Aquaculture Nutrition
2011 17; 469–481
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College of Animal Sciences, Zhejiang University, Hangzhou, China
An 8-week feeding trial was conducted to determine the effects
of dietary methionine level on juvenile black sea bream Sparus
macrocephalus. Fish (initial body weight: 14.21 ± 0.24 g)
were reared in eighteen 350-L indoors flow-through circular
fibreglass tanks (20 fish per tank). Isoenergetic and isonitro-
genous diets contained six levels of L-methionine ranging from
7.5 to 23.5 g kg)1 of dry diet in 3.0 g kg)1 increments at a
constant dietary cystine level of 3.1 g kg)1. Growth perfor-
mance and feed utilization were significantly influenced by
dietary methionine levels (P < 0.05). Maximum weight gain
(WG), specific growth rate (SGR), feed efficiency ratio, protein
efficiency ratio and protein productive value (PPV) occurred at
17.2 g methionine kg)1 diet, beyond which they showed
declining tendency. Protein contents in whole fish body and
dorsal muscle were positively correlated with dietary methio-
nine level, while muscle lipid content was negatively correlated
with it. Apparent digestibility coefficients (ADCs) of dietary
nutrients were significantly affected by dietary treatments
except for ADCs of crude lipid. Fish fed the grade level of
methionine demonstrated a significant improvement in whole-
body methionine content, total essential amino acids
P P
( EAA), total non-essential amino acids ( NEAAs) and
P P
EAA/ NEAA ratio (P < 0.05). Regarding serum charac-
teristics, significant differences were observed in total choles-
terol, glucose and free methionine concentration (P > 0.05),
while total protein level and triacylglycerol concentration kept
relatively constant among treatments (P < 0.05). Analysis of
dose response with second-order polynomial regression on the
basis of either SGR or PPV, the optimum dietary methionine
requirements of juvenile black sea bream were estimated to
be 17.1 g kg)1 of diet (45.0 g kg)1 methionine of protein) and
17.2 g kg)1 of diet (45.3 g kg)1 methionine of protein) in the
presence of 3.1 g kg)1 cystine, respectively.
KEY WORDS: growth, L-methionine, requirement, Sparus
macrocephalus juvenile
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 2010 Blackwell Publishing Ltd
doi: 10.1111/j.1365-2095.2010.00823.x
Received 18 April 2010, accepted 26 July 2010
Correspondence: Shao Qingjun, Aquaculutre Nutrition Laboratory,
College of Animal Sciences, Zhejiang University, Hangzhou 310029,
China. E-mail: qjshao@zju.edu.cn
Protein, especially when derived from fish meal, is the most
expensive nutrient in the preparation of diets for aquatic
organisms (El-Saidy & Gaber 2002). Therefore, it is important
to incorporate inexpensive protein ingredients in the
formulation of fish feed by taking care of essential amino acids
(EAA) balances (Kaushik et al. 2004; Azaza et al. 2008;
Martı́nez-Llorens et al. 2009; Sardar et al. 2009). Lysine and
methionine are the most limiting amino acids in feed ingredi-
ents used in diets for fish, especially, when plant protein sources
are used to replace fish meal (Abimorad et al. 2009; Sardar
et al. 2009). Methionine is known as a precursor of choline and
various other metabolic processes (Ruchimat et al. 1997;
Kasper et al. 2000). As cystine can only be synthesized from a
methionine precursor, a portion of the methionine require-
ment can be spared by cystine in some fish species (approxi-
mately 40–60%) (Moon & Gatlin 1991; Kim et al. 1992; Griffin
et al. 1994; Goff & Gatlin 2004). Therefore, the requirement
for total sulphur amino acids (TSAA) can be met by either
methionine alone or the proper mixture of methionine and
cystine (Moon & Gatlin 1991). So it is important to consider
the dietary cystine content to quantify the methionine
requirement of the cultured species for maximum growth and
efficient feed utilization (Luo et al. 2005). In a previous study,
we have evaluated the optimal dietary lysine requirement for
black sea bream (Sparus macrocephalus) (Zhou et al. 2010a).
However, so far, the quantitative dietary requirement for
methionine is still unknown for this fish species.
Black sea bream is a valuable commercial fish species
cultured in China, Japan, Korean and some other countries
of Southeast Asia (Nip et al. 2003; Gonzalez et al. 2008). It is
highly appreciated as an excellent aquaculture species for
intensive culture because of its resistance to diseases, ability
to tolerate a wide range of environmental conditions, high
stocking densities and relative fast growth rate under inten-
sive culture and good quality meat (Hong & Zhang 2003;
Shao et al. 2008). However, trash fish was used for cultured
black sea bream in China, which could not meet the nutri-
tional requirements, and was difficult to store and easy to
pollute aquaculture environment. Thus, the formulation of a
nutritionally adequate and cost-effective feed is most
important for successful and sustainable culture of this fish
species. Up to now, a few studies have been conducted on the
nutrient requirements of black sea bream (Ma et al. 2008;
Shao et al. 2008; Zhou et al. 2010a,b; Zhang et al. 2010). The
purpose of this study was to quantify the dietary methionine
requirement at a constant dietary cystine level for black sea
bream juveniles.
Ingredients and proximate composition of the experimental
diets are presented in Table 1, amino acid compositions
(g kg)1 dry diet, L-form) of dietary ingredients in Table 2
and the analysed EAA contents for each diet are given in
Table 3. Six diets were maintained isonitrogenous by
decreasing the levels of glutamic acids as the methionine level
was increased. Experimental diets contained 380 g kg)1
crude protein, which was slightly lower than the optimum
protein requirement suggested in our preliminary experiment
(410 g kg)1, Zhang et al. 2010) to ensure the maximum uti-
lization of methionine for growth and limited catabolism for
energy (Wang et al. 2005; Luo et al. 2005). The 280 g kg)1
dietary protein was supplied by fish meal and soybean pro-
tein concentrate, and the remaining by a mixture of crystal-
line amino acids (CAAs) without methionine to simulate an
amino acid profile found in 380 g kg)1 whole-body protein of
black sea bream. The basal diet (diet 1) contained the mini-
mum level of methionine from fish meal and soybean protein
concentrate. The final levels of methionine were confirmed by
amino acid analysis, and the values were 7.5, 10.9, 14.1, 17.2,
20.6 and 23.5 g kg)1, respectively, by adding incremental
levels of crystalline methionine ranging from 0 to 15 g
methionine kg)1 diet (Table 3).
The CAAs were coated by carboxymethylcellulose (CMC)
and j-carrageenan as described by Wang et al. (2005). In
preparing experimental diets, all dry ingredients as well as
coating CAA mixture were finely ground, weighed, mixed
manually for 5 min and then transferred to a food mixer for
another 15 min mixing. Chromic oxide (Cr2O3), which was
used as external indicator for digestibility determination,
was dissolved in 100 mL of distilled water (about 40%, v/w)
and sprayed with an atomizer on the mash during mixing,
then fish oil and corn oil were added slowly while mixing.
During mixing, 6 N NaOH was added to establish a pH
level of 7–8 (Wilson et al. 1977). Distilled water was added
to achieve a proper consistency, and the mixture was further
homogenized and extruded through a 3-mm die by food
processor (Modle L-2730026; EHSY, Co., Shanghai, China).
The noodle-like diets were dried at 23 C for 72 h with air
condition and electrical fan. Dried noodles were broken into
particles, sieved to remove particles above 2 mm and then
stored in a refrigerator at )20 C. A representative sample
was taken for proximate analysis (Table 1).
Black sea bream were obtained from Marine Fisheries Re-
search Institute of Zhejiang province in Zhoushan, China.
Prior to initiation of the feeding trial, all fish were kept in
800-L circular fiberglass tanks and fed with diet 1 for
2 weeks. At the beginning of the experiment, 20 uniform-
sized and healthy fish (initial mean weight: 14.21 ± 0.24 g,
mean ± SD, n = 360) in good health and condition were
stocked in each fiberglass tank (350-L water volume). Each
experimental diet was randomly assigned to triplicate tanks
in a completely randomized design. Each flow-through tank
was supplied with sand-filtered aerated seawater at a flowing
rate of 2 L min)1. Fish were maintained under a natural
photoperiod, and the temperature, ammonia-nitrogen and
salinity of the seawater in tanks were 28 ± 1 C, 0.02–
0.04 mg L)1 and 29 g L)1, respectively. pH was 8.1–8.3, and
dissolved oxygen concentrations were above 5.0 mg L)1 at
any point during the experiment by using air stones with
continuous aeration. Experimental fish were fed by hand
twice daily (0800 h and 1600 h), which were fed slowly little
by little to prevent waste of feeds. When the experimental
feeds were supplied, the fish would swim to the water surface
to ingest the feeds. As long as fish were fed to satiation, they
would never come up to water surface again. Hence, their
apparent satiation could be judged by feeding behaviour
observation, and the feed losses could also be avoided almost
completely. The experiment lasted for 8 weeks and feed
consumption was recorded daily. Tanks were thoroughly
cleaned as needed and mortality was checked daily.
Faeces were collected using a faecal collection column
similar to the one described by Cho & Kaushik (1990). All
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Aquaculture Nutrition 17; 469–481  2010 Blackwell Publishing Ltd
 Table 1 Composition and proximate
Diets no.
analysis of experimental diets (g kg)1
diet) Ingredients Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6

