by mortality, anaemia,

increased blood clotting time and histopathological changes

in liver and gills However, one should assess both inherent

and supplemented forms of vitamin K in feeds for exact

determinations, as relevant novel feed ingredients of plant

origin may be sufficient to meet the requirement for vitamin

K The current review gives an overview of the biochemical

role of vitamin K, and discusses vitamin K requirement in

fish in light of updated literature, with special emphasis on

salmonids

requirement, vitamin K

Received 31 January 2011; accepted 28 July 2011

Correspondence: Christel Krossøy, Department of Biology, University of

Bergen, PBox 7803, N-5020 Bergen, Norway E-mail: Christel.Krossoy@

bio.uib.no

Fish, like all other animals, need a certain amount of

vita-mins for optimal growth and proper health that vary

according to factors like nutritional status, external stressors,age and health status Vitamin requirements published by theNRC (1993) usually designate minimum requirements as thevitamin level required to avoid clinical deficiency signs andsupport normal growth (Woodward 1994) There is a dis-tinction between minimum requirement and requirement foroptimal growth or optimal health, which could lead to thedefinition of higher requirement or recommendation levelsadapted to a specific function or to certain conditions Inintensive commercial fish farming, the last decade hasbrought with it changes in genetics, husbandry and dietcomposition leading to increased growth rates and subse-quently changes in the minimal requirement of micronutri-ents (Waagbø 2008) However, detailed evaluations of thenutrient requirements for fish have not kept pace with thechanges as most of the vitamin requirements of salmonidswere determined more than 30 years ago It is thus unclear ifthe given requirements are appropriate for modern diet for-mulations The earliest requirement studies on fish wereperformed in an effort to increase the survival of the stock injuvenile stages Test diets and growth rates were not com-parable to commercial rearing, and the response criteria usedwere mostly survival, weight gain, absence of deficiency signsand maximum tissue storage The latter resulted in relativelyhigh requirement estimates, but the cost of adding too highlevels of vitamins were lower than the cost of suffering highmortalities As commercial farming became more efficient,more sensitive response criteria for vitamins were used, somemeasuring metabolically active forms and specific enzymeactivity This lowered the recommendations for most vita-mins (Woodward 1994)

Historically, vitamin K is best known for its essential role

in blood coagulation (Olson 1999), being responsible for theposttranslational modification and activation of the vitaminK-dependent (VKD) proteins (Knapen et al 1993; Luo et al.1997; Boskey et al 1998; Lee et al 2007), and the first VKD .

Ó 2011 Blackwell Publishing Ltd

2011 17; 585–594 . doi: 10.1111/j.1365-2095.2011.00904.x

Aquaculture Nutrition

proteins identified were those involved in vitamin K

hae-mostasis (Nelsestuen et al 1974; Stenflo et al 1974; Ferland

1998) In the past few decades, it has become clear that

vitamin K plays an important role in other biological

pro-cesses, such as bone metabolism and growth control (Price

1988; Manfioletti et al 1993) The diverse range of functions

of VKD proteins implicates a broad biological impact of

vitamin K (Berkner 2008), but the exact roles of vitamin K

and VKD proteins have been difficult to assess, and the

physiological consequences of non-carboxylated and

under-carboxylated proteins are unknown Estimates of dietary

vitamin K requirement differ widely among fish species, and

the quantitative requirement of vitamin K for most fish is still

unknown (NRC 1993) In the current review, we will give an

overview of the biochemical role of vitamin K, and discuss

vitamin K requirement in fish in light of updated literature

with special emphasis on salmonids However, differences in

experimental design, fish species, developmental stage,

bio-markers, as well as inclusion level and forms makes the

published studies challenging to compare Overall, the

min-imum requirement of vitamin K has been difficult to estimate

owing to natural occurrence in feed ingredients, feed

pro-cessing and storage stability of inherent and added vitamin

K, vitamin leaching, variable feed intakes and variable

bio-availability of the different K vitamers

Lipid soluble vitamin K was first discovered by the Danish

scientist Henrik Dam in 1929 as an antihaemorrhagic factor

in chicks (Olson 1999) The factor was later shown to be

related to the absence of prothrombin activity in plasma For

decades, it was believed that the only function of vitamin K

was in the coagulation cascade, but several vitamin K

dependent proteins have now been isolated from bone,

den-tin, cartilage, kidney, atherosclerotic plaque and numerous

soft tissues (Vermeer et al 1995, 1996; Shearer et al 1996;

Booth 1997; Ferland 1998)

Vitamin K refers to a family of compounds derived from

quinone, that share a common 2-methyl-1,4-naphthoquinone

ring, but differ in the side chain at the C3-position (Lambert

& De Leenher 1992) All vitamers K are insoluble in water,

slightly soluble in alcohol and readily soluble in non-polar

organic solvents (Koivu-Tikkanen 2001) They have a

rela-tively high thermostability (Lambert & De Leenher 1992),

but are sensitive to light and alkaline conditions

(Koivu-Tikkanen 2001) There are at least two naturally occurring

Fig 1a) is synthesized by plants, and is mainly found in greenleafy vegetables (Booth & Suttie 1998) Phylloquinone has aphytyl group with one double bond in the side chain Vitamin

-1,4-naphtho-quinone, Fig 1b), on the other hand, is primarily of bial origin, and is found in fermented products and in foods

micro-of animal origin (Booth & Suttie 1998) Menaquinonesinclude a range of vitamin K forms, named according to thenumber (n) of prenyl groups in the unsaturated side chain,thus designated MK-n, with n ranging from 2 to 14 (Lambert

& De Leenher 1992) Menaquinone-4 (MK-4) and MK-7 arethe most relevant nutritional menaquinones (Fodor et al

2010) Of these, MK-4 is unique as it is the product of certaintissue-specific conversions directly from dietary phylloqui-none (Thijssen & Drittij-Reijnders 1994; Ronden et al 1998;

Okano et al 2008) Menaquinones may be synthesized bybacteria in the gut (Conly & Stein 1993), and the requirement

of vitamin K in mammals is met by a combination of dietaryintake and intestinal bacterial synthesis Both diet composi-tion and the use of antibiotics are known to affect intestinalproduction (Mathers et al 1990) The quantitative signifi-cance and role of menaquinones produced by the intestinalmicroflora in maintaining vitamin K status is still unknown(Conly & Stein 1993; Suttie 1995; Vermeer et al 1995), butbacterially derived long-chain menaquinones have beenfound in human liver (Usui et al 1989; Thijssen & Drittij-Reijnders 1996) However, the importance of intestinal pro-duction of vitamin K or the effect of antibiotics has not beenestablished in fishes or crustaceans (Tan & Mai 2001) Vita-

are chemically synthesized vitamin K compounds used incommercial feeds for domestic animals It is a vitamin Kderivate in the form of water soluble salts, like menadione

(a)

.

sodium bisulphite (MSB) and menadione nicotinamide

bisulphite (MNB) Menadione has no side chain, and is

chemically unstable compared to the naturally occurring

vitamin K forms (Marchetti et al 1995, 1999) It is not itself

biologically active and is easily excreted, but can at least be

partly alkylated enzymatically to MK-4 in tissues when

present in animal feeds (Dialameh et al 1971; Udagawa

2000; Graff et al 2002, 2010; Okano et al 2008; Krossøy

Most of the work within vitamin K research has been

con-ducted on humans and laboratory animals It was long

thought that the role of vitamin K was limited to the

syn-thesis of factors within the coagulation system, but the

dis-covery of vitamin K as a cofactor and the identification of

additional VKD proteins, significantly expanded the

under-standing of its physiological roles (Stenflo et al 1974; Suttie

1992; Ferland 1998; Vermeer et al 1998) Key VKD proteins

include coagulation proteins, anticoagulation proteins and

bone proteins, in addition to the VKD growth factor

growth-arrest-specific-6 (Gas6, Table 1; Suttie 1992; Ferland 1998)

Calcium binding is essential for the activation of the seven

VKD proteins that mediate blood coagulation and

antico-agulation Coagulation factors II (prothrombin), VII, IX and

X make up core actors of the coagulation cascade, while

proteins C, S and Z belong to the anticoagulation proteins

With the exception of protein S, which is also synthesized by

osteoblasts, these proteins are produced exclusively in the

liver (Ferland 1998) Blood clotting follows the same

fun-damental pattern in both mammals and teleosts, generating

thrombin by pathways involving VKD factors (see

Hanu-manthaiah et al 2002 and Jiang & Doolittle 2003; and

ref-erences cited therein) In addition to protein S, the VKD

proteins found in bone are bone Gla-protein (BGP; synonym

for osteocalcin) and matrix Gla-protein, MGP (Vermeer

not clear, it is suggested to function as a regulator of boneformation and bone mineral maturation (Ducy et al 1996;Boskey et al 1998) BGP is produced by osteoblasts andodontoblasts only (Dimuzio et al 1983) The protein wasoriginally isolated from bovine bone where it was shown toinhibit the formation of hydroxyapatite (Price et al 1976).Vitamin K is involved in the posttranslational modification

of VKD proteins and acts as a cofactor for the enzymec-glutamylcarboxylase (GGCX) GGCX catalyses the car-boxylation of glutamic acid (Glu) residues in VKD proteinsresulting in its conversion to c-carboxyglutamic acid (Gla)residues (Stenflo et al 1974) Although VKD c-carboxyla-tion occurs only on specific Glu-residues in a small number ofproteins, it is critical for the functionality of these proteins(Suttie 1992) Both phylloquinone and menaquinones act asco-factors in the GGCX mediated carboxylation (Buitenhuis

for the carboxylation reaction (Shea & Booth 2008) As afirst step, vitamin K is reduced to vitamin K hydroquinone

carboxylation reaction, leading to formation of Gla residuesand vitamin K epoxide (KO) KO is subsequently reduced byKO-reductase to vitamin K, in a process commonly calledthe vitamin K cycle (Ferland 1998; Berkner 2000; Stafford2005) which conserves the available vitamin K very effi-ciently The resulting Gla domain formed from the carbox-ylation is a calcium-binding amino acid moiety required forthe function of VKD proteins In the presence of calciumions, these proteins undergo a structural transition leading tothe exposure of a phospholipid (membrane) binding site.Vitamin K deficiency leads to the occurrence of under-carboxylated proteins with Glu-residues, and are most oftenbiologically inactive Lower VKD enzymatic activities ordegree of VKD protein carboxylation can be used as markersfor suboptimal vitamin K nutrition (Ferland 1998; Vermeer

Bone Gla-protein missing one or more Gla residues istermed under-carboxylated osteocalcin (ucOC), and the ratiobetween fully carboxylated and ucOC has been suggested as asensitive marker for vitamin K deficiency (Vermeer et al.1995; Ferland 1998) In humans, a correlation betweenosteoporosis and ucOC has been found (Szulc et al 1994,1996) When supplemented with vitamin K, the level ofucOC, bone resorption and urinary calcium secretion isreduced, while bone formation increases (Braam et al 2003).MGP, originally purified from mammalian bone (Price &Williamson 1985), is a small VKD protein synthesized

by osteoblasts and a wide variety of other cells, like

Table 1 Vitamin K-dependent (VKD) proteins

Factor VII Factor IX Factor X

Protein S Protein Z

Matrix Gla Protein

Gla-rich Protein

.

Aquaculture Nutrition 17; 585–594 Ó 2011 Blackwell Publishing Ltd

chondrocytes and vascular smooth muscle cells It contains

five Glu-residues that need modification to Gla for its

acti-vation (Schurgers et al 2007) Animal studies suggest that

MGP is a physiological inhibitor of tissue calcification (Luo

gene structure, amino acid sequence and tissue distribution

are similar among examined animal species (Laize´ et al

2005) MGP is also important in chondrocyte differentiation

and maturation, regulating endochondral and

intramem-branous ossification (Luo et al 1997; Newman et al 2001)

As in mammals, studies have shown that MGP expression

and function is associated with regulation of mineralization

in turbot, Scophthalmus maximus (Roberto et al 2009) InAtlantic salmon (Salmo salar L.), BGP and MGP are ex-pressed in vertebrae, as well as in fin, gills and scales, con-firming the presence of vitamin K in bone, and suggestinginvolvement of vitamin K in bone metabolism of Atlanticsalmon (Krossøy et al 2009b) The latest addition to theVKD family, is Gla-rich protein isolated from sturgeon(Acipenser nacarii) cartilage This VKD protein is highlyexpressed in chondroblasts, chondrocytes, osteoblasts andosteocytes, and is suggested to regulate calcium in theextracellular environment (Viegas et al 2008)

Bone and spinal deformities represent a recurring problemfor commercial fish farming, and have raised ethical concerns

in animal welfare issues in recent years Suggested risk factorsare nutrition, genetics, environment, vaccination and fastgrowth (Waagbø et al 2005; Waagbø 2008) The importance

of vitamin K in bone health has been established in mammals(Vermeer et al 1995, 1996; Shearer et al 1996; Booth 1997;

Ferland 1998), and the interest in vitamin K requirement fornormal bone development in fish has recognized that thevitamin K supply may be suboptimal for bone but sufficient

to maintain normal growth and prevent mortality (Udagawa2000) To date, there is no information on the form or thelevels of vitamin K required to achieve optimal bone healthneither in humans nor in fish Only a few reports have dealtwith the impact of vitamin K deficiency on fish bone health(Udagawa 2001, 2004; Graff et al 2002; Roy & Lall 2007;

Krossøy et al 2009a) Studies on mummichog (Fundulusheteroclitus) larvae have shown that diets without vitamin Ksupplementation caused a higher incidence of deformities inthe vertebrae and caudal skeleton (Udagawa 2001) Further,the effect of parental vitamin K deficiency on bone structurewas examined in the developing mummichog larvae (Udag-awa 2004) The author concluded that the offspring from fishfed a vitamin K deficient diet had abnormal vertebral for-mation 5 days posthatching compared to larvae from fish fed

a vitamin K rich diet with significantly lower incidences ofmalformations More specifically, vitamin K deficiencycaused the formation of thin and weak bone, and inducesbone structure abnormalities such as vertebral fusion androw irregularity, both in early development and during latergrowth in mummichog (Udagawa 2001, 2004) Radiologicaland histological findings in haddock (Melanogrammus aeg-

decreased bone mineralization and increased the occurrence

of bone deformities, without affecting the number of

Figure 2 The vitamin K cycle: The vitamin K-dependent (VKD)

c-carboxylation system consists of the vitamin K-dependent enzyme

c-glutamylcarboxylase (GGCX) which requires the reduced vitamin

en-zyme vitamin K 2,3-epoxide reductase (KO-reductase) Vitamin K is

acid (Glu) residues in VKD proteins to c-carboxyglutamic acid (Gla)

to vitamin K 2,3-epoxide (KO) coincide with the c-carboxylation.

