Methionine requirement of juvenile Japanese ¯ounderParalichthys olivaceus estimated by the oxidation ofradioactive methionine

M.S. ALAM, S. TESHIMA, M. ISHIKAWA, S. KOSHIO & D. YANIHARTO Laboratory of Aquatic

Animal Nutrition, Faculty of Fisheries, Kagoshima University, Shimoarata, Kagoshima, Japan

Abstract

Growth and amino acid oxidation studies were conducted

to estimate methionine requirement of juvenile Japanese

¯ounder, Paralichthys olivaceus, by using the puri®ed diets

containing 500 g kg±1 crude protein from casein, gelatine

and crystalline amino acids (CAA). Diets with six graded

levels of methionine (5.3, 8.3, 11.3, 14.3, 17.3 and

20.3 g kg±1 diet) were fed to triplicate groups of the

juvenile (initial weight 2.8 � 0.05 g) twice a day for

40 days. To prevent leaching losses, CAA were precoated

using carboxymethylcellulose (CMC), and further diets

were bound by CMC and j-carrageenan. Based on

broken-line analysis of percentage weight gain and feed

conversion e�ciency, the methionine requirements of Jap-

anese ¯ounder in the presence of 0.6 g kg±1 of cystine were

14.9 and 14.4 g kg±1 dry diet, respectively. After the growth

study was ®nished, a direct estimate of methionine require-

ment was made by examining the in¯uence of dietary

methionine level on 14C-methionine oxidation by determin-

ing radioactive carbon dioxide, protein and nonprotein

fractions of the whole body. The dose±response curve

between expired radioactive CO2 and dietary methionine

levels showed that the optimum methionine level for the

¯ounder was estimated to be within the range of 14.3±

17.3 g kg±1 of diet in high agreement with values obtained

from the growth study.

KEYWORDSKEYWORDS: amino acid oxidation, 14CO2, Japanese ¯ounder,

methionine, Paralichthys olivaceus, requirement

Received 11 September 2000, accepted 9 February 2001

Correspondence: Shin-ichi Teshima, Laboratory of Aquatic Animal Nutri-

tion, Faculty of Fisheries, Kagoshima University, Shimoarata 4-50-20,

Kagoshima 890-0056, Japan. E-mail: teshima@®sh.kagoshima-u.ac.jp

Introduction

Some nutritional strategy for ®sh fed with arti®cial diets is to

provide indispensable amino acids at a level su�cient to meet

the demands for maximum protein retention without avoid-

ing an excessive supply. Methionine is the sulphur-containing

amino acid, which is required for normal growth of many

®shes (Wilson 1989). Methionine was the most limiting

amino acid in diets for some warm water ®sh (Lovell 1989)

and in some semipuri®ed diet for the red drum (Moon &

Gatlin 1989). Methionine is converted to cystine in animals,

therefore, the presence of cystine spares the requirement of

methionine for maximum growth. The cystine replacement

value for methionine has been determined for many ®shes,

such as channel cat®sh Ictalurus punctatus (Harding et al.

1977), red drum Sciaenops ocellatus (Moon & Gatlin 1991)

and rainbow trout Oncorhynchus mykiss (Kim et al. 1992a).

Methionine is also a precursor of choline and Kasper et al.

(2000) reported that when methionine is not in excess in the

diet of Nile tilapia, Oreochromis niloticus, choline is required

for growth. Cataracts were observed in the rainbow trout fed

with methionine-de®cient diets (Walton et al. 1982; Rumsey

et al. 1983).

Amino acid requirements of ®shes are usually determined

and based on the growth rates of ®shes fed with graded levels

of the particular amino acid (Wilson et al. 1978; Nose 1979).

As growth is a�ected by more complex factors than the

adequacy of the dietary amino acid content and balance,

many physiological responses to changes in amino acid levels

in the diets have been used to de®ne amino acid requirement.

Direct oxidation studies on amino acids have been carried

out at the end of dose-response feeding trials for con®rma-

tion of the results of the growth curve (Cowey 1995).

Compared with growth measurement, amino acid oxidation

technique has given reliable results for several ®sh (Walton

et al. 1984a; Kaushik et al. 1988; Anderson et al. 1991; Lall

201

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Ó 2001Blackwell Science Ltd

et al. 1994). Amino acid oxidation technique is based on the

measurement of expired 14CO2 when an intraperitoneally

injected pulse or oral administration of 14C-labelled amino

acid under test is given at the di�erent levels of test amino

acid intake. When an amino acid is limiting or de®cient in a

diet, the major proportion will be utilized for protein

synthesis, and little will accumulate in the plasma or be

oxidized to CO2, whereas when the quantity of an amino acid

is supplied in excess, and is thus not a limiting factor for

protein synthesis, plasma levels will increase and more will be

available for oxidation (Brookes et al. 1972; Aguilar et al.

1974).

