Aquaculture nutrition, tập 19, số 5, 2013

Plant oil can replace fish oil without affect-ing growth provided that the requirement of marine long chain LC n-3 fatty acids is met, but the altered dietary fatty acid profile in diet

NIFES, National Institute of Nutrition and Seafood Research, Bergen, Norway

The present paper gives an overview on the use of plant

protein and plant oils as replacers for fish meal and fish oil

in diets for Atlantic cod Gadus morhua L In focus are

effects on growth, feed utilization, digestibility, gut health,

muscle and liver uptake and retention of nutrients, and

muscle quality Plant oil can replace fish oil without

affect-ing growth provided that the requirement of marine long

chain (LC) n-3 fatty acids is met, but the altered dietary

fatty acid profile in diet will be reflected in both muscle

and liver This can reduce the value of cod liver as an oil

source for cod liver oil production For the fish itself, there

are more challenges replacing fish meal than fish oil, due to

the amount of fibre and antinutrients in plant protein

meals However, A cod seems to tolerate a wide range of

plant types and their inclusion levels provided that the

amino acids requirements are met It is our view that there

is sufficient knowledge to be able to design an A cod diet

based on a mixture of plant and marine ingredients and be

able to predict performance such as growth, feed

utiliza-tion, digestibility, liver size and fish health in general

KEY WORDS: A cod, plant protein, plant oil, growth, protein

utilization, digestibility, gut health

Received 30 November 2012; accepted 27 March 2013

Correspondence: G.-I Hemre, NIFES, National Institute of Nutrition

and Seafood Research, Box 2029 Nordnes, 5817 Bergen, Norway.

E-mail: ghe@nifes.no

Until recently, the protein and lipid in farmed fish diets

have been based on fish meal and fish oil Currently, most

marine resources used in fish meal and fish oil production

are exploited to the highest maximum level, simultaneously

as the global production of farmed fish is increasing (FAO2010) It has therefore been essential to evaluate the poten-tial for utilizing plants as protein and oil sources in dietsfor farmed fish

In Northern Europe, interest in farming of A cod(Gadus morhus L.) has increased steadily over the past dec-ade stimulated by the decline in landings from fisheries and

a more predictable supply of hatchery reared juveniles forongrowing From 2000 to 2009, the production quantity offarmed A cod in Norway increased from 169 to

18 052 tonnes (FAO-Fisheries and Aquaculture tion and Statistics Service), and in 2010, 19 700 tonneswere slaughtered (Mugaas Jensen et al 2010) How-ever, due to economic problems, disease problems,increased landings of wild A cod and low prices(Torskenettverksmøte 2011, Bergen) in 2012, there wasonly one main producer left in Norway This means thatthe cost of feed must be as low as possible, simultaneously

Informa-as fish health and performance must be acceptable

A cod is a carnivorous lean fish species containing

& Diamond (1987) found that 69% of the gut area wasrepresented by the pyloric ceca giving a gut length/forklength ratio of 10, being 2.5 times higher than, for example,rainbow trout, increasing the absorptive surface of theintestine considerably Compared to the front and mid sec-tions of the intestine, the distal part of the cod intestinehas a clear distinguished appearance with a thick intestinal

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Aquaculture Nutrition

wall and a valve like structure The distal chamber has also

a high colonization of bacteria in the brush border and is

therefore probably a fermentation chamber, fermenting

indigestible intestinal content (Refstie et al 2006b; Seppola

et al 2006) This characteristic of A cod intestine can

affect the ability to cope with plant ingredients, as its

appearance is similar to the intestine of herbivorous fish

species (Krogdahl et al 2005)

This review aims to summarize the research carried out

in replacing fish meal and oil with plant ingredients in diets

for A cod

The dietary lipids constitute an important energy source as

well as being a source of essential fatty acids in A cod

diets Fish oil is characterized by high level of the marine

long chain n-3 fatty acids docosahexaenoic acid (DHA;

22:6n-3) and eicosapentaenoic acid (EPA; 20:5n-3), while

plant oils are characterized being high in the n-6 [mainly

li-noleic acid (LA; 18:2n-6)] and n-9 fatty acids [mainly oleic

acid (OA; 18:1n-9)] and the saturated fatty acids palmitic

acid (16:0) and stearic acid (18:0), and does not contain

DHA and EPA Replacement of fish oil with plant oil is

widely studied in fatty fish like salmonids that have a

higher lipid utilization capacity than the lean A cod

(Turchini et al 2009) There are a few studies reported for

A cod, where fish oil is used exclusively or partly as the

dietary oil source (Lie et al 1986, 1992; Waagbø et al

1995; Bell et al 2006; Mørkøre 2006; Mørkøre et al 2007;

Jobling et al 2008)

The only negative effect on growth and feed intake in

A cod was seen with use of dietary peanut oil (Lie et al

1986) Total or partial substitution of fish oil with linseedoil, echium oil or soybean oil in the diet gave no negativegrowth effects (Lie et al 1992; Bell et al 2006; Mørkøre2006; Mørkøre et al 2007; Jobling et al 2008) The trialwith peanut oil was also the only trial where squid mantle,not fish meal, was used as the dietary protein source andhad the lowest levels of marine n-3 fatty acids (Table 1)

Essential fatty acid deficiencies are unlikely when the dietcontains a relatively large amount of fish meal (Turchini

et al 2009), as fish meal usually contains 50–130 g kg 1lipid, whereas 20–35% of the lipid is marine LC n-3 fattyacids (NRC 2011) When comparing A cod trials that gave

no effect on the growth of dietary DHA level ranged from7.7 to 9.9 g kg 1 and the EPA level ranged from 4.2 to7.8 g kg 1(Bell et al 2006; Mørkøre 2006), giving LC n-3levels of 9.4–17.7 g kg 1

diet (Table 1) This indicates thatthese levels of DHA and EPA were sufficient to supportgrowth This was in line with what was reported to be therequirement for other marine fish species (5.0–25.0 g LCn-3 kg 1 feed) (Turchini et al 2009) In the diet with pea-nut oil, the level of LC n-3 was only 3.4 g kg 1(Lie et al

1986) and was probably deficient in LC n-3, as the first

Table 1 Level of long chain (LC) n-3 fatty acids in Atlantic cod diets with different plant oil sources

Oil source

Inclusion

(% of oil)

DHA (% of fatty acids)

EPA (% of fatty acids)

Sum LC n-3

Changed tissue FA profile Reduced prostaglandin

activity in gill cells

Bell & Waagbø (2008)

Changed tissue FA profile

Lie et al (1992)

Changed tissue FA profile

Lie et al (1986)

.

sign of deficiency normally is reduced growth, which was

reported in this study (Lie et al 1986) In this trial, feed

intake was reduced by 20% compared to a diet with cod

liver oil Lipid digestibility was also reduced, which may

partly explain the reduced growth (ibid)

In Atlantic cod, Jobling et al (2008) found a tendency to

increased HSI and liver lipid level when fed a diet with

rapeseed or palm oil compared to fish oil (equal lipid level

in all diets) However, this was not seen with use of

soybean or echium oil (Bell et al 2006; Mørkøre 2006;

Mørkøre et al 2007) The HSI was even reduced with the

use of only soybean oil compared to fish fed a diet with

only capelin oil in one trial (Waagbø et al 1995) The

vari-able results between studies might be due to different fatty

acids stimulating theb-oxidation capacity and thereby lipid

storage differently, as found for other species of fish

(Stubhaug et al 2007)

The fatty acid profile of the diet influences both muscle

and liver fatty acid profiles of A cod, where soybean,

rape-seed and palm oil fed fish had higher levels of LA and sum

of n-6 fatty acids in muscle and liver than fish oil–fed fish

(Lie et al 1992; Mørkøre 2006; Jobling et al 2008)

Feed-ing A cod fat soybean meal or a combination of

full-fat soybean, corn gluten and wheat gluten meal resulted in

muscle and liver reflecting the fatty acid profile of the diet

(Hemre et al 2004; Albrektsen et al 2006; Karalazos et al

2007) (Fig 1) The liver reflecting the fatty acid profile of

the diet can have consequences for the use of farmed

A cod liver in the production of cod liver oil, as the level

of DHA and EPA will decrease when dietary LC n-3

decrease This can be solved by feeding a finishing diet high

in LC n-3 fatty acids; however, reduction of LA in fish

ear-lier fed plant oil is found to be low (Mørkøre et al 2007;

Jobling et al 2008)

There have been some concerns regarding technical quality

of fillet due to altered fatty acid profile of muscle when

using diets high in plant oils As A cod muscle only

con-tains 5–10 g kg 1

lipid, most of the lipid is in the form ofphospholipids in the cell membranes, and changes in the

fatty acid profile of the membranes can change the

charac-teristics of the membrane Changes in dietary lipid level did

not result in changes in muscle lipid level in A cod,

how-ever, it affected muscle vitamin E and C concentrations,

and which showed a negative correlation with EPA andDHA and the ratio n-3/n-6 in muscle (Hemre et al 2004).Changes in dietary fatty acid profile do not affect muscle

pH, liquid losses or texture after frozen storage, however,soybean oil fed fish had significant lower degree of gaping(Mørkøre 2006; Mørkøre et al 2007) A trained test panelcould taste a difference between soybean oil and fish oil fedAtlantic cod and preferred no one over the other (Mørkøre

et al.2007)

There have also been some concerns regarding fish health,when the levels of DHA and EPA in the diet are reduceddue to inclusion of plant oil This concern is connected tothe positive effects of DHA and EPA seen in mammals oncoronary heart disease, but also to general disease resis-tance (Turchini et al 2009) Waagbø et al (1995) showedlower specific antibody response against V Anguillarum insoybean oil fed A cod compared to marine oil fed fish,indicating reduced disease resistance On the other hand,Bell et al (2006) indicated a positive effect on someimmune parameters with use of dietary echium oil, whichresulted in reduced prostaglandin F2 a production in gillcells together with reduced macrophage activity This wasprobably connected to the increases of ARA/EPA ratio inthe fish when fed echium oil, as ARA is the primary

35.0 20.0 –5.0 10.0 25.0

14:0

16:0 18:0

16:1n-7

18:1n-9 18:1n-7

20:1n-9 22:1 24:1n-9

18:2n-6

20:2n-6 18:3n-3

18:4n-3 20:4-n-3

20:5n-3 22:5n-3

eicosanoid precursor in fish, and macrophages are activated

by eicosanoids (Tocher 2003) Changing the LC n-3 level in

Atlantic salmon diets has shown both positive and negative

effects on the immune system (Turchini et al 2009),

indi-cating immune effects will be dependent on how large and

which changes in fatty acid composition the plant oil

causes

In conclusion, there is great potential to replace fish oil

with plant oil without compromising performance;

how-ever, this will reduce the value of the A cod liver as a

mar-ine LC n-3 source in, for example, cod liver oil production,

if not using a ‘finishing’ diet

There is a wide variety of plant protein ingredients being

good alternatives to fish meal as protein sources in fish

feeds This includes oilseeds (soybean, sunflower,

cotton-seed and rapcotton-seed/canola), legumes (lupins, beans, peanut

and pea) and grails (corn and wheat glutens) There are

several different products and qualities of those plant

pro-tein ingredients, varying in propro-tein, fat and antinutrient

contents (NRC 2011)

To obtain acceptable growth and feed utilization,

simul-taneously avoiding large liver sizes, A cod needs diets with

500–600 g kg 1

protein, 130–200 g kg 1

lipid and ate carbohydrate levels (Lied & Braaten 1984; Jobling

moder-1988; Lie et al moder-1988; Hemre et al 1989; Dos Santos et al

1993; Morais et al 2001; Rosenlund et al 2004;

Grisdale-Helland et al 2008; Hansen et al 2008) Lower dietary

protein levels do not result in lowered growth, but

increased FCR, as A cod is found to be able to

compen-sate with higher feed intake to reach its optimal level of

protein and energy intake to meet its growth potential

(Hemre et al 1989; Lekva et al 2010) To achieve a high

dietary protein level in plant-based diets (above

500 g kg 1), processed plant ingredients like wheat and

corn gluten and soy protein concentrate need to be used

A cod, like other animals, has a requirement for

indispens-able amino acids (IAA), for optimal growth and protein

utilization The requirement for IAAs has been shown to

highly correlate with the amino acid pattern of the fish

(Wilson & Poe 1985; Mambrini & Kaushik 1995), and this

pattern is similar between fish species (Mambrini &

Kaus-hik 1995) High-quality fish meal is therefore regarded as a

good protein source that covers the need for all amino

acids On the other hand, plant proteins differ from fish

meal in several IAAs Further, some plant proteins have

high levels of undesirable components like ANFs and fibre,

which can alter intestinal function and influence ity coefficients in fish (Francis et al 2001)

digestibil-Fish meal has been replaced by plant protein in several als with A cod (Table 2) (von der Decken & Lied 1993;

tri-Albrektsen et al 2006; Refstie et al 2006a; Hansen et al

2007a,b; Karalazos et al 2007; Walker et al 2010) Effects

on growth are dependent on the plant ingredients, whichmixture is used, and the inclusion level Replacing fish mealprotein with 30% protein from full-fat soybean meal indiets for A cod induced reduced growth (von der Decken

& Lied 1993; Karalazos et al 2007) This was due toreduced feed intake, maybe a consequence of reduced pal-atability or ANFs, but it was also speculated to be caused

by the shortage of one or several amino acids triggering ametabolic mechanism of food intake regulation (von derDecken & Lied 1993) On the other hand, no effects ongrowth and feed intake were observed by Albrektsen et al