Fish meal 280 280 280 280 280 280

Soybean protein concentrate 120 120 120 120 120 120
Carboxymethylcellulose 40 40 40 40 40 40
Crystalline amino acid premix 106 106 106 106 106 106
Methionine 0 3 6 9 12 15
Glutamic acid 15 12 9 6 3 0
Fish oil 40 40 40 40 40 40
Corn oil 80 80 80 80 80 80
Mineral premix1 15 15 15 15 15 15
Vitamin premix2 15 15 15 15 15 15
Others3 289 289 289 289 289 289
Total 1000 1000 1000 1000 1000 1000
Proximate analysis (g kg)1 dry matter)4
Crude protein 382.6 385.7 383.7 381.0 385.8 380.9
Crude lipid 149.6 145.2 147.6 145.2 150.6 146.6
Ash 139.1 137.9 140.3 135.8 133.6 134.3
Moisture 100.1 99.1 99.6 99.3 100.2 100.3
Methionine 7.5 10.9 14.1 17.2 20.6 23.5
Cystine 3.1 3.2 3.1 3.1 3.0 3.2
Gross energy (kJ g)1 diet) 15.7 15.6 15.7 15.5 15.8 15.5
1
Mineral premix (g kg)1 of premix): Na2SiO3, 0.4; CaCO3, 350; NaH2PO4ÆH2O, 200; KH2PO4, 200;
MgSO4Æ7H2O, 10; MnSO4ÆH2O, 2; CuCl2Æ2H2O, 1; ZnSO4Æ7H2O, 2; FeSO4Æ7H2O, 2; NaCl, 12; KI, 0.1;
CoCl2Æ6H2O, 0.1; Na2MoO4Æ2H2O, 0.5; AlCl3Æ6H2O, 1; and KF, 1.
2
Vitamin premix (mg kg)1 diet): retinyl acetate, 40; cholecalciferol, 0.1; DL-a-tocopheryl acetate,
80; menadione, 15; niacin, 165; riboflavin, 22; pyridoxine HCl, 40; thiamin mononitrate, 45; D-Ca
pantothenate, 102, folic acid, 10; vitamin B-12, 0.9; inositol, 450; ascorbic acid, 150; Na mena-
dione bisulphate, 5; thiamin, 5; choline chloride, 320 and p-aminobenzoic acid, 50.
3
Others (%): a-starch, 180; sodium dihydrogen phosphate, 25; j-carrageenan, 25; a-cellulose, 54;
Cr2O3, 5.
4
Values for the proximate analysis of the test diets are means of triplicate analyses.