The requirements of all the essential amino acids are

known only for a limited number of juvenile species such as,

rainbow trout Oncorhynchus mykiss (Ogino 1980), catla

Catla catla (Ravi & Devaraj 1991), chinook salmon

Oncorhynchus tshawytscha, channel cat®sh, common carp

Cyprinus carpio, Japanese eel Anguilla japonica, Nile tilapia

(NRC 1993) and white sturgeon Acipenser transmotanus

(Ng & Hung 1995). On the other hand, research on the

requirements of marine ®sh for essential amino acids is scarce

except for the studies on the requirements of a couple of

amino acids in commonly cultured species such as yellow tail

(Ruchimat et al. 1997). Japanese ¯ounder, a popular food

®sh in Japan, but few studies have been conducted using

di�erent alternative protein sources which mainly focus on

the speci®c nutritional requirements (Kikuchi et al. 1994a,b;

Kikuchi 1999). Except for lysine (Forster & Ogata 1998),

there have been no studies with regard to the essential amino

acid requirements of the juvenile Japanese ¯ounder. The

purpose of the present experiment was to evaluate optimum

dietary methionine level for the Japanese ¯ounder, Paralich-

thys olivaceus by growth response and the oxidation of a

tracer dose of 14C-methionine.

Materials and methods

Growth study

The details of the preparation of diets and rearing conditions

of the growth studies have been described in our recent

publication (Alam et al. 2000). The juveniles (2.8 � 0.05 g)

were fed the six isonitrogenous test diets (Table 1) containing

500 g kg)1 crude protein in triplicate at a ration size 5% of

their body weight (BW) twice a day for 40 days. Casein,

gelatin and crystalline amino acids (CAA) were added to the

test diets to provide an amino acid pattern similar to that of

the juvenile Japanese ¯ounder whole body protein except for

methionine. Diet 1 (basal diet) contained minimum level of

methionine, 5.3 g kg)1 of diet or 10.6 g kg)1 of protein, from

intact protein and gelatin. The other diets were supplemented

with incremental levels of 3 g kg)1 of crystalline methionine

to the diet 1, resulting in methionine concentrations ranging

from 8.3 to 20.3 g kg)1 of diet or 16.6 to 40.6 g kg)1 of

protein. These dietary methionine levels were below and

above the methionine level of the juvenile ¯ounder whole

body protein (Table 2). Diets were prepared according to

Millamena et al. (1996) with slight modi®cation. To prevent

leaching loss of CAA, they were precoated with carboxy-

methyl cellulose (CMC) and j-carrageenan.

Amino acid oxidation study

Experimental ®sh At the end of the growth experiment, six

®sh from each dietary group were randomly collected,

transferred to the radioisotope laboratory and the ®shes

were placed in three 10 L glass aquarium, called feeding

chambers (each chamber contained two ®shes). Triplicate

groups of the ®sh for each dietary treatment were fed with the

respective test diets for 7 days to acclimatize to the environ-

ment. Fifty percent of water in the feeding chambers was

exchanged daily and aeration was supplied.

Radioactively labelled diets [1±14C]LL-Methionine [speci®c

activity, 55 mCi mmol±1 (2.03 G Bq mmol±1)] was purchased

from American Radiolabeled Chemicals Inc. (St Louis, MO

63146, USA) in ethanol:water (7:3). The compositions of the

radioactive diets were the same as for growth trials, except

for the inclusion of 14C-methionine. To prepare radiolabelled

diets, 1 g of the respective cold diets were ground in a small

mortar adding water (1:1 w/v), 14C-Methionine was added

and mixed well to ensure that all particles of diets were

completely mixed with 14C-methionine. The dough was

extruded through a 10-mL plastic disposable syringe with a

3.0-mm diameter outlet. The spaghetti-like strands were

dried and then broken up into pellets. The speci®c activity of

the pellets was approximately 3 lCi g±1 (111 K Bq g±1).

Administration of 14C-methionine diets and 14CO2 collection

To determine oxidation of 14C-methionine, expired 14CO2

were collected according to Querijero et al. (1997) with the

following modi®cation. The juveniles were starved for 24 h

before feeding the radiolabelled diets. The ®shes were fed

with the respective labelled diet at the dose of 20 000 dpm g)1

BW. The feeds were consumed by the juveniles within few

minutes except those fed with diets 1 and 6, which left very

small amounts of uneaten feeds. After 5 min of feeding, the

juveniles were removed from the chamber, washed with sea

M.S. Alam et al.

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water and transferred to the metabolic chamber, a 4-L glass

container containing 2.5 L sea water. The opening of each

metabolic chamber was covered with the plastic cover with

an inlet and outlet tube. Air entering the inlet tube into the

water of metabolic chamber passed through 250 mL of 1.0 MM

NaOH solution to remove CO2 from air. The air leaving

from the metabolic chamber passed through four-test tube

traps in series to collect all the expired 14CO2, each

containing 2 mL of 0.36 MM NaOH solution to absorb

metabolic 14CO2 leaving the metabolic chamber. The air

tube leaving the metabolic chamber was ®tted with a three-

way junction cork to enable alternate collection of metabolic14CO2 at various collection periods, without permitting the

escape of metabolic 14CO2 at the end of each period. The

®shes were kept in the metabolic chambers for 48 h and

expired 14CO2 was sampled at 24 and 48 h. After 48 h, the

®shes were removed from the metabolic chambers, freeze

dried and stored for radioactivity measurements. After the

®shes were removed from the metabolic chambers, 30 mL of

0.5 MM HCl was added in chambers to decrease the pH of the

water to 5. Further collection of 14CO2 that might have been

trapped in the water was carried out for another 12 h.

Ingested radioactivity was obtained by determining the sum

of the total radioactivity of expired 14CO2, whole ®sh body,

and water from the metabolic chambers. The radioactivity of

the feeding chamber was not analysed.

