Aquaculture nutrition, tập 18, số 6, 2012

1,2 1 1 1 1 1 1 1University, Guangzhou, China A 63-day growth trial was undertaken to estimate the effects of supplemented lysine and methionine with differ-ent dietary protein levels on

1 1,2 1

des Sciences, Ancien logement des maıˆtres, La Rosie`re, Lamentin, Guadeloupe, FWI, France

One of the bottlenecks for the queen conch, Strombus

gigas, aquaculture is the lack of well-adapted formulated

food for optimal growth The goals of this study were to

analyse the digestive gland structure of conchs fed with

dif-ferent diets using histochemical techniques and to evaluate

the growth and survival of S gigas juveniles with nine

acido-philic granules were detected in the digestive cells The

abundance of both granule types was variable, according

to the nutritional state of the animals The granular

con-tent of the digestive cells of conchs fed with artificial diets

was scarce when compared with conchs fed on natural

food Of the nine formulated feeds, the diet with 365 and

with digestive cells in the best condition as determined

his-tologically Histochemical analysis of the digestive gland

differentiated with Alcian blue staining determines the

nutritional status much better than a simple growth index

and is therefore more useful in assessing adjustments to the

feed formulation to meet the real needs of conchs

mol-luscs, physiology

Received 29 January 2011; accepted 14 October 2011

Correspondence: D.A Aranda, CINVESTAV IPN Unidad Me´rida,

Laboratorio de Biologı´a y Cultivo de Moluscos, Km 6 antigua carretera

a Progreso CP 97310 Me´rida, Yucata´n, Me´xico E-mail: daldana@

mda.cinvestav.mx

The queen conch, Strombus gigas, (Linnaeus, 1754) is amarine resource of ecological and economic importance inthe Caribbean (FAO 2007) Since pre-Columbian time,queen conchs have been an important source of food forthe inhabitants of Caribbean coasts and islands (Wing2001) However, queen conch meat that was a popular sta-ple food is now mostly consumed as a tourist delicacy It is

an important source of income in several exporting tries and is an overexploited fishery (Theile 2001) As popu-lations have been declining for several decades, much of thecurrent research focuses on aquaculture, restocking andtransplanting techniques to help replenish wild conch popu-lations Queen conch aquaculture has been developed in theTurks and Caicos (to expand conch production farm and tolicense grow-out farms throughout the Caribbean) and inFlorida Harbor Branch Oceanographic Institution (Davis2000; Shawl et al 2008) Even though queen conch aquacul-ture is a success in terms of hatchery spat production,growth still depends on the use of large areas of wild envi-ronment (Davis 2000) One of the bottlenecks for intensiveconch farming is the lack of formulated food for optimalgrowth in hatchery at a reasonable price (Shawl & Davis2006; Shawl et al 2008) There is a need to improve hus-bandry techniques for the grow-out of juveniles with dietsthat allow a growth rate equal to or higher than that of wildjuveniles Moreover, the use of prepared feeds can be verypractical as formulations can be manipulated to obtain anoptimum nutritional value Furthermore, they are available

coun-on demand and if properly prepared may be stored for alonger time period (Bautista-Theurel & Millamena 1999).Aldana Aranda et al (1996) reported a growth rate of

Glazer et al (1997) fed conchs with Koi fish food and

,respectively Rathier (1987), Iversen & Jory (1997) and

.

Aquaculture Nutrition

Aldana Aranda et al (2005) obtained growth rates of 0.16,

conch fed on natural food Most studies on the digestive

tract and digestive gland of microphagous prosobranch

Gastropods have been performed on species living in

inter-tidal environments in order to investigate the influence of

tidal variations upon the digestive gland cycle (Nelson &

Morton 1979) Queen conch natural feed is a complex

mix-ture of macroalgae, microbenthos and biofilm involving

ingestion of sediment (Stoner & Waite 1991; Shawl et al

2008; Serviere et al 2009) However, real nutrient

require-ments for queen conch, in terms of energy level, protein and

micronutrients, are unknown (Amber et al 2011)

Further-more, shell growth and weight are not precise indices of

optimal growth and assimilation (Lucas & Beninger 1985)

The goals of this study were to compare the status of the

digestive gland of juvenile queen conch fed with different

formulated diets with increasing levels of proteins and lipids

using histochemical techniques and to evaluate the growth

and survival of conchs fed with these diets

An experimental aquaculture facility was set up at Xcaret

Marine Park, south of Cancun (Mexico), to raise juvenile

queen conch received from Ocean Reef Aquarium society

(ORA) in Fort Pierce, Florida Experimental cultures were

which was continuously oxygenated using an air pump Sea

cleaned twice a week to avoid the development of

microal-gae and biofilm which could be used as a food source

Juve-nile queen conchs were fed 150 mg of diet per conch each

day for 3 months, and the uneaten food was removed daily

Two experimental treatments and a control were set up

The experimental treatments were as follows: (i) conchs

were given formulated feed (diets 1, 3, 4, 5, 6 and 9, see

Table 1) for 84 days and (ii) conchs were given formulated

feed (diets 2, 7 and 8) for 84 days and then returned to a

natural diet (a biofilm of 50% of red and green algae and

50% of seagrass and sand) for 21 days The main

ingredi-ents used in these formulated diets were fish meal, soy

flour, wheat flour, spiruline, corn starch, fish oil, vegetable

oil and soy lecithin Each of the nine diets was tested using

three replicates of 30 conchs each, giving a total of 810

conchs on experimental diets The control comprised two

replicates of 30 conchs each (n = 60 conchs), which were

kept on a natural food diet (a biofilm of 50% of red and

green algae and 50% of seagrass and sand) for 84 days

The nine formulated diets were tested (Table 1), containing

conchs was analysed using the following indices: siphonallength, flesh weight and histological features of the diges-tive gland Juveniles of S gigas were measured and

days 21, 42, 63 and 84 At the beginning of the experiment,juveniles from the wild, in the same size range as theconchs from ORA, were dissected and prepared for the his-tological analysis of the digestive gland Likewise, threejuvenile queen conch from the Florida hatchery were alsoanalysed before the start of the experiment, and anotherthree juveniles of each replicate receiving one of nine diets(n = 9 conchs) were examined at the end of the experiment

Histological examination involved cutting the visceralmass of each individual into two sections: a distal part, con-taining only digestive gland and connective tissue, and a midpart also containing stomach Sampled sections were fixed inalcoholic Bouin fluid and processed using standard histolog-ical techniques (Gabe 1968; Luna 1969) After dehydration

in ethanol series, and clearing with Clarene, the sections

were stained with a trichrome stain following Gabe (1968)which included Alcian blue (Hycel de Mexico, SA de Cv

Zapopan, Jalisco, Mexico) at pH 2.5 to differentiate glycans Slides were also treated by the Periodic acid-Schiff(PAS) reaction for glucide detection (Gabe 1968) The slideswere examined and pictures were taken with a Nikon DXm1200F digital camera mounted on a Nikon microscope Allthe pictures were corrected for contrast and colour (Photo-shop software, Adobe Photoshop CF 2 version 9.0, SanJose, CA, USA) For every juvenile, two microscope slideswere prepared with five sections per slide (distal part) The

proteo-Table 1 Biochemical composition of experimental diets used to feed juveniles of Strombus gigas

incidence of blue granules (Gros et al 2009) was obtained

by counting the total number observed in three fields of the

mean and standard deviation for each diet A feed index was

established as the sum of blue granules counted in the large

cells of adenomers These counts were transformed into

), using the circle area formula for the

) observed on

blue colour of the granules observed in the digestive cells

after staining with Alcian blue indicated that they were

pro-teoglycan components Such conspicuous secretory granules

are good markers that demonstrate the digestive cell

secre-tion being delivered to the stomach One microscope slide

was prepared with the mid part (containing stomach) as a

control of histochemical analysis The stomach epithelium

that contains mucocytes stained blue A non-parametric

Tukey test (Sokal & Rohlf 1995) was used to test for

signifi-cant differences (P < 0.05) among diets in the feed index

and growth rates in siphonal length and whole body weight

In wild juvenile conchs, the digestive gland has an array

of adenomers (Fig 1a) similar to those described in

adults All these secreting structures are connected tosmall ducts, which join larger ducts attached to the stom-ach The small secondary ducts are lined with a simpleepithelium composed of a single cell type The larger pri-mary ducts have two areas (Fig 1b), one similar to thesmall duct epithelium devoid of cilia and another com-posed of ciliated cells and mucocytes The connective tis-

characteristic cell types, small round amoebocytes stainedred by PAS and blue granules in the large cells stainedblue by Alcian blue Both may play a role in the transfer

of metabolites The functional glandular structure

These cells alternate with vacuolated cells which arealways occupied by brown inclusions These inclusions aresporozoa-like microorganisms belonging to the Apicom-plexa group The observation of unstained sections dem-

sporozoa-like microorganisms are their natural colours.The mucous lining of the stomach as well as a number ofconnective tissue cells are stained by Alcian blue (Fig 1c)giving a positive control to the histochemical reaction inthe digestive gland The crystalline style is stained red bythe PAS reaction even after amylase digestion and stainedblue by Alcian blue, demonstrating that it contains glyco-proteins as well as proteoglycans

(a)

(b)

Figure 1 (a) Digestive gland from a

wild juvenile with primary ducts and

adenomers; (b) primary duct with two

types of epithelium; low and simple ( )

similar to the secondary ducts

epithe-lium and plicate ciliated epitheepithe-lium ( Δ)

typical of primary ducts; (c) stomach

wall with mucocytes ( ◊) stained by

Alcian blue that constitutes a positive

control for various diets as mucocytes

are stained blue even if the digestive

gland granules are not; (d) adenomers

of a digestive gland section containing

with the two cell types Large digestive

or secreting cells with large blue

gran-ules (star) and crypt cells with

sporo-zoa-like microorganisms belonging to

the Apicomplexa group (arrow).

The status of the digestive gland from three individuals

immediately after being received from the hatchery was

exhib-ited digestive cells with fewer and smaller blue granules

than in wild conchs (Fig 2b) The blue granules were not

similar in structure to those of wild juveniles as the blue

colour was restricted to the centre of the granules The

spo-rozoa-like microorganisms were also different from those

described in wild juveniles, being a light creamy colour and

numerous

Table 2 shows the initial and final siphonal length, weightand daily growth of juvenile S gigas fed on nine diets for

84 days, and values of a non-parametric Tukey test The

The control group had an initial and final siphonal length

of 54.69 ± 7.3 and 75.98 ± 4.4 mm, respectively, (n = 60)

among diets in the mean growth per day in siphonal length

Figure 2 Comparison of digestive glands of Strombus gigas juveniles among reared animals fed various formulated diets, including control

animals fed on a natural diet and wild juveniles All the pictures are of digestive glands at the same magnification with obj 940 (a)

Diges-tive gland of a wild juvenile showing large digesDiges-tive or secreting cells with large blue granules (star) and crypt cells with sporozoa-like

microorganisms (arrow); (b) laboratory-reared individual from ORA showing smaller blue granules and a less homogenous structure than

the wild juvenile; (c) reared juvenile fed for 84 days on diet 8 showing that the digestive gland cells are completely destroyed although the

duct appears normal; (d) reared juvenile fed for 84 days on diet 2 showing intracellular unstained by Alcien blue, whereas the stomach

epithelium contains some mucocytes stained blue (black arrow); (e) reared juvenile fed for 84 days on diet 7 showing blue granules

approxi-mately half the size of those observed in the wild juveniles; (f) reared juvenile fed for 84 days on diet 7 and then on a natural food diet for

21 days showing blue granules typical of wild juveniles Diet formulations are given in Table 1.