Table 2 Amino acid composition

Supplied by Supplied by 120 g Supplied by 380 g kg)1
(g kg)1 diet) of dietary ingredients for
Amino 280g fish soybean protein crystalline whole-body
experimental diets (excluding trypto- acids meal kg)1 diet concentrate kg)1 diet amino acids Total protein
phan)
EAAs
Val 10.6 4.8 9.5 24.9 24.9
Leu 15.0 6.9 10.6 32.5 32.5
Ile 9.2 4.2 8.7 22.1 22.1
Phe 8.7 4.7 2.6 16.0 16.0
Thr 7.1 2.8 8.5 18.4 18.4
His 4.4 2.0 1.0 7.4 7.4
Arg 13.0 6.0 8.6 27.6 27.6
Lys 14.7 5.2 13.0 32.9 32.9
Met 4.4 1.3 Variable Variable 14.5
NEAAs
Glu 24.5 14.5 Variable Variable 59.5
Gly 14.7 3.4 6.4 24.5 24.5
Ala 13.0 3.5 2.5 19.0 19.0
Tyr 4.9 2.2 9.6 16.7 16.7
Asp 16.7 9.1 15.5 41.3 41.3
Ser 5.6 2.8 6.7 15.1 15.1
Pro 10.4 3.6 2.8 16.8 16.8
Cys 1.6 1.5 – 3.1 3.0

EAAs, essential amino acids; NEAAs, non-essential amino acids.

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Aquaculture Nutrition 17; 469–481  2010 Blackwell Publishing Ltd

Table 3 Analysed essential amino acid (excluding tryptophan) con- Moisture concentration was determined by drying minced
tents in the experimental diets samples for 6 h in a forced-air oven maintained at 105 C.
Diets Ash content was analysed by incinerating samples at 600 C
EAAs
for 24 h in a muffle furnace. Crude protein was estimated as
(g kg)1 diet) Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Diet 6
Kjeldahl-nitrogen using factor 6.25, and crude lipid was
Val 30.4 31.5 30.6 30.8 31.2 31.6
determined by Soxhlet extraction with petroleum ether for
Leu 41.1 41.3 40.9 41.2 41.2 40.9
Ile 21.1 20.9 20.9 21.3 20.7 20.9 6 h. The concentrations of total protein, total cholesterol,
Phe 12.6 12.4 12.3 12.2 12.5 12.7 triacylglycerol and glucose in the serum of juvenile black sea
Thr 21.2 21.1 21.6 22.1 21.1 21.3 bream were all measured within 3 days, using the Diagnostic
His 9.8 10.2 10.2 9.7 9.9 9.9
Arg 17.2 17.2 17.6 17.3 17.5 17.4 Reagent Kit purchased from Nanjing Jiancheng Bioengi-
Lys 31.7 32.2 32.2 32.1 31.9 31.7 neering Institute (Nanjing, Jiangsu Province, China)
Met 7.5 10.9 14.1 17.2 20.6 23.5 according to the manufacturers instructions. Faecal samples
EAA, essential amino acid. were oven dried at 105 C to a constant weight for the
determination of dry matter. Then the dried samples were
possible care was taken during feeding so that feed losses finely ground with a mechanical mortar and pestle and sieved
could be avoided almost completely. Two hours after the using a 1-mm screen prior. Chromic oxide (Cr2O3) content in
final feeding of the day, the drain pipe and faecal collection diets and faeces was determined by the method of Furukawa
columns were thoroughly cleaned with a brush to remove the & Tsukahara (1966). Gross energy in feeds and faeces was
residual feed and faeces from the system. Faeces were then determined by Automatic Isoperibol Calorimeter (Modle
allowed to settle overnight, and faecal samples were collected 6300; Parr Instrument Company, Moline, IL, USA) in the
at 06:00 each morning before the next feeding. Faeces col- laboratory in the Institute of Feed Science, Zhejiang Uni-
lected from the settling columns were immediately filtered versity.
with filter papers to separate other materials such as dirt The amino acid compositions of all samples including
particles and then stored at )20 C for chemical analysis. ingredients, diets, whole fish body and serum were analysed
Faeces from the same tank were pooled over the sampling following acid hydrolysis using an automatic amino acid
period to provide sufficient faecal matter for analysis. analyser (Hitachi 835-50, Tokyo, Japan) with a column
(Hitachi custom ion exchange resin no. 2619) in the labora-
tory in Institute of Feed Science, Zhejiang University. In
brief, performic acid oxidation was performed prior to
Ten fish at the start of the feeding trial were sampled and hydrolysis to oxidize methionine at )10 C for 3 h to obtain
stored frozen ()20 C) for the analysis of proximate carcass methionine sulfone. Then sodium metabisulfite was added to
composition. At the termination of the 8-week experiment, decompose surplus performic acid. Subsequently, amino acid
approximately 24 h after the last feeding, all fish were was liberated from protein by hydrolysis with 6 N HCl for
counted and individually weighed. Three fish from each tank 24 h. Hydrolysed samples were diluted with sodium citrate
were anesthetized (MS-222, Sigma, St Louis, MO, USA at buffer, pH was adjusted to 2.2 and individual amino acid
80 mg L)1) and then stored at )20 C for subsequent whole- components were separated by ion exchange chromatogra-
body proximate analysis. Blood samples were drawn from phy. Cystine content in diets was determined from the same
the caudal vein of five fish per tank with a 27-gauge needle acid hydrolysate after treatment with dithiothreitol and so-
and 1- mL syringe. Blood samples were immediately centri- dium tetrathionate (Inglis & Liu 1970). Tryptophan could
fuged at 836 g for 10 min (4 C) to obtain serum to measure not be detected after acid hydrolysis and it was excluded
nutrient levels (Ai et al. 2006), and serum was stored at from analysis at the present experiment.
)20 C until use. Dorsal muscles were obtained from all the
remaining fish in each tank and stored at )20 C for sub-
sequent proximate analysis.
Pooled fish samples (including whole body, dorsal muscle, All data were subjected to the analysis of variance and cor-
serum and faeces) in each tank were analysed in triplicate for relation analysis when appropriate using the software of SPSS
proximate composition. Moisture, ash, crude protein and for Windows (ver16.0; Chicago, IL, USA). Differences
crude lipid were determined following methods of the Asso- between the means were tested by Tukeys multiple range test.
ciation of Official Analytical Chemists (AOAC 1995). Differences were considered significant at P < 0.05. Broken-
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Aquaculture Nutrition 17; 469–481  2010 Blackwell Publishing Ltd