Separation of protein and free amino acid fraction from the

whole body Fish samples were separated into protein, non-

protein (mainly free amino acids), and remainder fractions by

treatment with trichloroacetic acid (TCA) as follows: the

whole body samples were freeze dried (LABCONCO, Ashahi

life Science Freeze Dry System, Japan), cut into small pieces

and homogenized with 10 volumes of distilled water using a

Polytron homogenizer (KINEMATICA, Gmbh LITTAU,

Switzerland). The homogenate was separated into the TCA-

precipitable (protein) and TCA-soluble (mostly free amino

acids) fraction after addition of 10 volumes of 10% TCA by

centrifugation (3000 ´ g, 10 min). The radioactivity of

TCA-soluble fraction was measured after removing TCA

Table 1 Composition of the test dietsTest diets (g kg)1 dry diet)

Ingredient 1 2 3 4 5 6

Casein 170 170 170 170 170 170Gelatine 80 80 80 80 80 80Amino acid mixture1 253 250 247 244 241 238Squid liver oil2 50 50 50 50 50 50Soya bean lechithin3 50 50 50 50 50 50Vitamin mixture4 50 50 50 50 50 50Mineral mixture5 50 50 50 50 50 50a-Starch 120 120 120 120 120 120Carboxymethyl cellulose (CMC) 44 44 44 44 44 44j-Carrageenan 25 25 25 25 25 25a-Cellulose 88 88 88 88 88 88Attractants6 10 10 10 10 10 10LL-methionine 0 3 6 9 12 15

Total methionineg kg)1of diet 5.3 8.3 11.3 14.3 17.3 20.3g kg)1of protein 10.6 16.6 22.6 28.6 34.6 40.6

Crude protein (g kg)1, dry basis) 502.2 503.3 495.7 504.2 507.3 500.8Crude lipid 97.2 97.3 98.5 94.7 96.0 96.3Crude ash 54.3 55.0 55.2 56.2 53.3 55.1

1SeeTable 2..2 Feed oil W, RikenV|tamin,Tokyo, Japan.3 Kanto Chemical Co., Inc.,Tokyo, Japan.4 (g kg)1diet) q-Amino benzoic acid,1.60; biotin, 0.02; inositol,16.02; nicotinic acid, 3.20; Ca-pantothenate,1.12; pyridoxine-HCl, 0.19; ribo£avin, 0.80; thiamine-HCl, 0.24; menadione, 0.19; vitamin A-palmitate, 0.77;a-tocopherol,1.60; cyanocobalamine,1.10; calciferol, 0.04; ascorbyl-2-phosphate-Mg, 0.28; folic acid, 0.06and cholin chloride, 32.75.5 (g kg)1 diet) NaCl, 1.838; MgSO4á7H2O, 6.850; NaH2PO4á2H2O, 4.360; KH2PO4, 11.990; Ca(H2PO4)2á2H2O,6.790; Fe-citrate, 1.485; Ca-lactate, 16.350; AlCl3á6H2O, 0.009; ZnSO4á7H2O, 0.179; CuCl2, 0.005;MnSO4á4H2O, 0.040; KI, 0.008 and CoCl2, 0.050.6 (g kg)1diet) Taurine, 5; betaine, 4; and inosine-5-monophosphate,1.

Methionine requirement ofJapanese flounder

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with diethyl ether treatment. The TCA-precipitable fraction

was washed with 5 volumes of cold ethyl alcohol, followed by

cold ethyl alcohol and chloroform (3:1), and cold di-ethyl

ether. The protein fractions so obtained were freeze dried.

The washings obtained by these treatments were considered

as the remainder fraction.

Measurement of radioactivity Radioactivities were measured

as described by Querijero et al. (1997). To measure the

radioactivity of 14CO2, TCA-soluble fraction and sea water,

2 mL of sample was added to a 10-mL of scintillation

cocktail SCSII (Amersham, USA) in a scintillation vial. The

diets, whole body, protein and remainder fraction were

digested in SOLUENE-350 (Packard, USA) for 24 h in a

55°C water bath with continuous stirring, then were added

8 mL of toluene based scintillation cocktail containing 3.2 g

PPO (2,5 diphenyloxazole), 0.25 g POPOP {1.4-bis-2

(5-phenyloxazolyl) benzene}, in a mixture of 500 mL toluene,

200 mL triton X-100 (polyoxyethylene octylphenyl ether)

and 100 mL distilled water. After adding the scintillation

cocktail, all samples were set aside for 6 h, then radioactivity

was read using a liquid scintillation counter (Aloka LSC

3000, Aloka, Japan).

Others chemical and statistical analysis Amino acid analysis

was performed by HPLC as described by Teshima et al.