(2006) using a mixture of full-fat soybean meal and corngluten up to 54% of total dietary protein Similarly, noeffect on growth was found using 24% (of total protein)solvent-extracted or bioprocessed soybean meal (Refstie

et al 2006a), a combination of solvent-extracted soybeanmeal and corn gluten meal (70–310 g kg 1

) or where 58%

of protein came from a 1:1 combination soy protein centrate and wheat gluten (Hansen et al 2007b) Also, nonegative effects on growth were seen when 100% of the fishmeal was replaced with soy protein concentrate (Walker

con-et al.2010) Using a regression design from an all fish meal

to an all plant protein diet (500 g kg 1 wheat ten+ 140 g kg 1

glu-bioprocessed soybean meal+ 360 g kg 1soy protein concentrate) resulted in reduced growth at aplant protein inclusion level of 50% and higher, with amean reduction in SGR of 16% when given 75% plantprotein, and 43% when given 100% plant protein (Hansen

et al.2007a) Increased feed intake was reported for A codwhen including plant protein (Albrektsen et al 2006;

Refstie et al 2006a; Hansen et al 2007a) This is in trast to what is seen with total (de Francesco et al 2004;

con-Espe et al 2006) or partial (Gomes et al 1995; Refstie

et al.1998; Torstensen et al 2008) replacement of fish mealfor salmonids, where a reduction in feed intake was seenespecially in the first period of the trials In the trial byHansen et al (2007b), the plant protein inclusion in coddiets also resulted in reduced protein utilization Theincreased feed intake registered could therefore be due to acompensation for the lowered protein utilization, as it has

.

been found that A cod increase its feed intake to meet

die-tary demands, for example, for protein and energy (Hemre

et al.1989; Lekva et al 2010)

Plant proteins in fish diets may lead to reduced protein

retention (Kikuchi 1999; Regost et al 1999; Carter &

Hauler 2000; Refstie et al 2000; Opstvedt et al 2003; de

Francesco et al 2004; Lim et al 2004) This is possible due

to deficiency of one or several amino acids Espe et al

(2007) showed that 95% replacement of fish meal with

plant protein could be used without compromising growth

in diets for A salmon, provided that the amino acid levels

mimicked that of a fish-meal-based control diet and equal

feed intake This shows that differences in amino acid levels

may explain the reduction in growth when exceeding 50%

plant protein in diets for A cod (Hansen et al 2007a) In

this latter trial, the plant-based diets methionine was

limit-ing when compared to fish meal Methionine concentration

in diet also highly correlated with plasma and muscle free

methionine concentrations when sampled 5 h after the last

meal At 75% and 100% plant protein replacement of fish

meal protein, free methionine was not detected in the

muscle free pool at all, showing that the free amino acid

pool was emptied of methionine (Fig 2) Further, adecrease in muscle free lysine of 67% was seen comparingthe fish meal group and the 100% plant group, correlatingwell with dietary levels of this amino acid However,lysine and methionine supplementation to diets holding

650 g kg 1 plant protein did not result in improved totalgrowth, feed intake or protein retention (Hansen et al.2011) This indicates that plant protein-based diets for

A cod need not be added lysine or methionine, or both,

to maintain total growth, foreseen that diets hold

Table 2 Studies where plant proteins replace fish meal in diets for Atlantic cod

Amino acid

Feed utilization

Gradually reduced

Gradually reduced

Hansen et al (2007a)

MV

MV G

G

Figure 2 Hind gut from Atlantic cod fed a fish meal diet (a) and a 100% plant protein diet (b) MV, micro villi; G, goblet cells (Olsen

et al 2007).

19.2 g lysine kg diet and 9.4 g methionine kg diet.

Grisdale-Helland et al (2011) tested a dietary lysine level

of 13.7–28.3 g kg 1 diet and did not find any effects on

growth, but the best lysine gain was achieved with the diet

containing 26.2 g lysine kg 1 diet Lysine requirement for

A cod is in line with requirements reported for other

marine species (17–26 g lysine kg 1

diet) (NRC 2011) Atthe same time, increased lysine intake affected lipid storage

resulting in reduced lipid retention, HSI and plasma TAG

(Hansen et al 2011) On the other hand, this was not

observed in other trials with plant protein in A cod diets

(Hansen et al 2007a,b), but a tendency of reduced HSI

when including plant protein was reported in some trials

(Albrektsen et al 2006; Refstie et al 2006a) Microarray

and qPCR data comparing fish meal fed A cod with fish

fed 75% plant protein of total protein, showed alteration

in hepatic expression of genes involved in protein turnover,

affecting both protein degradation and anabolic pathways

(Lie et al 2011)

Diets with plant protein can reduce digestibility of

nutri-ents in fish (Hilton et al 1983; Francis et al 2001)

Krog-dahl et al (2003) showed reduced mucosal enzyme

activities in Atlantic salmon fed up to 300 g kg 1

solvent-extracted soybean meal, mirroring reduced macronutrient

digestibility Plant protein can contain soluble fibres and

antinutrients that can interfere with nutrient digestibility

(Krogdahl et al 2005) Soluble fibres increase viscosity of

gut content, which potentially can reduce digestible enzyme

activities, and negatively affect nutrient digestion and

absorption (Storebakken 1985; Leenhouwers et al 2006)

Of the antinutrients protease inhibitors, tannins and lectins

are known to affect protein utilization and digestion

(Francis et al 2001) Protease inhibitors, especially high

levels in soybean, act by blocking the activity of trypsin

and chymotrysin Lectins, on the other hand, bind to

receptors on the intestinal cells and can cause damage to

the villi Tibbetts et al (2006) investigated the digestibility

of a number of plant protein ingredients in A cod and

found ADC values in the same range for plant protein

ingredients, like soybean meal, lupin meal, corn gluten and

wheat gluten meal as for fish meal However, other studies

have reported reduced ADC for protein and lipid in studies

with A cod when including solvent-extracted soybean meal

(Førde-Skjærvik et al 2006; Hansen et al 2006), and

Han-sen et al (2006) observed reduced ADC of protein, and fat

and starch when including corn gluten meal Some of the

discrepancy between studies may be due to different dient qualities and variable inclusion levels, for example,replacing 100% of the fish meal with plant protein results

ingre-in a fibre level ingre-increase from 30 to 80 g kg 1 (calculatedamount) (Hansen et al 2007a) The fibre fraction is shown

to disturb lipid micelle formation in the intestine and toincrease the viscosity of fish gut content, both factors thatcan explain a reduced fat digestion (Krogdahl et al 2005;

Refstie et al 2006b; Olsen et al 2007) The reduced lipiddigestion may also be linked to alcohol-soluble componentsfrom soybean, like saponins, that have been found to result

in altered gut function and reduced lipid digestibility in

A salmon (Olli & Krogdahl 1995; Knudsen et al 2007)and Japanese flounder (Chen et al 2011) Total saponinconcentration increased from 0.42 to 0.86 mg g 1 (DW)when increasing plant protein inclusion from 50% to 100%

of total protein, in A cod diets, and unfortunately the fishwas not able to digest these saponins (Olsen et al 2007) Inthe gastrointestinal tract of A cod, you find a high number

of bacteria (Hansen et al 2006; Refstie et al 2006a,b;

Seppola et al 2006), which changed dependent on soybeaninclusion in the diet (Ringø et al 2006) So, the reduceddigestibility of protein and starch observed in the cod stud-ies (Førde-Skjærvik et al 2006; Hansen et al 2006) mayalso be a consequence of changed intestinal microflora

Morphological changes in the intestine have been shown forsalmonids when including plant proteins, especially soybean(van den Ingh et al 1991; Baeverfjord & Krogdahl 1996;

Storebakken et al 2000) Typically, extensive endocytoticactivity and high numbers of intracellular vacuoles are found

in cells of the distal part of the gut Damages are often acterized by increases in number of mucus-producing gobletcells, intracellular absorptive vacuoles, changed cellularstructure of the lamina propria, amount of connective tissue,degree of mucosal folding and infiltration of the ephithelium

char-or lamina propia by inflammatchar-ory cells In extreme cases,massive necrosis occurs, a condition referred to as soybean-induced enteritis (van den Ingh et al 1991; Baeverfjord &

Krogdahl 1996) In A cod, some effects in gut morphologywere induced by plant protein, especially when dietary plantinclusion exceeded 75% of protein, but the alterations weremoderate and involved mostly goblet cells (Olsen et al

2007) The main plant protein ingredient in those diets was,however, highly refined bioprosessed soybean meal with lowlevels of ANFs One would therefore not expect any majorchanges caused by bioprocessed soybean meal in A cod

.

gastrointestinal (GI) tract This was supported by Refstie

et al.(2006b) where an inclusion of 240 g kg 1biopresessed

soybean meal did not cause any alterations in intestinal

mor-phology In A cod fed diets, where 100% of the fish meal

was replaced with soy protein consentrate, no gut damages

were seen (Walker et al 2010) In salmonids,

soybean-induced gut damage is usually related to distal parts of the

GI-tract affecting cell types with endocytotic activity and

high levels of intracellular vacuoles A cod does not have

the same differentiation of cell types, and most of the

intes-tine contains cells that do not appear to be endocytotic

(Odense & Bishop 1966) The difference between A cod and

A salmon may have implications related to the gut

sensitiv-ity to ANFs

Little research has been carried out regarding micronutrient

requirements in on-growing A cod There are some

con-cerns when replacing fish meal with plant proteins

regard-ing the water-soluble vitamins riboflavin, niacin,

pantothenic acid and vitamin B12 (Bell & Waagbø 2008)

Hansen et al (2007a) showed a reduction in vitamin B12

concentration from 0.12 to 0.01 mg kg 1 in diets, when

replacing 100% of fish meal with a plant protein blend

Because the requirements of these vitamins are not

estab-lished for A cod, addition levels have to be based on what

is known for other marine species (NRC 2011) The only

water-soluble vitamin studied in juvenile A cod is vitamin

C, showing that 1.5 g A cod had a qualitative requirement

for vitamin C (no vitamin C versus 500 mg vitamin C

Kg 1diet) (Sandnes et al 1989), but the exact level needed

to be present to avoid deficiency was not determined

Major shifts in fish feed ingredient profiles, especially

from fish meal to plant protein meals, call for increased

attention to mineral contents and availability in diets, due

to fish meal being a good source of several minerals (NRC

2011) Diets high in plant ingredients are expected to

increase the need for supplementation The two minerals

with highest concentration in fish meal are phosphorus (P)

and calcium (Ca) (NRC 2011) The P level in soybean is

mostly in the form of phytate which is less bio-available,

phytate is also regarded as an ANF Kousoulaki et al

(2010) studied growth and tissue mineralization in A cod

fed diets holding 4.7–10.4 g P kg 1

diet, supplemented with

Ca giving a dietary Ca level of 4.1–11.9 g kg 1

The resultsshowed that 7.6–10.4 g P kg 1

diet had positive effects onfish performance, growth and tissue mineralization A cod

diets containing <7.6 g P kg diet should therefore besupplemented with P, placing A cod in the upper range of

P requirement among other marine fish species(6.0–8.0 g P kg 1

diet) (NRC 2011) However, Ca level of11.9 g kg 1or higher had negative effects and suggests that

Ca contents should be minimized, so addition of Ca whenreplacing fish meal with plant proteins may not be neces-sary (NRC 2011) Not only can minerals be absorbedthrough the intestine (from both the diet and the water),but also some absorption may take place via the gills andskin A cod lives in seawater which is rich in ‘bone miner-als’ and electrolytes such as Ca, magnesium, sodium, potas-sium and chloride, and in addition contains about

60lg iodine L 1

water The main function of iodine, aspart of the thyroid hormones thyroxine (T4) and triiodo-thyronine (T3), is to participate in the control of intermedi-ary energy metabolism, growth and development Inmarine halibut larvae, it is identified that water iodine cov-ers up to 90% of total iodine uptake (Moren et al 2008)

No similar data exist for cod but are expected to be in thesame range The only studies on mineral requirement for

A cod are performed on larvae and live feed, and no dataexist for on-growing stages A cod larvae fed rotifersenriched with iodine, copper, manganese and zinc showed

no beneficial effects of the iodine enrichment, while Curesulted in increased metallothionein mRNA expression,indicative of a coping mechanism to deal with excessive

Cu When the larvae was fed, Mn and Zn in addition to

Cu, Mn and/or Zn ameliorated the effect of excessive Cu.Further, increased Zn whole body concentration wasfound, and increased Mn-SOD (superoxide dismutase)activity was identified (Pengalese, S 2013, unpublishedresults) Enriching rotifers with selenium has also provenbeneficial to cod larvae SOD activity but had no influence

on survival or growth of A cod larvae (Pengalese et al.2010) Fish meal is rich in selenium, so when plants exertmajor part of the on-growing diet for A cod, it might benecessary to add selenium to meet requirement The ter-tiary symptoms observed from selenium deficiency in fishare related to selenium as a functional part of the antioxi-dant enzyme glutathione peroxidase (GLPx) The minimumrequirement can be estimated from optimal activity ofplasma GLPx (Lorentzen et al 1994) A cod given dietswhere plant protein exerted 75% of the protein did, how-ever, not show any reduction in GLPx mRNA expression,

or other enzymes related to energy turnover (Lie et al.2011), which indicates sufficient selenium in those diets.Often when requirement studies lack, tissue or wholebody concentrations of minerals have been used as

indicators of requirement Because the major part of

miner-als is not stored in the lipid fraction, deposition of lipids

will be a dilution factor to mineral concentrations in whole

body (Shearer 1984), as will be the case in farmed A cod

with large livers and lipid deposits up to 70% of total liver

weight (Rosenlund et al 2004) To compensate for such

dilution factors, often mineral content relative to ash

con-tent has been used as an indicator of mineral requirement

(Knox et al 1981)