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Aquaculture Nutrition 18; 581–588 ª 2012 Blackwell Publishing Ltd

and flesh weight (P < 0.0001) Tukey test (P  0.05)

showed significant differences among diets 2, 3 and 7

Survival was 100% for conchs fed with natural food and

diets 2, 3, 5, 6, 7, 8 and 9, and 97.9% for those fed with

diets 1 and 4 The digestive glands of juveniles reared with

various formulated diets have a different appearance All

these experimental animals are characterized by the lack of

either blue granules or granules unstained by Alcian blue,

therefore, lacking their proteoglycan component The PAS

reaction demonstrates that the granule glycoprotein in these

experimental conchs is similar to that of wild juvenile

diges-tive granule content For example, conchs fed diets 2 and 5

showed a normal structural appearance of the cells in the

digestive gland However, the large granules of digestive

cells were not stained blue by the histochemical

proteogly-can reaction (Fig 2c) With diet 8, the digestive duct

epithe-lium and the stomach contained a number of mucocytes

stained blue (Fig 2d), and the digestive and crypt cells of

the digestive gland are completely destroyed Here, the cell

membranes and intracellular granules have almost

com-pletely disappeared giving the cell a destroyed appearance

(Fig 2d) Diet 7 appears to be the best of the 9 tested, as

the digestive gland structure still possesses some blue

gran-ules (Fig 2e) However, these grangran-ules are much smaller

than in the digestive gland of wild juveniles

The highest occurrence of blue granules was registered in

conchs fed with natural food with a feed index of 30.0%

Conchs fed with diet 7 showed a feed index of 14.5%,

conchs from ORA had a value of 10.0% on arrival and

those fed with diet 7 and then replaced with natural food

showed an increase in the mean value to 17.9% The

diges-tive gland was restored showing the digesdiges-tive gland status

observed in the digestive gland of the queen conch control

with digestive cells again containing numerous blue

(Fig 2f ) The lowest feed index was registered with diets 2,

6, 8 and 9 with no blue granules being found for any of

signifi-cant differences among diets in the median value of feed

significant differences among feed index of diets 5, 7,conchs fed with diet 7 plus natural food (Fig 3) All theindividuals fed with formulated food have few sporozoa-like microorganisms included in the vacuolated cells of thedigestive gland These are generally light coloured withoutevident sporulation

Most histological studies of the digestive gland structure ofmolluscs deal with bivalves (Purchon 1977; Morse & Zar-dus 1997) Gastropod digestive glands have been described

by comparison with bivalve digestive glands (Fretter &Graham 1962) However, feeding methods and nutritionalrequirements are not similar in these two groups Studiedbivalves are suspension feeders and ingest small unicellularalgae submitted to intracellular digestion in the digestivegland cells (digestive cells), whereas the prosobranch gas-tropod S gigas is a grazer, usually feeding on macro algaewhen available and on biofilm by grazing on Thalassialeaves or by ingesting sand Stoner & Waite (1991) studiednatural feed of S gigas in the Bahamas and observed feed-ing differences at two sites (one with high seagrass biomassand one with bare sand) At the site with low seagrass bio-mass, these authors found that macroalgae made upapproximately 50% of the stomach content, with detritusand small infauna making up the other 50% Despite large

Table 2 Growth parameters of the of juveniles of Strombus gigas fed for 84 days on one of nine formulated diets

Diets

Initial siphonal length (mm)

End siphonal length (mm)

Growth rate day 1

(mm)*

Initial weight (g)

End weight (g)

Growth rate day 1 (g)** n

Tukey test (P  0.05)

amounts of seagrass detritus in the stomachs of conch from

all size classes, stable isotope ratios of carbon and nitrogen

indicated that the food of conchs cannot be based only

upon detritus at any of the habitats Macroalgae,

particu-larly Laurencia spp and Batophora oerstedi, were the

standing crops Differences in stomach contents were

higher between sites than ontogenetic or seasonal

varia-tions Serviere et al (2009) studied natural feed in juveniles

and adults of S gigas from San Pedro, Belize These

authors observed different kinds of food in stomach

con-tents from both categories of conchs They reported a total

of 22 items in the stomach contents of juvenile and adult

conchs The most diverse phylum was Rodophyta The

sec-ond largest groups present in the stomach contents were

Cyanophyta and Protozoa Seagrass also constituted an

important component in the stomachs of adults

Aquacul-ture for queen conch has been established for several

dec-ades However, there is a need to improve husbandry

techniques for the grow-out of juveniles with diets that

allow a growth rate equal to or higher than that of wild

1997) Aldana Aranda et al (1996) fed conchs (0.9 mm)

, tively The highest growth rate obtained by Shawl et al

respec-(2008) when supplementing an artificial juvenile queenconch diet with various macroalgae (Agardhiella added to

determined the growth of juvenile queen conchs fed on

containing a soy protein isolate protein substitution of15% or less, showing the importance of the protein source

in the artificial diets of juvenile queen conchs (Table 3) In

Britz & Hecht (1997) studied a herbivorous Gastropod(Haliotis midae) and concluded that diets containing the

signifi-cantly lower growth rates and efficiencies of protein tion in comparison with abalone fed on diets containing

more marked among the small abalone size class From theanalysis of data of Table 3, the growth of juvenile S gigas

and growth with different diets used to fed juvenile queen

Figure 3 Box plots (n = 85–90 in each plot) showing feed index established of area of blue granules counted in the adenomers of a digestive

gland of juvenile queen conch divided by the image area (37 368 lm 2 ) observed at 40 9 magnification, and multiplied by 100 In juvenile

queen conchs fed nine different diets (see Table 1) for 84 days, including control animals fed on a natural diet (a biofilm of 50% of red

and green algae and 50% of seagrass and sand), conchs fed diet 7 for 84 days and then returned to a natural food diet for 21 days and

lab-oratory-reared The box contains 50% of the data (90% of data when whiskers are included), while dots indicate extreme values Data are

given as medians (horizontal line within each box) ± SD Letters indicate signi ficant differences between dietary treatments (Tukey test,

P  0.05).

.

Aquaculture Nutrition 18; 581–588 ª 2012 Blackwell Publishing Ltd

fairly similar to that obtained with natural food However,

it has been shown that siphonal length is not a sufficient

measure to accurately determine the nutritional status of

molluscs (Lucas & Beninger 1985) This was also the case

in the present study, where data of growth rates

demon-strate similar growth for conch fed with different diets The

histological appearance of the digestive gland, particularly

the digestive cells, appears to be a more sensitive feed index

with which to evaluate the efficiency of diets for juvenile

queen conch Histochemical analysis of the digestive gland

and the feed index proposed in this study will be a useful

tool to test micronutrients issued from algae to adapt the

formulated feed and feeding rate to the real needs of

juve-niles The feed index proposed in this study, which provides

be considered adequate The structure of the digestive

gland in wild adults of S gigas appears more complex than

in wild juveniles Crypt cells containing spherocristals

described by Gros et al (2009) which have not been

identi-fied in the digestive gland of wild juveniles or coming from

ORA for this study These cells may differentiate with age

and maturation of the nutritional or excretory function of

the digestive gland However, an ultrastructural study of

the digestive gland will be necessary to ascertain the

absence of these crypt cells and spherocristals Brown

inclusions observed in vacuolated cells in this study have

been described in the digestive gland of adult queen conchs

(Baqueiro Ca´rdenas et al 2007; Gros et al 2009; Volland

Feeding on formulated pellets is a very important

modifi-cation of the feeding habits of queen conchs, although the

pellets are ingested and have been identified in the stomach

We have identified a number of synthetic foods stuck tothe crystalline style in histological sections The food pelletsseem to be digested in the stomach and reduced to a finehomogenous powder that is transported to the digestivegland tubules where it may be absorbed The presence inthe stomach of large quantities of secretion granules origi-nating from the digestive cells demonstrates that these cellshave a secreting function different from the functionidentified in suspension feeders which is only intracellulardigestion Volland (2010) and Volland et al (2010) demon-strated enzyme activity (arylsulphatase and acid phospha-tase) associated with the apical region of digestive cellswhich contained the blue granules in various Strombidaefed with natural and artificial food

For the experimental trial of nine formulated pellets, theoptimal nutritional status that resulted in the best digestivegland structure as well as the best shell growth corre-

pro-teins) However, the histological structure of the digestivegland demonstrates that this diet, even though it appears

to be the best of the nine formulations tested, is not reallysatisfactory and could be improved as demonstrated by theimproved appearance of the digestive gland of the individu-als returned to a natural algal diet

The authors wish to thank Xcaret aquarium facilities fornursery work (G Quintana, A Cordova, R Raigoza,

E Briones, E Rios, R Torres), the Ichtyology laboratory

of CINVESTAV IPN Merida and Marine laboratory ofthe University of French Antilles, for histology and digital

Table 3 Summary of available growth

rate estimates for Strombus gigas

juve-niles reared on various diets Site Food

Initial shell length (mm)

Growth rate (mm day 1) References

Martinique FWI

et al (1996)

Bahamas Natural food 0.08 –0.16 Iversen & Jory (1997) Punta Gavila´n,

Me´xico

Natural food 99 0.14 De Jesu´s & Oliva (1997)

Laboratory Agardhiella added

to catfish chow

0.11 Shawl et al (2008) Laboratory Catfish chow (60%),

Agardhiella (22%), soy protein (15%) and fish oil (2%)

0.10 Amber et al (2011)

photomicroscope facilities, respectively, Adriana Zetina

and Teresa Cola´s for histology work, and Erick Baqueiro

and Gemma Franklin (a native English speaker) for

Eng-lish revision Thanks to three anonymous reviewers for

their comments that enriched this work Grant:

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1,2 1 1 1 1 1 1 1

University, Guangzhou, China

A 63-day growth trial was undertaken to estimate the

effects of supplemented lysine and methionine with

differ-ent dietary protein levels on growth performance and feed

utilization in Grass Carp (Ctenopharyngodon idella) Six

plant-based practical diets were prepared, and 32CP, 30CP

and methionine supplementation In the supplementary

group, lysine and methionine were added to formulate

accord-ing to the whole body amino acid composition of Grass

Carp In the groups without lysine and methionine

supple-mentation, weight gain (WG, %) and specific growth rate

higher than that of fish fed 30CP and 28CP diets, but no

significant differences were found between 30CP- and

28CP-diet treatments WG and SGR of the fish fed 32AA

and 30AA diets were significantly higher than that of fish

fed 28AA diets, and the performance of grass carp was also

significantly improved when fed diets with lysine and

between dietary protein level and amino acid

intake (FI) was significantly increased with the increase in

dietary protein level and the supplementation of lysine and

after total ammonia nitrogen (TAN) concentration test, the

values of TAN discharged by the fish 8 h after feeding

body weight for fish fed 32CP, 32AA, 30CP, 30AA, 28CPand 28AA diets, respectively TAN excretion by grass carpwas reduced in plant-based practical diets with the increase

in dietary protein level and the supplementation of lysineand methionine (P < 0.05) The results indicated thatlysine and methionine supplementation to the plant proteinsources-based practical diets can improve growth perfor-mance and feed utilization of grass carp, and the dietary

protein level

protein reduction, total ammonia nitrogenReceived 29 January 2011; accepted 13 November 2011 Correspondence: Yong-Jian Liu, School of Life Science, Sun Yat-Sen University, Guangzhou 510275, China E-mail: edls@mail.sysu.edu.cn

Grass carp (Ctenopharyngodon idella) represent the secondlargest aquaculture industry in the world inferior to silvercarp Hypophthalmichthys molitrix, constituting 14.7% ofthe world aquaculture production, with an average annualincrease of 14% in China (FAO 1999) One of the majorreasons for the increase in production is the use of pelleted

mono-culture of this species to be achieved

Protein is a major component in fish feeds, because it vides the essential and nonessential amino acids to synthesize

.