 line regression analysis was performed on specific growth rate
(SGR) and protein productive value (PPV) to establish the
dietary requirement of methionine for black sea bream. The
equation used in the model is Y = L + U (R ) X), where Y
is the parameter (SGR or PPV) chosen to estimate the
requirement, L is the ordinate and R is the abscissa of the
breakpoint. R is taken as the estimated requirement and U is
the slope of the line for X (Robbins et al. 1979).
Growth performance, body-organ indices and feed utilization
for juvenile black sea bream given graded levels of methionine
for 8 weeks are shown in Table 4. Survival was the highest for
fish fed the diet containing 17.2 g kg)1 methionine and
showed no significant difference among dietary treatments,
and no other nutritional deficiency signs were observed in
black sea bream fed methionine-deficient diets. Mean feed
intake was significantly lower in fish fed 7.5 g methionine kg)1
diet than those of fish fed 14.1 and 17.2 g methionine kg)1
diets, and the values were insignificantly different among the
other groups (P > 0.05). WG and SGR increased with dietary
increasing methionine level up to 17.2 g kg)1, but further
additions of methionine decreased grow rate of black sea
bream. Fish fed diet 1 showed the lowest feed efficiency ratio
(FER) (0.78), protein efficiency ratio (PER) (2.10), nitrogen
gain (1.05) and PPV (0.35), and those indices of fish were
significantly improved by methionine supplementation
Table 4 Growth performance, body-organ indices and feed utilization of black sea bream juvenile fed the diets with graded levels of methionine
for 8 weeks
(P < 0.05), although there were different decline extent for fish
fed excessive methionine level diets. The highest FER (0.89),
PER (2.35) and PPV (0.43) were recorded in the fish fed diet
containing 17.2 g kg)1 methionine. Hepatosomatic index
(HSI) was higher in fish fed 23.5 g methionine kg)1 diet than
that of in fish fed diet without methionine supplement, but
differences among the other treatments were not significant
(P > 0.05). Increasing dietary methionine level decreased
condition factor (CF) values although there was no significant
difference (P > 0.05).
Based on SGR, the optimum requirement of dietary
methionine was estimated to be 17.1 g kg)1 of diet (45.0
g kg)1 of protein) using break-point regression method
analysis (Fig. 1). When PPV was plotted against dietary
methionine, the optimum dietary methionine requirement
was 17.2 g kg)1 of diet (45.3 g kg)1 of protein) (Fig. 2). Be-
cause the experimental diets contained 3.1 g kg)1 of cystine,
the corresponding requirements of this fish for TSAA
(Met + Cys, TSAA) were calculated to be 21.1 g kg)1 of
diet (53.2 g kg)1 of dietary protein) and 21.5 g kg)1 of diet
(56.6 g kg)1 of dietary protein), respectively.
The effects of graded levels of dietary methionine on
whole-body and dorsal muscle composition were described in
Table 5. Protein content of whole body showed an increasing
trend with increasing dietary methionine levels (P < 0.05),
but there was a slight decline for fish fed the diets over
17.2 g kg)1 methionine. No significant differences were
observed in body lipid, ash or moisture among the dietary

Diets (methionine level, g kg)1)

Diet 1 (7.5) Diet 2 (10.9) Diet 3 (14.1) Diet 4 (17.2) Diet 5 (20.6) Diet 6 (23.5)