The epoxide is subsequently reduced back to vitamin K by

KO-reductase, ready to enter another cycle (Enzyme nomenclature

adapted from http://www.chem.qmul.ac.uk/iupac/iupac.html).

.

osteoblasts (measured by histomorphometry) in the

verte-brae This indicates that vitamin K is necessary for bone

mineralization in haddock (Roy & Lall 2007) Investigations

of bone health, performed by mechanical testing and

radio-logical and/or visual examination, revealed no signs of

vertebral deformities in juvenile Atlantic salmon (Krossøy

given an un-supplemented diet Moreover, neither

phyllo-quinone nor MK-4 were detected in samples of vertebrae

(Graff et al 2010), but both bgp and mgp were expressed in

vertebrae, gills and pectoral fin as analysed by in situ

hybridization and qPCR (Krossøy et al 2009a,b)

Further-more, gene expression of ggcx was found in vertebrae, scales,

operculum and fin of adult Atlantic salmon, indicating

GGCX activity in bony tissues of Atlantic salmon (Krossøy

pro-teins BGP and MGP remains unknown, they may be

important in regulation of bone growth (Dimuzio et al 1983;

Boskey et al 1998) Together these latter results suggested

the involvement of vitamin K in bone metabolism of Atlantic

salmon (Krossøy et al 2009b)

Lately, studies in mammals have proposed multiple roles of

vitamin K beyond coagulation that are both dependent and

independent of its classical role as an enzyme cofactor, as

reviewed by Booth (2009) A novel mechanism of vitamin K

function in transcriptional regulation of osteoblastic cells was

demonstrated by Tabb et al (2003), showing that

menaqui-none is a transcriptional regulator of bone markers, such as

alkaline phosphatase and MGP in osteoblastic cells It has

been shown that menaquinone is a ligand for the nuclear

pregnance X receptor (PXR; also known as steroid xenobiotic

receptor or SXR), suggesting a role of menaquinone in

reg-ulation of bone homoeostasis (Tabb et al 2003; Zhou et al

2009) and collagen formation (Ichikawa et al 2006)

Men-aquinones potentially contribute to improved bone quality by

gene regulation (Ichikawa et al 2006; Horie-Inoue & Inoue

2008) in addition to its role as an enzymatic co-factor Gas6 is

involved in regulating cell survival and proliferation, and

protecting against cellular apoptosis (see review by Hafizi &

Dahlba¨ck 2006) Gas6 is found throughout the nervous

sys-tem, as well in the heart, lungs, stomach, kidneys and cartilage

(Ferland 1998; Hafizi & Dahlba¨ck 2006) It affects vascular

smooth muscle cell movement and apoptosis (Danziger 2008),

and appears to play important physiological roles in

inflam-mation, energy metabolism, renal disease, sepsis and

neo-plasia (Manfioletti et al 1993; Arai et al 2008; Booth 2009)

Lastly, a role of vitamin K in prevention of oxidative damage

of the brain and sphingolipid synthesis has been suggested,

as reviewed by Shearer & Newman (2008)

The minimum requirements given by NRC (1993) are marily determined for small fish and the studies are per-

purified, synthetic or semi-synthetic diets produced underconditions causing minimal losses These studies andrequirements are obviously not valid for commercial condi-tions using practical diets Normally, most vitamins aresupplemented at levels above the NRC minimum require-ments to compensate for factors influencing the vitamin level.Thus, practical vitamin allowances correct for losses underfeed production and storage (Marchetti et al 1999), andshould take into consideration the bioavailability of vitaminforms, challenging rearing conditions and the developmentalstage of the fish (Hamre et al 2010) Practical dietary vitamin

K recommendations given for optimum health and tivity of farmed fish are therefore often several folds abovethe minimum requirement

produc-In fish, typical vitamin K deficiency signs include increasedblood coagulation time, reduced growth, anaemia, haemor-rhages, loss of fin tissue, weak bones, and occurrence ofspinal curvature, short tails and increased mortality (Tave-ekijakarn et al 1996; Udagawa 2004; Lall & Lewis-McCrea2007) The earliest vitamin K requirement studies in fin fishwere based on increased blood coagulation time and mor-tality as the primary criteria (Kitamura et al 1967; Poston1976; Murai & Andrews 1977) Studies with vitamin Kdeficient feed caused no detectable deficiency symptoms inrainbow trout (Kitamura et al 1967) and channel catfish,

growth and increased mortality in amago salmon,

in-creased mortality in mummichog (Udagawa & Hirose 1998).Lately, more sensitive biomarkers have been used As themajor function of vitamin K is to act as co-factor for GGCX,the activity of this enzyme may provide a biomarker fordeficiency Results from recent studies in juvenile Atlanticsalmon confirmed that GGCX activity is a sensitive markerfor evaluating vitamin K status and intake (Krossøy et al.2009a, 2010) However, altered enzyme activity does notnecessarily represent a deficiency state and because therewere no indications of deficiency in any of the otherparameters measured, Krossøy et al (2009a) concluded thatthe minimum requirement in salmon juveniles was at, or less .

Aquaculture Nutrition 17; 585–594 Ó 2011 Blackwell Publishing Ltd

than, the basal level of phylloquinone found in the diets

where no signs of deficiency were recorded

Current estimates of dietary vitamin K requirement differ

in what is considered adequate levels in the feeds for fish

(Table 2) In NRC (1993) recommendation, the minimum

requirement for growing lake trout (Salvelinus namaycush) is

in Halver (2002) the vitamin K recommendation for growth

compre-hensive review of vitamin requirement studies in fish

sug-gested that vitamin K concentrations equivalent to 0.45 mg

(Woodward 1994) In addition, Kaushik et al (1998) showed

that supplementation of practical diets with 1.5 mg

deficiency signs in juvenile rainbow trout (Oncorhynchus

mykiss), Chinook salmon (Oncorhynchus tschawytscha) and

European seabass (Dicentrachus labrax) In the same period,

Grahl-Madsen & Lie (1997) suggested that <0.2 mg vitamin

cod (Gadus morhua) Recently, Krossøy et al (2009a) found

the minimum requirement for normal growth, health and

bone strength in juvenile Atlantic salmon fed a diet without

vitamin K supplementation from start feeding Confounding

factors in this study may have been the transfer of vitamin K

to offspring from the broodfish, as analyses show that salmon

0.016 ± 0.005 lg MK-4 per egg (R Ørnsrud, unpublished

data) Considering that the vitamin K cycle efficiently

recy-cles vitamin K, this endogenous source may cover the

mini-mum requirement for start feeding fry

There may be several reasons for the variation between fishspecies in vitamin K requirement research First, differences

in biological efficacy among the chemical vitamin K forms(phylloquinone, menaquinones and menadione) have to betaken into consideration In addition, different outcomesmay arise from differences in absorption, metabolism andexcretion, feed intakes, and choice of biomarkers Lastly, theuse of the very labile forms of menadione as a vitamin Ksource in the feed, and challenges with the vitamin K anal-ysis, might have led to an overestimated requirement (Graff

Menadione has been reported to cause toxicity symptomslike abnormalities in liver, kidney and lungs, as well ashaemorrhage and haemolytic anaemia in mammals (Smith

tox-icity have been reported for phylloquinone and nones Available data for fish is contradictory As an

trout after 20 weeks of feeding (Grisdale-Helland et al

Atlantic cod after 23 weeks of feeding (Grahl-Madsen & Lie1997) In a study by Udagawa (2001), the upper tolerance

(Krossøy et al 2009a; Graff et al 2010), no signs of toxicity

in growth, mortality or health measures were recorded,showing high dietary tolerance of MNB However, safeupper levels of vitamin K for fish have not been established

Currently, the most common menadione forms added tofeeds for farmed fish are MSB and MNB Only a limited

Table 2 Overview over published vitamin K requirement and recommendations in fish (1970–2011)

K vitamer

Recommendations/

Requirement

coagulatin time

Lie (1997)

bone health

n.g.,not given; *, vitamin recommendation for growth.

.

number of the papers published on vitamin K requirement in

fish include analysis of menadione in the feed (Grahl-Madsen

& Lie 1997; Roy & Lall 2007; Graff et al 2010), and thereby

validate if the levels of menadione in the feed are in

accor-dance with the targeted amounts Graff et al (2010) reported

that the analysed levels of menadione in the experimental

feeds were very low compared to the target levels (added

with findings of Tavcˇar-Kalcher & Vengusˇt (2007) showing

that up to 90% of the menadione in premixes may be lost

during 12 months of storage The instability of menadione

may be increased by addition of choline in the vitamin

pre-mix (Marchetti et al 1995, 1999; Tavcˇar-Kalcher & Vengusˇt

MSB was added to Atlantic cod diets, Grahl-Madsen & Lie

(1997) were able to analyse the same amount added in the

feed Roy & Lall (2007), on the other hand, reported a 50%

reduction from added to analysed dietary menadione content

using levels of MSB corresponding to 0–40 mg menadione

between the works of Grahl-Madsen & Lie (1997) and Roy &

Lall (2007) compared to Graff et al (2010) may be related to

several factors First, the feeds may have been produced by

different processing methods (not described in the papers)

Secondly, feeds for cod and haddock contain less fat

thus be easier to analyse Thirdly, the stability of MSB and

MNB may differ

Knowledge about the availability of inherent and synthetic

K vitamers in fish feed is scarce (Lall 2005) Menadione must

be alkylated enzymatically to MK-4 in animal tissues to

become biologically active (Udagawa 2000), and is thought

to have lower bioavailability than the naturally occurring K

vitamers, as shown in mummichog (Udagawa 2001;

Udaga-wa & Murai 2001) Results from Krossøy et al (2010)

con-firmed that menadione did not act directly as a co-factor for

GGCX in Atlantic salmon, despite its structural similarities

with the inherent vitamin K forms (Lambert & De Leenher

1992) This is also in line with earlier studies by Sadowski

only phylloquinone and menaquinones can function as a

co-factor for GGCX However, menadione is known to be

converted to MK-4 in the tissues of several fish species

including sardine, Sardinops melanosticus (Udagawa et al

1993), Atlantic cod (Grahl-Madsen & Lie 1997), mummichog

(Udagawa & Hirose 1998), ayu (Plecoglossus altivelis)

(Udagawa et al 1999) and Atlantic salmon (Graff et al 2002,

2010; Krossøy et al 2009a), and can thus be used as a

vitamin K source in aquaculture feeds A dose–response

relationship has been found between menadione intake andthe liver MK-4 concentration in Atlantic salmon (Krossøy

con-version and retention of MK-4 It has to be clarified if theconversion of menadione to MK-4 is severely rate limited, or

if the low liver MK-4 levels are solely related to extremelylow stability and intake from feed In the study by Graff et al.(2010), a comparison between MNB and phylloquinone fedsalmon showed a considerably higher retention of phyllo-quinone compared to menadione, necessitating validation ofactual levels of menadione in the feeds after feed production.Additional knowledge about retention of K vitamers in fishfillets is also an important step to tailor fish fillets towardsimproved nutritional value (Bell & Waagbø 2008)

Recommendations for vitamin requirements must be seen inrelation to factors like species, life stage, overall feed com-position and farming conditions (Waagbø 2008) Welfareand quality of farmed fish is a serious issue of debate, andmore scientific investigations should be directed towardsproviding properly formulated feeds, securing optimalnutrient content throughout all production stages Theincreased global production of fish through farming andthe worldwide shortage of marine resources, has led toreplacement of fish meal and fish oil by novel protein andlipid sources of plant origin and from marine by-products.Some relevant alternative oils in fish feed, like soybean oil

higher levels of natural vitamin K forms compared to

(Oster-meyer & Schmidt 2001, Wollard et al 2002; Suttie 2006)

To reduce unnecessary vitamin supplementation and feedcosts, information is needed on the concentration and bio-availability of naturally occurring vitamers in the feedingredients used

Based on the points mentioned, it is necessary to define amore precise dietary requirement for vitamin K The stability

of vitamin K in processing and storage should be furtherinvestigated, and a focus on the bioavailability of inherentand synthetic forms, is needed Traditionally, estimation ofvitamin requirements is mostly performed on the basis of onevitamin, but it has become clear that it is necessary to have amore integrated approach with multiple variables This isrelated to the fact that different vitamins show interactionswith each other As an example, some of the non-infectiousdiseases and deformities causing trouble for the aquacultureindustry have been related to suboptimal production .