(1986). Approximately 2 mg dry sample was weighed and

hydrolysed with 4NN-methanesulphonic acid for 22 h at

110°C. The pH of the hydrolysate was adjusted to 2.2 and

injected into a HPLC unit with an ion exchange resin

column. Norleucine was used as an internal standard

(0.06 ng 100 lL±1). The crude protein of the diets was

determined by Kjeldahl method with a Tecator Kjeltec

System (1007 Digestion system, 1002 Distilling unit, and

Titration unit) using boric acid to trap ammonia, and the

crude lipid was determined using Bligh & Dyer (1959)

method. Ash and moisture contents were analysed as per

Association of O�cial Analytical Chemists (AOAC 1990)

methods. Data on growth performance and the radioactive

measurements were tested using one-way analysis of variance

(ANOVAANOVA). Signi®cant di�erences between means were evalu-

ated by Duncan New Multiple Range Test (package Super

Supplied by

Amino acidsCasein(170 g kg)1)

gelatine(80 g kg)1) CAA1 Total

50% flounder wholebody protein

EAA2

Arginine 6.0 6.5 18.8 31.3 31.3Histidine 4.9 0.8 6.9 12.6 12.6Isoleucine 8.1 1.2 12.8 22.1 22.1Leucine 15.0 2.4 21.4 38.8 38.8Lysine3 16.0 2.4 32.9 51.3 51.3Methionine 4.8 0.5 variable4 variable4 14.2Phenylalanine 8.4 1.6 11.0 21.0 21.0Threonine 6.5 1.7 13.1 21.3 21.3Tryptophan ND ND 3.6 3.6 3.65

Valine 9.9 2.0 13.7 25.6 25.6

NEAA6

Aspartic acid 10.6 4.8 32.2 47.6 47.6Glutamic acid 38.4 8.7 34.7 81.8 81.8Serine 7.1 2.0 10.2 19.3 19.3Proline 17.6 10.0 0 27.6 23.4Glycine 2.4 17.6 11.4 31.4 31.4Alanine 4.3 8.3 20.4 33.0 33.0Tyrosine 10.1 0.2 9.7 20.0 20.0Cystine 0.57 0.17 ^ ^ NDHydroxyproline ^ 9.2 ^ 9.2 ^

1The mixture of crystalline LL-amino acids (Ajinomoto Co., Inc., Japan).2 Essential amino acids.3 Supplied as LL-LysineáHCl.4 SeeTable 1.5 Kanazawa et al. (1989).6 Nonessential amino acids.7 NRC (1993), ND = Not detected.

Table 2 Amino acid composition of

ingredients used in the test diets and the

whole body protein (g kg)1 dry weight)

of the Japanese ¯ounder

M.S. Alam et al.

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ANOVAANOVA, Abacus Concepts, Berkeley, CA, USA). Probabilities

of P<0.05 were considered signi®cant. The optimum dietary

methionine requirement based on weight gain data was

determined using the broken-line regression method (Zeitoun

et al. 1976; Robbins et al. 1979). Regression analysis was

performed using software package StatViewTM (Abacus

Concepts). The requirement was estimated to be that level

eliciting 95% of the maximum responses.

Results

Growth performance

The results of the growth performance of the Japanese

¯ounder were already reported in details elsewhere (Alam

et al. 2000). In brief, as shown in Table 3, the lowest weight

gain, survival and feed e�ciency (FE), were obtained in the

group fed diet without supplemental methionine and the

highest values were recorded with diet 5 containing

17.3 g kg)1 of methionine in diet. Survival rate was also

signi®cantly a�ected by methionine supplementation. The

optimum dietary level of methionine for the juvenile Japan-

ese ¯ounder based on broken-line was estimated to be

14.9 g kg)1 of diet or 29.8 g kg)1 of protein from the weight

gain data.

Oxidation of 14C-methionine

Data on oxidation of labelled methionine are presented in

Table 4. Signi®cantly lower 14CO2 was released in the ®sh

which had received the two lowest levels of methionine, 5.3

and 8.3 g kg±1, than in those ®sh receiving higher levels

of methionine, 14.3, 17.3 and 20.3 g kg±1. An addition of

methionine to the basal diet up to the level 11.3 g kg±1 of diet

enhanced slightly the oxidation of 14C-methionine but no

statistical di�erences were observed among the groups

receiving methionine at the levels of 5.3, 8.3, 11.3 g kg±1 of

diet. Juveniles fed with the diets containing more than

11.3 g kg±1 methionine showed signi®cantly higher expired14CO2 than those fed lower levels of methionine. No

statistical di�erences were observed among the groups

receiving methionine at the levels of 14.3 and 17.3 g kg±1 of

diet. The production of radioactive CO2 rapidly increased

above a level of 17.3 g kg±1 of diet (Table 4). Therefore, from

the ANOVAANOVA of expired 14CO2, the requirement of Japanese

¯ounder for methionine was suggested to be within the range

of 14.3±17.3 g kg±1 of diet (28.6±34.6 g kg±1 of protein).

Distribution of the whole body radioactivity after 48 h

The radioactivity retained in the whole body, TCA-preci-

pitable, TCA-soluble and remainder fractions are presented

in Table 4. The radioactivity of the whole body (% of

ingestion) decreased with increasing levels of dietary methi-

onine. The highest radioactivity in the whole body protein

fraction (% of the whole body) was obtained in the

juveniles fed with the basal diet (diet 1). There was a trend

that the recovered radioactivity in the protein fraction

decreased with increasing dietary methionine supplement.

On the other hand, supplementation of methionine to the

basal diet led to an increase in the radioactivity of the TCA-

soluble fraction.