IBW 14.01 ± 0.48 14.20 ± 0.18 14.30 ± 0.26 14.27 ± 0.10 14.23 ± 0.19 14.25 ± 0.18
FBW 52.28 ± 0.73c 58.02 ± 1.47b 59.68 ± 1.48ab 62.27 ± 2.70a 60.60 ± 1.29ab 59.40 ± 1.71ab
Survival 95.00 ± 5.00 96.67 ± 2.87 96.67 ± 5.77 100.00 ± 0.00 96.67 ± 5.77 91.67 ± 2.89
WG 273.44 ± 10.56d 308.64 ± 4.44c 318.39 ± 4.25bc 335.50 ± 6.03a 326.08 ± 5.07ab 316.86 ± 4.83bc
SGR 2.49 ± 0.05c 2.66 ± 0.02b 2.69 ± 0.03b 2.78 ± 0.05a 2.73 ± 0.04ab 2.69 ± 0.01b
CF 2.01 ± 0.06 1.96 ± 0.07 1.96 ± 0.03 1.99 ± 0.13 1.93 ± 0.05 1.91 ± 0.25
HSI 2.20 ± 0.16b 2.27 ± 0.06ab 2.38 ± 0.06ab 2.27 ± 0.13ab 2.23 ± 0.08ab 2.51 ± 0.10a
MFI 0.88 ± 0.01b 0.95 ± 0.03ab 0.96 ± 0.03a 0.97 ± 0.03a 0.95 ± 0.03ab 0.95 ± 0.04ab
FER 0.78 ± 0.02d 0.82 ± 0.02c 0.84 ± 0.01bc 0.89 ± 0.01a 0.87 ± 0.01ab 0.85 ± 0.02b
PER 2.09 ± 0.03d 2.20 ± 0.02c 2.27 ± 0.03b 2.32 ± 0.03a 2.27 ± 0.02b 2.23 ± 0.01bc
N gain 1.05 ± 0.01c 1.25 ± 0.05b 1.31 ± 0.05b 1.42 ± 0.06a 1.37 ± 0.03a 1.37 ± 0.02a
PPV 0.30 ± 0.01c 0.35 ± 0.01bc 0.37 ± 0.02b 0.43 ± 0.02a 0.40 ± 0.04ab 0.40 ± 0.01ab

Values are presented as mean ± SD (n = 3); values with different superscripts in the same row differ significantly (P < 0.05).
Survival (%) = 100 · final fish number/initial fish number; weight gain (WG) (%) = 100 · (FBW ) IBW)/IBW; Specific growth rate (SGR) (%
day)1) = 100 · (ln FBW ) ln IBW)/day; CF (Condition factor) (g cm)3) = 100 · (live weight, g)/(body length, cm)3; hepatosomatic index
(HSI) = 100 · (liver weight, g)/(body weight, g); intraperitoneal ratio (IPR) = 100 · (intraperitoneal fat weight)/(body weight); mean feed
intake (MFI) = g fish)1 day)1; feed efficiency ratio (FER) = 100 · wet weight gain in g/dry diet fed in g; protein efficiency ratio (PER) =
weight gain in g/protein intake in dry basis in g; N gain (%) = 100 · (FBW · Nf ) IBW · Ni); protein productive value (PPV) = protein gain in
g/protein fed in dry basis in g; where IBW is initial mean body weight and FBW is final mean body weight; Nf and Ni are the N contents of
the carcass at the end and at the beginning of the feeding trial.
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Aquaculture Nutrition 17; 469–481  2010 Blackwell Publishing Ltd

treatments (P > 0.05). Muscle protein content was positively were more variable and could not be related to dietary
correlated with dietary methionine level, while lipid content treatments (P > 0.05).
was negatively correlated with it. Ash and moisture contents Data on nutrients digestibility of black sea bream fed the
different experimental diets are presented in Table 6. Fish fed
the 17.2 g methionine kg)1 diet had higher apparent digest-
2.85 Y = 2.782 – 0.0282 (17.1 – X) (X ≤ 17.1)
ibility coefficients (ADCs) of dry matter than that of fish fed
2.8 R2 = 0.9029
the 7.5 g methionine kg)1 diet (P < 0.05), but no significant
2.75
differences were found among fish fed the other diets. ADCs
2.7
SGR (%/day)

2.65
of crude protein was lower in fish fed the diet without
Y = 2.782 – 0.0137 (X – 17.1) (X > 17.1) methionine supplement (P < 0.05), while the values in the
2.6
R2 = 0.9106
2.55 rest groups were very stable (P > 0.05). ADCs of gross en-
2.5 ergy increased significantly with increasing methionine level
Xopt = 17.1
2.45 up to 17.2 g methionine kg)1 diet and then decreased slightly
2.4 (P < 0.05). However, ADCs of crude lipid were unaffected
7 9 11 13 15 17 19 21 23 25
Dietary methionine level (g kg–1 dry diet) by dietary treatments (P > 0.05).
Increasing dietary methionine level increased EAA and
Figure 1 The relationship between weight gain (SGR, %/day, y) and non-essential amino acids (NEAA) contents of whole fish
P P
dietary methionine levels (g kg)1, x) of juvenile black sea bream fed body, and the EAA/ NEAA ratio was also increased
with the experimental diets for 8 weeks. SGR, specific growth rate. significantly (P < 0.05) (Table 7), and the values peaked at
fish fed 23.5, 20.6 and 23.5 g methionine kg)1 diets, respec-
0.5 tively. The contents of Val, Leu and Ile in different groups
Y = 0.0132X + 0.1993
2
Y = 4.13 were very stable (P > 0.05); however, the levels of rest EAAs
0.45 R = 0.9275
in whole body showed increasing tendency (P < 0.05) with
0.4 increasing dietary methionine level. Body methionine con-
centration increased significantly with dietary methionine
PPV