Aquaculture Nutrition

body protein and in part provides energy for maintenance.

Dabrowski (1977) found the protein requirement of grass

crude tein content Protein, especially when derived from fishmeal

pro-and soybean meal, is the most expensive nutrient in the

prep-aration of diets for grass carp The protein sources of grass

carp commercial diets almost are canola meal and cotton

meal Lysine and methionine are the two most limiting

amino acids in the diets for grass carp (Wang et al 2005;

Yang et al 2010) It has been demonstrated that grass carp

can efficiently utilize crystalline amino acid (Yang et al

2010) Supplementation of lysine-deficient diets with lysine

also improved WG in common carp (Viola et al 1992a) and

channel catfish (Robinson et al 1980; Robinson & Li 1994;

Zarate & Lovell 1997) Botaro et al (2007) reported that

reducing 2.7% of dietary digestible crude protein (from

no negative impact on growth performance of Nile tilapia

Recently, Gaylord & Barrows (2009) also found dietary

crude protein content of plant-based diet for rainbow trout

supple-menting lysine, methionine and threonine with no reduction

in growth In addition, the imbalance of dietary amino acid

profile can lead to eutrophication of receiving water by

nitro-gen excretion (Cheng et al 2003; Conceicao et al 2003)

Therefore, the present studies were carried out to

evalu-ate the effects of dietary protein reduction with lysine and

methionine supplementation on growth performance and

total ammonia nitrogen (TAN) excretion of grass carp

under laboratory conditions These results could be useful

for the formulation of low-cost feed for grass carp

Formulations of the experimental diets are shown in

Table 1 Three practical experimental diets (32CP, 30CP

and 28CP) that were formulated with 32% (DM), 30%

(DM) and 28% (DM) crude protein without

supplementa-tion of crystalline amino acid, lysine and methionine were

deficient for grass carp In the other three experimental

diets (32AA, 30AA and 28AA), lysine and methionine were

added to the 32CP, 30CP and 28CP diets and were made

to equal the levels calculated to be present in the grass carp

whole body of 32% protein All diets were made

isoener-getic The amino acid compositions of six experimentaldiets are shown in Table 2

All dry ingredients were finely ground, weighed, mixedmanually for 5 min and then transferred to a Hobart mixer(A-200T Mixer Bench Model unit, Resell Food EquipmentLtd, Ottawa, ON, Canada) for another 15-min mixing

Soya lecithin was added to a preweighed premix of soy oiland mixed until homogenous The oil mix was then added

to the Hobart mixer slowly while mixing was still uing All ingredients were mixed for another 10 min Then,

mixture-form dough The wet dough was placed in a screw extruder (Institute of Chemical Engineering, SouthChina University of Technology, Guangzhou, China) andextruded through an 1.25-mm die The diets were dried

used

Grass carp (Ctenopharyngodon idella) juvenile from our ities were used in this experiment, and their initial wet

were acclimated to the experimental conditions for 2 weeks

randomly distributed to each of the 18 experimental

200 L) connected to a recirculation system Water exchange

oxygenated, passed through artificial sponge (3 cm ness), coral sand (25 cm thickness) and active-carbon filter(25 cm thickness) to remove chlorine During the trial per-iod, the diurnal cycle was 12-h light/12-h dark Water qualityparameters monitored weekly as follows: temperature,

were collected daily during the last 2 weeks as described by

In the growth experiment, the fish were fed three timesper day and 7 days per week to apparent satiation for

9 weeks After the growth experiment, ten healthy fish

distrib-uted to each of the 12 experimental fibreglass tanks

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Aquaculture Nutrition 18; 589–598 ª 2012 Blackwell Publishing Ltd

fed with the diets from the growth experiment for 2 days.

After fish were fed at 0800 h with 2.5% of body weight, all

tanks were cleaned, the water supply to the tanks was shut

off, and oxygen was supplied to the tanks At 0800 h and

1600 h, water samples were collected for TAN analyses for

2 days

At the beginning of the feeding trial, 18 fish were randomly

sampled from the initial fish and killed for analyses of

whole body composition At the end of the 63-day

experi-ment, 12 fish from each tank were randomly collected for

proximate analysis, four for analysis of whole body

compo-sition and 8 were anaesthetized with tricaine methane

individual whole body, viscera, liver and mesenteric fat.White muscle from both sides of the fillets without skinand liver was dissected and frozen immediately in liquid

Diets and fish samples (including white muscle and liver)were analysed in triplicate for proximate composition Crudeprotein, crude lipid, moisture, crude ash and gross energy(GE) were determined following standard methods (AOAC

Kjel-dahl method after acid digestion using an Auto KjelKjel-dahl tem (1030-Auto-analyzer, Tecator, Sweden) Crude lipid wasdetermined by the ether extraction method using a Soxtec

Sys-Table 1 Formulation and approximate composition of practical diets for grass carp

Digestible energy, gross energy, and digestive protein were measured (Wang et al 2005).

1 Zhuhai Shihai Feed Corporation Ltd, Zhuhai, China.

2 Mineral mix(mg kg 1 of diet): MgSO 4 ·7H 2 O,315; ZnSO 4 ·7H 2 O,285; CaHPO 4 ·2H 2 O,250; FeSO 4 ·7H 2 O,200; MnSO 4 ·H 2 O,25; CoSO 4 ·7H 2 O,25; CaIO 3 ,25; CuSO 4 ·5H 2 O,15; Na 2 SeO 3 ,10 (Guangzhou Chengyi Aquatic Technology Ltd, Guangzhou, China).

3 Vitamin mix (mg kg 1 of diet): thiamin,3; riboflavin,8; vitmin A,1 500 IU; vitamin E,40; vitamin D3,2 000 IU; menadione,6; pyridoxine,4; cyanocobalamin,2; biotin,2; calcium pantothenate,25; folic acid,2; niacin,12; inositol,50 (Guangzhou Chengyi Aquatic Technology Ltd, Guangzhou, China).

4

L -Lysine SO 4 contained L -Lysine  51% (CJ Co., Ltd., Liaocheng, China).

5 MHA-Ca contained DL-HMTBA (2-hydroxy-4-methylthio butanoic acid)  84% (Novus International Inc., Zhibo, China).

6 Y 2 O 3 (Yttrium oxide), analytical pure (Weibo Chemical Ltd, Guangzhou, China).

System HT (Soxtec System HT6, Tecator, Sweden) Moisture

ash was determined by incineration in a muffle furnace at

bomb calorimeter Amino acids were analysed following acid

(HPLC; Hewlett Packard 1090, Palo Alto, CA, USA) The

by inductively coupled plasma atomic emission

spectropho-tometer [ICP; model: IRIS Advantage (HR), Thermo Jarrel

Ash Corporation, Boston, MA, USA] after perchloric acid

digestion (Bolin et al 1952)

Water samples were analysed for TAN concentration by

Nessler’s reagent colorimetric method (Zang 1991) Light

absorbance of water samples at 420 nm wavelength was

recorded on a UV-spectrophotometer (UV250), and TAN

concentration was determined using a standard curve

ver 13.0 for Windows of GLM procedure (SPSS Inc.,

determine the effects of dietary protein content, crystal

amino acid supplementation and interaction of the two

fac-tors When interaction between protein level and amino

acid supplementation was statistically significant for a ticular response, differences among protein levels withineach diet type were determined using Tukey’s mean separa-tion Treatment effects and interactions were considered

Fish readily accepted the experimental diets At the end of

were no significant differences in survival among fish fed allthe diets (Table 3) Weight gain, specific growth rate (SGR),feed conversion ratio (FCR), FI, nitrogen retention (NR),lipid retention (LR) for grass carp after 9-week feeding trialare presented in Table 3 FI and NR were significantlyincreased with the increase in dietary protein level and the

WG and SGR also showed the same trends, but there wasinteraction found between dietary protein level and the

significantly decreased with the increase in dietary proteinlevel and the supplementation of lysine and methionine

Table 2 Amino acid composition of experimental diets for grass carp (g kg 1

dry diets) Amino acids

Diet

Essential amino acids

Tryptophan is calculated from NRC.

As an analog of methionine, MHA-Ca cannot be detected by the amino acid analyser, so

methionine value was analysed the sum of MHA-Ca and Methionine.

.

Aquaculture Nutrition 18; 589–598 ª 2012 Blackwell Publishing Ltd

The proximate compositions of the whole body, white

mus-cle and liver of the grass carp are shown in Table 4 No

significant differences were found in the whole body

mois-ture, protein and ash contents of fish among all the diet

showed decreasing trends when fish was fed diets with

The highest muscle lipid content of fish was found in

28CP-diet treatment, while the lowest muscle lipid content

of fish was found in 32AA-diet treatment 32CP, 30AA,

30CP and 28AA gave the intermediate results of muscle

lipid content No significant differences were found in

mus-cle moisture and protein contents of fish among all the diet

Liver moisture significantly increased with the increase in

dif-ferences were found in liver protein contents of fish among

(HSI), intraperitoneal fat ratio (IPF) and viscerasomatic

index (VSI) of grass carp fed experimental diets are presented

in Table 4 VSI, IPF, CF and HSI decreased with increasing

dietary protein levels among diet treatments without amino

acids supplementation VSI, IPF, CF and HSI showed a

decreasing trend among diet treatments with amino acids

supplementation

The amount of TAN discharged into water among different

diet treatments are shown in Fig 1 Fish fed 32CP diet

dis-charged more TAN than that fed 30CP and 28CP diets,

indicating higher discharges of TAN at higher dietary

protein level, fish fed lysine and methionine supplemented

diets discharged less TAN, demonstrating that lysine and

Fish were fed to apparent satiation, and feed consumption

was affected by both the energy and protein content of the

diets FI of grass carp was increased by dietary protein

con-tent An increase in FI due to an increase in dietary protein

content, was also observed by Kim et al (2001) for the dock (Melanogrammus aeglefinus L), and by Luo et al.(2004) for the grouper Epinephelus coioides Compared withmost aquaculture fish species, grass carp has a low energyrequirement, grass carp preferentially adjusted intake toprotein before energy (Du et al 2005) FI of grass carp wasalso significantly increased with addition of lysine andmethionine Amino acids deficiency causes loss of appetite,resulting in low FI as shown in Chanos chanos (Borlongan

had-& Benitez 1990), Labeo rohita (Khan had-& Jafri 1993),

indicated that Oncorhynchus mykiss showed a preferencefor the balanced amino acid diet over the imbalanced aminoacid, and Oncorhynchus mykiss can also discriminatedeficiency of lysine in diets and show a rapid reduction in theconsumption of the amino acid imbalanced diet (Yamamoto