Aquaculture Nutrition 17; 585–594 Ó 2011 Blackwell Publishing Ltd

conditions and nutrition, and nutritional factors like the

lipid-soluble vitamins A, D and K have been proposed as

impor-tant for development and mineralization of bone (Waagbø

bone proteins and minerals are controlled by bone cells Bone

cells react on external stimuli, such as the hormone like

compounds vitamins A and D Vitamin A may accumulate in

fish and lead to hypervitaminosis (Ørnsrud et al 2002) and an

increase in the occurrence of both craniofacial and spinal

deformities (Ørnsrud et al 2008), while vitamin D is shown to

be an important regulator of mineral homoeostasis (Fraser

1995; Lock et al 2010) Both vitamins A and D influence

expression and synthesis of VKD proteins (Lian et al 1989;

Fu et al 2008; Darias et al 2010) and have shown strong

regulatory effects on the activity of bone cells (Lall &

Lewis-McCrea 2007), while vitamin K is responsible for the

posttranslational modification and activation of the VKD

proteins (Knapen et al 1993; Luo et al 1997; Boskey et al

1998; Lee et al 2007) Intensive rearing, the use of novel feed

ingredients and exposure to diseases and contaminants may

alter the relative requirement of some vitamins, especially in

the juvenile stages Therefore, a re-evaluation of the vitamin

K requirement in farmed fish species is needed

This project is part of the research programme ÔRoles of fat

soluble vitamins in bone development and mineral

metabo-lismÕ, funded by the Research Council of Norway (project

number 153472)

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1,2 1 1,2 1,2 1,2 1,2 1 1,2

1

Nutrition of China Ministry of Education, Yaan, China

Oxidative damage and antioxidant status of intestine and

hepatopancreas for juvenile Jian carp (Cyprinus carpio var

Jian) fed graded levels of methionine hydroxy analogue

enzymes activities such as superoxide dismutase (SOD),

catalase (CAT), glutathione-S-transferase (GST), glutathione

peroxidase (GPX) and glutathione reducase (GR), as well as

glutathione (GSH), protein carbonyl (PC) and

malondial-dehyde (MDA) contents were assayed in these tissues

Results indicated that anti-superoxide anion capacity in

intestine and anti-hydroxyl radical capacity in

hepato-pancreas significantly improved with dietary MHA levels

decreased (P < 0.05) SOD, CAT, GST, GPX, GR activities

in intestine and hepatopancreas, as well as GSH content in

hepatopancreas significantly increased with optimal MHA

thereafter decreased (P < 0.05) Meanwhile, MDA and PC

contents in these tissues together with GOT and GPT

activities in plasma significantly decreased with optimal

and thereafter increased (P < 0.05) These results suggested

that MHA improved antioxidant status and depressed lipid

and protein oxidation in intestine and hepatopancreas

KEY WORDS

KEY WORDS::antioxidant enzymes, Cyprinus carpio var Jian,

lipid peroxidation, MHA, oxygen radicals, protein oxidation

Received 3 July 2010, accepted 12 January 2011

Correspondence: Xia-Qiu Zhou, Animal Nutrition Institute, Sichuan

Agricultural University, Yaan 625014, China E-mail: zhouxq@sicau.edu.cn

Methionine has been demonstrated to be a dietary essentialamino acid for normal growth of juvenile Jian carp (Cyprinus

(Cirrhinus mrigala) (Ahmed et al 2003) As a common thetic methionine source, supplement methionine hydroxyanalogue (MHA) to methionine-deficient diets can partly

trout (Oncorhynchus mykiss) (Poston 1986), juvenile sunshine

Gatlin 1995) and juvenile red drum (Sciaenops ocellatus)(Goff & Gatlin 2004) Our previous study also indicated that

equal-sulphur basis made no significant difference in growthperformance of Jian carp (Xiao et al 2010a) By other hand,

it has been reported that animal growth is dependant ondigestion and absorption ability (Hakim et al 2006) Ourprevious research found that MHA promoted digestion andabsorption ability and thus growth of Jian carp by improvingintestine and hepatopancreas function (Xiao et al 2010b).Shoveller et al (2005) reviewed that intestinal growth andfunction are usually involved in its antioxidant status Pre-vious studies with Jian carp also indicated that improvedgrowth and function of intestine and hepatopancreas werepositively related to antioxidant status in these organs bynutrients such as glutamine (Lin & Zhou 2006; Chen et al.2009), pyridoxine (He et al 2009; Hu et al 2011) and myo-inositol (Jiang et al 2009b, 2010) Reactive oxygen species

cellular metabolism, and these reactive species in turn maypromote the production of many other reactive molecules

.

 2011 Blackwell Publishing Ltd

2011 17; 595–604 . doi: 10.1111/j.1365-2095.2011.00853.x

Aquaculture Nutrition

and peroxynitrite (HOONO) (Storz & Imlayt 1999) Like all

aerobic organisms, antioxidant defence systems in fish also

consist of low-molecular-weight antioxidants and

antioxi-dant enzymes (Martınez-Alvarez et al 2005) When the ROS

generation rate exceeds that of their removal, oxidative stress

occurs which may produce deleterious effects including

pro-tein oxidation, DNA strand-break damage and peroxidation

of unsaturated lipids (Martınez-Alvarez et al 2005)

Peroxi-dation of membrane lipid often initiates the loss of

mem-brane integrity, which may induce injury of tissues so as to

the leak of enzymes or ions (Veena et al 2006) According to

this, serum glutamate-oxaloacetate transaminase (GOT) and

glutamate-pyruvate transaminase (GPT) act often as markers

of liver injury in terrestrial animals (Yoshikawa et al 2002)

Humtsoe et al (2007) also implied that reduced GOT and

GPT in muscle and liver of rohu carp (Labeo rohita) when

exposed to oxidative stress might be related to the leak of

them into serum Therefore, GOT and GPT activities in

serum warrants investigation to further reflect the

antioxi-dant status of tissues influenced by MHA

Methionine as an important sulphydryl-containing

com-pound is particularly susceptible to oxidation by ROS and

then converted to methionine sulphoxide (MeSOX) by

bio-chemical assays in vitro (Levine et al 1996) However, studies

with yeast (Moskovitz et al 1997), bovine (Moskovitz et al

1996) and human tissue (Kuschel et al 1999) implied that the

enzyme MeSOX reductase could reduce MeSOX back to

methionine if the MeSOX was not further oxidation It

appears that the cycle of methionine oxidation and reduction

represents a natural scavenging system for ROS

Further-more, studies had demonstrated that MHA was converted

(Dibner & Knight 1984) and chicken small intestine

(Martı´n-Venegas et al 2006) By other hand, methionine is not only a

substrate for protein synthesis (Me´tayer et al 2008), but also

a regulator that can modulate phosphorylation and protein

synthesis in avian myoblast cell line (Tesseraud et al 2003),

and simultaneously can affect DNA methylation and further

influence gene expression in mouse as an important source of

methyl group (Tremolizzo et al 2002; Dong et al 2005)

Based on these observations, methionine may influence

antioxidant enzymes activities in vivo Studies with rats

indicated that methionine improved SOD, GPX and CAT

activities in the heart of male Sprague–Dawley rat

(Sene-viratne et al 1999), as well as GR and GST activities in

tert-butylhydroperoxide induced brain synaptosomes of albino

rat (Slyshenkov et al 2002) A few research have been

conducted to study the antioxidant defence of methionine

increased glutathione (GSH) in juvenile sunshine bass liver(Keembiyehetty & Gatlin 1995) and declined thiobarbituricacid reactive substances (TARS) in hybrid striped bass

However, few studies have evaluated effects of methioninesources on free radical generation and antioxidant enzymes

in fish, which needs further experimental investigation

This study was a part of a larger study that involved in thedetermination of the effects of MHA on digestive andabsorptive capacity in Jian carp (Xiao et al 2010b), andprovides a first insight into the possible effects of MHA onfree radical generation and antioxidant enzymes response infish The results can provide partial theoretical evidence forthe effect of MHA on digestive and absorptive capacity infish

Formulation of the basal diet is presented in Table 1 Exceptmethionine, dietary components (amino acids, vitamins andminerals) were supplemented to meet the requirements ofjuvenile Jian carp according to our previous studies (Zhou

et al 2009b; Wen et al 2009; Huang et al 2011; Ling et al

2010; Tan et al 2010) and reported nutritional requirementsfor common carp (NRC 1993) Six experimental diets wereformulated according to MHA supplementation: 0 (control),

(Sumitomo-chemical, Tokyo, Japan) was added to the testdiets to provide different concentrations, and the amount ofcorn starch was reduced to compensate final amount Themethionine concentration in the basal (control) diet was 6.9 g

previously reported (Xiao et al 2010b)

Hatchery-reared juvenile Jian carp were obtained from theTongWei Hatchery (Sichuan, China) Before starting theexperiment, fish were acclimatized to the aquaria system andfed six times daily (08:00, 10:30, 13:00, 15:30, 18:00, 20:30)

diet, which was similar to that of basal experimental diet Atotal of 900 fish with an initial weight of 8.24 ± 0.03 g were .

randomly distributed to each of 18 experimental aquaria

ran-domly assigned to triplicate aquaria The aquaria system,

culture system, and water quality were the same as our

pre-vious study (Xiao et al 2010b) The experimental fish were

hand-fed with the respective diet to apparent satiation for

60 days six times daily (08:00, 10:30, 13:00, 15:30, 18:00,

20:30) from day 1 to 30 and four times daily (08:00, 12:00,

16:00, 20:00) from day 31 to 60 Thirty minutes after thefeeding, uneaten feed were removed by siphoning and thenair dried The experimental units were under a natural lightand dark cycle and water temperature was 25 ± 1C

At the end of the feeding trial, fish were anaesthetized with

Immedi-ately, blood of 15 fish from each aquarium was drawn from

)20 C for further glutamate-oxaloacetate transaminase(GOT) and glutamate-pyruvate transaminase (GPT) activityassays Intestine and hepatopancreas of the same 15 fishwere quickly removed, weighed and frozen in liquid nitrogen

fish per tank were homogenized on ice in 10 volumes (w/v)

analysis

ability) were determined by the method described by (Zhang

capacity was determined by using the superoxide anion freeradical detection Kit (Nanjing Jiancheng Bioengineer Insti-

of xanthine and xanthine oxidase With the electron acceptoradded, a coloration reaction is developed by using the Griessreagent The coloration degree is directly proportional to thequantity of superoxide anion in the reaction Tissue ASA

defined as the amount that scavenged superoxide anion freeradical in 40 min per milligram of tissue protein whichequalled to per microgram of vitamin C scavenging at thesame condition Anti-hydroxy radical (AHR) capacity wasdetermined by using the hydroxyl free radical detection Kit(Nanjing Jiancheng Bioengineer Institute) It was on the basis

reac-tion With the electron acceptor added, a coloration reaction

is developed by using the Griess reagent The colorationdegree is directly proportional to the quantity of hydroxylradicals in the reaction Tissue AHR capacity was expressed

rice gluten meal were used as dietary protein sources Fish oil,

soybean oil and wheat flour were used as dietary lipid and

car-bohydrate source respectively Lysine, threonine, available

phos-phorus, n-3 and n-6 calculated contents were 20.0, 17.0, 6.0, 10.0

(1984).

-a-tocopherol acetate, 20.00 (50%); menadione, 0.20 (50%);

thiamin nitrate, 0.10 (98%); ascorhyl acetate, 7.23 (92%); niacin,

2.51 (98%); riboflavine, 0.63 (80%); pyridoxine hydrochloride, 0.76

(98%) All ingredients were diluted with corn starch to 1 kg.

MHA, and the amount of corn starch was reduced to compensate.

Six MHA premix were elaborated according to different proportion

liquid MHA (g) and corn starch (g): 0/1000.0, 193.3/806.7, 290.0/

710.0, 386.7/613.3, 483.3/516.7 and 580.0/420.0.

.

Aquaculture Nutrition 17; 595–604  2011 Blackwell Publishing Ltd

Superoxide dismutase (SOD) and glutathione peroxidase

(GPX) activities were assayed as described by Zhang et al

One U means 50% of inhibition by SOD of nitric ion

protein One U was defined as the amount that reduced

Catalase (CAT) activity was determined by the

decomposi-tion of hydrogen peroxide (Aebi 1984) The result was

of tissue protein Glutathione-S-transferase (GST) activity

was measured by monitoring the formation of an adduct

between GSH and 1-chloro-2,4-dinitrobenzene (Lushchak

in 1 min per milligram of tissue protein Glutathione

reduc-tase (GR) activity was measured according to described by

1 min per milligram of tissue protein GSH content was

determined by the formation of 5-thio-2-nitrobenzoate

fol-lowed spectrophoto-metrically at 412 nm (Vardi et al 2008)

protein and commercial GSH was used as standard

GOT and GPT activities in serum were determined by the

method of Bergmeyer & Bernt (1974a) and Bergmeyer &

Bernt (1974b) respectively Both GOT and GPT activities

malondialdehyde (MDA) content was assayed as described

by Livingstone et al (1990) using the thiobarbituric acid

The protein carbonyl content was determined according to

the method described by Armenteros et al (2009) The

pro-tein carbonyl content was calculated from the peak

absor-bance at 370 nm, using an absorption coefficient of

protein concentration of samples was determined by themethod of Bradford (1976)

All data were subjected to one-way analysis of variance(ANOVA) followed by the Duncan method to determine sig-nificant differences among treatment groups All results wereexpressed as mean ± SEM The parameters with significantdifferences were subjected to quadratic regression analysiswith dietary MHA level

SOD, CAT, GST, GPX, GR activities and GSH content inintestine are displayed in Table 2 SOD activity significantly

then the data significantly decreased maintaining a plateau

CAT activity also significantly increased as dietary MHA

significant gradual decrease When MHA level was up to 5.1 g

plateaued GST activity in fish fed diet 1 and 6 were cantly lower than that of fish fed the other four diets GPXactivity was significantly increased with higher MHA levels

but a significant decreased activity was manifested in fish fed

lowest in fish fed the MHA-unsupplemented diet, and the

diet Intestinal CAT, GST and GR activities manifestedquadratic responses to the increasing dietary MHA levels

are significantly different (P < 0.05).