Discussion

The optimum dietary methionine level estimated by the

oxidation method using the ®sh fed with di�erent levels of

methionine was found to be within the range 14.3±17.3 g kg±

1 of diet or 28.6±34.6 g kg±1 of protein. This range was close

to the requirement value obtained from data on weight gain

and FE (Fig. 1, Alam et al. 2000). The optimum dietary

methionine level for the Japanese ¯ounder was less than that

the requirement value of 40 (g kg±1 protein) reported for the

species such as gilthead sea bream Sparus aurata (Luquet &

Sabaut 1974) and chinook salmon (Halver et al. 1959), but

were close to those for most of the commercially important

®n®sh species, namely, rainbow trout (30, Rumsey et al.

1983), common carp (31, Nose 1979), Japanese eel (32, NRC

1993), Nile tilapia (32, Santiago & Lovell 1988), milk-®sh

Chanos chano (32, Borlongan & Coloso 1993), coho salmon

Oncorhynchus kiutch (27, Arai & Ogata 1993), yellowtail

Table 3 Weight gain, feed e�ciency (FE) and survival of the juvenile

Japanese ¯ounder fed diets graded levels of methionine for 40 days.

Values are means of three replicate groups. Mean with di�erent letter

in the same column di�er signi®cantly (P < 0.05)

Methionine levelWeight gain1

g kg)1of diet g kg)1of protein (%) FE (%)2 Survival (%)

53 106 46.7a 28a 78a83 166 133.8b 48b 79a113 226 196.9c 67c 78a143 286 344.5d 74cd 88ab173 346 345.2d 78cd 98b203 406 338.1d 82d 88abPooled SEM 21.9 4.0 3.2

1Weight gain (%) = (Mean ¢nal body weight ) Mean initial bodyweight) / Mean initial body weight ´ 100.2 Feed e¤ciency (FE) = weight gain (g) / total feed intake in dry basis(g) ´ 100.

Methionine requirement ofJapanese flounder

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Seriola quinqueradiata (25.6, Ruchimat et al. 1997) and red

drum (26.9, Moon & Gatlin 1991). Lower dietary require-

ments of methionine have been reported for Mossambic

tilapia Oreochromis mossambicus (9.9, Jauncey et al. 1983)

and sea bass Dicentrarchus labrax (20, Thebault et al. 1985)

as compared with that for ¯ounder in this present study.

There are many factors which may a�ect amino acid

requirements, including species and age, dietary protein

sources, crystalline amino acids, environmental conditions

and experimental design (Tacon & Cowey 1985).

The results of the present study also showed that the

Japanese ¯ounder is capable of utilizing crystalline methion-

ine supplements for growth as indicated by Alam et al.

(2000). The high e�ciency of crystalline methionine in

improving the growth of the Japanese ¯ounder is caused by

the improvement of its water-stability by coating with CMC

and j-carrageenan. Cho et al. (1992), when quantifying the

arginine requirement of rainbow trout, added agar-coated

free amino acids to the diet and gave high rates of growth

comparable with the complete test diet.

The amino acid oxidation technique has been successfully

applied to determine amino acid requirement or verifying

dietary amino acid requirements estimated by growth study

for sheep (Brookes et al. 1973), pigs (Chavez & Bayley 1976;

Kim et al. 1983), rainbow trout (Walton et al. 1984a,b;

Kaushik et al. 1988) and Atlantic salmon (Lall et al. 1994).

In the present experiment 2 for the estimation of methionine

requirement by the oxidation technique, increase in dietary

methionine levels from 5.3 to 11.3 g kg±1 had no marked

increases in the production of CO2 (Table 4). In other words,

the Japanese ¯ounder expired CO2 slightly when they were

fed with the diets de®cient in methionine. This con®rmed that

the levels of essential amino acids in¯uence the release of14CO2 from 14C-labelled amino acid (Kim et al. 1983;

Kaushik et al. 1988).

Figure 1 Expired radioactive carbon dioxide and growth response of

Japanese ¯ounder fed graded levels of dietary methionine. Values

shown are means � SE of triplicate groups. The methionine

requirement for Japanese ¯ounder was estimated to be within the

range of 14.3 to 17.3 g kg)1 of diet from the expired radioactive

carbon dioxide.

Test diets

1 2 3 4 5 6 SEM1

Administration of 14C-methionineLevel of cold methionine (g kg^1) 5.3 8.3 11.3 14.3 17.3 20.3Radioactivity of prepared diet 3.11 3.23 3.51 3.16 3.31 3.52

(dpm ´ 103 mg^1)Given radioactivity 20 20 20 20 20 20

(dpm ´ 103 g^1BW)Uneaten diets (% of diet given) 6.26 0 0 0 0 2.20Ingested radioactivity 59.24 61.07 58.48 57.08 57.76 56.19 3.09

(% of diet given)

Radioactivity recovered (% of ingestion)Total expired CO2 6.19a 6.29a 8.52a,b 9.95b 9.97b 14.40c 0.7Whole fish body 88.2 83.9 86.3 81.7 77.7 79.1 2.8Water in metabolic chamber 5.5 10.1 5.1 9.0 12.3 6.5 2.7

Distribution of radioactivity in the whole body (% of whole body)Protein fraction 51.7 43.6 38.6 31.3 22.4 20.6 2.7

(TCA-precipitable)Nonprotein fraction 22.9 37.0 46.0 53.8 57.8 60.2 4.3

(TCA-soluble)Remainder fraction 25.4 19.4 15.4 15.0 19.8 19.2 3.4

1SEM = Pooled standard error of the mean.