Dabrowski (1977) found there were a linear relationshipbetween dietary protein level and body protein and growth

low-protein diets induced adverse effects on growth mance of grass carp, there were significant differences ingrowth performance of fish fed 32CP, 30CP and 28CPdiets, which indicated that grass carp need to be fed dietwith higher protein content

perfor-In the present experiment, the growth performance of fishfed 32AA and 30AA diets were significantly higher thanthat of fish fed 32CP and 30CP diets Results of the present

growth and feed utilization of grass carp can be achieved by

187.53 207.08 157.31 170.64

128.87 141.31

0 50 100 150 200 250

32AA 32CP 30AA 30CP 28AA 28CP

Figure 1 Total ammonia nitrogen discharged into water (mg kg 1

body weight, mean ± SD) after fed 8 h.

researchers also have demonstrated that Indian Major Carp

(Mukhopadhayay & Ray 1999; Sardar et al 2009), Nile

tilapia (Furuya et al 2004), Rainbow trout (Cheng et al

2003), Red sea bream (Pagrus major) (Takagi et al 2002)

and Pacu (Abimorad et al 2009) fed diets supplemented

with lysine and methionine had better growth performance

Addition of multiple amino acids to reduce dietary protein

content have been widely studied and used in the

produc-tion animal industry The dietary digestible crude protein of

(Botaro et al 2007) Gaylord & Barrows (2009) found that

plant-based dietary protein content for rainbow trout could

supplement-ing lysine, methionine and threonine without growth

reduc-tion The dietary protein content for common carp also

supple-menting lysine without growth reduction (Viola et al

1992a) In the present experiment, fish fed 30AA diet had a

higher growth than fish fed 32CP diets, 32CP diet was

defi-cient of lysine and methionine for grass carp, and 30AA

diet was balanced by providing lysine and methionine So

we can slightly reduce the dietary crude protein in the

prac-tical diets through balancing amino acids profile But the

growth performance of grass carp fed 32AA diet was

signifi-cantly higher than that of fish fed 30AA diet, which

indi-cated that dietary protein could not be reduced by

supplementing lysine and methionine if amino acids meet or

exceed the requirement The present results indicated that

no significant differences were found in growth performance

of grass carp fed 28CP and 28AA diets Research on the

Blue catfish Ictalurus furcatus also demonstrated that the

growth performance of catfish fed low-protein diets was not

improved by supplementing lysine and methionine (Webster

fish meal-based diets with low protein to energy ratios can

improve the protein utilization in rainbow trout

(Yamamot-o et al 2005), because the relative pr(Yamamot-op(Yamamot-orti(Yamamot-on (Yamamot-of the

ingredi-ents containing protein, that is, fish meal, soybean meal and

wheat flour, were the same between diets having different

protein to energy ratios, and therefore, the essential amino

acid balance of the test diets excluding the supplemental

amino acids is the same In our study, 28CP diet fed to the

grass carp had lower soybean meal content and higher

maize meal content than in the high-protein diets, which

resulted in inferior essential amino acid balances The effect

of supplemental limited amino acids to the lower-protein

diets for grass carp as well as for pig noted above was

con-sidered to be the result from the improvement in dietary

amino acid balance, not the same effect as found in the

low-protein diets with the same essential amino acid balance

as the high-protein diet (Kerr & Easter 1995)

Dietary protein levels also affected the morphologicalmeasurements of grass carp (Table 4), VSI, IPF and HSIwere inversely related to dietary protein levels This rela-tionship has also been reported in several other studies(Brown et al 1992; Yang et al 2002; Gaylord & Barrows2009) In our study, VSI, IPF and HSI showed a signifi-cant decreasing trend with lysine and methionine supple-mentation Gaylord & Barrows (2009) also found HSI andIPF of rainbow trout (Oncorhynchus mykiss) were signifi-cantly reduced with multiple amino acid supplementation

in plant-based feeds Brown et al (1992) suggested thatIPF and HSI reflect the proportional accumulation ofenergy in both the abdominal cavity and the liver, and ithas been widely acknowledged that feeding diets deficient

in amino acid results in excess energy deposition as fat inthe liver, fillet or abdominal cavity The results indicatedthat muscle, liver and whole body lipid contents of fish fed32CP diet were slightly lower than that of fish fed 30CPand 28CP diets, and muscle, liver and whole body lipidcontents showed a reducing trend with increasing lysineand methionine supplementation Several studies havedemonstrated that supplementation of lysine-and methio-nine-deficient diets with lysine and methionine reduced car-cass lipid content of Indian Major Carp (Labeo rohita H.)and rainbow trout (Cheng et al 2003; Sardar et al 2009)

But there was no differences in the body composition ofchannel catfish fed diet with supplemental lysine andmethionine (Li & Robinson 1998) The reduction in carcasslipid content of fish may be related to enhanced proteinsynthesis as a result of lysine and methionine supplementa-tion The NR was increased with lysine and methioninesupplementation

Total ammonia nitrogen was directly related to dietarynitrogen and protein intake in teleosts (Rychly 1980; Beam-ish & Thomas 1984; Engin & Carter 2001; Yang et al

2002) Our results indicated TAN excretion of grass carpincreased with increasing dietary protein levels regardless

of amino acid supplementation In the present experiment,the diets were isoenergenic, the diets with less dietary pro-tein levels had higher dietary carbohydrate levels (i.e

maize; Table 1), and conversely, the diets with higher tary protein levels had lower dietary carbohydrate levels

die-Some researchers found that increasing the dietary level

of non-protein digestible energy could increase NR bydecreasing nitrogen losses (Kaushik & Oliva Teles 1985;

Me´dale et al 1995) In the present study, high crudeprotein diets resulted in higher excretion of ammonia,

.

Aquaculture Nutrition 18; 589–598 ª 2012 Blackwell Publishing Ltd

which is in agreement with other reports (Savitz et al.

1977; Chakraborty et al 1992; Cheng et al 2003) TAN

excretion of grass carp was also reduced with lysine and

methionine supplementation in every dietary CP levels

Viola & Lahav (1991) reported that feeding common carp

excre-tion per unit WG by 20% Viola et al.(1992b) further

reported that common carp could reduce nitrogen excretion

methionine (0.3%) Cheng et al (2003) also found that

TAN excretion by rainbow trout was reduced with lysine

supplementation In this study, protein retention of grass

carp was slightly increased with lysine and methionine

sup-plementation, and ammonia is the major end product of

protein catabolism (Elliott 1976), so TAN excretion of

grass carp was reduced with lysine and methionine

supple-mentation

In conclusion, results of the present investigation indicated

the growth performance of grass carp can be improved with

supplementation of lysine and methionine in practical diets,

acids profile TAN excretion of grass carp was also reduced

with lysine and methionine supplementation

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pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro- pro-.

Aquaculture Nutrition 18; 589–598 ª 2012 Blackwell Publishing Ltd

1 2 2 1

Atlantic salmon fed diets devoid of fishmeal but added

growth and lipid deposition without affecting protein

accre-tion as compared to fish fed a fishmeal-based control diet

The aim of the current study was to assess whether higher

inclusion of FPC improved the growth and lipid deposition

of Atlantic salmon (initial body weight 380 g) fed high

plant protein diets Quadruplicate groups of fish were fed

79 days The rest of the diet protein was a mixture of plant

proteins The lipid source used was fish oil A

fishmeal-based diet was included as a positive control for growth

performance None of the test diets differed from the

posi-tive control-fed fish in voluntary feed intake, growth

per-formance or nutrient accretion Thus, the test diets were

found appropriate to assess the effect of FPC inclusion

Replacement of fishmeal with increasing concentration of

FPC did not affect voluntary feed intake (P = 0.56), but

growth performance decreased (P = 0.02) resulting in an

increased feed conversion ratio (P = 0.003) Viscerosomatic

index decreased as diet FPC inclusion increased (P = 0.012)

without affecting the dress out weight (P = 0.08) Thus, the

FPC inclusion was because of a higher visceral mass Possible

reasons for the reduced visceral mass following addition of

FPC to high plant protein diets are discussed

plant proteins, taurine, visceral mass

Received 15 June 2011; accepted 13 November 2011

Correspondence: Marit Espe, National Institute of Nutrition and Seafood

Research (NIFES), PO Box 2029, Nordnes N-5817, Norway E-mail:

marit.espe@nifes.no

Any increase in farmed fish production requires the use ofalternative protein sources as the wild fish catch will notincrease to any extent (Tacon 1995; Tacon et al 2006) Gen-erally, fish fed diets with high inclusions of plant proteiningredients shows poorer performance than fish fed marineingredients (Gomes et al 1995; Kaushik et al 1995, 2004;Mambrini et al 1999; de Francesco et al 2004) When thetotal or a major proportion of diets are replaced by plantproteins and the diets are added hydrolysed fish protein con-centrate (FPC), voluntary feed intake and growth improve(Fournier et al 2004; Espe et al 2006, 2007) Low inclusion

of FPC in fishmeal-based diets has been reported to improvethe voluntary feed intakes and growth, while higher inclu-sion levels decreased the growth (Espe et al 1999; Refstie

FPC, the growth was reduced because of a reduced lipidaccretion, while protein accretion was unaffected (Espe

the addition of FPC to plant-based diets might be due tothat FPC also supplies non-amino acid nitrogen compoundshaving an attractive smell and supplies taurine that is not aconstituent of plant ingredients (Espe & Lied 1999; Liaset &Espe 2008) Atlantic salmon synthesize taurine providedthat they are fed adequate methionine (Espe et al 2008,

2010, 2011) Recently, Espe et al (2012) reported that

diet reduced the body lipid without affecting the body tein in juvenile Atlantic salmon and increased both liver andmuscle pools of free amino acids In addition, liver concen-tration of polyamines increased in fish fed the taurine sup-

supplemented with taurine Atlantic salmon fed limiting diets had reduced liver taurine and stored more tri-acylglycerol (TAG) in liver as compared to fish fed diets