0.35
levels from 7.5 to 20.6 g kg)1, but kept relatively constant
0.3
Xopt = 17.21 thereafter (P > 0.05).
0.25 In serum profile, total cholesterol concentration was
0.2
highest in fish fed 7.5 g methionine kg)1 diet (P < 0.05), but
7 9 11 13 15 17 19 21 23 25 showed no significant differences for fish fed other diets
Dietary methionine level (g kg–1 dry diet) (Table 8). Fish supplied with 7.5 and 23.5 g methionine kg)1
diets showed higher glucose content (P < 0.05), compared to
Figure 2 The relationship between protein productive value (PPV, y)
and dietary methionine levels (g kg)1, x) of juvenile black sea bream that of fish fed the diets containing 10.9 and 20.6 g kg)1
fed with the experimental diets for 8 weeks. methionine. Serum free methionine concentration signifi-

Table 5 Effect of dietary methionine level on proximate compositions of whole body and muscle of juvenile black sea bream (% on wet matter
basis)

Diets (methionine level, g kg)1)

Diet 1 (0.75) Diet 2 (10.9) Diet 3 (14.1) Diet 4 (17.2) Diet 5 (20.6) Diet 6 (23.5)

Whole body
Protein 16.94 ± 0.16b 17.52 ± 0.18ab 17.66 ± 0.10a 18.03 ± 0.23a 18.05 ± 0.31a 17.78 ± 0.37a
Lipid 13.27 ± 0.34 13.36 ± 1.60 13.29 ± 0.87 13.50 ± 1.27 13.63 ± 1.51 13.35 ± 1.52
Ash 4.78 ± 0.29 4.80 ± 0.37 4.62 ± 0.29 4.79 ± 0.21 4.86 ± 0.17 4.66 ± 0.18
Moisture 63.77 ± 0.16 64.08 ± 1.01 64.17 ± 3.01 63.47 ± 0.15 63.40 ± 1.03 64.18 ± 1.82
Dorsal muscle
Protein 19.27 ± 0.53b 19.76 ± 0.43ab 20.36 ± 0.70ab 20.49 ± 0.30a 20.57 ± 0.14a 20.46 ± 0.16a
Lipid 5.04 ± 0.42a 4.57 ± 0.11ab 4.48 ± 0.18ab 4.03 ± 0.50b 4.16 ± 0.09b 4.19 ± 0.18b
Ash 1.45 ± 0.08 1.41 ± 0.03 1.39 ± 0.03 1.43 ± 0.02 1.41 ± 0.03 1.41 ± 0.02
Moisture 73.48 ± 1.11 73.52 ± 0.73 73.52 ± 0.48 73.89 ± 0.33 73.19 ± 0.54 73.19 ± 0.57

Values are presented as mean ± SD (n = 3); values with different superscripts in the same row differ significantly (P < 0.05).
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 Table 6 Effect of dietary methionine level on apparent digestibility coefficients (ADCs) of main nutrients in diets for juvenile black sea bream
for 8 weeks

Diets (methionine level, g kg)1)

Diet 1 (7.5) Diet 2 (10.9) Diet 3 (14.1) Diet 4 (17.2) Diet 5 (20.6) Diet 6 (23.5)
b ab ab a ab
Dry matter 85.17 ± 1.24 87.86 ± 1.29 88.18 ± 1.68 90.21 ± 0.77 88.04 ± 0.54 87.47 ± 1.39ab
Protein 95.32 ± 0.50b 97.15 ± 0.42a 96.65 ± 0.48a 97.30 ± 0.63a 96.78 ± 0.22a 96.63 ± 0.35a
Lipid 96.59 ± 0.36 97.82 ± 0.51 97.98 ± 0.35 97.95 ± 0.34 97.28 ± 1.26 98.10 ± 0.52
Gross energy 88.17 ± 0.40c 89.12 ± 0.43bc 90.29 ± 0.30b 91.83 ± 0.21a 91.45 ± 0.72ab 90.19 ± 0.53b

ADC of dry matter (%) = 100 · [1 ) (dietary Cr2O3)/faecal Cr2O3].

ADCs of nutrients or energy (%) = 100 · [1 ) (F/D ) DY/FY)], where F is the per cent of nutrients or energy in faeces, D is the per cent of
nutrients or energy in diet, DY is the per cent of Cr2O3 in diet and FY is the per cent of Cr2O3 in faeces.
Values are presented as mean ± SD (n = 3); means with different superscript letters in the same row differ significantly (P < 0.05).