.

P= 0.109; YGST= 36.4970 + 1.6786X) 0.1000X, R =

,

SOD, CAT, GST, GPX, GR activities and GSH content in

hepatopancreas are presented in Table 3 SOD activity of fish

that it significantly decreased GSH content was the highest in

but no significant difference was obtained with dietary MHA

whereupon it significantly decreased GPX activity

then a decreased activity was sustained for the remaining

groups GR activity of fish fed diets containing MHA from

Hepatopan-creas CAT, GST and GR activities were quadratic responses

MDA, protein carbonyl content, anti-superoxide anion

and anti-hydroxy radical capacities in intestine of juvenile

Jian carp fed graded levels of MHA are presented in Table 4

MDA content, which was the highest in fish fed the

MHA-unsupplemented diet, decreased with dietary MHA levels up

diet Protein carbonyl content was decreased as dietary MHA

capacity significantly increased as dietary MHA levels was up

maintaining a plateau No significant difference was fested for anti-hydroxy radical capacity among six diets.MDA, protein carbonyl content, anti-superoxide anionand anti-hydroxy radical capacities in hepatopancreas ofjuvenile Jian carp fed graded levels of MHA are displayed inTable 5 MDA content was the highest in fish fed the MHA-unsupplemented diet, and decreased with increasing dietary

increased with dietary MHA concentration further up to 12.7

then gradually increased Fish fed on six experimental dietsshowed no significant difference for anti-superoxide anioncapacity Anti-hydroxy radical capacity gradually improved

are significantly different (P < 0.05).

containing graded levels of methionine hydroxy analogue (MHA g

Dietary MHA

group Mean values within the same column with different superscripts are significantly different (P < 0.05).

.

Aquaculture Nutrition 17; 595–604  2011 Blackwell Publishing Ltd

with increasing dietary MHA levels up to 10.2 g kg)1diet,

after that significantly decreased Furthermore, anti-hydroxy

radical capacity showed quadratic response with dietary

GOT and GPT activities in serum of juvenile Jian carp fed

graded levels of MHA are shown in Table 6 GOT activity

was the highest for fish fed the MHA-unsupplemented diet,

and decreased with increasing dietary MHA levels up to 7.6 g

obtained for serum GPT activity Both GOT and GPT

activities showed quadratic response with dietary MHA

low-a threlow-at to cell integrity (Scherz-Shouvlow-al &low-amp; Ellow-azlow-ar 2007)

Therefore, the main purpose of this study was to investigatethe effects of methionine hydroxy analogue (MHA) on

diets containing graded levels of methionine hydroxy analogue

group Mean values within the same column with different

superscripts are significantly different (P < 0.05).

in serum of juvenile Jian carp fed diets with graded supplemental

group Mean values within the same column with different

superscripts are significantly different (P < 0.05).

0.0 3.0 6.0 9.0 12.0 15.0 18.0 MHA supplemental levels (g kg –1 diet)

–1 protein)

(a)

(b)

MHA supplemental levels (g kg –1 diet)

Figure 1 Quadratic regression analysis of protein carbonyl content

in intestine (a) and hepatopancreas (b) for juvenile Jian carp fed diets with graded levels of MHA for 60 days [Each point represents the mean of three groups (n = 3), with six fish per group Optimal supplemental levels of MHA for protein carbonyl content in intestine

.

antioxidant response and oxidative damage in intestine and

hepatopancreas by evaluating lipid peroxidation and protein

oxidation, to determine whether MHA can protect their

structure integrity

The scavenging ability of MHA against superoxide radical

) are strongly involved in

when-ever molecular oxygen chemically oxidizes electron carrier

of oxidative damage within the cell (Hoshi & Heinemann

2001) In this study, intestine superoxide radical-scavenging

ability was improved by dietary optimal MHA

supplemen-tation, whereas intestine hydroxyl radical-scavenging ability

showed no alterations Nevertheless, the trend of superoxide

and hydroxyl radical-scavenging ability in hepatopancreas

was opposite displaying a curious different behaviour

depending on tissue assayed The reason for these interesting

results was not clear However, these data implied that MHA

would have an antioxidant role in enhancing the superoxide

or hydroxyl radical-scavenging ability

In the present study, antioxidant enzymes and GSH

con-tent response were also measured to further determine the

mechanism of MHA-induced inhibition of radical generation

in intestine and hepatopancreas SOD is the first enzyme

involved in antioxidant defence systems to clear superoxide

radical (Visner et al 1990), while CAT is an essential defence

against the potential toxicity of free radical like hydroxyl

2008) In our study, the data displayed that SOD and CAT

activities in intestine and hepatopancreas significantly

improved with increasing dietary MHA level up to 5.1, 7.6 or

MHA can reduce the superoxide and hydroxyl radical in

tissues GPX, GST and GR are three important enzymes

dependent on GSH (Rudneva 1997) Among them, GPX can

1998), and GST is able to detoxify compounds containing

reactive electrophilic centres to facilitate their excretion from

cells (Elia et al 2006) The results in present study indicated

that a significant enhancement of GPX and GST activities

were obtained in intestine and hepatopancreas with dietary

supporting that MHA can increase enzymatic antioxidant

capacity in fish intestine and hepatopancreas to prevent

oxidative damage in these tissues Studies had demonstrated

utilization in chicken liver (Dibner & Knight 1984) and small

intestine (Martı´n-Venegas et al 2006) Through converting

synthesis, and thus influence antioxidant enzymes activities inintestine and hepatopancreas Meanwhile, a few studies hadimplied that excess methionine intake might make DNAhypermethylated, which appeared to down-regulate somegenes expression in mouse (Waterland 2006) This discoverymay interpret the depression of all antioxidant enzymesactivities in intestine and hepatopancreas when dietary MHAlevel was high

GSH is a major low-molecular-weight antioxidant in vivothat acts as a substrate for GPX and GST, enzymes thatcatalyse the reactions for detoxification of xenobiotics andROS (Atmaca 2004) The present study indicated that GSHcontent in intestine and hepatopancreas all significantly in-

result was found in liver of juvenile sunshine bass fed on dietwith MHA supplementation (Keembiyehetty & Gatlin 1995).GSH homeostasis is maintained through de novo synthesisfrom precursor methionine and cysteine or regeneration fromits oxidized form GSSG (Shoveller et al 2005) GR catalysesthe reduction of GSSG back to GSH by the expense of theNADPH (Elia et al 2006) In the present study, GR activities

in intestine and hepatopancreas increased with 5.1 or

-methio-nine in male Ross chicken small intestine, after that diverting

to the transsulphuration pathway to the synthesis cysteine,which is the precursor for GSH synthesis (Martı´n-Venegas

in digestive organs by MHA may be due to the increment ofprecursor for GSH synthesis, and the elevated GR activityfor GSH regeneration Furthermore, studies with humancolon epithelial cells indicated that cysteine availability andlocal GSH concentration have a direct influence on epithelialcell proliferation and survival (Shoveller et al 2005) In ourprevious study, MHA improved the intestine and hepato-pancreas weight and intestine length (Xiao et al 2010b),which may be related to the enhancement of GSH content inintestine and hepatopancreas cells However, this hypothesisneeds further investigation

Most components of cellular structure and function arelikely to be the potential targets of ROS, polyunsaturatedfatty acids in the biomembrane being the most susceptiblesubstrates for oxidation, which undergo peroxidation rapidly(Zhang et al 2004) MDA is one of the most readily assayedend-products of both enzymatic and non-enzymatic lipidperoxidation reactions (Requena et al 1996) The presentstudy showed that optimal level of MHA significantlydeclined MDA content in intestine and hepatopancreas, .

Aquaculture Nutrition 17; 595–604  2011 Blackwell Publishing Ltd

suggesting that lipid peroxidation in these organs was

de-pressed by MHA According to this, Li et al (2009a,b)

indicated that MHA level was negatively correlated with

thiobarbituric acid reactive substances (TBARS) in hybrid

striped bass liver Peroxidation of membrane lipid often

ini-tiates the loss of membrane integrity, which may lead to the

leak of ions or enzymes from tissue cells (Veena et al 2006)

Therefore, GOT and GPT activities in serum were further

investigated to study tissue damages associated to lipid

per-oxidation With the increasing of MHA levels, GOT and

GPT activities in serum decreased to a point, and then

increased in this study The trend was opposite with GOT

and GPT activities in muscle and hepatopancreas of Jian

crap in our previous study (Xiao et al 2010b) Humtsoe et al

(2007) implied that GOT and GPT in muscle and liver of

rohu carp might release into serum when exposed to

oxida-tive stress Therefore, the reduced GOT and GPT activities in

serum further demonstrated that lipid peroxidation was

depressed by MHA in tissues Also, protein oxidation

dam-age can be directly induced by ROS (Berlett & Stadtman

1997), and can also be led by lipid peroxidation end products

such as MDA and 4-hydroxynonenal (Negre-Salvayre et al

2008) Protein carbonyl content is the most widely used

biomarker for oxidative damage to proteins, and reflects

cellular damage induced by ROS (Baltacıog˘lu et al 2008) In

our study, we observed that optimal level of MHA

signifi-cantly declined protein carbonyl content in intestine and

hepatopancreas, suggesting that protein oxidation was also

depressed in these organs by MHA The optimal MHA

supplemental level for protein carbonyl content in intestine

and hepatopancreas by the quadratic regression analysis was

results, we also found that MDA and protein carbonyl

content enhanced, while all antioxidant enzymes activities

decreased in intestine and hepatopancreas when dietary

MHA levels were high It seems that excess levels of dietary

MHA have some bad effects on antioxidant status in fish

intestine and hepatopancreas

In conclusion, MHA could promote the antioxidant

de-fence in fish intestine and hepatopancreas by increasing

enzymatic antioxidant capacity, GSH content and clearance

of oxygen radicals, thus protecting the structure and function

of these organs Therefore, the result of this study can

pro-vide some theoretical epro-vidence for our previous research

about the improvement of digestive and absorptive capacity

by MHA in Jian carp (Xiao et al 2010b) Nevertheless, the

specific molecule mechanism that MHA mediates antioxidant

defence in fish needs further investigation

This study was financially supported by Sumitomo-chemical(Japan), National Science Foundation of China (30771671and 30871926), Programme for New Century Excellent Tal-entsin University (NCET-08-0905) and the Key Project ofChinese Ministry of Education (208120) The authors wouldlike to thank the Sumitomo-chemical for providing financialassistance, and thank the personnel of these teams for theirkind assistance

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1,2 3 3 4 4 1 1,2

1

von Thu¨nen-Institut, Federal Research Institute of Rural Areas, Forestry and Fisheries; Institute of Fisheries Ecology,Wulfsdorfer Weg, Ahrensburg, Germany

The potential of rapeseed protein concentrate as fish meal

alternative in diets for wels catfish (initial average weight

86.5 ± 1.9 g) was evaluated Sixteen fish were stocked into

each of 12 experimental tanks being part of a freshwater

recirculation system Fish were organized in triplicate groups

50% and 75% of fish meal replaced with rapeseed protein

period, weight gain, standard growth rate, feed intake, feed

conversion ratio and protein efficiency showed no significant

difference between control group and fish fed on diets with

25% reduced fish meal content by inclusion of rapeseed protein

concentrate Higher dietary fish meal replacement negatively

affected diet quality and palatability resulting in reduced feed

intake, feed efficiencies and fish performance However, blood

serum values of triglycerides, glucose and protein were not

significantly different between treatment groups, still

indicat-ing a favourable nutrient supply from all experimental diets

KEY WORDS: feed evaluation, fish meal, growth trial, rapeseed,

rapeseed protein concentrate, wels catfish

Received 24 September 2010, accepted 4 February 2011

Correspondence: Hanno Slawski, Institute of Fisheries Ecology,

Wulfs-dorfer Weg 204, 22926 Ahrensburg, Germany E-mail: hanno.slawski@

vti.bund.de

In 2009, worldwide production of rapeseed (including

cano-la) was 61.6 Mio t Thus, rapeseed, commonly produced in

temperate regions, ranked as number three oilseed wide, only surpassed by soybean (222.2 Mio t) and cottonseed (64.0 Mio t) (FAO 2010) For soybean, a crop mainlycultivated in warm regions, efforts and research have beenundertaken to make it a commonly accepted fish feedingredient and fish meal alternative (Gatlin et al 2007) Theusage of rapeseed products as fish feed ingredients, however,

world-is limited Either simple oilcakes or rapeseed meals withincreased protein content produced from oilcakes that werede-oiled with organic solvents have been tested as proteinsources in feeding trials with several fish species, among them

(Web-ster et al 1997), Cyprinus carpio (Dabrowski & Kozlowska1981), Pagrus auratus (Glencross et al 2004) and Psetta

results showed that the nutritional quality of simple rapeseedproducts is below that of fish meal although they contained awell-balanced amino acid profile Particularly, antinutritionalfactors (ANF) determine the quality of rapeseed products forfish nutrition Prominent ANF in rapeseed products areglucosinolates, phytic acid, phenolic constituents (e.g tan-nins) and indigestible carbohydrates (Mawson et al 1995;Francis et al 2001) Several processing techniques can beadapted to reduce the level of antinutrients in rapeseedproducts and improve their value for fish nutrition Dehul-ling of seeds and utilization of high temperatures and organicsolvents (hexane) during oil extraction as well as sieving ofmeal decrease content of glucosinolates, phytate, fibre, cel-lulose, hemicellulose, sinapin and tannins (Fenwick et al.1986; Anderson-Hafermann et al 1993; Tripathi et al 2000)and increase protein level in meals (Mwachireya et al 1999).Protein extraction from meals by methanol–ammonia .