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Aquaculture Nutrition

being adequate in methionine (Espe et al 2010) On the

other hand, Dias et al (2005) reported lower activity of the

lipogenic enzymes and decreased TAG accumulation in the

marine teleost European seabass (Dicentrarchus labrax) fed

soy protein containing low methionine as compared to a

fishmeal-fed control Gaylord et al (2007) reported that

die-tary methionine but not taurine affected lipid metabolism

and viscera mass in juvenile rainbow trout (Oncorhynchus

mykiss) Rats fed proteins containing low taurine and

gly-cine contained more visceral fat as compared to rats fed

pro-tein sources with higher concentrations of these amino acids

(Liaset et al 2009) As our previous experiments in which

Atlantic salmon were fed high plant protein diets only tested

and growth improved by higher FPC inclusion in

fishmeal-based diets (Espe et al 1999), the current study aimed to

fishmeal present affected voluntary feed intake and growth

As a positive control for voluntary feed intake and growth,

was used

Five 6-mm extruded diets were prepared in which the 200 g

of FPC such that the final diets contained 0, 37.5, 75,

from blue whiting (Micromesisticus poutasou) FPC mirrors

fishmeal protein and amino acid composition, while its

sol-ubility may differ dependent on storage time and

tempera-ture (Espe & Lied 1999) Therefore, the protein solubility

was analysed in the diets, and the higher the inclusion of

FPC the higher is the solubility of dietary proteins The

lowest fishmeal inclusion was set to 5% as Atlantic salmon

fed diets without any fishmeal grew less than the

fish-meal was included to such diets, growth did not differ from

the fishmeal-based control diet (Espe et al 2007) The

remaining diet protein was a blend of plant protein

ingredi-ents As a control for growth performance and nutrient

with-out any FPC was included in the trial The lipid source

used in all diets was fish oil All test diets contained the

same energy content as well as an amino acid profile to

ful-fil the requirement of Atlantic salmon The diet

composi-tion and amino acid profiles are listed in Tables 1 & 2

Fifty Atlantic salmon with a mean body weight (BW) of

380 g were used in each of 24 tanks (size of the tanks of

Each tank was supplied with running seawater (salinity

7.2 °C from November 2006 to February 2007) at a flow rate

to maximize growth Each experimental diet was randomlyallocated among the tanks, and each diet was fed to quadru-plicate tanks of fish The fish were fed three times daily toapparent satiation using automatic belt feeders (HøllandTeknologi, Sandnes, Norway) All tanks were equipped withfeed collectors (Excess Fish feed collector; Hølland Tekno-logi) to measure the actual feed intake Uneaten feed was col-lected daily At the start and end of the experiment, fish wereindividually weighed A pooled sample of 10 fish was col-lected at the start of the experiment to be analysed for chemi-cal composition At the end of the growth experiment, sixfish from each tank were sampled 5 h postprandial and killedwith a sharp blow to the head Individual BW to the nearest

g and fork length to the nearest 0.5 cm were recorded in each

of the sampled fish and the condition factor (CF = BW in

caudal vein into heparinized syringes and centrifuged at

1800 g for 10 min before plasma was collected Viscera andliver were collected and weighed for calculation of indexes

Table 1 Composition of diets (g kg ) Fishmeal (g kg
1) 350 50 87.5 125 162.5 200 FPC (g kg
1) Control 150 112.5 75 37.5 0

Micronutrient mixture is proprietary of EWOS Innovation but contains vitamins and minerals added to fulfil the requirement of Atlantic salmon (NRC 1993) in addition to the inert marker, 0.1%

yttrium oxide Plant protein was soy protein concentrate and pea protein concentrate blended 1 : 1.14 by weight.

FPC, fish protein concentrate.

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Aquaculture Nutrition 18; 599–609 ª 2012 Blackwell Publishing Ltd

relative to BW Liver and white epaxial trunk muscle was

dissected and flash frozen in liquid nitrogen Remaining fish

in the tanks were then fed their respective diets for a period

of 1 week after which faeces was collected by stripping

Thereafter, feed was withheld for 2 days until the rest of the

fish were individually weighed and the length recorded

Evis-cerated fish were also weighed and used to calculate dress

out weight (10 fish per tank) Before handling, fish were

sedated with AQUI-S (4 ppm) and fully anaesthetized with

approved by the Norwegian Board of Experiments with

Liv-ing Animal

All chemical analyses were carried out in duplicate

Nitro-gen was determined after total combustion using a NitroNitro-gen

Analyser (Perkin Elmer, 2410 Ser II, Norwalk, CT, USA).The total lipid in feed and faeces was determined gravimetri-cally as the sum of free and bound lipid Free or looselybound lipid was extracted with petroleum ether and dried at

103 ± 1 °C The samples were thereafter hydrolysed withHCl in a Tecator Soxtec Hydrolysing unit to release thebound lipid, which was extracted with petroleum ether anddried at 103 ± 1 °C Dry weight and ash content were deter-mined gravimetrically after freeze-drying the samples anddried to constant weight in an oven at 550 °C, respectively.Gross energy was analysed by Parr Bomb Calorimetry(Moline, IL, USA) Muscle lipid content was analysed gravi-metrically after extraction with ethyl acetate Yttrium wasdetermined in both the feed and faeces by the use of ICP-

MS as described (Espe et al 2006) Dietary and faecalamino acid concentration was determined after acidichydrolysis in 6 N HCl at 110 °C for 22 h and prederivatiza-

according to the study by Cohen & Strydom (1989) Dietarytryptophan was determined after basic hydrolyses in Ba

column) as described (Liaset et al 2003) Amino acid position in de-proteinized plasma, liver and muscle wasdetermined on the Biochrom 20 plus Amino Acid Analyzer(Amersham Pharmacia Biotech, Uppsala, Sweden) equippedwith a lithium column using postcolumn derivatization withninhydrin as described (Espe et al 2006) The degree ofhydrolysis in the feeds was determined as the free alphaamino groups relative to the total after being hydrolysed in

com-6 N HCl at 110 °C for 22 h as described (Espe et al 1999).Lipid classes in liver were determined as described by Bell

2010) S-adenosylmethionine (SAM) and

detected at 254 nm as described (Wang et al 2001) Thepolyamines putrescine, spermine and spermidine were

dansyl chloride and separated on a C18 reverse phase umn and detected at 254 nm as described (Liaset & Espe2008) The quantification of SAM, SAH and the polyamineswas performed by standards from Sigma (Sigma Aldrich,Munich, Germany)

col-Feed conversion ratio (FCR) was calculated from theamount of diet fed (kg dry matter) and the total biomass(kg) gained:

Table 2 Amino acid composition (g per 16 g N) of the

Tau is taurine, and NAA-N (non-amino acid N) is nitrogen not

accounted for by the amino acid analyses Tau is not included in

the IAA : DAA ratios Solubility was analysed as the % free of total

alpha amino acid N in diets analysed after reaction with TNBS.

FPC, fish protein concentrate.

FCR¼ ðkg diet fedÞ  ðkg final biomass

Specific growth rate (SGR) was calculated as % daily

growth increase

in grams, respectively

Protein efficiency ratio (PER) was calculated as weight

gain (g) for each gram protein fed/consumed

Protein productive value (PPV) and energy productive

value (EPV) were calculated as retained nutrient (g) of fed

intakes and performance between fish fed the test diets and

the fishmeal-fed control Homogeneity in variation was

tested using Levenes test To determine the effect of diet

(i.e FPC inclusion) on performance and tissues amino acid

concentrations, regression analyses were applied according

to the statistical design of the experiment Any differencesbetween the liver lipids, SAM, SAH and polyamines in fishfed the control feed and those fed the test diet added 0 or

Tukey’s test All tests were performed using the statistical

USA), and P < 0.05 was accepted significantly different

To determine whether FPC improved voluntary feed intakeand growth, Atlantic salmon were fed high plant proteindiets where up to 75% of the dietary fishmeal present wasreplaced with FPC Growth increase relative to initial BWwas 187% in the fishmeal-fed control, while the corre-

significant differences existed between fish fed the fishmealcontrol diet and any of the test diets in either mean volun-tary feed intake or growth performance Neither did thetest diets differ from the control-fed fish in protein accre-

were digested equally well as was the reference fishmeal

Table 4) Thus, the test diets supported the growth andnutrient accretion equally well as did the fishmeal-basedcontrol diet containing from two to seven times more fish-meal Based on these results, the test diets were foundacceptable to be used to evaluate the effect of increasingthe FPC inclusion in the Atlantic salmon using the regres-sion design

Table 3 Start and end BW (g), growth increase (DG,% of start wt), mean feed intake (MFI, g per fish per day), feed conversion ratio

(FCR), hepatosomatic index (HSI), visceasomatic index (VSI), condition factor (CF), protein efficiency ratio (PER) and protein productive

value (PPV) in Atlantic salmon fed the respective diets for a period of 79 days

Values are tank means ± SE, n = 4.

BW, body weight; FPC, fish protein concentrate.

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Aquaculture Nutrition 18; 599–609 ª 2012 Blackwell Publishing Ltd

The dietary amino acid profiles were similar in all of the

test diets where the fishmeal present was replaced with

increased FPC inclusion increased dietary taurine anddecreased tryptophan as these amino acids were higherand lower in the FPC as compared to the fish meal,

Table 4 Apparent digestibility (%) of crude compounds and amino acids in Atlantic salmon fed the test diets The control diet is listed for comparison

Values are tank means ± SE.

na, not analysed; FPC, fish protein concentrate.

Table 5 Effect of dietary FPC

inclu-sion (x, %) on growth performance

Daily protein gain (g kg
1ABW) y = 1.8
0.01x 0.27
0.26 Daily lipid gain (g kg
1ABW) y = 1.6
0.01x 0.18
0.31

ABW is arbitrary body weight (initial BW+end BW/2) VFI is the mean voluntary feed intake Dress out weight is the BW-viscera weight The other abbreviations are given in Material and methods, and bold letters indicate statistical difference ( P < 0.05).

respectively The higher the FPC inclusion, the higher the

nitrogen solubility becomes (Table 2) Apparent amino

acids digestibility of all diets were high (Table 4), and

replacement of the dietary fishmeal with FPC had no effect

on digestibility Replacement of the fishmeal with FPC had

no significant effect on voluntary feed intake (P = 0.56),

but reduced the end BW (P = 0.02) As a consequence,

these fish had a higher feed conversion (FCR, P = 0.003,

Table 5) Replacement of fishmeal with increasing amounts

of FPC had no significant effect on protein utilization

(PER and PPV, Table 5) The body lipid-to-protein ratio

was not affected by treatment (P = 0.64) neither was the

lipid nor protein gain Increasing the dietary FPC inclusion

significantly reduced VSI (P = 0.012), while HSI and CF

did not significantly decreased (Table 5) The dress out

weight was not significantly affected (P = 0.08) by the tary FPC inclusion (Table 5)

die-Plasma, liver and muscle pools of free amino acids ally decreased when dietary fishmeal was replaced withFPC (Table 6) However, the decrease in amino acid con-centration was only significant for methionine and gluta-mine in plasma and muscle, histidine in plasma and liver,phenylalanine in plasma, and glycine and proline in muscle,while tyrosine was significantly reduced in plasma, liverand muscle (Table 6) Free tryptophan was only found inplasma, while the tryptophan concentration in both liverand muscle was below the detection level In plasma, tryp-tophan decreased as dietary FPC increased The only freeamino acids that increased when dietary FPC inclusionincreased were arginine, serine and alanine (Table 6) These

gener-Table 6 Effects of dietary FPC inclusion (x, % inclusion) on postprandial amino acids and nitrogen metabolites in plasma, liver and white

muscle (y, lmol per 100 g or lmol dL
1 )

Bold letters indicate statistical differences.

FPC, fish protein concentrate.