In diets, where up to 100% of the fish meal was replaced

with a plant protein blend, the ash content decreased from

100 g kg 1 in the fish meal diet to 50 g kg 1in the 100%

plant protein diet (Hansen et al 2007a) High dietary ash

levels (180 g kg 1) from fish bone and crab by-products

seemed to be beneficial for Atlantic cod, increasing growth

with 10% compared to a fish meal reference diet (Toppe

et al.2006) Hansen et al (2012) found equal growth when

adding a diet plant protein (50% of total protein) with

200 g kg 1crab or 100 g kg 1 shrimp shell compared to a

control without crab or shrimp shell meal inclusion The

fish compensated for the reduced protein/energy level of the

diet by increasing feed intake resulting in equal protein and

energy intake as the control diet, and thereby equal growth

A 200 g kg 1shrimp shell meal inclusion on the other hand

caused damages in the intestine, for unknown reasons

There is high potential to replace fish meal and fish oil with

plant ingredients Plant oil can replace fish oil without

affecting growth provided that the requirement of marine

LC n-3 fatty acids are met, but the altered dietary fatty

acid profile will be reflected in the muscle and liver, and

can reduce the value of A cod liver as a commercial

prod-uct There are more challenges replacing fish meal than fish

oil, due to the amount of fibre and antinutrients present in

plant protein meals However, A cod seems to tolerate a

wide range of types and inclusion levels of plant proteins

provided that the amino acids requirements are met There

are no trials with A cod where a combination of plant

proteins and plant oils replaces marine ingredients This is

recommended as synergistic effects of replacing both of

these simultaneously in diets for A salmon was revealed

(Torstensen et al 2008) Also, requirement studies for

mi-cronutrients naturally present in fish meal and fish oil lack

for A cod, so the necessity to add should follow

recom-mendations for other marine fish species (NRC 2011) Still

the knowledge base is, in our view, sufficient to design a

A cod diet based to a major extent on a mixture of plantingredients

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1 1 1 2 3

1

National Institute of Nutrition and Seafood Research, Bergen, Norway; 2 Department of Biology, University of Bergen,Bergen, Norway;3 Matre Research Station, Institute of Marine Research (IMR), Matredal, Norway

The vitamin A (VA) concentration in salmon aquaculture

feeds is varying and may lead to sublethal adverse effects

In this study, 135 g Atlantic salmon postsmolts were given

eight diets in duplicates with 6, 12, 26, 55, 82, 112, 360 and

749 mg retinol (ROL) kg 1 for 116 days Subsequently,

fish given 6, 82 and 749 mg ROL kg 1were transferred to

a common net pen and given a standard commercial diet

for further 28 weeks Feed conversion rate, liver

functional-ity and markers of VA homoeostasis were not negatively

affected by dietary VA level, but chronic high VA intakes

led to adverse effects on growth and bone health In

plasma, there was an antagonistic effect of dietary ROL on

circulating 1,25 (OH)2 vitamin D3 (calcitriol) Moreover, a

dose–response of VA on craniofacial deformities, condition

factor and vertebral morphometry and mechanical strength

was observed Vertebral deformities were observed after

28 weeks on a standard diet and not immediately after the

116 days on the experimental diet Elevated VA is a risk

factor for bone deformities, and the dietary intake of VA

should not exceed 37 mg ROL kg 1 body weight day 1 in

Atlantic salmon postsmolts

KEY WORDS: Atlantic salmon, benchmark dose,

Hypervitam-inosis A, vertebral deformities, vitamin D

Received 11 May 2012; accepted 11 October 2012

Correspondence: R Ørnsrud, National Institute of Nutrition and Seafood

Research, PO Box 2029 Nordnes, 5817 Bergen, Norway E-mail: Robin.

ornsrud@nifes.no

Vitamin A (VA) is a generic term comprising all

sub-stances with the qualitatively similar biological activity of

retinol (ROL) Formally, the term includes those

provita-min A carotenoids that serve as precursors of ROL

because VA cannot be synthesized de novo by animalsand can only be formed from precursor compounds such

as b-carotene or astaxanthin Chemically, VA is ered a subgroup of the retinoids The term retinoidincludes naturally occurring forms of VA, its metabolitesand the many synthetic analogues of ROL, disregarding ifthe compound exhibits VA activity (IUPAC-IUBMB1992) Intracellularly, ROL is oxidized to retinal (RAL), aprosthetic group of opsin photopigments and essential forvision, and may undergo further oxidation to retinoic acid(RA) that performs all other (non-visual) functions of VA(Ross 2006) RA regulates transcription of several hun-dred genes by binding to nuclear transcription factors(Balmer & Blomhoff 2002; Blomhoff & Blomhoff 2006)and is crucial for reproduction, embryonic development,the immune response and cellular differentiation andgrowth

consid-The feed composition in salmon aquaculture of todaygenerally contains high lipid (300–400g kg 1

) and protein(350–550g kg 1) contents and low carbohydrate content(100g kg 1) (Einen 2001) Fish oil and fish meal are stillcommonly used as main ingredients in the commercial fishfeed, even though shortage of global marine resources hasopened for use of new feed ingredients, mainly of plantorigin which does not contain VA as ROL The VA level

in fish feed will therefore vary according to which ents that were used in the feed Fish feed containing VA-rich fish oil and fish meal may subsequently contain high

ingredi-VA levels Indeed, the average ingredi-VA concentration in fishfeed produced in Norway between 2003 and 2008 was

.

Aquaculture Nutrition

and reduced growth indicating that the maximum

accept-able VA level for first-feeding fry is well below this VA

concentration (Ørnsrud et al 2002) Grisdale-Helland et al

(1991) found no effect on growth although a slight

mortal-ity increase was indicated in Atlantic salmon first-feeding

fry fed 34 mg ROL kg 1 (~113 000 IU) for 30 weeks

Hilton (1983) suggested a maximum tolerable level of

271 mg ROL kg 1 (~903 000 IU) as retinyl palmitate in

rainbow trout juveniles (Oncorhynchus mykiss) after

16 weeks of feeding although increased activity of plasma

alkaline phosphatase and reduced iron stores in liver and

kidney were found already at 31 mg ROL kg 1

(~103 000 IU) In Japanese flounder (Paralichthys

olivac-eus), larvae fed different dietary ROL levels for 6 weeks,

VA inclusion above 15 mg ROL kg 1 (50 000 IU kg 1)

reduced growth and increased occurrence of vertebral

deformities (Dedi et al 1995) In Tilapia (Oreochromis

nil-oticus) fingerlings fed different dietary ROL levels for

18 weeks, 3 mg ROL kg 1 (10 000 IU kg 1) caused

slightly reduced weight gain, while 12 mg ROL kg 1

(40 000 IU kg 1) caused growth depression, abnormal

bone formation and fin necrosis, increased liver and spleen

size and mortality (Saleh et al 1995) Although most

stud-ies on VA toxicity in fish have focused on early life stages,

novel findings suggest that VA may affect bone metabolism

also in later life stages by interfering with the vitamin D

endocrine system and Ca homoeostasis (Ørnsrud et al

2009) Thus, the sensitivity to hypervitaminosis A seems to

be species and life stage dependent in fish, as has also been

shown for mammals (Nau et al 1994) So far, no studies

have been performed investigating the tolerance level of

VA for Atlantic salmon after transfer to sea water Thus,

the purpose of the trial was to establish the safe upper level

of VA intake for postsmolt Atlantic salmon

Eight diets with increasing inclusion of retinyl acetate(Rovimix A 500, DSM Nutritional Products, Basel, Switzer-land) were produced by EWOS Innovation, Dirdal, Norway,containing (in g kg 1) Norse LT-94 herring fish meal (280),NorSalmOil fish oil (203), wheat gluten (80), wheat grain(135) soy protein concentrate (300), vitamin premix (1) andmineral premix (1) The proximate composition, energy andretinol contents of experimental diets are given in Table 1

The trial was performed at the Institute of MarineResearch, Matredal, Western Norway (60°N′5°E,) Atlanticsalmon (S salar L.) 0+ postsmolts of the AquaGen strainwere distributed randomly into 16 fibreglass tanks(1.59 1.5 m) and fed eight levels of VA in duplicates for

116 days from 12 November 2007 to 3 March 2008 The

VA concentrations were 6, 12, 26, 55, 82, 112, 360 and

749 mg ROL kg 1 (Table 1) for diet numbers 1–8, tively Starting weight was approximately 135 g, and thefish were fed to apparent satiation using automated fishfeeders Feed intake per tank was measured by collectingfeed waste twice daily The fish were reared in sea water(34 g L 1, 8.9°C) under continuous light The O2 satura-tion of the outlet water was always above 80% Mortalitywas recorded on a daily basis

respec-At the end of the trial period, a total of 300 fish that were feddiet numbers 1, 5 and 8 (100 per diet group; 50 per tank) werefin-clipped to tank to preserve the duplicate structure of thetrial, transferred to one 1.59 5 m sea cage and given a stan-dard commercial diet for 28 weeks, until 17 September 2008

Table 1 Proximate composition, energy and retinol contents of experimental diets

.

Samplings were performed at 0, 41, 82 and 116 days of

feeding for all diet groups and 28 weeks after transfer to a

common net pen for diet groups 1, 5 and 8 Feed was

with-held for 2 days prior to each sampling point After

benzo-caine anaesthesia (40 mg L 1), tissue pools of liver, muscle

and plasma were made from five fish per tank Individual

spinal columns of five fish per tank were dissected, the

neu-ral and haemal arches were removed and the column

brushed free of adhering tissue All samples were

immedi-ately frozen on liquid N2 Vertebrae numbers 40–43 from

five fish per tank were dissected at 116 days of feeding and

used for morphometric measurements and mechanical

strength testing Individual body weights and fork lengths

were measured on 50 fish per replicate tank per sampling

point

Retinol was measured in diets, liver and muscle

homogen-ates using an HPLC procedure with UV detection as

described in Ørnsrud et al (2002) Protein was calculated

as N9 6.25 and total nitrogen measured on homogenized,

freeze-dried samples using a nitrogen analyser (LECO,

FP-428, St Joseph, MI, USA) Total lipid was measured

gravimetrically after ethyl acetate extraction Dry matter

and ashes were measured gravimetrically after freeze-drying

and incineration at 550°C, respectively Plasma total

pro-tein, albumin, aspartate transaminase (AST), alanine

trans-aminase (ALT), total Ca and total P were analysed in a

diagnostic autoanalyzer with corresponding kits for the

analyte in question (Maxmat PL analyzer, Montpellier,

France) Plasma RA was analysed using LC/MS/MS as

described in Gundersen et al (2007) Plasma

1,25-di-hydroxyvitamin D3, calcitriol (CTR), was analysed using

the LC MS-MS as described in Fjelldal et al (2009) Cyp26

gene expression was measured as described in Ørnsrud

et al (2009) with elongation factor 1A (EF1AA) as

refer-ence gene (Olsvik et al 2005) Briefly, the Q-PCR primers

for cyp26 were GAG GAC TCG TCG CGT TTT AAC T

(Forward) and TTG GCG AAC TCT TTC CCT ACA

(Reverse), while the Q-PCR primers for EF1AAwere CCC

CTC CAG GAC GTT TAC AAA (Forward) and CAC

ACG GCC CAC AGG TAC A (Reverse) All samples

were run in triplicates with accompanying no template

controls and no activity controls All qPCR products

showed a single melting curve indicating that no primer

dimer formation occurred

Vertebral columns from 20 fish per tank were dissected attermination of the tank sea-cage periods, radiographed(Fjelldal et al 2009) and evaluated for vertebral deformities

at 116 days of feeding and after 28 weeks in a common netpen for diet groups 1, 5 and 8 Individuals with one or moredeformed vertebrae were classified as deformed Com-pressed vertebrae with normal intervertebral spaces wereclassified as vertebral compression, and compressed verte-brae without intervertebral spaces were classified as verte-bral ankylosis and compression as described in Fjelldal

et al (2007) Using the classification system established byWitten et al (2009), the compressed phenotypes found inthis study included type 2, 3, 4, 5 and 13 deformities, whiledeformities classified as ankylosis and compression includedtype 6, 7 and 8 deformities Vertebrae numbers 40–43 weredissected out from five fish per tank at termination of theperiod on different experimental diets These vertebrae hadtheir length, and dorsoventral and lateral diameters mea-sured by a slide calliper Further, their mechanical strengthwas tested using a compression jig (Fjelldal et al 2009) andthe modulus of elasticity calculated according to Hamilton

et al.(1981) Fish with snout, jaw or operculum deformitieswere classified as craniofacial deformities (Ørnsrud et al.2004) and evaluated on 65 fish per tank at the end of thefeeding period and 50 fish per tank group (100 per dietgroup) at the termination of the sea-cage period Alkalinephosphatase (ALP) and tartrate-resistant acid phosphatase(TRACP) activity was measured in spinal column homogen-ates from five fish per tank at the end of the feeding period

as described in Fjelldal et al (2012b)