 2011 Blackwell Publishing Ltd

2011 17; 605–612 . doi: 10.1111/j.1365-2095.2011.00857.x

Aquaculture Nutrition

treatment or ethanol treatment will increase protein level and

effectively remove glucosinolates, phenolic compounds,

sol-uble sugars, such as sucrose, and some oligosaccharides (e.g

raffinose and stachyose) (Naczk & Shahidi 1990; Chabanon

a cost-effective method for removing glucosinolates from

rapeseed meals (Tyagi 2002) Sporadically, rapeseed protein

products of high quality are being produced in different

countries for application in animal nutrition However, these

products are produced for test purposes in small volumes

until their potential as protein source in animal nutrition is

clarified Besides nutritive quality, their costs of production

will have to become low enough to make rapeseed protein

products available at a competitive price compared to other

protein sources, especially fish meal In this study, a

high-quality rapeseed protein concentrate containing 710 g CP

were fed to wels catfish (Silurus glanis L.), a carnivorous

species that is believed to have potential for indoor

recircu-lation farming in Europe as a high-value product for local

markets (Mazurkiewicz et al 2008) Fish performance and

blood serum parameters were investigated to evaluate

rape-seed protein concentrate as fish meal alternative in diets for

wels catfish

Four experimental diets were formulated in which fish meal

was replaced with rapeseed protein concentrate (RPC) at

0%, 25%, 50% and 75% level (designated as R0, R25, R50

or R75, respectively) Solvent extracted RPC was obtained

from the Pilot Pflanzeno¨ltechnologie Magdeburg e.V.,

Magdeburg, Germany For the production of RPC, a batch

of rapeseed (variety Lorenz; Norddeutsche Pflanzenzucht,

Hohenlieth, Germany) was conditioned in a vacuum dryer

Then, rapeseed was cold-pressed To remove residual oil

crushed into 1- to 5-mm particle size followed by a hexane

treatment The treatment lasted for 2 h, and the incubation

extract was desolventized under pressure to remove hexane

(<300 ppm), then rapeseed meal extract was further crushed

to a particle size of 0.2–0.1 mm A four-step treatment using

glucosinolates from rapeseed meal extract This resulted in a

residual oil content of the nearly glucosinolate-free rapeseed

li-quid water extraction (rapeseed meal extract 1 : 15 water)

2 h of constant agitation Afterwards, the suspension wasdecanted Following decantation, the solvent was collected,and residue material was secondly extracted (residue 1 : 10

constant agitation Following extraction, the suspension wasdecanted Solvent was collected, and residue prepared for athird extraction Then, solvents of extraction 1, 2 and 3 werecollected to remove low-molecular compounds and to con-centrate dissolved proteins by dia- and ultrafiltration Duringfiltration, conductivity was checked Protein washing ended,

Vitamins and minerals were added to diets to meet thedietary requirements of freshwater fish [NRC (NationalResearch Council) 1993] The diets were formulated to be

protein concentrate (RPC) and concentration of antinutritional factors detected in RPC

fat + ash + fibre).

2

.

0.3 MJ kg)1) Essential amino acid concentrations did

not differ considerably between experimental diets The

diets were manufactured to give pellets of 4 mm in diameter

(L 14-175; AMANDUS KAHL, Reinbek, Germany) Diet

formulations, proximate compositions and amino acid

profiles are given in Table 2

The growth trial was conducted at the Johann Heinrich

von Thu¨nen Institute of Fisheries Ecology, Ahrensburg,

Germany Juvenile wels catfish (Silurus glanis L.) were

obtained from the Ahrenhorster Edelfisch GmbH & CO KG

(Ahrenhorst, Germany) Two weeks before the experiment

started, 17 fish were stocked in each of nine experimental

), being part of a

recir-culation system Tanks were provided with freshwater at

In respect of the fishesÕ light sensitivity, tanks were covered with translucent plastic lids For a 2-week adapta-tion period, fish were fed the control diet in four daily mealsuntil apparent satiation After the adaptation period, initialaverage fish weight was determined (86.5 ± 1.9 g) For anexperimental period of 63 days, triplicate groups of fish werefed the experimental diets in four daily meals (8:00 and 11:00a.m., 2:00 and 5:00 p.m.) until apparent satiation At thebeginning and at end of the experiment, two fish per tank

initial and final body composition

At the end of the feeding period, blood samples from thecaudal vein and artery of eight fish per experimental treat-ment were taken with a heparinized syringe (1 mL) Bloodhaematocrit percentage was determined after centrifugation(10 000 g, 6 min) of glass tubes filled with fresh blood in ahaematocrit centrifuge (Haematokrit 210; Andreas HettichGmbH & Co KG, Tuttlingen, Germany) Remaining freshblood was filled in Eppendorf tubes and centrifuged(1000 g, 5 min) Supernatant blood plasma was separated

Diets and homogenized fish bodies were analysed in cate for proximate composition Dry matter was calculated

constant weight Fat content was determined after HClhydrolysis (Soxtec HT6; Tecator, Ho¨gana¨s, Sweden) andtotal nitrogen content by the Kjeldahl technique (pro-

Sweden) Ash content was calculated from weight loss after

Dietary amino acid concentrations were analysed by infrared reflectance spectroscopy according to van Kempen

near-& Bodin (1998) Blood plasma concentrations of rides, glucose and protein were determined using a micro-

Switzerland, CH) and commercial kits (Triglycerides GPOand Glucose GOD-PAP; Greiner Diagnostic GmbH, Bah-

Co.KG, Karlsruhe, Germany)

mat-ter) of experimental diets

Fish performance was determined, using the following

for-mulae:

consumption per fish per day as a percentage of the daily fish

body weight for the experimental period The daily fish body

weight was calculated using daily SGR values equal to the

final SGR of each tank

Feed conversion ratio (FCR) = g dry feed intake/g wet

body weight gain

Protein efficiency ratio (PER) = g wet body weight gain/

All diets were assigned by a completely randomized design

Biological and analytical data were checked for normal

dis-tribution using the Kolmogoroff–Smirnov test and eventually

subjected to transformation Data were subjected to

17.0 for Windows (SPSS Inc., Chicago, IL, USA) When

differences among groups were identified, multiple

compari-sons among means were made using TukeyÕs HSD test

Statistical significance was determined by setting the

aggre-gate type I error at 5% (P < 0.05) for each set of

compari-sons

No significant differences in growth performance parameters

and feed efficiencies were detected between control diet and

R25 diet-fed fish Compared to the control group, fishgrowth performance, voluntary feed intake and feed effi-ciencies declined at fish meal replacement levels above 25%

Feed intake as per cent body weight was not affected up to50% fish meal replacement level (Table 3) While fish growthperformance, voluntary feed intake and feed efficiencies sig-nificantly correlated with the dietary inclusion level of RPC(Table 3), no correlation was found between feed intake asper cent body weight and dietary inclusion of RPC No sig-nificant differences in whole-body composition were detectedbetween fish fed on the control diet and fish receiving RPCdiets (Table 4) Significant correlations were found betweendietary RPC and phosphorus level and whole-body moisture

Haematocrit values as well as blood serum values determinedshowed no significant difference between treatment groupsand were not correlated to the dietary inclusion level of RPC(Table 5)

While usability and limitations of simple rapeseed products

as fish feed ingredients have been widely investigated(Dabrowski & Kozlowska 1981; Davies et al 1990; Webster

Table 3 Growth response, feed intake, feed efficiencies, condition factor (CF) and survival of wels catfish fed experi- mental diets

Values are given as mean ± standard deviation Values in the same row with common superscript

letters are not significantly different (P < 0.05).

FCR, Feed conversion ratio; PER, Protein efficiency ratio; SGR, Specific growth rate.

wels catfish fed the experimental diets

.

et al.1997; Burel et al 2000a,b,c, 2001; Thiessen et al 2003,

2004; Glencross et al 2004; Shafaeipour et al 2008), lack of

information exists about the benefits of high-quality products

originating from rapeseed oilcakes with protein contents

comparable to or above that of fish meal Higgs et al (1982)

successfully replaced 25% of dietary protein from a fish meal

control diet for juvenile Oncorhynchus tshawytscha with

reducing growth rate and food (protein) utilization In the

study, however, higher fish meal replacement levels with

rapeseed protein concentrate were not evaluated

The results of our study demonstrate that 25% of dietary

fish meal can be replaced with RPC in diets fed to wels

catfish without negative effects on feed efficiencies and fish

growth When 50% of dietary fish meal was replaced with

RPC, the feed intake as per cent of fish body weight was not

significantly different from the control group but feed

effi-ciencies and fish growth were reduced At 75% fish meal

replacement level, fish showed reduced diet acceptance and

reluctant feed intake as a result of unfavourable diet taste It

appears, therefore, that the level of blood meal incorporated

into diets as feed attractant did not effectively counteract the

negative effects on diet taste resulting from rapeseed protein

concentrate It is known that the bitter taste exuded by

glucosinolate metabolites, such as isothiocyanates and

vi-nyloxazolidinethiones, present in rapeseed meals can

poten-tially retard diet acceptance by fish This was found in

Because the RPC used in our study contained 0.2 lmol

(Table 2) This value is far below the level when

glucosino-lates become detrimental on food intake of O mykiss and

typical mustard smell of glucosinolates was still noticeable in

diets R50 and R75 It seems, therefore, that wels catfish is

more sensitive towards a bitter diet taste than other

car-nivorous fish species This goes together with the fishÕs

excellent developed olfactory organ (Jakubowski & Kunysz1979) Reduced feed intake in fish fed on diets R50 and R75resulted in lower growth rates and reduced feed conversioncompared to the control group (Table 3) For prospectivefeeding trials with rapeseed protein products in wels catfish,

it appears recommendable to use other feed attractants thanblood meal Fish protein hydrolysate, squid hydrolysate,stick water or krill meal at dietary levels from 30 to

sources of amino acids and minerals when diets low in fishmeal were fed to carnivorous fish (Espe et al 2006, 2007;Torstensen et al 2008; Kousoulaki et al 2009) As fish be-haved calm in all treatment groups, increased energyexpenditure because of feed-searching activity in high RPCgroups did not deplete feed conversion Thus, lower feedefficiency might be a result of reduced diet digestibility be-cause of RPC inclusion

In this study, we did not determine the digestibility ofnutrients and minerals from RPC in wels catfish We dis-covered that faeces collection from wels catfish to determinenutrient and mineral digestibility appears hardly possible Onthe one hand, faeces of wels catfish are slimy and rapidlydilute in water This precludes faeces collection with anautomatic collector On the other hand, faeces stripping, evenwhen fish are anaesthetized, will stress the sensitive fish As aresult, wels catfish will stop feed intake for days Killing fish,

as a last alternative to gain faeces, requires a high number ofindividuals to collect enough faeces for laboratory analysis

In this study, fish count was not sufficient to gain requiredamounts of faeces for laboratory analysis Therefore,assumptions regarding nutrient and mineral digestibility ofRPC in wels catfish are based on studies conducted withrapeseed protein products in other fish species Mwachireya

with phytate, can have greatest adverse effects on thedigestibility of canola protein products for O mykiss Theauthors reported that among different canola products

nutrient digestibility coefficients corresponding to fish meal

In our study, the applied processing techniques to produceRPC from rapeseed oilcake led to relatively low levels of

cal-culated dietary phytic acid concentrations originating from

respec-tively In fish nutrition studies, phytic acid concentrationsthat negatively influence mineral and nutrient availability arecommonly higher Spinelli et al (1983) observed decreased

synthetic phytic acid Synthetic phytic acid at concentrations

Table 5 Blood haematocrit content and blood serum values of wels

catfish fed experimental diets

of 5 and 10 g kg)1feed resulted in lower growth performance

in common carp (Hossain & Jauncey 1993) As a result of

insignificant phytic acid concentrations in diets R50 and R75,

we assume that diet digestibility was mainly reduced by fibre

and other complex carbohydrates However, lack of

infor-mation exists about the influence of complex carbohydrates

on nutrient digestibility in wels catfish But it is known from

other carnivorous fish that complex carbohydrates can

greatly reduce mineral and nutrient availability from

aqua-feeds, thereby reducing feed efficiencies as observed in Salmo

diets R50 and R75 were less digestible than the control diet

for wels catfish, because of increased dietary fibre and NfE

contents originating from RPC In contrast, Hajen et al

(1993) found that rapeseed protein concentrate (600 g CP

to O tshawytscha According to our findings, it seems that

wels catfish is highly sensitive towards dietary fibre and NfE

The amino acid requirement of wels catfish, to our

knowledge, is not known We therefore assume that it is

comparable to other carnivorous fish such as rainbow trout

Accordingly, experimental diets were formulated to contain

amino acid concentrations above established requirement

levels [NRC (National Research Council) 1993] However,

because of antinutritional factors present in RPC,

digest-ibility of amino acids could have been negatively affected as it

is known from other protein sources of vegetable origin

(Francis et al 2001) In particular, lower dietary levels of

lysine in diets R50 and R75 compared to the control diet

together with reduced lysine digestibility might have

nega-tively influenced feed efficiencies and fish growth in this

study

Fish body composition was not significantly different

between treatments (Table 4) Regression analysis, however,

revealed a correlation between the dietary level of RPC and/

or phosphorus and the moisture, fat and ash content in fish

bodies Sinking ash levels in fish body indicate that the levels

of available phosphorus in diets were not sufficient to meet

the dietary requirement of wels catfish It is known that

whole-body ash can be reduced when carnivorous fish are fed

a diet deficient in available phosphorus (Skonberg et al

1997; Shao et al 2008) Although dietary levels of

estab-lished requirement levels for many fish species [NRC

(National Research Council) 1993], it seems possible that

phosphorus availability from RPC was lower than from fishmeal Antinutritional factors such as phytic acid, fibre andother complex carbohydrates present in RPC are known toinfluence phosphorus availability in fish (Francis et al 2001)

However, as shown above, phytic acid concentrations in dietsR50 and R75 were insignificant We assume, therefore, thatphosphorus availability was mainly reduced by fibre andother complex carbohydrates The often increased whole-body lipid content with high dietary levels of vegetable pro-teins that has been reported in several fish species (Adelizi

In-stead, the whole-body lipid level tendentially decreased withincreasing dietary level of vegetable protein as reported byEspe et al (2006) when feeding Atlantic salmon a diet devoid

of fish meal According to Espe et al (2006), it seems sible, therefore, that the substitution of fish meal with plantproteins may not give the same results in different fishspecies

pos-In prospective feeding trials with wels catfish and rapeseedprotein concentrate, it appears advisory to supplement dietswith a phosphorus source such as dicalcium phosphate toovercome problems regarding phosphorus availability Thishas been shown to positively affect dietary phosphorussupply, feed efficiencies and fish growth when diets rich inplant-based proteins are applied (Lee et al 2010)

In this study, blood serum parameters were not cantly different between treatment groups, and no correlationwith dietary RPC levels was found (Table 5) As suggested byCaruso & Schlumberger (2002), blood serum parameters can

signifi-be used to estimate the health status of fish Our resultsindicate that fish did not suffer from malnutrition and thatdietary nutrient supply was sufficient to support growth andmaintain average body development in all feeding groups

However, found individual blood values of wels are highlyvariable as attested by a high standard deviation This wasalso found by Caruso & Schlumberger (2002) who estab-lished a baseline blood haematocrit value of 0.25 ± 0.01 forwels catfish The fish (individual weight 55–250 g) were

fed at 1% of their biomass per day The haematocrit baselinevalue corresponds to the value determined in our study(Table 5) Detected blood serum values of triglycerides,glucose and protein, however, differ from values cited byJira´sek et al (1998) They monitored blood serum values of1-year-old wels catfish (individual weight 752 and 1288 g)held in heated effluent water of a power station Fish were fed

.