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Aquaculture Nutrition 18; 599–609 ª 2012 Blackwell Publishing Ltd

changes in amino acid profiles occurred even though the

dietary concentration of these amino acids were similar

(Table 2) and the mean voluntary feed intake was

unaf-fected by treatment (Table 5) The nitrogen-containing

metabolites generally declined as dietary FPC increased

(Table 6) However, the postprandial taurine concentration

in plasma, liver or white trunk muscle was not affected by

the FPC inclusion (Table 6) even though FPC inclusion in

the diets increased the diet taurine (Table 2) Cystathionine

and urea were significantly reduced in plasma, liver and

white trunk muscle, while citrulline only was significantly

decreased in the plasma (Table 6) The imidazole

muscle rendering a significant increase in plasma 1-methyl

histidine (Table 6)

The altered amino acid profiles thus indicate that the

inclusion of FPC in the diets caused changes in the

metab-olism Therefore, the control-fed fish and the fish fed the

further to determine any differences in lipid classes,

poly-amines or the methylation status (i.e SAM, SAH, SAM/

SAH ratio) in the liver The dietary FPC inclusion did not

affect the liver TAG, diacylglycerol (DAG) or

non-esteri-fied fatty acids (Table 7) Neither did these values differ

from the control-fed fish Total cholesterol concentration

was lower in liver of fish fed the diet containing 150 g

(Table 7, P = 0.06) SAM and SAH in liver were not

sig-nificantly different in fish fed either the fishmeal control

However, the higher mean SAM and lower SAH in fish fed

sig-nificantly higher (P = 0.001) ratio of SAM to SAH in liver

of fish fed this diet as compared to the control-fed fish andthose fed the diet without any FPC (i.e the diet containing

higher FPC inclusion showed an increased ratio of arginine

to lysine in plasma and liver (Table 6), and as arginineavailability might affect the polyamines, these were analy-

spermine (P = 0.055), but did not differ significantly inputrescine and spermidine concentrations (Table 7) How-ever, the higher mean spermine resulted in a significantlylower ratio (P = 0.02) of spermidine to spermine in the

compared to fish fed both the control diet and the dietwithout FPC (Table 7)

Fish fed higher levels of FPC apparently spent more energy

on growth and metabolism compared with fish fed the dietswith lower FPC inclusion as the fish consumed equalamounts of feed, but grew less Increasing the dietary FPCincreased the solubility of the dietary proteins Previously

we reported that lower inclusion of FPC improved growthperformance, while higher inclusion of FPC reducedgrowth in Atlantic salmon fed fishmeal-based diets (Espe

Table 7 Neutral lipid classes, taurine-to-methionine ratio, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), SAM-to-SAH ratio, arginine-to-lysine ratio, the polyamines putrescine, spermine and spermidine and the spermidine-to-spermine ratio as occurring 5 h postprandial in liver of fish fed the control diet and the test diets containing 0 and 150 g FPC kg
1

SAH was not homogeneous in variance, and Kruskall–Wallis test was used Values are mean ± SE, n = 4.

DAG, diacylglycerol; FPC, fish protein concentrate; NEFA, non-esterified fatty acids; SAH, S-adenosylhomocysteine; TAG, triacylglycerol Row means followed by different letters are significantly different (p < 0.05).

et al.1999) We speculated whether that was because of the

different absorption profiles present in fish fed hydrolysed

proteins as compared to fish fed the protein-bound proteins

(Espe et al 1993) Therefore, the solubility of the protein

and their absorption may be one of the contributors for

the reduced growth observed in the current study As

apparent digestibility was unaffected by the FPC inclusion

(regressions not shown), and only some of the tissue free

amino acids were significantly reduced while arginine and

alanine increased, it is not likely that the reduced growth

was because of protein solubility Dietary FPC increased

diet taurine concentration, which increased the taurine

P = 0.00001) The increase in the dietary FPC reduced the

con-centration was, however, not affected by FPC inclusion,

and no significant correlation between liver taurine

concen-tration and VSI was observed (P = 0.13, r = 0.36) Thus,

the reduction in VSI following increased FPC inclusion

cannot be explained by any changes in liver taurine

concen-tration The improved growth in fish fed low dietary FPC

diets was because of a higher viscera mass as the dress out

weight was unaffected by FPC consumption As viscera

were not collected in the current study, neither metabolites

nor enzymes could be analysed to verify whether these were

affected by the FPC inclusion

Previously we reported that methionine limitation reduced

tissue taurine concentration and increased the relative liver

weight in Atlantic salmon (Espe et al 2008) Furthermore,

methionine limitation increased liver TAG, increased liver

lipogenic enzymes and reduced bile acid in plasma and

fae-ces (Espe et al 2010) without affecting carcass

lipid-to-protein ratio Contrary to methionine limitation, taurine

supplementation in diets fed to juvenile Atlantic salmon

reduced whole-body lipid-to-protein ratio (Espe et al 2012)

Gaylord et al (2007), on the other hand, reported that

methionine limitation affected the visceral mass in juvenile

rainbow trout, while taurine supplementation had no effect

on viscera mass These differences may be due to the

differ-ent concdiffer-entrations of taurine tested in the rainbow trout

and Atlantic salmon studies In the current study,

methio-nine was not limiting, and only plasma-free methiomethio-nine

con-centration (P = 0.050) was reduced when FPC inclusion

increased Thus, it is unlikely that limitations in methionine

caused the effects observed in this experiment

In addition to providing taurine, FPC also contains other

compounds that might be limiting when plant proteins

replace fishmeal (i.e betaine, choline, carnitine and

crea-tine) These metabolites might be synthesized in the mals, but common for several of these is the requirement ofmethyl groups from SAM (Noga & Vance 2003; Brosnan

are fed diets low in fishmeal, some of these non-proteincompounds might become limiting or the synthesis of themmight deplete cellular SAM and/or cost more energy, thusreducing the growth or increase lipid deposition (Wilson &

Poe 1988; Griffin et al 1994; Ji et al 1996; Chawla et al

1998; Kasper et al 2000; Slow & Garrow 2006; Huang

syn-thesizing SAM in animals, and the availability of SAM isknown to interact with lipid metabolism (Mato et al 2002;

Anstee & Goldin 2006; Fukada et al 2006; Hirsche et al

2006; Oda 2006; Zhan et al 2006) The ratio of SAM toSAH has been used as an indicator for the capacity ofmethylation within cells (Kerr 1972; Cantoni & Chiang

had a higher SAM/SAH ratio, indicating a higher capacity

of methylation as compared to the control fish and thosefed the diet without FPC inclusion Atlantic salmon fedmethionine deficient diets had reduced liver SAM (Espe

Methionine enrichment to a fishmeal-based diet, on theother hand, had no effect on either liver SAM or TAG, butincreased liver cystathionine and taurine (Espe et al 2011)

Thus, the availability of methyl is likely to be one of thefactors contributing to the lower viscera mass In the cur-rent study, no differences in liver neutral lipid were found

in fish fed the control feed or the diets added 150 or 0 g

the liver taurine concentration affected by dietary ment Thus, it is unlikely that inclusion of FPC as used inthe current study would affect lipogenesis in the liver How-ever, this does not rule out that FPC inclusion may affectthe lipogenesis in visceral adipose tissues as reported in therat being fed diets in which the protein source was low intaurine and glycine (Liaset et al 2009)

treat-FPC inclusion increased the arginine-to-lysine ratio inboth plasma and liver (Table 6), rendering more arginineavailable for other metabolic pathways such as polyaminesynthesis (Berge et al 2002) Previously we reported anincreased arginine-to-lysine ratio in liver of juvenile Atlanticsalmon fed plant-protein-based diets supplemented withtaurine even though dietary arginine-to-lysine ratio wasequal between treatments Concomitantly, the polyamineconcentration in liver was higher (Espe et al 2012) The die-tary taurine concentration in the juvenile feed was similar to

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Aquaculture Nutrition 18; 599–609 ª 2012 Blackwell Publishing Ltd

(0.2–0.3 g per 16 gN) Moreover, in the current trial, the

spermine concentration was higher in fish fed the diet

spermidine to spermine indicative of interactions between

diet FPC inclusion and the polyamine metabolism In a

study involving the transgenic mouse model where the

poly-amine turnover is increased, less white adipose tissue and

reduced energy stores were present (Pirinen et al 2007)

Obese rats up-regulated several genes associated with the

lipid synthesis and had higher concentrations of polyamines

in white adipose tissue as compared to their lean

counter-parts (Jamdar et al 1996) Thus, the reduced viscera mass

found when Atlantic salmon are fed FPC probably are

because of the fact that FPC contained several non-amino

acid nitrogen compounds in addition to taurine of which

affect viscera mass production The changes in the pools of

amino acids in fish fed the FPC diets may reflect a change in

the endogenous synthesis of these non-amino acid nitrogen

compounds Thus, any possible lipid-reducing mechanisms

of these compounds should be studied in more details to

unravel which one and in which concentration reduce the

visceral lipid accretion most efficient to be able to produce a

healthy fish without influencing growth and dress out

weight

Increasing the dietary level of FPC reduced white trunk

muscle concentration of the dipeptides carnosine and

anserine, while plasma concentrations of their constitute

free amino acids increased In contrast to an earlier report

by Aksnes et al (2006) which showed that neither taurine

nor anserine in muscle or the whole body of rainbow trout

differed by different inclusion levels of hydrolysed proteins

These differences may be due to sampling at different

post-prandial times, size of the fish and the composition and the

incorporation of the hydrolysed protein used As anserine is

the major buffering agent in white trunk muscle of Atlantic

salmon (VanWaarde 1988), this might have implication for

the function and stability of muscle cells including the

quality of the harvested fish (Ruiz-Capillas & Moral 2001;

Ogata 2002; Førde-Skjærevik et al 2006) and protection of

the muscle from fatigue development after longer periods of

swimming (Takahashi et al 2008) However, these aspects

were not investigated in the current study

Replacement of fishmeal with FPC in plant-protein-based

diets fed to Atlantic salmon did not affect either voluntary

feed intake or improve growth Instead the fish fed diets

with increasing FPC inclusion showed lower growth The

apparently improved growth in fish fed the diets with lowFPC inclusion was because of an increased viscera mass asdress out weight was unaffected The mechanisms under-pinning these observations require further investigationsbecause an increase in viscera mass is associated with obes-ity and may affect the health of the farmed fish

Liv Oma, Ewos Innovation AS is thanked for taking care

of the experimental fish and sampling thereof JosephMalaiamaan, NIFES is thanked for technical assistance inanalysis and sampling Parts of the work were supported

by the Norwegian Research Council

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1 2 1 2 3

1

This study investigated the combined effects of photoperiod

and dietary P level on bone osteoclast and osteoblast

activ-ity, and morphology, as well as plasma vitamin D status in

Atlantic salmon post-smolts The fish were reared under

continuous light (LL) or 12 h light/dark (LD) per day, and

die-tary P, HP) for 79 days in seawater LL significantly

resis-tant acid phosphatase (TRACP) activity, and decreased

level and bone alkaline phosphatase activity, but decreased

bone TRACP activity and matrix metalloproteinase 13

(mmp13) mRNA transcription, and vertebral mineral

con-tent, stiffness and length/dorso-ventral diameter (l/d) ratio

A significant interaction between light and P was only

observed on the l/d ratio, and the LP-LL group was the

only group that developed vertebrae with a compressed

morphology (significantly lowest l/d ratio) In practical

terms, these results show that Atlantic salmon post-smolts

under continuous light may need P supplemented diets to

support normal bone development

photoperiod, vertebrae, vitamin D

Received 7 April 2011; accepted 18 November 2011

Correspondence: P.G Fjelldal, Institute of Marine Research (IMR),

Matre Aquaculture Research Station, N-5984 Matredal, Norway.