Condition factor (CF) and specific growth rate (SGR) werecalculated as described in Gil Martens et al (2012), and feedconversion efficiency (FCR) and hepatosomatic index (HSI)were calculated as described in Lock et al (2011) ROL dosewas calculated as mg ROL per kg body weight (b.w.) per dayusing tank feed intake and biomass per tank Bone deformityincidence was scored as presence of deformities divided bytotal number of fish examined The location of deformitywas recorded by dividing the vertebral column into fourmain regions (R) based on Kacem et al (1998): R1 (cranialtrunk), comprising vertebrae (V) V1-V8; R2 (caudal trunk),comprising vertebrae V9-V30; R3 (tail), comprising V31-V49and R4 (tail fin), comprising V50-V58 Individuals with one

or more deformed vertebrae were classified as deformed

Severity of deformity (%) was scored as number of deformed

vertebrae divided by total number of vertebrae per fish

Data were submitted to linear and polynomial regression

analyses using log (x + 1) of ROL intake (mg ROL kg 1

body weight day 1) as the abscissa and any given parameter

as the ordinate Two models were suitable for the data set: a

linear regression model (y= B1x + B0) and a quadratic

model (y= B0 + B1x + B2x2

) where y= physiologicalresponse, x= ROL intake, B1 and B2 = regression coeffi-

cients and B0= intercept on response (Y) axis Linear dose–

response regression curves were considered applicable when

the slope was significantly different from zero (P< 0.05) and

curvilinear dose–response curves when goodness of fit >0.75

Deformity occurrence was compared using a contingency

table analysis followed by the chi-square test Growth data

(weight, CF and SGR) after 28 weeks in a common net pen

for diet groups 1, 5 and 8 were subjected to nested ANOVA

with 40 fish nested in each replicate Levene’s test was used

to assess homogeneity of variance, and Tukey’s unequal n

post hoc test was used to test for significant differences

between groups Gene expression data for cyp26 were

analy-sed using one-way ANOVA The benchmark dose (BMD)

approach was used to derive guidance values for the upper

level of intake of VA In the BMD approach, the dose that

causes a low but measurable effect on any parameter

consid-ered as an adverse effect is termed the BMD (Fig 1) By

cal-culating the lower confidence limit of that dose (BMDL), the

uncertainty and variability of the data are taken into

account Dose–response modelling was performed using the

US EPA BMDS 2.2 software (version 2.2.0.67, available for

free at the US Environmental Protection Agency website:

http://www.epa.gov/ncea/bmds/index.html) at default

set-ting with a benchmark response of 10% and the associated

BMDL with 95% one-sided confidence level No

model-spe-cific restrictions were applied in the analysis, and the most

suited model was chosen based on best fit achieved by

maxi-mizing the log-likelihood (EFSA 2009)

The fish grew from approximately 136 g at trial start to

approximately 468 g (overall mean) after 116 days of

feed-ing After 28 weeks in a common net pen for diet groups

number 1, 5 and 8, the final weights were 2.2, 2.2 and 1.8 kg,

SGR was 0.78, 0.76 and 0.72, and the CF was 1.31, 1.30 and1.26, respectively The group that had received diet 8 differedsignificantly from diet groups 1 and 5 in terms of final weightand from diet group 1 in terms of CF (P< 0.05, nestedANO-

VA) There was a dose–response of ROL on the SGR (Fig 2)

at 116 days of feeding where SGR decreased linearlywith increasing dietary ROL (y= 0.1x + 0.9, r2=0.46,

P= 0.004) There was no effect on FCR, although the fishreceiving diet 8 exhibited a slightly higher FCR at the end ofthe feeding period (Table 2) There was a dose–response ofROL on the CF, where CF decreased in a curvilinear fashionwith increasing dietary ROL throughout the trial (Table 2)

The best-suited dose–response model was the quadraticregression model where the dose–response for CF was

y= 0.1x2+ 0.1x + 1.15 (r2=0.86), y = 0.07x2+ 0.06x + 1.21(r2=0.89) and y = 0.03x2+ 0.02x + 1.22 (r2=0.85) for days 41,

82 and 116 of feeding, respectively Mortality was low, mately 0.2% for the whole population (eight of 3600 fish), andthere were no mortalities related to experimental treatment

approxi-There was a dose–response of ROL in liver and muscle

to dietary VA inclusion level (Table 3) Liver ROL at

Figure 1 Illustration of key concepts for the benchmark dose

response curve determines the BMD (point estimate), which is the dose that gives a low but detectable change in response, denoted

as the benchmark response (BMR) The dashed curves represent the upper and lower 95% confidence bounds (one sided) Their intersections with the BMR (horizontal line) are at the lower (BMDL) and upper (BMDU) bounds of the BMD (Figure taken

.

the end of the feeding period ranged from 166 to

5570lg g 1 in the groups given lowest and highest VA

inclusion levels, respectively (Table 3) The groups

receiving diets number 1 and 2 decreased their liver

ROL concentration during the feeding period (Table 3)

Muscle ROL at the end of the feeding period ranged

100-fold from 0.2 to 19.4lg g 1

in the groups givenlowest and highest VA inclusion levels (Table 3) There

was no effect of diet on plasma RA at any sampling

points and the concentration ranged between 3 and

18 ng mL 1 (Table 3) There was an effect of diet on

plasma ROL, the concentration of ROL was elevated in

groups receiving diets number 7 and 8 at all sampling

points and the concentration ranged between 121 and

1440 ng mL 1 (Table 3) Liver mRNA expression of the

RA catabolizing enzyme CYP26 at the end of

the feeding period was >60-fold higher (P = 0.0002) in

the group receiving diet number 8 compared to the

other diet groups (Table 3)

Dietary VA decreased liver/body weight ratio (HSI)

HSI decreased linearly with increasing dietary VA

(y= 0.1x + 1.1, r2=0.45, P = 0.004) at 82 days of feeding,

but not significantly at 116 days of feeding (P= 0.08)

Val-ues for the plasma clinical analyses total protein, albumin,

ASAT and ALAT showed no dose–response and did not

indicate liver damage (Table 4)

Figure 2 Dose–response curve showing a linear relationship

spe-cific growth rate (SGR) as the ordinate after 116 days of feeding

eight diets with increasing levels of ROL to Atlantic salmon

(Salmo salar) postsmolt.

Dietary VA decreased plasma calcitriol (Table 5) At

116 days of feeding, calcitriol decreased linearly withincreasing dietary VA (y= 0.3x + 1.4, r2=0.59,

P= 0.0005) There was no effect of dietary VA on the centration of circulating 25 (OH)2 D3 Plasma P increasedlinearly with dietary VA at 41 days of feeding(y= 0.2x + 2.3, r2= 0.42, P = 0.006), while plasma Caincreased linearly with dietary VA both at 41(y= 0.2x + 2.6, r2= 0.25, P = 0.049) and 116 days of feed-ing (y= 0.2x + 2.6, r2=0.44, P = 0.005)

con-Bone ALP increased with dietary VA at 116 days of feeding(y= 0.01x + 0.08, r2=0.20, P < 0.0001, Table 6) There was

no effect of dietary VA on bone TRACP activity but theALP/TRACP ratio, an indicator of bone formation, showedelevated values for the group receiving diet 8 (Table 6) Bonemorphology was affected by dietary VA The ratio betweenlateral and dorsoventral diameter decreased linearly withincreasing dietary VA (y= 0.01 + 1.0, r2=0.60, P = 0.0004,Table 6) There was no effect of dietary VA on the ratiobetween length and dorsoventral diameter The modulus ofelasticity of the vertebral bodies increased linearly with dietary

VA at 116 days of feeding (y= 0.0009x + 0.03, r2=0.34,

P= 0.018, Table 6) There was a dose–response on cial deformities with a curvilinear increase with increasingROL dose (y= 20x2

craniofa-32x+ 13, r2=0.93) at 116 days of ing; craniofacial deformities increased approximately fourfoldand 10-fold for the two highest dietary inclusion levels of VAcompared to the remaining groups (Table 6) There was nodose–response for the incidence or severity of spinal deformi-ties at 116 days of feeding (Table 6) At 44 weeks after com-mencement of feeding, diet groups number 1, 5 and 8 weresampled for spinal deformities Fish that had received dietnumber 8 had an approximately 2.5-fold increase in the inci-dence of spinal deformities (diet number 1: 26%, diet number5: 28%, diet number 8: 63%), caused by deformities in thepostcranial region of the vertebral column (region 1, Table 7).The most common deformity in this region in fish that hadreceived diet number 8 was a fusion between the most anteriorvertebrae and the occipital bone of the skull (Fig 3) No cra-niofacial deformities were observed at 44 weeks after com-mencement of feeding

feed-Craniofacial deformities were chosen as the critical point and subjected to BMD analysis The analysis resulted

in a BMD of 52 mg ROL kg 1 b.w day 1and an

associ-ated BMDL (with 95% one-sided confidence level) of

37 mg ROL kg 1b.w day 1 In this study, this dose

corre-sponded to a feed concentration of 183 mg ROL kg 1feed

fed for a duration of 116 days

Hypervitaminosis A may be of concern in salmon

aquacul-ture due to the varying and sometimes high concentrations

of VA in fish meal and fish oil commonly included in

for-mulated diets Although acute toxicity resulting in lethal

effects is probably not relevant in practical fish farming,

sublethal adverse effects as a consequence of long-term

exposure to high concentrations may be of importance in

salmon aquaculture In the present study, chronic high VA

intakes led to adverse effects on growth and bone health in

Atlantic salmon postsmolts although liver functionality and

markers of VA homoeostasis were not negatively affected

by high dietary VA intake

Bone has long been established as a tissue sensitive to

toxic effects of VA in fish Several studies have been

performed on marine larvae exposed to VA resulting in

vertebral compression and malformed jaw and fin bone

structures (Dedi et al 1995, 1997; Takeuchi et al 1995,

1998; Martinez et al 2007), particularly in flatfish

(Fernandez & Gisbert 2011) Similarly, first-feeding fry of

Atlantic salmon fed chronic high concentrations of ROL

showed abnormal vertebral growth (Ørnsrud et al 2002)

Although the underlying mechanism of VA toxicity

through disruption of expression of central genes involved

in early life stage bone formation has been described

(Laue et al 2008; Spoorendonk et al 2008; Fernandez

et al 2011), our understanding of the mechanism at which

VA interferes with bone growth in later life stages has notbeen elucidated so far

Bone is a dynamic tissue that undergoes constant elling through bone formation and bone resorption.Osteoblasts are responsible for bone formation throughproduction of a proteinaceous extracellular matrix (ECM),mainly consisting of collagen but also non-collagenous pro-teins such as osteocalcin (BGP) and matrix gla protein(MGP) (Krossøy et al 2009) The ECM mineralizes bybinding of hydroxyapatite crystals to collagen fibres, in aprocess regulated by the osteoblast, and involves alkalinephosphatase (ALP), a commonly used marker for osteo-blast activity Osteoclasts perform bone resorption by dis-solving both minerals and the ECM (Witten & Huysseune2009), and tartrate-resistant acid phosphatase (TRACP) is

remod-a commonly used mremod-arker of osteoclremod-ast remod-activity In ourstudy, we used the ratio of ALP/TRACP activity as a mar-ker of whether the equilibrium of bone formation was in a

‘build up’ (favouring ALP activity) or resorption phase(favouring TRACP activity) Overall, TRACP activity didnot change in response to increasing ROL intake Anincrease in bone TRACP activity has been suggested as apossible reason for the deleterious effects of VA on bone(Rohde & Deluca 2003), but there was no indication of anincreased bone resorption in our study On the contrary,ALP activity increased at the highest dietary inclusion ofROL indicating an increase in osteoblast activity and/ormineralization of the vertebrae The increase in ratio ofALP/TRACP activity at the highest dietary inclusion level

of ROL augmented this assumption, the equilibrium ofbone formation was shifted towards more osteoblast activ-ity and mineralization Indeed, vertebral stiffness increasedwith increasing dietary ROL, possibly indicating anincrease in mineral content as was seen by Fjelldal et al.(2006) Contradicting results on the effects of RA on min-eralization have been reported Whereas increased minerali-zation has been reported in murine osteoblast andchondrocyte cell cultures (Wang & Kirsch 2002; Song et al.2005; Wan et al 2007), other studies have indicated adecrease in mineralization (Nuka et al 1997; Cohen-Tanugi

& Forest 1998; Iba et al 2001) In fish, information isscarce on the effect of RA on mineralization Hilton (1983)did not find any increase in concentration of Ca or P invertebrae However, a zebrafish (Danio rerio) knockout forcyp26b1, stocksteif, lacking the ability to degrade RA,showed overossification of developing centra with subse-quent fusion of vertebrae Furthermore, this phenotype was

Table 7 Occurrence (%) of fish with one or more deformed

increasing levels of retinol (ROL) for 116 days to Atlantic salmon

(Salmo salar) postsmolt The examined fish had been fed 6 (diet 1),

water transfer before being individually tagged and transferred to

a common net pen for 28 weeks

reproduced in wild-type zebrafish given excess RA or a

blocker of CYP26 activity showing that tight regulation of

intracellular RA concentrations is crucial to prevent

over-ossification (Laue et al 2008; Spoorendonk et al 2008) In

the present study, systemic RA concentrations were well

regulated at all dietary levels showing only a fourfold

vari-ation between 4 and 18 ng RA mL 1 in plasma

Further-more, hepatic cyp26 expression was only elevated at the

highest dietary inclusion level of ROL indicating that RA

homoeostasis was not compromised However, the

concen-tration of the circulating vitamin D metabolite calcitriol

was reduced as a consequence of increasing intake of ROL

(Table 5) Vitamin D (cholecalciferol) is biologically

inac-tive, and hydroxylation of this compound is required to

form 1,25 dihydroxyvitamin D3 (calcitriol, the active

metabolite of vitamin D) This is in line with previous

find-ings where intraperitoneal injection of RA led to a

three-fold reduction in the concentration of circulating calcitriol

(Ørnsrud et al 2009) The classic function of vitamin D is

to maintain calcium and phosphorus homoeostasis in

verte-brates This is achieved mainly through increasing the

intestinal absorption of these minerals although renal

conservation of Ca is also of importance (Lock et al

2010) Although reduced concentrations of calcitriol couldimpede the ability to absorb dietary minerals, the circulat-ing concentrations of Ca and P did not decrease in thepresent study and even increased at high dietary concentra-tions of ROL Similarly, Ørnsrud et al (2009) found anincrease in intestinal Ca uptake, a positive correlationbetween intestinal P and Ca uptake and an increase in ecacmRNA expression in the gill of fish injected with RAimplying that similar to calcitriol, VA increases mineralabsorption Although the antagonistic effect of VA on cal-citriol did not lead to impaired plasma Ca or P, other neg-ative effects of reduced plasma calcitriol on bone arepossible Vitamin D has additional functions that do notinvolve maintenance of Ca and P concentrations, amongthem ECM formation (Van Leeuwen et al 2001; Nagpal

et al.2005) Calcitriol is a ligand to the vitamin D receptorand modulates gene expression by binding to a vitamin