49.39 mM L)1 and total protein 29.89 g L)1 We assume

that differences between values published by Jira´sek et al

(1998) and in the present study reflect the different

compo-sitions of used diets and possible differences in starvation

time before sampling It is known that blood serum values

generally represent the nutrient composition of a diet (Jira´sek

have a significant influence on plasma glucose, triglycerides

and protein concentrations (Shi et al 2010)

In conclusion, wels catfish accept diets formulated to

At higher dietary RPC, inclusion diet taste became

undesir-able for wels catfish, thereby reducing feed intake and fish

growth Antinutritional factors present in RPC might also

have reduced dietary phosphorus and amino acid availability

with negative effects on feed efficiencies and fish growth To

overcome difficulties with diet taste and nutrient availability,

we suggest the use of ANF-free rapeseed protein isolates in

prospective feeding trials

The project was financed by The European Fisheries Fund

and the Zukunftsprogramm Fischerei des Landes

Schleswig-Holstein

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.

1,2,3 3 1

Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Nubeena Crescent, Taroona, Tas., Australia;

2

University of Auckland, Warkworth, New Zealand

The effect of various carbohydrate sources (glucose,

su-crose, agar, wheat, tapioca, maize, potato and dextrin),

and inclusion levels of gelatinized maize starch (0, 70, 170,

performance [growth, survival, food consumption (FC),

enzyme activity and glycogen content of the digestive gland

(DG)] of spiny lobster juveniles was investigated in a

12-week culture experiment There was no difference in

with fresh mussel grew significantly faster (specific growth

tested produced a significant improvement in growth or

survival over a basal control diet However, the diet

highest moulting (mean = 2.1 moults per lobster),

concentra-tions among lobsters fed with the formulated diets,

suggesting a superior utilization of this source of

other diets), found in the DG of lobsters fed with fresh

mussel indicated a metabolism strongly directed to the

utilization of glycogen

KEY WORDS: carbohydrate, digestion, digestive enzyme,

glu-cose, glycogen, utilization

Received 5 August 2010, accepted 15 February 2011

Correspondence: Cedric J Simon, Tasmanian Aquaculture and Fisheries

Institute, University of Tasmania, Nubeena Crescent, Taroona, Tas 7053,

Australia E-mail: cedricjsimon@gmail.com

The development of a cost-effective and nutritionally quate formulated diet is fundamental to the future viability

ade-of spiny lobster aquaculture (Williams 2007) At present, adiet of fresh mussel has consistently produced better growththan formulated diets in spiny lobsters from temperate watersincluding for Jasus lalandii (Dubber et al 2004), Panulirus

James 2007; Simon & Jeffs 2008) Recent research suggeststhat cultured J edwardsii juveniles have a limited ability toincrease feed intake in response to poorly digestible dietsbecause of a small foregut capacity (2.5–3% BW), slow rates

of filling (1–2 h) and evacuation (10 h) of the foregut, and areduced appetite revival (>18 h) (Simon & Jeffs 2008; Simon2009a) The poor appetite revival is consistent with the lack

of improvement in food consumption (FC) at higher feedingfrequencies (Simon & Jeffs 2008), which could relate to thedigestive system of lobsters being unable to efficiently processthe arrival of formulated food immediately after the evacu-ation of a former meal (Simon 2009b) Together, these resultssuggest that improving the digestibility and utilization offormulated diets to maximize the amount of nutrient assim-ilated per meal is essential to improve their performance forspiny lobster culture

Carbohydrates are the most economical source of energy

in formulated aquaculture diets (Rosas et al 2000) and havethe best potential to produce cost-effective spiny lobster diets(Williams 2007) However, they are thought to be one of theleast digested and utilized components of experimental dietsfor lobsters (Bordner et al 1983; Johnston et al 2003; Ward

support the contention of a protein-sparing effect of dietarycarbohydrate in the American lobster, Homarus americanus .

 2011 Blackwell Publishing Ltd

2011 17; 613–626 . doi: 10.1111/j.1365-2095.2011.00861.x

Aquaculture Nutrition

(Capuzzo & Lancaster 1979; Brown 2006) and the spiny

lobster, J edwardsii (Radford et al 2008), indicating that a

potential exists for selecting better performing carbohydrates

for spiny lobster diets (Johnston et al 2003; Radford et al

2005; Simon 2009c) Early lobster studies have used native

corn starch as a source of carbohydrate in experimental diets

for clawed lobsters (Capuzzo & Lancaster 1979; Boghen

poorly digested (Bordner et al 1983; Simon 2009d), and

therefore little energy is provided to spare protein for growth

Most of the recent studies on spiny lobsters have used

pre-gelatinized maize starch as a carbohydrate source (Glencross

2003, 2005; Ward et al 2003), but despite being more

digestible than native maize starch (Simon 2009d), it may not

be as well utilized as it results in a fast rise in postprandial

haemolymph glucose and prolonged hyperglycaemia (Simon

2009c) Several carbohydrate sources that are digestible (e.g.,

dextrin) and that are potentially better utilized than

pregel-atinized maize starch (e.g., native wheat starch and

carb-oxymethyl cellulose), have been identified in J edwardsii in

previous research (Simon 2009c; d) However, at present no

growth experiment has focused specifically on finding an

optimum carbohydrate source, or inclusion level, for spiny

lobster culture In fact, previous studies have produced

confounded results by varying the amount of protein at the

expense of gelatinized starch when investigating the effect of

protein inclusion level on growth (Glencross et al 2001;

Smith et al 2003, 2005; Ward et al 2003) It remains to be

tested whether there is a negative effect of increasing starch

inclusion level for a constant protein level A comparative

growth experiment would also have the potential to

deter-mine accurately the utilization and protein-sparing effect of a

selection of carbohydrate sources, as well as their overall

benefit to spiny lobster nutrition (Alava & Pascual 1987;

Cruz-Sua´rez et al 1994)

Studying the enzymes regulating the digestion of

carbo-hydrates and proteins in combination with growth

experi-ments is useful because they can help to reveal the variation

in carbohydrate digestion and utilization in response to the

source and inclusion level of dietary carbohydrates and

proteins (Cuzon et al 2000) Various crustaceans have

demonstrated the ability to adapt their production of

diges-tive enzymes to match long-term changes in dietary

compo-sition (Hoyle 1973; van Wormhoudt et al 1980; Lee et al

1984; Lucien-Brun et al 1985; Le Moullac et al 1994;

Cec-caldi 1997; Guzman et al 2001) Quantitative changes in

digestive enzyme activities under different formulated diet

regimes have not been determined in spiny lobsters

How-ever, there is recent evidence for a rapid and substantiala-amylase adaptation to a natural and formulated diet incultured J edwardsii juveniles (Simon 2009b) This indicatesthat the use of digestive enzymes as a tool to determine thenutritional quality of key components of diets has potentialwith spiny lobster (Simon 2009b) Glycogen deposition in thedigestive gland (DG) is another important indicator of car-bohydrate digestion and utilization in Crustacea (Rosas et al

2000), which has been used successfully in the spiny lobster

The aim of this study was to assess the effect of variouscarbohydrate sources, and inclusion levels of gelatinizedmaize starch, on the culture performance of early J ed-

mea-sured were 1) growth and survival, 2) food consumption,3) glycogen concentration in the DG, and 4) digestiveenzyme (i.e., a-amylase, a-glucosidase, total protease andtrypsin) activities of the DG at the end of the growthexperiment

Approximately 700 J edwardsii pueruli and early settledjuveniles caught in July-August 2007 in crevice collectorsdeployed in Gisborne, New Zealand (lat 3839¢S, long

17805¢E), were ongrown communally on opened fresh

the experiment Lobsters of a similar initial size (i.e., meanweight ± SD = 1.53 ± 0.42 g; n = 560) were then stocked

were fed with 14 different dietary treatments allocated domly to four replicate tanks The tanks were fitted withairstones and received partially recirculated seawater via a

Tanks remained free of shelters, to facilitate food recoveryfor feed consumption measurements, and because previousstudies have shown no beneficial effect of shelter on growth

of early J edwardsii juveniles (Crear et al 2000; James et al

2001) Lighting was provided on a 12 : 12 dark : light cycleand the water quality, checked fortnightly, remained similar

to previous growth experiments (Crear et al 2000);

.

A benchmark diet of fresh mussel (Simon & James 2007) and

13 semi-purified diets formulated to contain different sources

of carbohydrate and different inclusion levels of

pregelati-nized maize starch were used for the experiment (Table 1)

These semi-purified diets were the same as used previously to

measure haemolymph glucose fluxes in larger juveniles

(Simon 2009c) The fresh mussels, P canaliculus, used for

feeding lobsters were purchased from an aquaculture

oper-ation in 25 kg bags and held alive at ambient temperature

(14–18C) in a large flow-through tank for a maximum of

3 weeks The nutritional quality of the fresh mussels was

satisfactory for feeding as the specific growth rate (SGR)

achieved in this study (1.80) was in agreement with previous

studies on J edwardsii juveniles (SGR = 1.32, Crear et al

2000; SGR = 0.92, Ward et al 2003; SGR = 2.24, Ward &

Carter 2009)

performance of juvenile lobsters on different dietary

carbo-hydrate sources, a control semi-purified diet was produced

based on the current state of knowledge of the nutritional

requirements of juvenile lobsters as outlined by Williams

diatomaceous earth by dry weight) that was substituted with

either sucrose, agar, native plant starches from potato, maize,

tapioca and wheat, dextrin and pregelatinized maize starch

(BO11C) to make up eight isonitrogenous (approximately

inter-fered with dietary binding and overall diet integrity The bulk

inert ash filler All diets contained a small quantity of othercarbohydrates which were present in the ingredients used to

con-trol diet (0% BO11C) and the 27% BO11C diet produced insection ÔCarbohydrate sourceÕ, two isonitrogenous diets wereproduced with 17% BO11C and 7% BO11C (i.e., the bulk

dry weight of the inert ash filler respectively) to test the effect

of pregelatinized starch inclusion level (n = 4) at a constant

BO11C/HP diet was formulated to contain a higher protein

substituting 20% of the supplemented BO11C (7%) with20% sodium caseinate This allowed assessing the effect ofthe main protein source (i.e., sodium caseinate) on thegrowth response of lobsters (Table 2)

(by dry weight) of low-heat dried (hydrolysed) squid meal(Co´rdova-Murueta & Garcı´a-Carren˜o 2002), 7% of freeze-

Table 1 Formulated diets and the source

Image Holdings Ltd., Auckland, New Zealand

Ltd., Auckland, New Zealand

dried mussel meal, 6% of wheat gluten, and 4.7% of a

selection of CMC-casein-gelatin coated amino acids

(argi-nine, 44%; methio(argi-nine, 20%; threo(argi-nine, 10%) as per Alam

deficiencies of the sodium caseinate as the main protein

source (Mente et al 2002) Dry ingredients were blended and

added to a mixture of fresh P canaliculus mussel liquor (i.e.,

liquid collected while shucking fresh mussels which served as

proportion depending on the type of carbohydrate included,

to reach a similar dough consistency The dough was kept

1996) The diets were left at room temperature for 2 h and

)20 C A second batch of diets was manufactured under the

exact same conditions after 8 weeks The high level of

available lysine and glutamine residues in the soluble sodiumcaseinate allowed fast cross-linking of the dietary ingredients(De Jong & Koppelman 2002) and the production of waterstable semi-purified diets with a mean ± SE moisture con-

to the experimental culture conditions and mortalitiesreplaced during that time only The growth experimentlasted 12 weeks with lobster wet body weight measured onday 0, 28, 56 and 84 by gently blotting lobsters dry andweighing on a Mettler PC 4400 balance (±0.01 g accuracy)

The tanks were siphoned daily in the morning and checkedfor moults and mortalities to calculate the number ofmoulting events and survival per tank Specific growth rate(SGR; % body weight increase per day) was calculated

Table 2 Ingredient composition and proximate composition of the formulated diets Values are based on dry weight

and ground (<700 lm) before use (Co´rdova-Murueta & Garcı´a-Carren˜o 2002).