Table 4 Oral administration of

radioactive diets containing14C-methionine and distribution of

radioactivity in the Japanese ¯ounder.

Values are means of six ®shes. Mean

with di�erent letters in the same column

di�er signi®cantly (P<0.05)

M.S. Alam et al.

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206

The results of the direct determination of methionine

requirement using 14CO2 released from 14C-methionine by

®sh receiving diets with graded levels of methionine did not

allow the construction of a broken-line plot to show

requirement. However, Table 4 indicated that production

of 14CO2 from orally administrated 14C-methionine

increased sharply when the methionine level exceeded

17.3 g kg±1 of diet. By the ANOVAANOVA, the requirement for

methionine was estimated within the range of

14.3±17.3 g kg±1 of methionine in the diet. The requirement

value mostly agreed with that estimated by the weight gain

(14.9 g kg±1) and FE (14.4 g kg±1). Walton et al. (1986) was

incapable of showing the break point for the rainbow trout

in the dose-response for 14CO2 from 14C-lysine and14C-arginine, whereas in the case of 14C tryptophan it was

successful. Also, Kim et al. (1992b) did not ®nd any clear

break point for rainbow trout when analysis 14C-phenylal-

anine oxidation. Walton et al. (1986) suggested that the

oxidation technique would not be suitable for use in the

absence of growth data because of its lack of precision in

determining requirement values from graphical plots. The

variability in the release of radioactivity as CO2 from14C-methionine after ingestion of feed may be explained by

variation in either the rate of absorption of the methionine;

or in the rates of uptake by the tissues which were observed

by Bali & Bayley (1984) in the case of piglets. The

experimental procedure in the present study was designed

to minimize variation in rates of ingestion between the

®shes; they had been fasted overnight, the ®shes were

adjusted by respective cold diets for 7 days in the feeding

chamber and feeds were consumed in less than 5 min.

Metabolic chambers were also ensured to be without any

leakage of 14CO2. In the present study, however, other

groups of ®shes were fed with respective graded diet

containing 14C-methionine to compare with the ingestion

rate of the treated ®shes. The ingestion rate was more or less

same for both studies.

The recovered radioactivity of the protein (TCA-precipi-

tate) fraction (% of whole body radioactivity) in the Japanese

¯ounder fed with de®cient methionine was higher than that

fed with supplemented methionine. This suggests that the ®sh

receiving methionine-de®cient diets utilize a large proportion

of dietary methionine for protein synthesis, thereby reducing

methionine oxidation. As the dietary supply of methionine

exceeds the ®sh needs for protein synthesis, part of dietary

methionine could be oxidized to CO2 for energy production.

However, Kim et al. (1983) reported that the partition of

amino acids between protein synthesis and amino acid

catabolism must depend on many factors, particularly

protein turnover, the combined e�ects of which may be too

complex to permit the de®nition of a single requirement for

each nutrient.

In summary, the Japanese ¯ounder juveniles are able to

utilize crystalline amino acid in the coated form and show

overall good growth of the ¯ounder. In the presence of

0.6 g kg±1 of dietary cystine, methionine requirement for

juvenile Japanese ¯ounder obtained by weight gain

(14.9 g kg±1 of diet or 29.8 g kg±1 of protein) and feed

e�ciency (14.4 g kg±1 of diet or 28.8 g kg±1 of protein (Alam

et al. 2000)) were supported by the close values (14.3±

17.3 g kg±1 of diet or 28.6±34.6 g kg±1 of protein) obtained

from the oxidation technique.

Acknowledgements

The authors wish to acknowledge Ajinomoto Co., Inc.,

Japan for donation of crystalline amino acids. The ®nancial

support received by the ®rst author for this research from the

Ministry of Education, Culture and Sports (Monbusho) of

Japan is gratefully acknowledged.

References

Aguilar, T.S., Benevenga, N.J. & Harper, A.E. (1974) E�ects of

dietary methionine level on its metabolism in rats. J. Nutr., 104,

761±771.

Alam, M.S., Teshima, S., Ishikawa, M. & Koshio, S. (2000)

Methionine requirement of juvenile Japanese ¯ounder Paralichthys

olivaceus. J. World Aquacult. Soc., 31, 618±626.

Anderson, J.S., Lall, S.P., Anderson, D.M. & McNiven, M.A. (1991)

Lysine requirement for Atlantic salmon (Salmo salar) ®ngerlings.

In: Fish Nutrition in Practice, Biarritz, France, June 24±27, 1991

(Kaushik, S.J. & Luquet, P. eds), pp. 867±872. INRA, Paris (Les

Colloques, no. 61).

AOAC (Association of O�cial Analytical Chemists) (1990) O�cial

Methods of Analysis. 15th edn (Williams, S. ed.). AOAC, Arling-

ton, Virginia, USA.

Arai, S. & Ogata, H. (1993) Quantitative amino acid requirements of

®ngerling coho salmon. In: Proceedings of 20th U.S.-Japan

Symposium on Aquaculture Nutrition. (Collie, M.R. & McVey,

J.P. eds), pp. 19±28. UJNR Department of Commerce, Newport,

Oregon, USA.

Bali, R.O. & Bayley, H.S. (1984) Time course of the total and

radioactive carbon dioxide production by piglets receiving

dietary [14C]phenylalanine. Can. J. Physiol. Pharmacol., 63,

1170±1174.