D–responsive element (VDRE) in the promoter region ofthe gene Genes involved in extracellular matrix formationthat are regulated by calcitriol include osteocalcin (Demay

et al 1989), osteopontin (Noda et al 1990), collagen(Pavlin et al 1994) and possibly also matrix gla protein(Ørnsrud et al 2009) Similarly, RA affects extracellular

Figure 3 Lateral radiographs of vertebral bodies numbers 1–5 at 28 weeks after cessation of feeding eight diets with increasing levels of

ret-inol (ROL) for 116 days to Atlantic salmon (Salmo salar) postsmolt (a) Individual with normal vertebrae (b) Individual with a fusion

between vertebra number 1 and the occipital bone of the skull, fusion and compression type deformity in vertebra number 1, compression

fusion between the occipital bone of the skull and vertebra number 1, fusion and compression type deformity in vertebra numbers 1 and 2

.

matrix formation both by inhibiting production of collagen

(Dickson & Walls 1985) and increasing degradation of

col-lagen (Connolly et al 1994; Varghese et al 1994) In

Atlantic salmon, a dual effect of RA on bone has been

indicated While RA inhibits matrix formation by

decreas-ing expression of col1a2, it also appears to activate genes

involved in matrix mineralization by increasing expression

of alp (Ørnsrud et al 2009) Whether the effect of RA on

ECM is exerted through direct effects or through its

antag-onistic effect on calcitriol is still an open question but may

open up new and intriguing avenues of research on the

cross-talk of RA with the vitamin D endocrine system in

Atlantic salmon

Alterations in gene expression of ECM proteins have

been associated with spinal deformities in salmon

(Warge-lius et al 2010b; Ytteborg et al 2010) In aquaculture,

spinal deformities are a fish welfare concern and also lead

to production losses due to quality downgrading of the

fish The most prevalent type is compression of the

verte-brae (Witten et al 2005), also referred to as platyspondylia,

in the caudal part of the vertebral column (Fjelldal et al

2009) However, many morphological changes in the

verte-bral column have been described in Atlantic salmon

(Witten et al 2009) Changes in vertebrae morphometrics

have been established as early markers of incipient

defor-mity (Fjelldal et al 2006, 2009) and have been associated

with a lower transcription of ECM genes (Ytteborg et al

2010) or degradation of ECM components (Wargelius et al

2010a), as well as low mineral content (Fjelldal et al 2007)

In the present study, the lateral/dorsoventral diameter of

the vertebra decreased with increasing dietary intake of

ROL, but not the length/dorsoventral diameter that has

earlier been used as a marker of early deformity (Fjelldal

et al.2006, 2009) It has been suggested that there is a

con-siderable time lag between induction and development of

vertebral deformities because deformities usually become

manifest in a late life stage (Witten et al 2005) although

they may have been established at a much earlier stage, for

example, during the egg incubation (Ytteborg et al 2010),

at the parr stage (Fjelldal et al 2012a) or at the smolt stage

(Grini et al 2011) In our study, fish were fed increasing

concentrations of ROL after sea water transfer and for

116 days followed by a radiological examination of spinal

deformities Subsequently, fish from selected VA diet

groups 1, 5 and 8 were transferred to a common sea cage

and were re-evaluated for spinal deformities 28 weeks later

For the group receiving the highest dietary level of ROL,

there was a twofold increase in the occurrence of spinal

deformities (Table 7), primarily in the cranial trunk region

(vertebra 1–8) Clearly, the deformity incidence and severitywere aggravated and showed that the condition continued

to develop after cessation of the VA feeding Interestingly,the spinal deformities were mainly located close to the skullarea although the tail region is normally a more typicallocation for spinal deformities that develop in sea water(Fjelldal et al 2009; Grini et al 2011) Furthermore,increasing dietary ROL led to a marked increase in cranio-facial deformities measured as malformed operculum andjaw deformities after 116 days of feeding Craniofacialdeformities as a result of VA toxicity have been reportedfor marine fish species, primarily during the larval periodwhere the fish undergoes physiologic and morphologicalterations ultimately leading to an adult phenotype.Villeneuve et al (2005) fed retinyl acetate at 12, 31, 62 and

196 mg ROL kg 1 diet to European sea bass chus labrax) larvae and found a linear dose–response rela-tionship between dietary ROL and malformation incidence,manifested as vertebral, neurocranial, operculum and jawdeformities Lower jaw defects have similarly been found inlarval summer flounder (Paralichthys dentatus) (Martinez

(Dicentrar-et al 2007) and larval Japanese flounder (P olivaceus)(Suzuki et al 2000; Haga et al 2002, 2003) However,Ferna´ndez et al (2009) did not find jaw deformities inSenegalese sole (Solea senegalensis) fed with VA-enrichedArtemia although vertebral deformities were found andsuggested that the VA exposure was applied at a non-criti-cal developmental stage for jaw skeletogenesis (Fernandez

& Gisbert 2011) These studies suggest that VA is crucialfor proper development of skeletal structures at early stages

of development In the present study, the appearance ofjaw and operculum deformities in adult salmon after seawater transfer showed that jaw deformities can be inducedindependent of life stage in Atlantic salmon although thedose that elicits adverse effects may be higher at later lifestages Interestingly, the VA-induced craniofacial deformi-ties in the present study were mild and reversible; therewere no craniofacial deformities at termination of the study

28 weeks after cessation of feeding the experimental diets.The VA-induced deformities in the vertebral column, onthe other hand, aggravated and got more severe over time.Still, these were mainly located at the junction between theneurocranium and the vertebral column, showing that VAseems to affect the skull and vertebral column differently

At present, a rationale for this phenomenon is difficult toconstruct

The liver is a central storage organ for VA where retinylesters are stored in perisinusoidal stellate cells VA storingstellate cells are not only found in liver but also in the

intestine, kidney, ovaries, testes and gills of fish (Blomhoff

& Wake 1991) In the present study, liver retinol

concentra-tions increased with dietary inclusion of VA at all time

points (Table 3) showing that salmon has a high storage

capacity for VA similar to other fish species such as

sun-shine bass (Hemre et al 2004), carp (Aoe et al 1968), red

sea bream (Hernandez et al 2004) and rainbow trout

(Hilton 1983; Fontagne´-Dicharry et al 2010; Gesto et al

2012) Additionally, VA also accumulated in muscle in the

present study because ROL concentrations were elevated at

the two highest dietary inclusion levels of ROL, probably

as a result of costorage with lipids Similarly, the

circulat-ing concentration of retinol was stable up to the two

high-est dietary inclusion levels of ROL Plasma ROL represents

a combination of RBP-bound ROL and lipoprotein-bound

ROL It has been suggested that toxic manifestations of

VA may occur when plasma ROL increases and is

non-specifically delivered by lipoproteins to membranes (Mallia

et al 1975) Liver damage resulting from hypervitaminosis

A may be caused by activation of hepatic stellate cells

resulting in a myofibroblast-like phenotype with subsequent

production of excessive extracellular matrix (Nollevaux

et al 2006) In fish, pale yellow and fragile livers and a

reduced hepatosomatic index have been reported as a result

of hypervitaminosis A (Hilton 1983) In the present study,

reduced hepatosomatic index was found at 82 days of

feed-ing, primarily at the highest VA concentration, but there

was no sign of discoloration or increased fragility of the

liver Furthermore, clinical markers of liver damage

mea-sured as plasma concentration of total protein, albumin

and activities of ASAT and ALAT (Table 4) showed no

indication of liver malfunction and were within normal

range (Waagbø et al 1988) Although postsmolt Atlantic

salmon seemed resilient to excessive dietary VA in the

pres-ent study, the reduced growth and increase in bone

malfor-mation with increasing VA intake should be considered

adverse effects When a nutrient has the potential to exert

both health benefits and health risks, it is important to

establish an upper safe level of intake of that nutrient The

BMD approach is used to characterize toxicity risk and is

the exposure level that corresponds to a specific increase in

the probability of an adverse response (EFSA 2009) Using

the experimental data to model a dose–response

relation-ship for any given endpoint, low but detectable increases in

an adverse response can be used to determine the dose

threshold for a toxic effect (Fig 1) The uncertainty in the

data is reflected in the confidence interval and the BMD

lower confidence limit (BMDL) is by default used as a

ref-erence point for guidance values, for example, a tolerable

upper level of intake for VA The critical endpoint used inthe present study was craniofacial deformity although alldata were subjected to BMD modelling Different dose–response models may fit the data equally well but result indifferent BMD/BMDLs, reflecting model uncertainty Sev-eral models were accepted for the craniofacial deformitydata set with an associated BMDL range of 37–

40 mg ROL kg 1 b.w day 1 The lowest BMDL value,that is, the most conservative BMDL of this study, waschosen as a reference point for the safe upper level ofintake of VA In the present study, this dose corresponded

to a feed concentration of 183 mg ROL kg 1 feed fed forduration of 116 days Considering the range of VA concen-trations in feed for Atlantic salmon (Ma˚ge et al 2010) andthe increasing use of plant feed ingredients containing no

VA, farmed salmon does not seem to risk ingesting sive amounts of VA in aquaculture today

exces-This work was supported by the Norwegian ResearchCouncil (project no 153472) We would like to thank AnneKarin Syversen, Eva Mykkeltvedt, Torill Berg and LeiknyFjeldstad at NIFES and Arnor Gullanger and Ivar HelgeMatre at the IMR for their expert technical assistance

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.

1 1 1 2 1 2

1

Fishery College, Huazhong Agricultural University, Wuhan, China; 2 Postgraduate Research Base, Panjin Guanghe Fishery

Co Ltd., Panjin, China;

Up to date, the carnitine palmitoyltransferase (CPT) system

in fish nutrition receives little attention The present study

compared CPT I kinetic behaviour of Synechogobius hasta

(carnivorous) and Ctenopharyngodon idella (herbivorous)

The optimal conditions (temperature, incubation time,

mito-chondrial protein concentration and pH) for maximum CPT

I activity showed no significant difference between S hasta

and C idella CPT I activities in S hasta were significantly

higher than those in C idella Affinity constants (Km) for

carnitine in liver, heart, white muscle and spleen of S hasta

were significantly higher than those in C idella Km for

palmitoyl-CoA in liver and heart of S hasta were

signifi-cantly higher than those in C idella Vmaxfor carnitine and

palmitoyl-CoA in S.hasta tended to be higher than those in

C idella Catalytic efficiencies (Vmax/Km) for carnitine in

liver, white muscle and spleen of C idella were significantly

higher than those in S hasta Vmax/Kmvalues for

palmitoyl-CoA in liver and heart of C idella were higher than those in

S hasta Our study demonstrated that the lower catalytic

efficiency for carnitine in liver of S hasta indicated that the

fish showed a low capacity for energy generation through

b-oxidation of long-chain fatty acids, which easily caused

fatty liver syndrome This is the first study in which, by using

carnitine and palmitoyl-CoA as the substrates, the complete

kinetic characterization of CPT I in fish has been described,

which increases our knowledge about lipid metabolism and

its critical role in lipid utilization in fish

KEY WORDS: carnitine palmitoyltransferase I,

Ctenopharyng-odon idella, enzymatic kinetic behaviour, nutrient

prefer-ence, Synechogobius hasta

Received 19 June 2012; accepted 4 October 2012 Correspondence: Z Luo, Fishery College, Huazhong Agricultural University, Wuhan 430070, China E-mail: luozhi99@yahoo.com.cn.

At present, Z Luo is acting as a visiting scholar in University of St Andrews, KY16 8LB, UK.

*J.-L Zheng and W Hu contributed equally to the work.

Carnitine palmitoyltransferases I and II (CPT I and CPTII), together with the acyl-carnitine translocator, mediatethe transfer of acyl groups into mitochondria (McGarry &Brown 1997) and play a major role in the regulation ofmitochondrial b-oxidation in all vertebrates, including fish.CPT I (EC 2.3.1.21), located in outer membranes of mito-chondria and frequently described as the ‘rate-limitingenzyme’ of b-oxidation flux, catalyses the carnitine-depen-dent esterification of palmitoyl-CoA to form palmitoylcar-nitine (Zammit et al 1997; Kerner & Hoppel 2000) CPT

II, located on the inner mitochondrial membrane, catalyses

a second esterification, generating palmitoyl-CoA and nitine inside the mitochondrial matrix (McGarry & Foster1980) In vertebrates, CPT I has a key function controllingthe flux through b-oxidation and therefore is the mainregulatory step of fatty acid oxidation (Morash et al.2008)

car-Estimating the enzymatic properties and kinetic stants is one of the most critical parts of the studies related

con-to enzyme-catalysed reactions Elucidation of the kineticmechanism of CPT I will increase our knowledge about thephysiology of lipid metabolism and its critical role in lipidutilization At present, studies regarding the kinetic charac-teristics of CPT I were reported only in several mammals,such as rats (McGarry & Brown 1997; Fraser et al 2001),

.