4

Transglutaminase TG-B (99% caseinate), Image Holdings Ltd., Auckland, New Zealand.

acid, 0.5; DL a-tocopherol, 20; choline, 50.0; inositol, 10; and ascorbic acid, 10.

CMC, 20; vitamin C-coated, 10; betaine, 7; Naturose (astaxanthin 1.5%), 5; vitamin E, 2; choline chloride, 2; ethoxyquin, 0.5.

the proximate composition was also analysed (values in parentheses) as follows: total crude protein (%N · 6.25; elemental analyser

.

to account for the exponential increase in wet weight

(Crear et al 2000) Biomass gain per tank was calculated to

compare lobster yield on the different dietary treatments, as

initial lobster number (n = 10) and wet body weight (1.5 g)

were the same

weightÞ100=84

 number of lobster surviving

semi-purified diets was thawed weekly, and each diet

hand-fed daily at 1600 h to four replicate tanks to above satiation

weight) Fresh mussels were opened daily and fed as

half-shell to above satiation level for the benchmark dietary

treatment A single daily ration was shown to produce the

best growth of J edwardsii juveniles in a previous study

(Simon & Jeffs 2008) Consumption measurements were

taken over 5 weeks (i.e., week 2, 4, 6, 8 and 12) During these

weeks, mussels were shucked and only their flesh fed daily,

with a portion of mussel flesh sampled to determine dry

matter content Uneaten food was collected by siphoning the

tanks the next day from 1000 to 1200 h (18–20 h

postfeed-ing) The remaining feed from each tank was collected on a

250-lm sieve, washed gently with distilled water to remove

sta-bility and dry matter loss over 20 h from known amounts of

diets (n = 4) (i.e., including fresh mussel flesh for the

benchmark diet) was calculated in the same tank conditions

(i.e., feeds covered by a grid to prevent lobsters feeding) The

dry weight of the diets fed was back-calculated accounting

for the diet dry matter content and losses caused by leaching

and the recovery procedure (i.e., water stability of diets)

Mean daily FC, in dry matter per lobster, was calculated for

each tank as follows:

t

where WWo is the wet weight of the feed offered every day,

DM is the dry matter content (%) of the feed, S is the water

stability (i.e., percentage of food remaining after leaching

uneaten feed recovered, t is the duration of the collection

(i.e., t = 7 in most instances) and N is the number of

individuals alive per tank at the time of the uneaten feedcollection

Mean specific food consumption (SFC), in percentage oflobster wet body weight, was calculated for each tank asfollows:

where BW is the mean lobster body weight for each replicatetank, measured on days 0, 28, 56 and 84 For consumptionmeasurements obtained during weeks between censuses (i.e.,weeks 2 and 6), BW was estimated as the average of the twoclosest censuses

Mean food conversion ratio (FCR) was calculated for eachtank as follows:

where FC is the food consumption in dry matter per lobsterover the entire growth experiment (84 days) estimated fromthe 5 weeks of measurements

At the completion of the growth experiment, lobsters wereleft unfed for 24 h, and three lobsters per tank (i.e., 12 lob-sters per dietary treatment) were euthanized (i.e., chill-coma

their DG dissected out Each DG was weighed to the nearestmilligram using a precision AG204 Mettler Toledo balance,and a DG index calculated as follows:

A similar amount of DG tissue from the three lobstersreared in the same tank was combined for DG glycogen (i.e.,approximately 100 mg wet weight in total) and digestiveenzyme activity (i.e., approximately 200 mg wet weight intotal) analyses (section ÔDigestive enzyme activitiesÕ) Glyco-gen concentration was measured following a modified pro-cedure from Burton et al (1997) Digestive gland sampleswere individually homogenized in 3 mL of ice-cold buffer(100 mM trisodium citrate, pH 4.85) for 1 min, boiled for

6 min, and re-homogenized for 30 s A 500-lL aliquot of thehomogenate was incubated with 500 lL of a 2% solution ofamyloglucosidase (from Aspergillus niger; Sigma Cat No.10115) in 100 mM trisodium citrate buffer (pH 4.85).Another 500-lL aliquot was incubated with the same volume

of buffer but without amyloglucosidase to calculate free

.

Aquaculture Nutrition 17; 613–626  2011 Blackwell Publishing Ltd

were prepared and treated identically to the tissue samples.

Appropriate enzyme standards and blanks were included with

each analysis Aliquots were incubated at room temperature

at 16 110 g for 5 min The glucose concentration of the

supernatants (enzyme treated and enzyme untreated) was

from Invitrogen New Zealand Ltd (Cat No A22189;

Auckland, NZ) The assay mixture contained a final

As-says (200 lL) were performed in duplicate with glucose

measuring absorbance at 560 nm over 30 min The amount of

free glucose present in the DG was calculated (mg per gram

wet tissue) and subtracted from the amount of glucose

liber-ated after enzyme treatment, which was expressed as DG

The combined DG tissue of the three lobsters from the same

tank was homogenized in 1.5 mL of chilled 100 mM Tris,

20 mM NaCl buffer (pH 7.5) using a Ultra Turrax electric

homogeniser (IKA-Werke) at 24 000 rpm (i.e., 6440 g) for

30 s The homogenates were centrifuged at 16 110 g for

5 min to pellet debris, and 200 lL aliquots of supernatant

were analysed for a-amylase, a-glucosidase, total protease,

and trypsin activity as per Simon (2009b) Specific activity

was defined as enzyme activity per mg of soluble protein (SP)

deter-mined by the method of Bradford (1976) using bovine serum

albumin as the standard

Significant differences between dietary treatments for the

various parameters measured (i.e., mean initial weight, final

weight, SGR, biomass gain, moult events, survival, FC, SFC,

FCR, water stability of diets, DGI, DG free glucose,

glyco-gen, SP concentration and digestive enzyme specific activity)

nor-mality of residuals and homogeneity of variances were tested

using the Shapiro–WilkÕs test and LeveneÕs test respectively

All percentage data (i.e., survival and water stability) were

trans-formed where necessary (i.e., moult events, water stability,

free glucose, glycogen and digestive enzyme specific activity)

(P < 0.05) between the means were determined by post hoccomparisons of means using the Tukey-Kramer test (Zar1999) Possible relationships between the various parametersmeasured for lobsters feeding on the formulated diets weretested with PearsonÕs correlation tests All analyses wereperformed using the Statistical & Power Analysis SoftwareNCSS 2007 (Kaysville, UT, USA)

Early J edwardsii juveniles fed with fresh mussel daily(benchmark diet) had a significantly greater (P < 0.05) final

biomass gain (43.6 ± 3.4 g) at the end of the experiment thanthose fed on any of the 13 experimental formulated diets(Table 3) There were no significant benefits in growth andsurvival in substituting the ash filler (control diet) by any ofthe carbohydrate supplements tested in this study, or by anadditional increase in protein content (7% BO11C/HP) (Ta-ble 3) However, there were some trends in the performance ofsome of the carbohydrate sources The survival and SGR ofthe lobsters over 12 weeks varied from 60% and 0.92%

was further confirmed by a significant difference in the meanbiomass gain between these two diets (i.e., 4.8 ± 2.7 g ondextrin; 16.8 ± 2.6 g on 7% BO11C) The number of moultevents was significantly higher for lobsters fed with the freshmussel benchmark diet (25.5 ± 1.7 moults per tank on aver-age, i.e., 2.7 moults per lobster on average) than for mostformulated diets (P < 0.05), except for the diets containingnative wheat starch (19.8 ± 2.2) and the lower inclusion levels

of pregelatinized maize starch (19.8 ± 1.5 for 7% BO11C,19.5 ± 0.5 for 7% BO11C/HP and 18.5 ± 1.0 for the 17%

BO11C diet) Lobsters fed on all of these diets were found tomoult at least twice on average over the 12 weeks and therewas also no apparent reduction in moulting rate through time

on any of the experimental diets The number of moultingevents in each tank was poorly, but significantly, correlated

Lobsters feeding on fresh mussel showed higher food

.

better food conversion ratio (FCR = 0.83 ± 0.06) than on

the formulated diets (P < 0.05) However, the specific food

mussel benchmark diet was not significantly better than for

period, indicating that the greater FC was simply because of

the greater size and food capacity of the lobsters on the fresh

mussel diet Food consumption increased slowly through time

on all the formulated diets in proportion to the relative

increase in biomass in each tank Significantly higher SFC was

former diets also tended to be more poorly utilized for growth

(FCR = 1.74 ± 0.24 for sucrose; 1.61 ± 0.19 for 7%BO11C/HP versus 1.08 ± 0.17 for the dextrin diet) The bestFCR (1.03 ± 0.06) was obtained on the 27% BO11C diet,with FC tending to increase with decreasing inclusion level ofBO11C The fresh mussel diet showed significantly betterwater stability over 20 h (94.3 ± 3.4%) than all the otherdiets (P < 0.05), which ranged from 47.8 ± 1.0% for thesucrose diet to 87.4 ± 1.4% for the agar diet (Table 4)

The DG index of lobsters at the completion of the growthexperiment varied from 3.3 ± 0.2% on the dextrin diet to

Table 3 Growth and survival response of early Jasus edwardsii juveniles fed with fresh mussel and 13 semi-purified formulated diets containing various carbohydrate sources or inclusion levels of pregelatinized maize starch (BO11C) over 12 weeks Significant differences between means (n = 4) within the same column are marked by different letters (P < 0.05)

Treatment

Initial weight (g)

Final weight (g)

Specific growth

Biomass gain (g)

Moult events

Survival (%)

Table 4 Food consumption (FC) and

food conversion ratio (FCR) of early

mussel and 13 semi-purified formulated

diets containing various carbohydrate

sources or inclusion levels of

12 weeks The water stability of the diets

used to calculate FC is included

(n = 4) within the same column are

marked by different letters (P < 0.05)

Treatment

FC

Specific food consumption

Water stability after 20 h (%)

.

Aquaculture Nutrition 17; 613–626  2011 Blackwell Publishing Ltd

5.6 ± 0.2% on the fresh mussel benchmark diet, with

lobsters on the dextrin diet having a significantly lower DG

index than the 7% BO11C (4.6 ± 0.3%) diet, the native

potato starch (4.8 ± 0.3%) diet, and fresh mussel (P < 0.05)

(Table 5) Lobsters fed with the fresh mussel benchmark diet

wet tissue) in their DG in comparison with the other diets

diet Glycogen concentration was significantly correlated

with the dietary inclusion level of pregelatinized maize starch

concen-tration did not correlate with significantly improved survival

or growth (P > 0.05) (Table 3) Free glucose concentration

was higher in lobsters fed with fresh mussel (2.0 ±

protein concentration of the DG did not differ significantly

mussel diet (Table 5)

All digestive enzymes tested were present in early J edwardsii

juveniles, with significant differences among diets in the

specific activity of total protease and a-amylase (P < 0.05),

but not trypsin and a-glucosidase (P > 0.05) Total protease

found to be positively correlated with biomass gain

benchmark diet showed significantly greater a-amylase

except for the control diet and the diets containing the lowerBO11C inclusion levels (i.e., 7% and 17% by dry weight)

The specific activity of a-amylase of lobsters fed with the

was negatively correlated with BO11C inclusion level

(Table 6)

Table 5 Index, glycogen, free glucose and soluble protein (SP) concentration

of the digestive gland (DG) of early

mussel and 13 semi-purified formulated diets containing various carbohydrate sources or inclusion levels of pregelati-

12 weeks Significant differences between means (n = 4) within the same column are marked by different letters (P < 0.05)

Treatment

DG index (%)

Figure 1 Relationship between pregelatinized maize starch (BO11C)

digestive gland of early Jasus edwardsii juveniles left unfed for 24 h after previously being fed with the diets daily over 12 weeks.

.