E-mail: pergf@imr.no

The use of continuous light (LD24:0) is common practice

in farming of Atlantic salmon (Salmo salar L.) due to itswide range of advantageous effects on salmon physiology

Continuous light is used to induce smoltification son et al 1991), stimulate growth (Berg et al 1992; Hansen

during on-growth (Hansen et al 1992) and time the finalstages of sexual maturation (Taranger et al 1998) Further-more several experiments have shown that continuous lightaffects the skeleton in Atlantic salmon differently than anatural photoperiod; it changes the regional growth pattern(Fjelldal et al 2005), mRNA transcription of the growthhormone receptor and insulin-like growth factor 1 genes(Nordgarden et al 2006) and osteoid incorporation (War-gelius et al 2009) of the vertebral column In salmonids,information about the day–length is conveyed throughblood levels of melatonin, which is produced and secreted

by the photo-sensitive pineal gland (Ekstrøm & Meissl1997) Interestingly, pinealectomy reduces bone mineralcontent and induces severe vertebral deformities (Fjelldal

continuous light have a reduced bone mineral content whencompared to under natural light (Fjelldal et al 2005) Thevertebral column of Atlantic salmon comprise on average

58 amphicoelous vertebrae (Kacem et al 1998) The rims

of adjacent vertebrae are interconnected via intervertebralligaments (Nordvik et al 2005), but the hydrostatic pres-sure of the notochord prevents direct contact between them(Grotmol et al 2003) Ca and P are the most abundantminerals, and collagen I the major structural protein in

in collagen formation (Ornsrud et al 2009), and in the

.

ª 2012 Blackwell Publishing Ltd

2012 18; 610–619 .doi: 10.1111/j.1365-2095.2011.00918.x

Aquaculture Nutrition

regulation of P and Ca homeostasis (Lock et al 2010).

7-dehydrocholes-terol under the influence of sunlight is well established in

mammals but there is no evidence suggesting that fish

post-smolts, sub-optimal P nutrition both elevates plasma levels

increases the risk for vertebral deformities (Baeverfjord

potent stimulator of osteoclastic bone resorption, 1,25

matrix degradation through collagenases such as MMP13

(Uchida et al 2001; Fratzl-Zelman et al 2003) Collagen

XI and V controls the diameter and amount of collagen

fibers (Wenstrup et al 2004; Fernandes et al 2007), and

may thus have an impact on the quality of the extracellular

where it is expressed in the osteoblasts and chordoblasts of

the vertebral column (Wargelius et al 2010) How the

vita-min D system and bone of fish under continuous light

responds to different dietary P levels when compared to

fish under a short day has never been studied To study

this, morphological and physical bone properties, markers

of bone resorption (tartrate resistant acid phosphatase

(TRACP), mmp13), growth and mineralization (alkaline

phosphatase (ALP)), extracellular matrix quality (col11a1)

were measured in quadruple groups of Atlantic salmon

reared under 12 h light/dark or 24 h continuous light per

for 79 days in seawater

(mean weight 230 g) were transferred to seawater and

50 fish per tank The smolt were produced according to

standard procedures for under-yearling smolt production

(for details, see Fjelldal et al 2006) In the period between

11th December 2007 and 27th February 2008, eight tanks

were reared under 24 h continuous light (LL), and eight

tanks under 12 h light/dark per day (LD) The water

satu-ration, measured in the outlet water, was always keptabove 80% Within each photoperiod regime, four tanksreceived a low phosphorous diet (LP-LL and LP-LD), andfour received a high phosphorous diet (HP-LL and HP-LD), leaving four tank per experimental group The dietswere produced by EWOS Innovation, Dirdal, Norway (seeTable 1 for feed composition) The amount of digestibleprotein and available P were calculated by the feed pro-ducer The fish were fed continuously for 12 h per day,

automatic feeders (ARVO-TEC T Drum 2000, Arvotec,Huutokoski, Finland) The feeding level was 2% of wet fishweight For illumination, two 18 W fluorescent daylighttubes (OSRAM L 18W/840 LUMILUX, OSRAM GmbH,

mea-sured under water in the centre of the tank Photoperiodand feeding were controlled automatically by a PC oper-ated system (Normatic AS, Norfjordeid, Norway)

The present experiment was approved by the NorwegianAnimal Research Authority and performed according toprevailing animal welfare regulations

Table 1 Feed formulation and composition of the low (LP) and high phosphorous diets (HP) fed to underyearling smolt for

79 days after transfer to seawater (pellet size 4.5 mm)

Three samplings were performed during the study; 11

December 2007 (day 0), 28 January 2008 (day 49), and 27

February 2008 (day 79) The fish were starved for 48 h

before each sampling point Hundred fish per group (25 per

mea-sured for fork length and body weight at the start of the

experiment (day 0) On day 49 and 79, 20 fish per group (5

per tank) were euthanized, measured for fork length and

body weight, and blood from the caudal vessel was sampled

using a heparinized tuberculin syringe fitted with a 23-gauge

needle Plasma was separated by centrifugation, pooled into

measurement of bone properties, and vertebrae number 1-39

and 44-58 for measurement of enzyme activity and mRNA

transcription

Plasma vitamin D metabolites were measured as described

concentrations were measured using a Maxmat PL

multi-purpose diagnostic analyzer (Maxmat SA, Montpellier,

France) and with Dialab Calcium and Phosphorus reagents

(Dialab, Wien, Austria)

On day 49, 20 fish per group (5 per tank) were euthanized

of bone properties while the remaining vertebrae were used

for TRACP, ALP and gene transcription analyses Neural

and hemal arches and adhering tissue were removed before

the remaining amphicoelous centra were flash frozen (liquid

mortar and pestle Next, 300 mg of bone tissue was dissolved

para-nitrophenyl-phosphate, pH 10.2) or 1 ml of freshly prepared TRACP

10 mM para-nitrophenylphosphate, pH 5.3) was added

Both ALP and TRACP assays were performed in triplicates

200 rpm) and covered from light After 1 h the reactions

centrifuged (10 min, 12.000g) One hundred and forty litres of the supernatant was measured in triplicates at

Total RNA was extracted from vertebrae using FastPrep

Nor-way) according to the manufacturer’s instruction GenomicDNA was eliminated from the samples by RQ DNase I (Pro-mega GmbH, Manheim, Germany) treatment Quantity and

spec-trophotometer (NanoDrop Technologies, Wilmington, DE,USA) Only samples with a 260/280 nm absorbance

synthes-ised from 250 ng of total RNA using a Reverse tion Core Kit using random hexamers according tomanufacturers instructions (Eurogenetec, Seraing, Belgium)

Transcrip-RNA was extracted from the same samples being used forTRACP and ALP analysis Primers for amplification anddetection of col11a1 and mmp13, in addition to the control

using the Primer Express 2.0 software (Applied Biosystems,Foster city, CA, USA), and are listed in Table 2 Real-timePCR was carried out on an ABI 7900HT Fast Real-TimePCR system (Applied Biosystems, Oslo, Norway) using

Foster city, CA, USA), with the following thermal cycling

1 min The samples were run in duplicates in a 96-well PCRplate No-template controls for each gene were run on eachPCR plate To determine which reference gene to use 18S

them displayed an equal efficiency in random samplescollected from the material To determine the efficiency oftargets in relation to reference genes we used the standardcurve method and a validation experiment described in ABIUser Bulletin #2 (ABI 7700 sequence detection system) Inthe validation experiment 250, 125, 62.5 and 31.25 ng of

.

Aquaculture Nutrition 18; 610–619 ª 2012 Blackwell Publishing Ltd

RNA was used for cDNA synthesis, and the slope of log

0.042, col11a1/ef1a = 0.06 and for mmp13/ef1a =
0.03

target and reference are approximately equal The relative

transcription level was calculated using the Comparative Ct

method (ABI User Bulletin #2, ABI 7700 sequence detection

system)

Vertebrae numbers V40-43 were dissected (day 49), the

neural and haemal arches were removed, and the

amphicoe-lus centra were used for mechanical testing and

measure-ment of mineral content The vertebrae were compressed in

the cranial-caudal direction using a texture analyser

(TA-XT2 Texture Analyser, Stable Micro Systems, Haslemere,

resulting load-deformation data were continuously recorded,

and the stiffness calculated for each vertebra according to

Fjelldal et al (2004) After mechanical testing, vertebrae

number V40-V43 of each fish were pooled, defatted in

then incinerated for 13.5 h in a muffle furnace (Mod L40,

(min-eral) weights of each individual were weighed to nearest

Vertebrae from day 79 were radiographed using a portable

X-ray apparatus (HI-Ray 100, Eickenmeyer

Tokyo, Japan) The film was exposed twice for 50 mAs

and 72 kV, and developed using a manual developer (CofarCemat C56D, Arcore (MI), Italy) with Kodak Professionalmanual fixer and developer (KODAK S.A., Paris, France).The pictures were digitalised by scanning (Epson Expres-sion 10000 XL, Seiko Epson Corp., Nagano-Ken, Japan).Vertebral length and dorso-ventral diameter were measured

by means of image analysing software (Image-Pro Plus,

stan-dard error (SEM) Group differences in length, weight,stiffness, mineral content, and dorso-ventral diameterwithin sampling points were tested by a 2-way nested

investi-gated further by the Student Newman-Keuls (SNK) posthoc multiple comparison test to locate any differencesamong treatments Statistical testing of stiffness andlength/dorso-ventral diameter is based on mean values

blood parameters and bone gene transcription and enzymeactivity within sampling points were tested by a 2-way

test A confidence level of 95% was used for all tests

(Table 3) Fish under LL were overall significantly lighter

Table 2 Sequences of oligonucleotide primers used in real-time PCR

difference had disappeared on day 79 (Table 3) The LP-LL

factor than the LP-LD and HP-LD groups on day 49, when

fish under LL had an overall significantly lower (2-way

(Table 3) On day 79, however, photoperiod had no

while fish fed the LP diet had an overall significantly higher

HP diet (Table 3)

There was no mortality, diseases or abnormal behaviors

during the experiment

On day 79, there was a significant effect (2-way nested

dorso-ventral diameter of the vertebrae, with the LP-LL

than the LP-LD, HP-LD and HP-LL groups (Fig 1)

Radio-graphic examples of vertebrae with a normal (high length/

dorso-ventral diameter ratio) and compressed (low length/

dorso-ventral diameter ratio) morphology are shown in

Fig 2a,b

On day 49, LP had significantly decreased (2-way nested

the HP-LL and HP-LD groups displayed significantly higher

HP-LL and HP-LD groups (Table 4) LP significantly

and the HP-LL group displayed a significantly higher (SNK,

than the LP-LL group (Table 4) LP significantly

transcrip-tion, and the HP-LD group displayed a significantly highertranscription than the LP-LL group (Table 4) Except forthe dorso-ventral diameter of the vertebrae, there were nosignificant interaction effects between diet and light on any

of the measured bone parameters on day 49

Table 3 Fork length (cm), body weight (g) and condition factor (CF) (mean ± SE) in Atlantic salmon post-smolts fed diets with a high

(HP) or low (LP) phosphorous level, under continuous light (LL) or 12 h light/dark per day (LD) for 79 days

.