Table 7 Effect of dietary methionine levels on amino acid contents of whole body (g kg)1 on a dry matter basis) in juvenile black sea bream fed
for 8 weeks

Diets (methionine level, g kg)1)

Diet 1 (7.5) Diet 2 (10.9) Diet 3 (14.1) Diet 4 (17.2) Diet 5 (20.6) Diet 6 (23.5)

EAA
Val 3.27 ± 0.03 3.28 ± 0.06 3.27 ± 0.05 3.30 ± 0.03 3.29 ± 0.04 3.35 ± 0.03
Leu 4.62 ± 0.05 4.66 ± 0.09 4.93 ± 0.04 4.80 ± 0.17 4.74 ± 0.06 4.75 ± 0.05
Ile 2.79 ± 0.03 2.86 ± 0.07 2.85 ± 0.07 2.84 ± 0.09 2.84 ± 0.09 2.89 ± 0.07
Lys 4.68 ± 0.02c 4.75 ± 0.01b 4.81 ± 0.02b 4.92 ± 0.01a 4.94 ± 0.03a 4.99 ± 0.04a
Phe 2.55 ± 0.02b 2.55 ± 0.05b 2.67 ± 0.10ab 2.71 ± 0.03ab 2.74 ± 0.07a 2.77 ± 0.05a
Thr 2.48 ± 0.07c 2.50 ± 0.05c 2.59 ± 0.02b 2.66 ± 0.03ab 2.74 ± 0.05a 2.72 ± 0.04a
His 1.07 ± 0.02b 1.09 ± 0.02b 1.10 ± 0.03b 1.09 ± 0.01b 1.17 ± 0.03a 1.19 ± 0.02a
Arg 4.12 ± 0.05b 4.13 ± 0.02ab 4.17 ± 0.03ab 4.18 ± 0.03ab 4.20 ± 0.01ab 4.23 ± 0.03a
Met 1.76 ± 0.03c 1.78 ± 0.05c 1.83 ± 0.02c 1.97 ± 0.06b 2.12 ± 0.01a 2.14 ± 0.02a
NEAA
Asp 4.37 ± 0.02b 4.37 ± 0.07b 4.42 ± 0.03ab 4.41 ± 0.04ab 4.49 ± 0.02a 4.42 ± 0.04ab
Tyr 1.75 ± 0.01 1.80 ± 0.04 1.81 ± 0.04 1.78 ± 0.04 1.76 ± 0.04 1.78 ± 0.04
Pro 3.03 ± 0.02 3.04 ± 0.07 3.07 ± 0.04 3.09 ± 0.06 3.14 ± 0.06 3.09 ± 0.02
Ser 1.90 ± 0.03 1.92 ± 0.01 1.88 ± 0.03 1.92 ± 0.03 1.96 ± 0.04 1.95 ± 0.04
Glu 7.68 ± 0.03c 7.73 ± 0.05c 7.87 ± 0.05b 7.90 ± 0.02ab 7.98 ± 0.06a 7.97 ± 0.01ab
Gly 5.74 ± 0.03c 5.81 ± 0.03c 5.93 ± 0.05b 5.95 ± 0.02ab 6.03 ± 0.05a 6.00 ± 0.03ab
Ala 4.29 ± 0.03b 4.35 ± 0.06ab 4.31 ± 0.02ab 4.35 ± 0.03ab 4.40 ± 0.02a 4.38 ± 0.05ab
P
EAA 27.34 ± 0.06d 27.60 ± 0.15cd 28.01 ± 0.26c 28.46 ± 0.21b 28.78 ± 0.11ab 29.05 ± 0.06a
P
NEAA 28.99 ± 0.08d 29.26 ± 0.27cd 29.54 ± 0.15c 29.62 ± 0.10bc 30.08 ± 0.19a 29.83 ± 0.04ab
P P
EAA/ NEAA 0.94 ± 0.09c 0.94 ± 0.01c 0.95 ± 0.01bc 0.96 ± 0.02ab 0.96 ± 0.01a 0.97 ± 0.01a

Values are presented as mean ± SD (n = 3); values with different superscripts in the same row differ significantly (P < 0.05).
P P
EAA, essential amino acids; NEAA, non-essential amino acids; EAA, total essential amino acids; NEAA, total essential amino acids.

cantly increased with increasing dietary methionine levels
(P < 0.05) although there was no statistical difference for fish
fed 20.6 and 23.5 g methionine kg)1 diets (P > 0.05). Serum
total protein content (ranging from 27.68 to 32.80 g L)1)
showed an insignificantly increasing trend with increasing
methionine level, while triacylglycerol concentration de-
creased from 5.47 to 3.93 mmol L)1, and no significant dif-
ferences were observed among treatments (P > 0.05).
Dose–response experiments with increasing supply of amino
acid are accepted in principle as a method for determining
..............................................................................................
dietary amino acid requirements (Cowey 1995), and the
model used to analyse the dose–response relationship will
influence the estimate of requirements. Because the SGR
response of fish in the current study was linear, a broken-line
model resulted in the lowest error term for estimating the
requirement appeared to give a more precise empirical figure.
In previous studies, some researchers have demonstrated that
cystine could spare dietary methionine portion about 60% in
channel catfish (Harding et al. 1977), 51% in yellow perch
(Twibell et al. 2000), 50% in red drum (Moon & Gatlin 1991;
Goff & Gatlin 2004), 42% in rainbow trout (Kim et al. 1992)
and 40% in hybrid striped bass (Griffin et al. 1994); the
presence of dietary cystine reduces the amount of methionine

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Table 8 Determination of serum parameters in juvenile black sea bream, fed with graded levels of methionine for 8 weeks

Diets (methionine level, g kg)1)