Early juvenile J edwardsii fed with fresh mussel daily

achieved significantly greater growth and biomass gain than

on any of the 13 formulated diets tested in the present study

No significant difference in lobster final weight was obtained

across the range of semi-purified diets, either suggesting that

none of the carbohydrate sources tested contributed

signifi-cantly to the nutrition of the juvenile lobsters and/or that

there is a fundamental issue in the nutritional quality of the

diets reducing the scope for growth As the present diets were

formulated to contain all the required essential nutrients for

crustaceans (Shiau 1998) and for what is currently known for

spiny lobsters (Williams 2007), it is unlikely the poor

per-formance was related to a gross nutrient deficiency

(Glen-cross et al 2001) Lobsters in this study showed growth and

survival which was similar, if not better, than a good number

of previous studies feeding best formulation practical diets

(Crear et al 2000; Ward et al 2003; Bruce et al 2004; Ward

& Carter 2009) Furthermore, some of the experimental

semi-purified diets provided in the current study resulted in good

survival over 12 weeks in some cases as high (87.5% for the

7% BO11C/HP; 82.5% for the control diet) as the fresh

mussel benchmark diet (85%) Also there was no indication

for a reduction in moulting frequency over the course of the

experiment on any of the experimental diets which is less

likely to occur in the presence of an acute nutrient deficiency

Occasional cases of Ômoult death syndromeÕ were observed in

lobsters fed with some of the worst performing semi-purified

diets (glucose, sucrose and agar) especially towards the end

of the experiment, suggesting that lobsters may have had

underlying nutrition-related problems on these diets, possibly

because of poor utilization of the dietary nutrients It may

also be possible that these lobsters received insufficient levels

of phospholipid (i.e., 1.5% lecithin) feeding on these diets(Conklin et al 1980; Holme et al 2007) As much as 100

earlier research to prevent moult death syndrome whenfeeding casein-based diet to homarid lobsters (Castell et al.1989), but it was shown that lecithin is not required in crab

1985) There is some recent evidence for an improvement in

practical fishmeal diets for spiny lobster, J edwardsii,

microbound diets for mud crab, Scylla serrata, megalopa(Holme et al 2007) Nevertheless, the specific grow rates(SGR) on the formulated diets of this study (i.e., 0.92–1.07%

in recent growth experiments feeding practical diets to early

56% squid meal and 60% fish meal respectively over

1.5–3.2 g in 12 weeks) when fed several times throughout thenight (Bruce et al 2004) with the best performing formulateddiet for the tropical spiny lobster, P ornatus (Smith et al.2005; Barclay et al 2006; Williams 2007) In agreement withprevious research (Crear et al 2000; Thomas et al 2003;Ward et al 2003; Simon & Jeffs 2008), these results stronglyindicate that formulated diets, whether semi-purified orpractical, including an advanced experimental diet whichshowed promise for P ornatus (Williams 2007), are consis-tently providing vastly inferior nutrition for early juvenile

P canaliculus Johnston et al (2007) also showed that thegrowth of early P cygnus juveniles fed with the P ornatus

Table 6 Specific activity of the digestive

enzymes (i.e., total protease, trypsin,

a-amylase and a-glucosidase) from the

digestive gland of early Jasus edwardsii

juveniles fed with fresh mussel and 13

semi-purified formulated diets

contain-ing various carbohydrate sources or

inclusion levels of pregelatinized maize

starch (BO11C) over 12 weeks

Signifi-cant differences between means (n = 4)

within the same column are marked by

experimental diet daily was significantly lower (0.03 g day)1)

than when the diet was supplemented 2 days per week with

suggest that there are species-specific requirements for spiny

lobster feeds (Johnston et al 2007), and findings from one

spiny lobster species may not be directly transferable to

an-other The better performance of the fresh mussel benchmark

diet in the present study was also not related to greater SFC

nutrients from the fresh mussel flesh are better utilized than

from the experimental formulated diets (Crear et al 2000)

to previous studies (Crear et al 2002; Johnston et al 2003;

Thomas et al 2003; Simon & Jeffs 2008; Simon 2009a) and

may be inherent to this spiny lobster species which displays

slow rates of gut evacuation and appetite revival (Simon &

Jeffs 2008) With limited FC, a high ingredient digestibility

and utilization is paramount to achieve optimum growth in

culture In a recent study, Simon (2009d) found that the 27%

BO11C diet had high apparent digestibility (86.4% dry

matter; 86.3% starch; 96.6% protein; 88.7% carbon) in

digestibility of the carbohydrate sources tested in the present

study were 99.4% for dextrin, 91.2% for wheat starch, 83.7%

pregelatinized starch (BO11C), 59.7% potato starch and

59.1% maize starch when incorporated at an inclusion level

of 35% in practical fishmeal-based diets These sources of

carbohydrate had no significant influence on the protein

digestibility (82–88%) (Simon 2009d) It can be assumed that

these digestibility values would be broadly applicable to the

smaller juveniles of the present study, as similar digestive

enzymes specific activities were found here for 1.5 g juveniles

as for larger 30 g juveniles (Simon 2009b), which is in

agreement with the ontogenic changes in digestive enzyme

activity previously reported for juvenile J edwardsii

(John-ston 2003) These results would suggest that the semi-purified

diets containing 27% dextrin, wheat starch and BO11C have

high digestibility (i.e., >86% dry matter, >86% starch,

>96% protein), and therefore, poor utilization of the diets

(rather than poor consumption or digestibility) is the main

factor for their reduced performance in comparison with

fresh mussel

The specific activity of a-amylase was significantly higher

in lobsters fed with fresh mussel than the formulated diets, a

result in agreement with previous research (Simon 2009b)

This would indicate that the metabolism of juveniles fed with

fresh mussel was strongly directed to the digestion and

utilization of glycogen, which accounts for around 21.5% of

the dry weight of the mussel flesh ingested (Simon & Jeffs2008) In contrast, the low overall a-amylase activity on the

with increasing BO11C inclusion level indicates a reducedrequirement for glucose originating from the dietary carbo-hydrate sources tested (van Wormhoudt et al 1980) The

hepatopan-creas of lobsters fed with fresh mussel compared with the

respec-tively) at the completion of the experiment is further evidencefor the greater utilization of glycogen from fresh mussel as asource of energy and potentially as carbohydrate source forchitin production used in carapace formation This concurswith previous research which showed that the high haemol-

during the digestion of glycogen from fresh mussel gonad isutilized rapidly within 24 h postprandial (Simon 2009c) Thereason for the better utilization of glycogen from musselgonad is unclear, but further study is recommended in thisarea to understand its potential protein sparing effect

Whether some fresh mussel constituent inhibits the sion of the crustacean hyperglycaemic hormone (CHH) andincreases the uptake of glucose in the muscles and glycogensynthesis needs to be tested (Simon 2009c) Alternatively, aconstituent of fresh mussel may promote carapace formationand lead to the rapid utilization of glycogen in chitin for-mation explaining the high number of moult events andfaster growth observed in lobsters fed with fresh mussel

on a dry weight basis of P canaliculus; Ben Winters, Aroma

NZ Ltd., personal communication), these un-branchedpolysaccharides containing nitrogen residues perhaps acting

as efficient precursors for glucosamine synthesis which isdirectly related to exoskeleton formation

The lack of clear growth differences among the formulateddiets in the present study makes it difficult to provide rec-ommendations about the optimum carbohydrate inclusionlevel and source to use in spiny lobster juvenile diets On thebasis of the relatively good performance of the control diet

and survival (82.5%), one conclusion may be that drates are not having a significant effect on the nutrition ofjuvenile lobsters and therefore should be omitted whenpossible to provide greater inclusion of protein Previousstudies have shown a linear growth response with increasingprotein inclusion in the spiny lobsters P ornatus (Smith et al

carbohy-2005) and P cygnus (Glencross et al 2001) This is also likely

to be the case for J edwardsii juveniles which would have a .

limited protein intake because of restricted FC (Simon &

Jeffs 2008) However, the present research shows that besides

feeding fresh mussel, low levels of BO11C (7%) and higher

levels of wheat starch (27%) in the diet provided the greatest

number of moulting events over the course of the experiment

an inclusion of 27% sucrose, agar, potato starch and dextrin

provided the lowest number of moulting events (all these

events has been found to have a direct relation to growth in

spiny lobster (James & Tong 1997; Thomas et al 2003)

despite the poor correlation with biomass gain in the present

study On this basis, it would appear that low levels of

pre-gelatinized starch and a higher level of native wheat starch

may have some benefits for promoting ecdysis and growth in

juvenile J edwardsii It has been suggested that 27% wheat

starch and 7% pregelatinized starch may be better utilized

than 27% dextrin or 27% gelatinized maize starch because

the former carbohydrate sources (at these concentrations by

dry weight) result in slower postprandial rises in

haemol-ymph glucose (Simon 2009c) A slower appearance of glucose

in the haemolymph could in turn result in more efficient

glucosamine production, a critical step before chitin synthesis

and ecdysis (Cuzon et al 2000) Among lobster fed with the

found in the hepatopancreas of lobsters fed with native wheat

starch, which would suggest the superior utilization of wheat

starch in J edwardsii juveniles (Simon 2009c) Increasing

dietary gelatinized starch (BO11C) inclusion level had a

positive effect on the glycogen concentration of the

hepato-pancreas in J edwardsii juveniles, as has also been found for

the shrimp Litopenaeus vannamei (Rosas et al 2002)

How-ever, glycogen deposition in the hepatopancreas did not

correlate with survival or growth in J edwardsii juveniles in

the present study despite its apparent importance during

moulting cycles of crustaceans generally, but especially spiny

lobsters (Schwabe et al 1952; Travis 1955, 1957, 1960)

Glycogen has been observed to be highly labile within

vari-ous body tissues of spiny lobsters including the

hepatopan-creas and muscles, suggesting periodic cycles of accumulation

and utilization, especially by the epidermis which is involved

in the synthesis and re-absorption of the organic matrix that

makes up the carapace (Travis 1955, 1957, 1960) The state of

moult cycle may also greatly influence the ability of the

hepatopancreas and other tissues to accumulate glycogen, or

even effectively utilize dietary sources of carbohydrate that

could contribute to glycogen accumulation (Renaud 1949;

Schwabe et al 1952) Indeed, the moulting hormone systemthat controls ecdysis is intimately linked with the control ofcarbohydrate flux in crustaceans, so much so that it wasinitially known as Ôdiabetogenic hormoneÕ of the crustaceaneyestalk (Wang & Scheer 1963) For example, Scheer &Scheer (1951) showed that removal of eyestalks from spinylobsters resulted in increased tissue utilization of exogenousglucose and a concomitant decrease in blood sugar level Ifendogenous accumulation of glycogen either in the tissues orthe carapace is of such importance in the synthesis of newcarapace for the moult then it is also feasible that dietaryavailability of appropriate carbohydrate source may alsofeedback into the hormone control of moulting This mayperhaps explain higher moult increments observed with freshmussel, wheat and some of the BO11C dietary treatments inthis current study In the same manner, previous studies havealso linked moult frequency with dietary induced differences

in serum protein level in H americanus (Castell & Budson1974)

Postprandial haemolymph glucose concentration is not thesole determinant of the value of dietary carbohydrate sour-ces Carbohydrates also have a strong effect on the waterstability of diets, as well as their attraction and palatability,which are also important factors influencing the quality ofdiets for spiny lobsters The growth rate and survival oflobsters was lower on the 27% dextrin diet than on the 27%gelatinized starch diet in the present study, despite these dietshaving a similar influence on haemolymph glucose fluxes(Simon 2009c) The poorer performance of dextrin comparedwith gelatinized maize starch may be because of the lack ofbinding properties of dextrin, resulting in poorer diet sta-bility in water (63%), and therefore greater leaching andlower protein content after immersion However, excessivelyhigh water stability, as for the agar diet (87%), can also lead

to suboptimal growth by having a detrimental effect on thepalatability and attractiveness of the diet (SFC = 0.85%

compounds such as amino acids, amines, nucleotides andorganic acids from the food has been found to be essentialfor chemical attraction in spiny lobsters (Derby 1984; Wil-liams et al 2005) This was shown here with the extra aminoacids from the 20% increment in sodium caseinate (7%BO11C/HP) having a positive effect on food consumption

found to act as an attractant in J edwardsii juveniles, thelobsters feeding response being more intense and prolonged

on the diet containing 27% sucrose (pers obs.), leading to

compared with other relatively less attractive semi-formulated .

Aquaculture Nutrition 17; 613–626  2011 Blackwell Publishing Ltd

diets such as dextrin (SFC = 0.78% BW day)1) Sucrose may

be used in small quantities in further studies as a mean to

improve the attractiveness of formulated diets, which is

cru-cial to achieve high feed intake for spiny lobster aquaculture

(Williams 2007) It is however a poorly digestible source of

energy for J edwardsii juveniles (Simon 2009c)

One plausible explanation for the lack of clear growth

differences between dietary carbohydrates could be because

of sufficient glucose and energy being derived principally

from gluconeogenesis of the sodium caseinate of the present

semi-purified diets Most crustaceans appear to have a clear

tendency to direct their metabolism to use protein rather

than carbohydrate (Santos & Keller 1993; Rosas et al 2002)

Rosas et al (2001) showed that gluconeogenic enzymes, such

as phosphoenolpyruvate carboxykinase (PEPCK), are

sig-nificantly activated by a decrease in dietary carbohydrate and

shrimp Litopenaeus vannamei The diets of the present study

energy derived from the breakdown of amino acids being

sufficient for maintenance as lobsters feeding on the control

diet showed excellent survival and low concentrations of

in their DG Further evidence for the utilization of sodium

caseinate for energy is seen when comparing the diets 7%

BO11C and 7% BO11C/HP, where the 20% increase in

sodium caseinate content in the latter diet tended to improve

survival and glycogen concentration, not growth or weight

gain Casein is often regarded as a poor protein source for

crustaceans owing to its limited arginine content (Mente

additional amount of coated arginine was intended to

address this deficiency while keeping dietary carbohydrate

content low Future studies should refine the potential

nutritional benefit of native wheat starch in formulated diets

containing a protein inclusion level below the optimum

small J edwardsii juveniles (Johnston et al 2003; Ward et al

2003), and with a greater proportion sourced from

high-quality marine products such as krill meal The measurement

of CHH titres and PEPCK activity may assist in furthering

the understanding carbohydrate metabolism in spiny lobster

The fate of the dietary carbohydrate sources (i.e., carbon)

could also be studied via stable isotope tracing in the

lob-sters Overall, the results suggest that the provision of

com-mon carbohydrate sources will have limited value for

incorporation in formulated feeds for juveniles of this species

of spiny lobster

Thanks to all the staff at the NIWA Mahanga Bay culture facility for assistance, especially Phil James andJohnny Wright for help in the building of the rearing system,Phil Heath for assisting with the collection of pueruli andKevin Green for help with the feeding during the growthexperiment Thanks to Anna Kilimnik and Debbie Hulstonfor their assistance with the enzymatic assays Lobsters werecollected under a special research permit granted under theFisheries Act and euthanized using methods approved by theNIWA animal ethics committee NIWA, the University ofAuckland, the Glenn Family Foundation and the ProvinceSud of New Caledonia (Prix dÕEncouragement a` la Recher-che) provided funding for this research

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Bordner, C.E., DÕAbramo, L.R & Conklin, D.E (1983) tion of nutrients by culture hybrid lobsters (Homarus sp.) fed experimental diets J World Maric Soc., 14, 11–24.

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Burton, S.A., Mackenzie, A.L., Davidson, T.J & Macnair, N (1997) Evaluation of a glucose oxidase/peroxidise method for indirect measurement of glycogen content in marine mussels (mytilus edu- lis) J Shellfish Res., 16, 435–439.

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