Aquaculture Nutrition 18; 610–619 ª 2012 Blackwell Publishing Ltd

There were no effects of either photoperiod or diet on plasma

levels of total Ca or P, and they showed similar values at the

sampling after 49 and 79 days (Table 5) The continuous

and LP-LL compared to in the HP-LD and LP-LD groups

at both sampling points (Table 5) LP had significantly

after 79 days of feeding, when the LP-LL group had a

(Table 5) There were no significant interaction effectsbetween diet and light on any of the measured plasmaparameters There was a significant positive correlation

of the study on day 79 (Fig 3)

In the present study, light regime and dietary P content both

metabolites in a different and distinctive manner ous light with sufficient dietary P, or LD12:12 with marginaldietary P did not result in bone deformities However, thecombination of continuous light with marginal dietary P led

Continu-to alterations in vertebral morphology that are indicative of

an incipient bone deformity This demonstrates that thecause of a bone deformity can have a multifactorial origin,where continuous light and marginal dietary P are predis-posing risk factors The current findings indicate that effects

of light and low dietary P may interact through the vitamin

D system

Continuous light increased bone TRACP enzyme activity

photoperiod are not previously reported in a teleost The

7-dehy-drocholesterol under the influence of ultraviolet (UV)

(a)

(b)

Figure 2 Radiograph examples of vertebrae (40–43) with high

(normal) length/dorso-ventral diameter ratios (a) or low length/

dorso-ventral diameter ratios (b) Scale bars = 10.0 mm.

Table 4 Bone parameters (mean ± SEM) after 49 days of feeding in Atlantic salmon post-smolts fed diets with a high (HP) or low (LP) phosphorous level, under continuous light (LL) or 12 h light/dark per day (LD)

1 Different lower case letters indicate significant differences (SNK, P < 0.05), with ‘a’ as highest value.

2 Values in italic indicates significant effects (2-way ANOVA , P < 0.05) of diet or photoperiod, or significant diet – photoperiod tions.

interac-irradiation with a subsequent increase in circulating

Although a photochemical conversion of

reported in vitro (Hidaka et al 1989), the consensus is that

exposure (Sugisaki et al 1974; Takeuchi et al 1986; Rao &

Raghuramulu 1996) Alternatively, alterations in circadian

rhythms could explain the observed effect of photoperiod

present study However, there was a positive correlation

was a tendency towards higher plasma levels under LLcompared to LD Furthermore, a positive correlation

in humans (Rejnmark et al 2008) An increase in plasma

least in part, explain the results of the present experiment

Furthermore, the pineal gland is central in regulation ofcircadian rhythms In Atlantic salmon, a reduction in bonemineral content has been shown after both pinealectomy(Fjelldal et al 2004) and exposure to continuous light(Fjelldal et al 2005) In Atlantic salmon, continuous lightinhibits the nocturnal rise in plasma melatonin (Porter

gold-fish (Carassius auratus Linneaus) scales in vitro (Suzuki &

Hattori 2002; Suzuki et al 2008) Continuous light reducedcol11a1gene transcription in the present study Opposite toour results, Wargelius et al (2010) found that continuouslight increased col11a1 gene expression In that study, fishwere subjected to an abrupt change from natural light tocontinuous light at low temperature in January, twomonths after sea transfer and investigated 21 days later,while our study fish were either reared on continuous light

or changed to a 12 : 12 LD photoperiod at elevatedtemperature at sea transfer and measured 49 days later

Table 5 Plasma parameters (mean ± SEM) after 49 and 79 days of feeding in Atlantic salmon post-smolts fed diets with a high (HP) or

low (LP) phosphorous level, under continuous light (LL) or 12 h light/dark per day (LD)

Figure 3 Plasma concentrations of 1,25(OH) 2 D 3 and 25(OH)D 3

after 79 days of feeding Atlantic salmon post-smolt diets with a

high (HP, square-shaped) or low (LP, diamond-shaped)

phospho-rous level, under continuous light (LL, closed) or 12 h light/dark

per day (LD, half-open).

.

Aquaculture Nutrition 18; 610–619 ª 2012 Blackwell Publishing Ltd

Thus, timing of the onset of continuous light may be of

importance and complicates a comparison between these

two studies The observed effect of photoperiod on

transcription in these cells by melatonin (Suzuki & Hattori

2002; Nordgarden et al 2006) Indeed, a relationship

between decreased igf-I transcription in the vertebral

col-umn and the development of vertebral deformities has been

shown in Atlantic salmon (Fjelldal et al 2010)

In the present study, a P deficient diet led to reduced

min-eral content and stiffness of vertebral bone with a

concur-rent increase in bone ALP activity, and reduced bone

TRACP activity and mmp13 transcription Plasma 1,25

likely as an adaptive response to restore plasma P

as a source for P; both anabolic and catabolic effects of 1,25

verte-bral bone resorption was shown in European eel (Anguilla

tila-pia (Oreochromis mossambicus Peters) (Wendelaar Bonga

in European eel (A anguilla Linneaus) (Lopez et al 1980),

emerald rockcod (Pagothenia bernachii Boulenger) (Fenwick

a recent study by Darias et al (2010) showed that low

sea bass (Dicentrarchus labrax) juveniles In the present

study, P deficiency led to an increase in osteoblast activity

and a decrease in osteoclast activity, favoring vertebral bone

growth and mineralization This response could involve

other stimulating factors in addition to the vitamin D

endo-crine system Hormones like melatonin (Suzuki & Hattori

2002), calcitonin (Wendelaar Bonga & Lammers 1982) and

parathyroid hormone related protein (Abbink & Flik 2007)

are also involved in regulating osteoclast and osteoblast

activity in teleosts Although a P deficient diet may lead to

insufficient bone mineralisation, it has been suggested that P

is usually not extracted from vertebral bone but rather from

scales (Carragher & Sumpter 1991; Skonberg et al 1997;

Persson et al 1999) However, Roy et al (2002) found an

increased number of osteoclasts and suggested increased

resorption in vertebral bone of haddock (Melanogrammus

aeglefinus) fed 0.4% P in the diet Furthermore, Yamada

(Anguilla japonica) vertebrae during fasting and sexual

mat-uration Thus, vertebral bone can clearly serve as an

accessi-ble reservoir for minerals in some species In our study, thereduced vertebral mineral content could be a result of insuf-ficient bone mineralization, since decreased TRACP activityand mmp13 transcription and increased ALP activity suggestreduced bone resorption and increased bone mineralization

In salmon farming, compressed vertebrae (Witten et al.2005) are a major cause for downgrading and economicloss and represent an ethical dilemma in terms of animalwelfare (Hansen et al 2010) A compressed vertebra has anormal central part and deformational changes in the ante-rior and posterior parts In the central part of the vertebraethe angle between the wall of the cone of the vertebrae andthe anterior-posterior axis is at approximately 45 degrees

In the deformed anterior and posterior parts of the brae this angle is approximately 90 degrees (Witten et al.2005; Berg et al 2006; Fjelldal et al 2007) In the presentstudy, combined effects of continuous light and low Pinduced a compressed vertebral phenotype, with a reducedlength/dorso-ventral diameter Furthermore, these speci-mens had low vertebral stiffness and mineral content, andbone col11a1 and mmp13 transcription, and high bone

result-ing from impaired mineralization by low dietary P and

continuous light may have hindered normal development,and induced a linear growth failure in the compact bone ofthe vertebrae in the present study This could haveincreased the angle between the wall of the cone of the ver-tebrae and the anterior posterior axis, and given theobserved compressed phenotype The fact that continuouslight also up-regulated osteoclast activity may have madenormal development even more difficult for the fish fed alow P diet under continuous light

The results of the present experiment show that dietary Paffects bone osteoblast and osteoclast activity, as well as

In addition, the results show that the combination of uous light and low dietary P can be detrimental for normalgrowth and development of vertebrae in Atlantic salmon

contin-This study was funded by The Research Council of way, project number 172483/S40 and 153472/I40 Theauthors wish to thank the staff at the Institute of MarineResearch, Matre and Mrs Torill Berg at NIFES, Bergenfor excellent technical assistance

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1,2 1 1 1 1,3

1

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, The Chinese Academy of

Divisions, E-Institute of Shanghai Universities, Shanghai, China

A 10-week feeding trial was conducted in a flow-through

system to determine dietary choline requirement for

Purified basal diet was formulated using vitamin-free casein

as protein source Choline chloride was supplemented to

the basal diet to formulate seven diets containing 76.1, 163,

methionine was 0.58%, less than the requirement (0.69%)

The results indicated that specific growth rate (SGR) was

group Feeding rate and feed efficiency were not

signifi-cantly affected Protein productive value increased as

was lower in the fish fed the diet containing 4400 mg

(HDL-C) and total cholesterol significantly increased with

dif-ferences were found with further increase Fish carcass fat

contents decreased significantly with increased dietary

cho-line Hepatic lipid contents increased with dietary choline up

SGR and plasma HDL-C indicted dietary choline

Received 6 April 2011, accepted 18 November 2011

Correspondence: Shouqi Xie, State Key Laboratory of Freshwater

Ecol-ogy and BiotechnolEcol-ogy, Institute of HydrobiolEcol-ogy, The Chinese Academy

of Science, Wuhan, Hubei 430072, China E-mail: sqxie@ihb.ac.cn

Choline, a vitamin-like nutrient, performs three major bolic functions It is required (i) for the synthesis of theneurotransmitter acetylcholine; (ii) for the synthesis ofphosphatidyl choline (lecithin) and other complex choline-containing phospholipids; (iii) as a source of methyl groups,via betaine, for the synthesis of various methylated metabo-lites (Halver 2002) Choline has been classified as a B-com-plex vitamin, but it does not satisfy the standard definition

meta-of vitamin Because there is no evidence that choline is anenzyme co-factor, it could be synthesized at adequate

are present in the diet for some animals, such as pig and rat(Kroening & Pond 1967; Anderson et al 1979) However,young rapidly growing fishes cannot sufficiently synthesizecholine to satisfy their metabolic requirement (Wilson &

Poe 1988; Craig & Gatlin 1996) Thus, choline is an tial nutrient for fish, which should be taken from the food

essen-The quantitative requirement of choline has been studied

in many fish species The dietary requirement has been

for hybrid tilapia, Oreochromis

tilapia, choline deficiency has been showed to decrease fishblood triglyceride, cholesterol, phospholipids and fat con-centration in liver (Shiau & Lo 2000) Deficient dietarycholine could also decrease fish plasma total lipid, triacyl-glycerol, total cholesterol and phospholipid in sturgeonwhen choline was deficient in the diet (Hung 1989) Other

.

ª 2012 Blackwell Publishing Ltd

2012 18; 620–627 .doi: 10.1111/j.1365-2095.2011.00930.x

Aquaculture Nutrition