Aquaculture Nutrition

pigs (Nicot et al 2001; Peffer et al 2007), dogs (Lin &

Odle 2003) and felids (Lin et al 2005), and lower

verte-brates (lamprey) (Stonell et al 1997) However, the kinetic

characteristics of CPT I in fish remain unknown Given

the taxonomic differences between teleosts and mammals,

then it is highly likely that the kinetic characteristics of

CPT I in fish are different; however, we do not know

this

Synechogobius hasta, a species of carnivorous fish, is

widely distributed over the southern coast of Liaoning

Province, China, and considered to be the most potential

candidate for aquaculture in China owing for their good

taste and high market value (Liu et al 2010) Normally,

for S hasta, their food contains high lipid content (low

carbohydrate) (Luo et al 2008) Ctenopharyngodon idella

(its common name: grass carp) are herbivorous and

consti-tute the largest aquaculture industry in China

Ctenophar-yngodon idellaexhibits lower capacity to utilize high dietary

lipid, and the carbohydrate content of commercial diets for

the fish species remains relatively high

At present, the precise explanations for the differences in

lipid utilization between the fish with different feeding

hab-its are not fully understood, and the property and kinetic

behaviour of CPT I in various fishes are not well described

in the literature In mammals, reports have shown that

changes in CPT activity and kinetics may be affected by

changes in dietary energy supply (dietary fat versus

carbo-hydrate energy sources) (Thumelin et al 1994; Lin & Odle

2003) However, little is known about these mechanisms in

non-mammalian vertebrates and about the responsiveness

of CPT to changes in dietary energy supply (fat versus

car-bohydrate) Thus, a study was conducted to compare the

properties and kinetic behaviour of CPT I in various

tis-sues between the two fish species, S hasta and C idella,

related to different nutrient preference Due to the role of

CPT I in lipid utilization, the fundamental knowledge

gained from our findings was imperative to understanding

lipid metabolism in fish

All chemicals were of analytical grade and purchased either

from Sigma Chemical, St Louis, MO, USA, or Amresco

Inc., Cleveland OH, USA, with the exception of

palmitoyl-CoA (purchased from Larodan Fine Chemicals, Malmo,

Sweden) This study was approved by Huazhong Agricultural

University Animal Care and Use Committee and had been

carried out in accordance with EU Directive 2010/63/EU for

animal experiments

The present study was conducted in an indoor static ium system of Panjin Guanghe Fisheries Co Ltd, Panjin,China Sixty S hasta (initial body weight: 35.83± 3.79 g)were obtained from a local pond (Panjin, China) and kept inthree, 300-L circular fibreglass tanks for 2-weeks acclimatiza-tion, with 20 fish for each tank During the acclimatizationperiod, they were fed trash fish to satiation twice a day withcontinuous aeration to maintain the dissolved oxygen levelnear saturation Water in each tank was replenished 100%

aquar-twice daily, before feeding Care was taken to ensure thatuneaten feed remained in the tanks during feeding Faecalmatter was also quickly removed during the experiment

Sixty grass carp C idella (initial body weight: 500 ± 15 g)were obtained from a local farmer They were maintained inthree, 300-L circular fibreglass tanks for 2-weeks acclimatiza-tion, with 20 fish for each tank During the acclimatizationperiod, they were provided commercial diets (high carbohy-drate and low lipid) to satiation twice a day and continu-ous aeration to maintain the dissolved oxygen level nearsaturation Dechlorinated tap water in each tank wasreplenished 100% twice daily, before feeding Care wastaken to ensure that no eaten feed remained in the tanksduring feeding Faecal matter was also quickly removedduring the experiment

The experiment was conducted at ambient temperatureand subjected to natural photoperiod (approximately 14-hlight/10-h dark) Water quality parameters were monitoredtwice a week in the morning Water temperature rangedfrom 18.2 to 20.4°C; dissolved oxygen 6.0 mg L 1; totalammonia-nitrogen 0.046–0.065 mg L 1

At the end of the 2-weeks experiment, fish were starved for

24 h before sampling Then, nine fish from each tank wereeuthanized by severing of the spinal cord Whole liver, heart,white muscle, spleen and intestine were isolated immediatelyusing sterile forceps in ice, frozen in liquid nitrogen andstored at 80°C (not longer than 2 weeks) for the subse-quent analysis Crude lipid was determined by the ether-extraction and expressed as g crude lipid kg 1dry weight

For extraction of mitochondria and the determination ofmitochondrial protein, mitochondria were isolated fromliver, heart, white muscle, spleen and intestine according toSuarez & Hochachka (1981) with modifications by Morash

et al (2008) Briefly, each tissue was immediately excised

Tissues were placed in chilled mitochondrial isolation

.

buffer (MIB) consisting of (in mM) 140 KCl, 10 EDTA, 5

MgCl2, 20 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic

acid (HEPES) and 0.5% bovine serum albumin (BSA) (pH

7.0) for white muscle, heart, intestine and spleen, and 250

sucrose, 1 EDTA, 20 HEPES and 0.5% BSA (pH 7.4) for

liver The tissue samples were minced with scissors, washed

twice with fresh chilled MIB and then homogenized on ice

with a Teflon pestle Homogenates were centrifuged at

800 g for 10 min at 4°C The supernatant was centrifuged

at 9000 g for 10 min at 4°C to obtain the mitochondrial

pellet The latter were re-suspended in a small volume of

the appropriate MIB lacking BSA The re-suspended

homogenate was collected into a 15-mL centrifuge tube

and centrifuged again at 9000 g for 10 min at 4°C The

mitochondrial pellet was re-suspended in an appropriate

volume of MIB lacking BSA The mitochondrial

suspen-sion was stored on ice before the CPT I assay

Mitochon-drial protein content was measured according to Bradford

(1976), using BSA as the standard

Before starting the kinetic assays, the effects of protein

concentration (0–250 lg protein mL 1

), incubation time(0–45 min), pH (4–10) and incubation temperature

(12–40 °C) on CPT I activity were analysed Based on these

analyses, the optimal conditions for protein concentration,

incubation time, pH and incubation temperature for

maxi-mum CPT I activity were obtained

Carnitine palmitoyltransferase I activity was analysed

using the method of Bieber & Fiol (1986), based on

mea-surement of the initial CoA-SH formation by the 5,

5′-di-thio-bis-(2-nitrobenzoic acid) (DTNB) reaction from

palmitoyl-CoA by mitochondria samples withL-carnitine at

412 nm Briefly, 50lL buffer solution (containing 116 mM

Tris, 2.5 mM EDTA, 2 mM DTNB, 0.2% Triton X–100,

pH 8.0) and 50 lL mitochondria suspension (0.5–1 mg

mitochondrial protein per 50lL) were added to four

semi-microcuvettes After 5-min pre-incubation at 25°C, 50 lL

palmitoyl-CoA (1 mM dissolved in double distilled water)

was added to three cuvettes The fourth cuvette was used

as a blank, adding 50lL water instead of palmitoyl-CoA

The reaction was then started by adding 5 lL carnitine

solution (1.2 mM dissolved in 1 M Tris, pH 8.0),

immedi-ately followed by photometric measurement at 412 nm at

25 °C for 3 min One unit of enzyme activity is defined as

1lmol of product formed per min at 25 °C

According to McGarry et al (1983), only CPT I was

sensitive to malonyl-CoA inhibition in vertebrates

There-fore, CPT I activity was recorded at increasing CoA concentrations until minimum activity was obtainedaccording to the procedure from the study by Morash

malonyl-et al (2008) Residual activity was ascribed to CPT IIisoform and subtracted from the total CPT activity(obtained in fractions not incubated with malonyl-CoA) so as to obtain CPT I activity According tothe preliminary experiment, we determined that 50lMmalonyl-CoA was sufficient for the complete inhibition

of CPT I activity in different tissues of S hasta and

C idella

For the kinetic studies, the ranges of substrate trations for carnitine were from 0.5 to 10 mM, and forpalmitoyl-CoA from 0.02 to 0.6 mM The enzymatic reac-tion was initiated by adding palmitoyl-CoA (100lM) andcarnitine (400 mM) to generate palmitoylcarnitine andincubated at 25°C (Hamdan et al 2001) Analysis of thekinetic data was performed as described by Hofstee(1952) The values of Michaelis–Menten constants (Km)and maximal reaction rates (Vmax) were analysed using anon-linear regression method described by the Michaelis–Menten equation Catalytic efficiency, defined as anenzyme’s efficiency in transforming its substrate, was cal-culated by the ratio between maximum enzyme activityand Km (Vmax/Km) This parameter related total enzymeconcentration to the interaction between the enzyme andthe substrate All measurements were taken in duplicate.Lineweaver–Burk graphs (Lineweaver & Burk 1934) weredrawn using 1/reaction velocity (v) versus 1/[substrate con-centration] ([S]) values The Arrhenius plots based on thetemperature dependence of initial velocity (v0) for CPT Ifrom liver of S hasta and C idella at saturating substrateconcentrations were also assayed to define ‘transition tem-perature’

concen-Results were presented as mean± standard error of mean(SEM) Prior to statistical analysis, all data were tested fornormality of distribution using the Kolmogornov–Smirnovtest The homogeneity of variances among the different tis-sues with the same fish species was tested using theBarlett’s test Then, the data were subjected to one-wayANOVAand Duncan’s multiple range test For analysing thedifferences of CPT I activity from the tissue with the twodifferent species, the data were subjected to independentsamples t-test Difference was considered significant at

P< 0.05 All statistical analyses were performed using theSPSS10.0 for Windows (SPSS, Chicago, IL, USA)

For S hasta, the mitochondrial protein contents in tissues

followed the order: heart >liver >intestine = spleen >white

muscle, whereas for C idella, the order was heart

>intes-tine = spleen >liver >heart (Table 1) Generally speaking,

mitochondrial protein contents in liver and white muscle of

S hasta were significantly higher than those in the

corre-sponding tissues of C idella However, for heart, spleen

and intestine, the opposite was true

For S hasta, lipid contents in several tested tissues

fol-lowed the order: liver >intestine >spleen = white muscle

>heart, whereas for C idella, the order was intestine >liver

>spleen> heart >white muscle (Table 1) Lipid content in

liver, white muscle and intestine of S hasta was

signifi-cantly higher than those in the corresponding tissues of

C idella, but lipid content in heart of C idella was higher

than that in S hasta No significant difference was found

in lipid content of spleen between S hasta and C idella

For S hasta, CPT I activities were the lowest in liver,

white muscle and spleen (21.0, 21.3 and 23.2 nmol

min 1mg prot 1, respectively) and the highest in heart and

intestine (41.6 and 34.0 nmol min 1mg prot 1,

respec-tively) (Table 2) For C idella, the highest and the lowest

CPT I activities were obtained in heart and white muscle

(31.4 and 6.4 nmol min 1mg prot 1), respectively For

S hasta and C idella, no correlations were found between

lipid contents and CPT I activities of the tested tissues

CPT I activities in tissues of S hasta were higher than

those in C idella For C idella, CPT I activities were

posi-tively correlated with mitochondrial protein concentration

(P < 0.001), whereas in S hasta, the correlation was not

significant (P> 0.05) (Fig 1) Compared to white muscle,spleen and intestine of S hasta, the liver contained highmitochondrial protein per gram wet weight, but hepaticCPT I activity was relatively low

As shown in Fig 2, the hepatic CPT I optimal conditionswere as follows: for C idella, temperature 37°C, pH 7.4,mitochondrial protein concentration 120lg mL 1

andincubation time 5–20 min; for S hasta, temperature

34–40 °C, pH 7.4, mitochondrial protein concentration

120lg mL 1

and incubation time 5–25 min The optimalprotein, incubation time, temperature and pH for CPT Iactivity were not significantly different between the two fishspecies In addition, at each temperature, the values ofCPT I activity and initial velocity (v0) in S hasta werehigher than those in C idella Example of the Arrheniusplot based on the temperature dependence of v0for hepaticCPT I of S hasta and C idella was showed in Fig 3,where the transition temperature was 30°C for S hastaand 25°C for C idella

Using the optimal conditions described above, the thesis of palmitoyl-carnitine was then assayed as a function

syn-of both substrates: palmitoyl-CoA and L-carnitine Thereaction followed a normal Michaelis–Menten kinetics inthe substrate concentration range as shown in the satura-tion plots (Fig 4) and Lineweaver–Burk plots (Fig 5) Thekinetic parameters obtained by Lineweaver–Burk plotswere showed in Table 3 For S hasta, the Kmfor carnitine

in tissues followed the order: white muscle

>liver = heart = spleen = intestine while Vmax for carnitinefollowed the order: heart >intestine >white muscle >spleen

>liver The Km values for palmitoyl-CoA in different

Table 1 Mitochondrial protein content (mg protein per g wet weight) and crude lipid contents (g lipid per kg dry weight) in different tissues

of Synechogobius hasta and Ctenopharyngodon idella

.