Bligh, E.G. & Dyer, W.J. (1959) A rapid method of total lipid

extraction and puri®cation. Can. J. Biochem. Physiol., 37,

911±917.

Borlongan, I.G. & Coloso, R.M. (1993) Requirements of juvenile

milk ®sh (Chanos chanos) for essential amino acids. J. Nutr., 123,

125±132.

Brookes, I.M., Owens, F.N. & Garrigus, U.S. (1972) In¯uence of

amino acid level in the diet upon amino acid oxidation by the rat.

J. Nutr., 102, 27±36.

Methionine requirement ofJapanese flounder

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Ó 2001Blackwell Science Ltd Aquaculture Nutrition 7;201^209

207

Brookes, I.M., Owens, F.N., Brown, R.E. & Garrigus, U.S. (1973)

Amino acid oxidation and plasma amino acids levels in sheep with

abomasal infusions of graded amounts of lysine. J. Anim. Sci., 36,

965±970.

Cho, C.Y., Kaushik, S.J. & Woodward, B. (1992) Dietary arginine

requirement of young rainbow trout (Oncorhynchus mykiss).

Comp. Biochem. Physiol., 102A, 211±216.

Chavez, E.R. & Bayley, H.S. (1976) Amino acid metabolism in the

piglet. 3. In¯uence of lysine level in the diet on energy metabolism

and in vivo oxidation. Br. J. Nutr., 63, 369±380.

Cowey, C.B. (1995) Protein and amino acid requirements: a critique

of methods. J. Appl. Ichthyol., 11, 199±204.

Forster, I. & Ogata, H.Y. (1998) Lysine requirement of juvenile

Japanese ¯ounder Paralichthys olivaceus and juvenile red sea

bream Pagrus major. Aquacuture, 161, 131±142.

Halver, J.E., Delong, D.C. & Mertz. E.T. (1959) Methionine and

cystine requirements of chinook salmon. Fed. Proc., 18, 2076.

Harding, D.E., Allen, O.W. Jr & Wilson, R.P. (1977) Sulfur amino

acid requirement of channel cat®sh: LL-methionine and LL-cystine.

J. Nutr., 107, 2031±2035.

Jauncey, K., Tacon, A.G.J. & Jackson, A.J. (1983) The quantitative

essential amino acid requirements of Oreochromis mossambicus. In:

International Symposium on Tilapia in Aquaculture (1st Proceed-

ings, Nazareth, Israel) (Fishelson, L. & Yaron, Z. eds), pp.

328±337. Tel Aviv University, Israel.

Kanazawa, A., Koshio, S. & Teshima, S. (1989) Growth and survival

of larval red sea bream Pagrus major and Japanese ¯ounder

Paralichthys olivaceus fed microbound diets. J. World Aquacul.

Soc., 20, 31±37.

Kasper, C.S., White, M.R. & Brawn, P.B. (2000) Choline is

required by tilapia when methionine is not in excess. J. Nutr.,

130, 238±242.

Kaushik, S.J., Fauconneau, B., Terrier, L. & Gras, J. (1988)

Arginine requirement and status assessed by di�erent biochemi-

cal indices in rainbow trout (Salmo gairdneri R.). Aquaculture,

70, 75±95.

Kikuchi, K., Furata, T. & Honda, H. (1994a) Utilization of feather

meal as a protein source in the diet of juvenile Japanese ¯ounder.

Fish. Sci., 60, 203±206.

Kikuchi, K., Furata, T. & Honda, H. (1994b) Utilization of soybean

meal as a protein source in the diet of juvenile Japanese ¯ounder,

Paralichthys Olivaceus. Suisanzoshoku, 42, 601±604.

Kikuchi, K. (1999) Partial replacement of ®sh meal with corn gluten

meal in diets for Japanese ¯ounder Paralichthys olivaceus. J. World

Aquacul. Soc., 30, 357±363.

Kim, K.I., Elliott, J.I. & Bayley, H.S. (1983) Amino acid oxidation

by young pigs receiving diets with varying levels of sulphur amino

acids. Br. J. Nutr., 50, 383±390.

Kim, K.I., Kayes, T.B. & Amundson, C.H. (1992a) Requirement

for sulfur amino acids and utilization of DD-methionine

by rainbow trout (Oncorhynchus mykiss). Aquaculture, 101,

98±103.

Kim, K.I., Grimshaw, T.W., Kayes, T.B. & Amundson, C.H.

(1992b) E�ect of fasting or feeding diets containing di�erent levels

of protein or amino acids on the activities of the liver amino acid-

degrading enzymes and amino acid oxidation in rainbow trout

(Oncorhynchus mykiss). Aquaculture, 107, 89±105.

Lall, S.P., Kaushik, S.J., Le Bail, P.Y., Keith, R., Anderson, J.S. &

Plisetskaya, E. (1994) Quantitative arginine requirement of Atlan-

tic salmon (Salmo salar) reared in sea water. Aquaculture, 124,

13±25.

Lovell, R.T. (1989) Nutrition and Feeding of Fish. Van Nostrand

Reinhold, New York, NY, USA.

Luquet, P. & Sabaut, J.J. (1974) Nutrition azotee et croissance chez

la daurade et la truite. In: Actes Des Colloques, pp. 243±253.

Colloques sur l'Aquaculture, 1. Brest.