tissues followed the order: liver >spleen = intestine >heart

>white muscle while Vmax for palmitoyl-CoA was heart

>intestine = liver >spleen >white muscle The catalytic

effi-ciency for carnitine was higher in heart and lower in liver,

white muscle and spleen Catalytic efficiency for

palmitoyl-CoA was higher in heart and lower in liver

For grass carp C idella, Km for carnitine was the

great-est in intgreat-estine and the lowgreat-est in white muscle, and no

sig-nificant differences were observed among other tissues

Vmax for carnitine in different tissues followed the order:

heart >intestine = liver >spleen >white muscle, and Vmax

for palmitoyl-CoA in different tissues followed the order:

heart >intestine = liver = spleen >white muscle The lowest

Km for palmitoyl-CoA occurred in heart, while the highest

was observed in white muscle Catalytic efficiency for

carnitine was higher in heart and lower in intestine,

whereas higher and lower catalytic efficiency for

palmitoyl-CoA was observed in heart and white muscle, respectively

When compared between the two fish species, Kmvaluesfor carnitine in liver, heart, white muscle and spleen of

S hasta were significantly higher than those in their terparts of grass carp C idella, whereas in the intestine, theopposite was true Km values for palmitoyl-CoA in liverand heart of S hasta were significantly higher than those

coun-in C idella, whereas white muscle Kmvalues for CoA were significantly lower in S hasta than those in

palmitoyl-C idella Spleen and intestine Kmvalues for palmitoyl-CoAshowed no significant differences between the two fish spe-cies Vmaxvalues for carnitine in heart, white muscle, spleenand intestine of S hasta were significantly higher thanthose in C idella, whereas the contrary phenomenon wasobserved in liver Vmax values for palmitoyl-CoA in all thetested tissues in S hasta were all significantly higher thanthose in grass carp C idella Catalytic efficiency of carni-tine in liver, white muscle and spleen was lower in S hastathan those in grass carp C idella Catalytic efficiencies ofpalmitoyl-CoA in liver and heart of S hasta were signifi-cantly lower than those in C idella, whereas in white mus-cle, spleen and intestine, the catalytic efficiencies werehigher in S hasta than in C idella

In the present study, for the first time, we compared theproperties and the kinetic behaviour of the important cellu-lar lipolytic enzyme, CPT I, from several important tissues(liver, heart, white muscle, spleen and intestine) betweentwo different feeding teleosts, S hasta and C idella Ourstudy demonstrated significant differences between specieswith different feeding habits and CPT I kinetic characteris-tics, indicating a dependence of the metabolic enzyme activ-ity on food composition in fish

The present study indicated a positive correlationbetween mitochondrial protein contents and CPT Iactivities in several tested tissues of C idella, suggestingthat the differences in CPT I activities of all tested tissues

of C idella were mainly owing to the protein content on

Figure 1 Relationship between carnitine palmitoyltransferase I

activities and mitochondrial protein content in different tissues of

(hollow shape) Square, Rectangle, diamond Phombus, circle,

Tri-angle were corresponding to heart, intestine, spleen, liver and white

muscle, respectively A significantly positive correlation was

the outer mitochondrial membrane However, this was not

true in S hasta, indicating that other factors might affect

hepatic CPT I activities rather than mitochondrial protein

contents Brandt et al (1998) suggested that the difference

in the CPT I activity could be considered to be the result

of different mitochondrial contents and the CPT I gene

expression No correlations were found between lipid

con-tents and CPT I activities of the tested tissues in S hasta

and C idella, which was in agreement with the report by

Yotsumoto et al (2000)

The present study clearly showed that the optimal

condi-tions for maximum CPT activities between the two fish

spe-cies were similar For example, the optimal temperature for

the highest CPT I activity was 34–40 °C for S hasta and

37 °C for C idella, which was similar to mammals (Kashfi

& Cook 1995) and prawns (Lavarias et al 2009) However,

it was higher than that of Atlantic salmon (30°C,

Froy-land et al 1998) Generally speaking, CPT I catalysed the

reaction with increasing rate when temperature increased,

until thermal inactivation occurred In many studies, 37°C

had been used as the temperature for assays of CPT I

activity (Stonell et al 1997) However, in several other oratories, assays were performed at lower temperatures,such as at room temperature (~22 °C) by Morash et al

lab-(2009) Such an effect of temperature could account for thediscrepancies between CPT I activities obtained by differentlaboratories even under otherwise comparable conditions

The present study showed that the optimal pH for S hastaand C idella was 7.4, respectively The pH played animportant role in the maintenance of active site architec-ture According to Napal et al (2003), minor fluctuations

of pH may disrupt a hydrogen-bonding network or a saltbridge at the substrate binding site pocket of CPT I, neu-tralize positive charge on the carnitine substrate or the neg-ative charge on the palmitoyl-CoA, and destabilize theenzyme–substrate complex, thereby inactivating CPT I Inprawns, Lavarias et al (2009) reported that CPT I optimal

pH conditions were 8.0 With two non-linear Arrheniusplots, our study indicated that the transition temperaturewas 30°C for S hasta and 25 °C for C idella Both activ-ity and inhibition of CPT I were characterized by transitiontemperature, which was probably associated with a mem-

Figure 2 Effect of incubation time, temperature, protein concentration and pH on the activity of mitochondrial carnitine

.

brane lipid-phase transition (Raison 1973) Compared to

S hasta, the flatter v0 slopes below 25°C and steeper v0

slope above 30°C would be physiologically advantageous,

ensuring the maintenance of stable and relatively high

reaction rates into lower regions of hypothermia, which

could provide an equally rapid recovery of higher levels of

metabolism during rewarming under favourable conditions,

as suggested by Andjus et al (2002)

Until now, few data on CPT I activity in fish tissues were

available Our study indicated that CPT I activities in C

id-ella followed the order: heart >intestine = spleen = liver

>white muscle Similarly, Gutieres et al (2003) indicated

that CPT I activity in rainbow trout was heart

>intes-tine = liver >white muscle Morash et al (2008) suggested

that the maximal activity of CPT I measured in isolated

mitochondria followed a similar pattern of red muscle

>heart >white muscle >liver Synechogobius hasta heart also

expressed the highest CPT I activity: approximately two

times higher than in liver CPT I played a crucial role in

regulating cardiac fatty acid oxidation, and higher CPT I

activity provided the primary source of energy for cardiac

muscle contraction, and a chronic inhibition of CPT I

caused cardiac hepertrophy (Bressler et al 1989) For

S hasta, the difference in CPT I activities between liver and

white muscle was minor Liver and muscle were major

non-esterified fatty acid-utilizing tissues More than 50% of the

total body mass of S hasta was composed of muscle(Luo Z, Bai HJ and Xi WQ, unpublished data); thus,muscle made a significant contribution to the overall fattyacid oxidation capacity in the fish On the other hand, thespecific activities in all the tested tissues in S hasta appearedhigher than those in C idella This may be considered as anadaptation to the feeding habits between the two fish species

As lipid played a more important role in the food of S hastathan of C idella, we speculated that the increase in CPT Iactivity observed in S hasta might be the adaptation to die-tary high fat Studies in rodents (Thumelin et al 1994) andfelines (Lin et al 2005) also inferred that hepatic CPT activ-ity was responsive to changes in dietary fat content On theother hand, studies across reptiles and mammals have shownthat the activity of mitochondrial membrane bound enzymeswas positively correlated to the unsaturation of the mito-chondrial membrane fatty acids (Hulbert & Else 1999),which could be also significantly modified by changing thedietary fatty acid composition (Luo et al 2008) Accord-ingly, the difference in CPT activity between S hasta and

C idellaalso partly resulted from the dietary fatty acid position which affected membrane fluidity

com-In the present study, for both fish species, the kineticparameters of CPT I for carnitine and palmitoyl-CoAchanged with the tissues For example, for S hasta, Kmvalues for carnitine in tissues followed the order: whitemuscle >liver = heart = spleen = intestine For C idella,

Kmfor carnitine was the greatest in intestine and the lowest

in white muscle Kmcould be a very useful index for ation of the substrate status in the tissue (Lin & Odle2003) The differences in kinetic behaviour among varioustissues might be associated with the expression of CPT Iisoforms Studies have shown that mammalian tissuesexpressed three isoforms of CPT I: a liver, L-CPT I, and aheart/skeletal muscle, M-CPT I, that were 62% identical inamino acid sequence (Weis et al 1994) and a brain iso-form, CPT Ic, that was 54% identical to L- and M-CPT I(Price et al 2002) The CPT I isoforms were expressed in avariety of tissues other than those for which they werenamed and in variable and unpredictable amounts (Weis

evalu-et al.1994) These CPT I isoforms possessed very differentkinetic properties and sensitivity to inhibitor malonyl-CoA(McGarry & Brown 1997) For example, the L-CPT I pos-sessed relatively low sensitivity to malonyl-CoA inhibitionand had a low Km for carnitine (~0.03 mM), and theM-form (muscle) showed very high sensitivity to malonyl-CoA and had a high Km (~0.50 mM) for carnitine(McGarry & Brown 1997) In fish, Morash et al (2010)reported five CPT I isoforms, which were expressed in rain-

Figure 3 Example of the Arrhenius plot based on the temperature

tem-peratures in triplicates to evaluate the temperature dependence of

kinetic parameters at saturating substrate concentrations; thin

ver-ticals define ‘transition temperature’ corresponding to breaks in the

bow trout at the mRNA level in muscle, heart, liver, kidney

and intestine Accordingly, it is reasonable to speculate that

different kinetic characteristics of CPT I from various tissues

might be related the number of CPT I isoforms expressed in

different tissues However, further experiments will be needed

in the aspect

In the present study, saturation plots of CPT I activityfor the substrates carnitine and palmitoyl-CoA for several

Figure 4 Kinetic analysis of

equation of the activity of carnitine palmitoyltransferase I with respect to its substrates carnitine and palmitoyl- CoA Values represented the mean of

.

tested tissues (liver, heart, white muscle, spleen and

intestine) of S hasta and C idella yielded hyperbolic

behaviours, in agreement with other animals (Stonell et al

1997; Lavarias et al 2009) The kinetic behaviour of CPT

I varied from species to species with different feeding its Similarly, Fraser et al (2001) also suggested that the

hab-Figure 5 Kinetic analysis of

with respect to its substrates carnitine

and palmitoyl-CoA v: initial velocity

Val-ues represented the mean of three

in liver, muscle, heart, intestine and

spleen were 0.98, 0.98, 0.99, 0.99 and

0.98 for carnitine and 0.99, 0.98, 0.98,

muscle, heart, intestine and spleen were

0.99, 0.99, 0.99, 0.98 and 0.99 for

carni-tine and 0.99, 0.97, 0.96, 0.98 and 0.99

for palmitoyl-CoA, respectively.

feeding habits or diet composition could alter kinetic

char-acteristics of CPT I because diet composition could change

the membrane environment, including the formation of

localized membrane microdomains of distinctive lipid and/

or protein composition, and changes in overall

phospho-lipid composition The Km for carnitine was higher in

S hasta than in C idella Carnitine played a very

impor-tant role in activating and controlling the

carnitine-depen-dent fatty acid transport system, and its availabilities were

very important for the optimal CPT activity According to

Lin & Odle (2003), Km values of the enzymes

approxi-mated the physiological concentration of their substrate in

the tissues Thus, the higher Km for the carnitine could

have been caused by the presence of more carnitine in the

solid food for S hasta compared to C idella, but this was

speculative because we did not measure dietary carnitine

concentrations Patterns of enzyme Vmax across tissues

were useful in revealing differences in fatty acid oxidation

capacity (Morash et al 2008) Synechogobius hasta was a

carnivorous species and had the higher lipid (low

carbohy-drate) food The low catalytic efficiency of CPT I for

car-nitine in liver of S hasta indicated that the fish had a low

capacity for b-oxidation of long-chain fatty acids, thus

causing lipid accumulation Compared to S hasta, the

low-est Km and the higher catalytic efficiency for carnitine in

white muscle of C idella indicated that the fish species had

a high capacity for b-oxidation of long-chain fatty acids,thus causing the lowest lipid content in white muscle Forthe first time, we provided here additional data on thekinetic parameters of CPT I in intestine and spleen in fish

In conclusion, although similarity in some tics, CPT I displayed properties and kinetic differ-ences between carnivorous and herbivorous fish Thekinetic parameters of CPT I with respect to substrates ascarnitine and palmitoyl-CoA indicated that thefeatures of S hasta CPT I were most suited to a lipid-adapted organism, to help to overcome many difficulties

characteris-in lipid utilization To our knowledge, this was the firststudy in which, using carnitine and palmitoyl-CoA as thesubstrates, the complete kinetic characterization of CPT Ihad been performed in fish The further studies onCPT I isoforms expressions in two fish are needed toserve as a useful tool for understanding the regulation offatty acid oxidation in fish in relation to the dietaryregime

This work was funded by program for New Century lent Talents in University, Ministry of Education, China(grant no NCET-08-0782), by Special Fund for CentralUniversity, Ministry of Education, China (grant no 52204-

Excel-Table 3 Kinetic parameters of carnitine palmitoyltransferase I in liver, heart, muscle, spleen and intestine of Synechogobius hasta and

.