Millamena, O.M., Bautista, M.N. & Kanazawa, A. (1996) Methi-

onine requirement of juvenile tiger shrimp Paneus monodon

Fabricus. Aquaculture, 143, 403±410.

Moon, H.Y. & Gatlin, D.M. III (1989) Amino acid nutrition of the

red drum (Sciaenops ocellatus): determination of limiting amino

acids and development of a suitable amino acid test diet. In:

Proceedings of Third International Symposium on Feeding and

Nutrition in Fish. (Takeda, M. & Watanabe, T. eds), pp. 201±208.

29 August±1 September 1989, Toba, Japan.

Moon, H.Y. & Gatlin, D.M. III (1991) Total sulfur amino acid

requirement of juvenile red drum, Sciaenops ocellatus. Aquaculture,

95, 97±106.

Ng, W.K. & Hung, S.S.O. (1995) Estimating the ideal dietary

essential amino acid pattern for growth of white sturgeon, Acipens-

er transmontanus (Richardson). Aquacult. Nutr., 1, 85±94.

Nose, T. (1979) Summary report on the requirements of essential

amino acids for carp. In: Fin Fish Nutrition and Fish Feed

Technology (Halver, J.E. & Tiews, K. eds), pp. 145±156. Heene-

mann, Berlin, Germany.

NRC (National Research Council) (1993) Nutrient Requirement of

Fish. National Academy Press, Washington D.C., USA.

Ogino, C. (1980) Requirements of carp and rainbow trout for

essential amino acids. Bull. Jap. Soc. Sci. Fish., 46, 171±174.

Querijero, B.V.L., Teshima, S., Koshio, S. & Ishikawa, M. (1997)

Utilization of monounsaturated fatty acid (18: 1n±9, oleic acid) by

freshwater prawn Macrobrachium rossenbergii (de Man) juveniles.

Aquacult. Nutr., 3, 127±139.

Ravi, J. & Devaraj, K.V. (1991) Quantitative essential amino acid

requirements for growth of catla, Catla catla (Hamilton).

Aquaculture, 96, 281±291.

Robbins, K.R., Norton, H.W. & Baker, D.H. (1979) Estimation

of nutrient requirements from growth data. J. Nutr., 109,

1710±1714.

Ruchimat, T., Masumoto, T., Hosokawa, H. & Shimeno, S. (1997)

Quantitative methionine requirement of yellowtail (Seriola quin-

queradiata). Aquaculture, 150, 113±122.

Rumsey, G.L., Page, J.W. & Scott, M.L. (1983) Methionine and

cystine requirements of rainbow trout. Prog. Fish-Cult., 45,

139±143.

Santiago, C.B. & Lovell, R.T. (1988) Amino acids requirements for

growth of Nile tilapia. J. Nutr., 118, 1540±1546.

Tacon, A.G. & Cowey, C.B. (1985) Protein and amino acid

requirements. In: Fish Energetics: and New Perspectives (Tytler, P.

& Calow, P. eds), pp. 155±183. Croom-Helm, London, UK.

Thebault, H., Alliot, E. & Pastoureaud, A. (1985) Quantitative

methionine requirement of juvenile seabass (Dicentrarchus labrax).

Aquaculture, 50, 75±87.

Teshima, S., Kanazawa, A. & Yamashita, M. (1986) Dietary value of

several proteins and supplemental amino acids for larvae of the

prawn, Penaeus japonicus. Aquaculture, 51, 225±235.

Walton, M.J., Cowey, C.B. & Adron, J.W. (1982) Methionine

metabolism in rainbow trout fed diets of di�ering methionine and

cystine content. J. Nutr., 112, 1525±1535.

Walton, M.J., Cowey, C.B. & Adron, J.W. (1984a) The e�ects of

dietary lysine levels on growth and metabolism of rainbow trout

(Salmo gairdneri). Br. J. Nutr., 51, 279±287.

Walton, M.J., Coloso, R.M., Cowey, C.B., Adron, J.W. & Knox, D.

(1984b) The e�ects of dietary tryptophan levels on growth and

metabolism of rainbow trout (Salmo gairdneri). Br. J. Nutr., 51,

279±287.

M.S. Alam et al.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Ó 2001Blackwell Science Ltd Aquaculture Nutrition 7;201^209

208

Walton, M.J., Cowey, C.B., Coloso, R.M. & Adron, J.W. (1986)

Dietary requirements of rainbow trout for tryptophan, lysine and

arginine determined by growth and biochemical measurements.

Fish. Physiol. Biochem., 2, 161±169.

Wilson, R.P., Allen, O.W., Robinson, E.H. & Poe, W.E. (1978)

Tryptophan and threonine requirements of ®ngerling channel

cat®sh. J. Nutr., 108, 1595±1599.

Wilson, R.P. (1989) Amino acids and proteins. In: Fish Nutrition.

(Halver, J.E. ed.), 2nd edn, pp. 112±153. Academic Press, Inc.,

New York.

Zeitoun, I.H., Ullrey, D.E., Magee, W.T., Gill, J.L. & Bergen, W.G.

(1976) Quantifying nutrient requirement of ®sh. J. Fish. Res. Board

Can., 33, 167±172.

Methionine requirement ofJapanese flounder

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Ó 2001Blackwell Science Ltd Aquaculture Nutrition 7;201^209

209