10078), by National Natural Science Foundation of China

(grant nos 30800850, 31072226), and by ‘973’ project

(grant no 2009CB118706)

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Key Laboratory of Aquatic Product Processing, Ministry of Agriculture, South China Sea Fisheries Research Institute, ChineseAcademy of Fishery Science, Guangzhou, China

A feeding trial was conducted to determine the dietary

methionine requirement of juvenile golden pompano (initial

body weight 12.40
0.02 g) Six diets were formulated

with six graded levels of methionine (8.6, 9.2, 10.4, 11.5,

13.2 and 14.5 g kg1) Each diet was randomly assigned to

triplicate groups of 20 juvenile fish in seawater floating net

cages (1.0 m 9 1.0 m 9 1.5 m) Fish were fed twice daily

(08:30 and 16:30) to apparent satiation for 56 days Weight

gain (WG), specific growth rate (SGR), feed conversion

ratio (FCR), feed efficiency (FE), nitrogen retention

effi-ciency (NRE), proximate body composition, morphometry

and haematology were significantly (P< 0.05) affected by

the dietary methionine levels WG, SGR and FE increased

with increasing levels of methionine up to 13.2 g kg1 diet

(P < 0.05) and remained nearly the same thereafter NRE

also increased with increasing levels of methionine up to

13.2 g kg1 diet (P< 0.05) and remained nearly the same

thereafter Linear regression analysis on WG and NRE

indicated that the recommended optimum dietary

methio-nine levels for optimal growth of juvenile pompano were

10.6 and 12.7 g kg1 diet, respectively, corresponding to

24.6 and 29.5 g kg1 dietary protein, respectively, so the

level of dietary methionine should be between 10.6 and

12.7 g kg1diet, corresponding to 24.6–29.5 g kg1dietary

protein Additionally, the estimated requirements for the

other essential amino acids were calculated from A/E ratios

of whole-body amino acid profile based on the methionine

requirement determined from the present experiment

KEY WORDS: A/E ratio, golden pompano Trachinotus ovatus,

methionine, requirement

Received 28 June 2012; accepted 18 October 2012

Correspondence: H.-Z Lin, South China Sea Fisheries Research tute, Chinese Academy of Fishery Sciences (CAFC), Guangzhou 510300, China E-mail: gzniujin2003@163.com, linheizhao@163.com

Insti-Golden pompano (Trachinotus ovatus, Linnaeus 1758)belongs to family carangidae, genus Trachinotus, other spe-cies of interest of the genus Trachinotus include Floridapompano (T carolinus), snubnose pompano (T blochii),longfin pompano (T goreensis), Atlantic permit (T falca-tus) and palometa (T goodie; Tutman et al 2004) It is awarm-water species (25–32 °C) and is widely distributed inChina, Japan, Australia and other countries (Yang 2006).Recently, high price in the market and resilience to salinityand temperature ranges make pompano a good candidatefor aquaculture Pompano is considered one of the mostdesirable food fishes, and it commands a significantlyhigher price than many other marine and freshwater species(Tutman et al 2004) However, up to now, only few dataare available on ‘pompano’ nutritional requirements andonly for Florida pompano (Tatum 1972; Williams et al.1985; Lazo et al 1998) And the crude protein and crudelipid levels of the commercial diet for T ovatus in Chinawere 400 and 120 g kg1as referenced in the trout or bassfeeds As a result, many feeds are now available to meetthe energy demands of highly active, rapidly growing, mar-ine species Nutrient requirements of T ovatus are stillpoorly known Development of diets is crucial to sustainthe rapidly expanding pompano farming More recently,Liu et al (2011) reported that the suitable dietary crudeprotein level was 430 g kg1 for 25 g golden pompanoreared in net pens; however, they did not present dietaryamino acid composition and feed intake of test fish Lin

et al (2011) declared that fermented soybean meal couldreplace white fish meal up to 100 g kg1 without negative

.

Aquaculture Nutrition

effects on growth of T ovatus Nutrient-balanced diet is of

serious importance faced to current position of T ovatus

farming in China So more research is needed to optimize

the level of inclusion of other nutrients in the diets such as

amino acids Little work has been carried out in amino

acids with T ovatus

Ten amino acids have been found to be essential for all

fish studied to date (Wilson 1985) An indispensable amino

acid deficiency may cause reduced growth and poor diet

conversion (Wilson & Halver 1986); therefore, satisfying

the essential amino acids (EAAs) requirements of a species

is very important in preparing well-balanced diets

Methio-nine is one of the first limiting EAAs in many fish diets,

especially those containing higher levels of plant protein

sources such as soybean meal, peanut meal and copra meal

(Goff & Gatlin 2004) Studies have demonstrated that

sup-plementation of methionine to plant protein diets is able to

improve growth response of many fish species (Alam et al

2001; Mukhopadhyay & Ray 2001; Takagi et al 2001;

Opstvedt et al 2003; Luo et al 2005; Mai et al 2006;

Zhou et al 2006; Yan et al 2007) Furthermore,

methio-nine and cystine in food or feed are the source of sulphur

amino acids for animals But cystine is not essential

because it can be synthesized from methionine (Bhagavan

1992) So methionine may be spared from cystine synthesis

if cystine is present in the diet Thus, the requirement for

total sulphur amino acids (TSAA) can be met by either

methionine alone or the proper mixture of methionine and

cystine (Moon & Gatlin 1991) Therefore, it is important to

consider the dietary cystine content to quantify the

methio-nine requirement of the cultured species for maximum

growth and efficient feed utilization Many investigators

have reported the quantitative methionine requirements of

commonly cultured fish species with a range from 18 to

40 g kg1 dietary protein (Wilson 2002; Luo et al 2005;

Mai et al 2006; Zhou et al 2006; Yan et al 2007)

How-ever, except for our published data of lysine (Du et al

2011), there have been no studies with regard to other

EAAs requirements of T ovatus

The present research was undertaken to study the

influ-ence of varying dietary methionine levels in isonitrogenous

diets on growth performance, body composition and

bio-chemical parameters so as to determine the optimum

die-tary methionine requirement at a constant diedie-tary cystine

level of 2 g kg1for juvenile T ovatus We also estimated

the requirements for the other EAAs based on the

methio-nine requirement by using A/E ratio [10009 (essential

amino acid content/total essential amino acid contents plus

cystine and tyrosine)]

Six isonitrogenous and isoenergetic diets (D1, D2, D3, D4,D5 and D6) were formulated with six graded levels ofmethionine (Table 1) Dietary methionine was quantita-

Table 1 Composition and proximate analysis of the experimental

glycine, 0.16%; alanine, 1.45%; valine, 0.70%; arginine, 1.31%;

isoleucine, 0.35%; leucine, 0.39%; lysine, 1.30% (Shanghai Feeel Technology Development Co., Ltd.).

4

et al 2006).

45 mg; pyridoxine HCl, 20 mg; vitamin B12, 0.1 mg; vitamin K3,

10 mg; inositol, 800 mg; pantothenic acid, 60 mg; niacin acid,

200 mg; folic acid, 20 mg; biotin, 1.20 mg; retinal acetate, 32 mg;

2000 mg; choline chloride, 2500 mg; ethoxyquin 150 mg; wheat middling, 14.012 g (Mai et al 2006).

.

tively increased at the expense of glutamic acid L

-Crystal-line amino acids mixture was used so that the levels of all

amino acids, except methionine, would simulate the

whole-body amino acid pattern of pompano (initial whole-body weight

12.40
0.02) The amino acid (AA) contents of

experi-mental diets are shown in Table 2 Because the crude

pro-tein contents of experimental diets were about 430 g kg1,

the AA contents of the experimental diets were made

com-parable with those of whole-body protein AA contents at

430 g kg1 All the dietary AA contents were maintained

nearly the same levels as the corresponding AA contents in

whole-body protein at 430 g kg1 except for methionine

and glutamic acid A satisfying increase in methionine

con-tent was obtained in the diets, although it was not identical

to the quantities added initially The final levels of

methio-nine were 8.6, 9.2, 10.4, 11.5, 13.2 and 14.5 g kg1 diet,

respectively, by adding crystalline DL-methionine, analysed

by reverse-phase high-performance liquid chromatography

(HPLC, HP1100; Agilent Technologies, Palo Alto, CA,

USA) The range of dietary methionine content covered the

methionine level (11.3 g kg1) in crude protein from the

whole-body tissue of this species (Table 2)

All dry ingredients were finely ground, weighed, mixed

manually for 5 min and then transferred to a Hobart mixer

(A-200T Mixer Bench Model unit; Resell Food Equipment

Ltd., Ottawa, Canada) for another 15-min mixing During

the mixing, 6 N NaOH was added to establish a pH level

of 7–7.5 The pH of the diet was obtained by

homogeniz-ing a 5-g portion of the diet with 50 mL of distilled water

with a glass electrode pH meter on the supernatant(Robinson et al 1981) Soya lecithin was added to apreweighed fish oil and mixed until homogenous The oilmix was then added to the Hobart mixer slowly while mix-ing was still continuing All ingredients were mixed foranother 10 min Then distilled water (about 30–35%, v/w)was added to the mixture to form a dough A dough ofeven consistency was passed through a pelletizer with a2.5-mm-diameter die (Institute of Chemical Engineering,South China University of Technology, Guangzhou,China) The diets were dried until the moisture wasreduced to <100 g kg1 The dry pellets were placed inplastic bags and stored20 °C until fed

The feeding trial was conducted out at an experimental tion of South China Sea Fisheries Research Institute ofCAFS (Sanya, Hainan) The fish were obtained from acommercial farm near Hongsha Bay, Sanya, Hainan prov-ince, China Prior to the feeding trial, the fish were reared

sta-in floatsta-ing sea cages (3.0 m9 3.0 m 9 3.0 m), and fed thecontrol diet (D1) for 2 weeks to acclimate to the experi-mental diet and conditions At the start of the experiment,the fish were fasted for 24 h and weighed after being anes-thetized with eugenol (1:10 000; Shanghai Reagent Corp.,China) Juvenile pompano (Trachinotus ovatus) with similarsize (initial body weight 12.40
0.02) were randomly allot-ted into 18 sea cages (1.0 m9 1.0 m 9 1.5 m; three cages

Table 2 Analysed essential amino acid

(EAA; excluding tryptophan) and

diet) of the experimental diets

per treatment) and each cage stocked with 20 fish The fish

were fed by hand twice daily at 08:30 and 16:30 h,

respec-tively Fish were hand-fed slowly little by little to prevent

waste of dietary pellets When the experimental diet was

supplied, the fish would swim to the water surface to ingest

the diet As long as fish were fed to satiation, they would

never come up water surface again Hence, their apparent

satiation could be judged by feeding behaviour

observa-tion The feeding trial lasted for 56 days During the

exper-imental period, feed consumption was recorded The water

temperature fluctuated from 28 to 31°C, salinity from 28

to 31 g L1 and dissolved oxygen approximately

8.33 mg L1during experiment At the end of experiment,

the fish were fasted for 24 h and fish in each cage were

weighed

At the beginning of the feeding trial, 18 juveniles were

ran-domly sampled from the initial fish and killed for analyses

of whole-body composition At the end of the 56-day

experiment, 10 fish from each cage were randomly collected

for proximate analysis: four for analysis of whole-body

composition and six were anesthetized with eugenol for

blood collection and to obtain weights of individual whole

body, viscera and liver White muscle from both sides of

the fillets without skin and liver were dissected and frozen

immediately in liquid nitrogen and stored at 70 °C until

analysed Blood samples were drawn from the sinus of six

anaesthetized fish with heparinized syringes, and plasma

separated by centrifugation and stored at 70 °C until

analysed

Diets and fish samples were analysed in triplicate for

proximate composition Moisture, crude protein, crude

lipid and ash were determined using standard methods

(AOAC 1995) Moisture was determined by drying in an

oven at 105°C for 24 h; crude protein (N 9 6.25) was

analysed by the Kjeldahl method after acid digestion

(1030-Auto-analyzer; Tecator, H€ogan€as, Sweden); crude fat

was determined by ether-extraction method by Soxtec

Sys-tem HT (Soxtec SysSys-tem HT6; Tecator) and crude ash by

incineration in a muffle furnace at 550°C for 24 h The

amino acid composition of all samples were analysed

fol-lowing acid hydrolysis using an automatic amino acid

ana-lyser (Hitachi 835-50, Tokyo, Japan) with a column

(Hitachi custom ion exchange resin no 2619) by a

profes-sional laboratory In brief, performic acid oxidation was

performed prior to hydrolysis to oxidize cystine and

methi-onine to cysteic acid and methimethi-onine sulphone Then

sodium metabisulphite was added to decompose surplusperformic acid Subsequently, amino acids were liberatedfrom protein by hydrolysis with 6 N HCl Hydrolysed sam-ples were diluted with sodium citrate buffer, pH wasadjusted to 2.2 and individual amino acid components wereseparated by ion exchange chromatography at 570 nm

Tryptophan was not determined The concentrations ofplasma protein, cholesterol, triacylglycerol, glucose, aspar-tate aminotransferase (AST) and alanine aminotransferase(ALT) were determined using an automatic analyser (Hit-achi 7170A) from a professional laboratory (Sun Yat-senUniversity of Medical Sciences)

The following variables were calculated:

Viscerasomatic index (VSI)ð%Þ

¼ 100  viscerosomatic weight (g)/fish body weight (g)

Hepatosomatic index (HSI)ð%Þ

¼ 100  liver weight (g)/fish body weight (g)where Wf and Wi were mean final and initial fish bodyweights; t is the experimental duration in days; Nt is num-ber of fish at the end of the trials and N0 at the start; Wt(g) is total final body weight (FBW) and W0(g) total initialbody weight; Wd(g) is total body weight of the dead fish;

CNt (g kg1) is protein content in whole fish body at theend of the trial and CN0(g kg1) at the start, CNf(g kg1)

is protein content in the feed; I (g) is total amount of thefeed fed on a dry weight basis

All data are presented as means
SEM and subjected

to one-way analysis of variance (ANOVA) to test the effects

of experimental diets using the software of the SPSS forwindows (ver 16.0; SPSS, Inc., Chicago, IL, USA) Dun-can’s new multiple range test was used to resolve the differ-ences among treatment means (Duncan 1955) Statistical

.