Diet 1 (7.5) Diet 2 (10.9) Diet 3 (14.1) Diet 4 (17.2) Diet 5 (20.6) Diet 6 (23.5)
)1
Total protein (g L ) 27.68 ± 1.06 30.56 ± 2.16 31.29 ± 1.94 32.79 ± 3.08 30.52 ± 2.10 32.80 ± 1.76
T-CHO (mmol L)1) 9.33 ± 0.70a 7.27 ± 0.42b 6.76 ± 0.21b 6.60 ± 0.11b 6.61 ± 0.54b 6.99 ± 0.52b
TG (mmol L)1) 5.47 ± 1.22 5.36 ± 0.47 4.94 ± 0.43 4.71 ± 0.49 3.93 ± 0.47 4.98 ± 0.96
GLU (mmol L)1) 9.66 ± 1.37a 6.52 ± 1.35b 6.43 ± 1.21b 6.61 ± 0.58b 6.39 ± 0.74b 7.45 ± 0.68a
Free met (mmol 100 mL)1) 8.22 ± 0.06e 8.39 ± 0.05d 8.61 ± 0.07c 8.79 ± 0.03bc 8.96 ± 0.11ab 9.14 ± 0.07a

Values are presented as mean ± SD (n = 3); values with different superscripts in the same row differ significantly (P < 0.05).
T-CHO, total cholesterol; TG, triacylglycerol; GLU, glucose concentration.

required for maximum growth. Thus, Wilson & Halver
(1986) suggested that fish have a TSAA (Met + Cys)
requirement rather than a specific methionine requirement.
Methionine or total sulphur amino acids requirements have
been reported varying from 22.0 to 65.0 g kg)1 of dietary
protein in different fish species (De Silva & Anderson 1995).
In the present study, the TSAA requirement of juvenile black
sea bream was determined to be 53.2 g kg)1 of dietary pro-
tein (in the presence of dietary 3.1 g kg)1 cystine) based on
growth performance. This value was higher than that re-
ported for some commercially important finfish species,
namely, milkfish (43.7 g kg)1, Borlongon & Coloso 1993),
Jian carp (42.9 g kg)1, Tang et al. 2009), large yellow croaker
(40.2 g kg)1, Mai et al. 2006), catla (35.5 g kg)1, Ravi &
Devaraj 1991), yellowtail (32.8 g kg)1, Ruchimat et al. 1997),
grouper (32.3 g kg)1, Luo et al. 2005), African catfish
(32 g kg)1, Fagbenro et al. 1998), Japanese flounder
(31 g kg)1, Alam et al. 2000) and yellow perch (30 g kg)1,
Twibell et al. 2000). But, the requirement of black sea bream
is nearer to Indian major carp (55.0 g kg)1, Ahmed et al.
2003) and rohu (55.8 g kg)1, Khan & Jafri 1993). The wide
variation observed in the methionine requirement among
species may be because of fish size, age, laboratory condition
including feeding regime, feed allowance, water temperature,
stock density and ingredients used for basal diet such as zein,
casein, gelatin, gluten, soybean meal, fishmeal and CAAs in
various combinations (Tacon & Cowey 1985; Rodehutscord
et al. 1997; Forster & Dominy 2006; Nguyen & Davis 2009b).
In the current work, higher levels of cystine were not tested
so that it is not apparent whether this dietary level of cystine
is adequate, and it could also be a possible reason for the
relatively higher methionine requirement in this study.
However, in the study of rainbow trout, Walton et al. (1982)
observed similar growth performance of fish fed diets with or
without cystine provided that the level of methionine was
adjusted to meet the requirement level. Because a portion of
dietary methionine is converted to cystine the presence of
dietary cystine reduces the amount of methionine required
for maximum growth. The replacement value of cystine for
methionine and the use of methionine to provide for the total
sulphur amino acid requirement of black sea bream should
be further evaluated.
Amino acid balance in diets is necessary for optimal
growth of fish (Wilson & Halver 1986), and most authors
reported that fish fed dietary increasing methionine showed
two different growth pattern: (i) increased with increasing
dietary methionine and then remained constant when dietary
methionine is higher than requirement (Ruchimat et al. 1997;
Coloso et al. 1999; Alam et al. 2001; Luo et al. 2005; Nguyen
& Davis 2009a) (ii) increased with increasing dietary methi-
onine level and then decreased significantly when dietary
methionine is higher than requirement (Murthy & Varghese
1998; Mai et al. 2006; Yan et al. 2007). A few studies
reported that none of the fish fed the test diets containing
increased methionine showed a significant difference in
growth (Sveier et al. 2001; Espe et al. 2008). In the present
study, the growth response black sea bream fed increasing
methionine diets is in accordance with the second pattern.
Reduction in the growth rate in fish fed diets with superflu-
ous level of TSAA may be because of toxic effects (Choo
et al. 1991), and excess amount of total sulphur amino acid in
fish body would lead to extra energy expenditure towards
deamination and excretion (Walton 1985; Murthy & Var-
ghese 1998; Sveier et al. 2001).
FER obtained in the present study ranged from 0.78 to 0.89,
which exhibited correlation with fish growth and seems satis-
factory. One possible reason may be that fish were slowly fed
by hand little by little till apparent satiation (visual observa-
tion of fish feeding behaviour) and prevent the waste of feeds.
On the other hand, generally high FER suggested that black
sea bream is able to utilize CAAs well in diets. However, there
was a marked decline in FER when fish fed methionine defi-
ciency diet or superfluous methionine diets, indicating that the
supplementing level of methionine should be optimized. Feed
intake was much lower when fish fed diet methionine-unsup-
plemented diets. These results are similar to those reported in
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 large yellow croaker (Mai et al. 2006), Indian major carp
(Ahmed et al. 2003) and yellow perch (Twibell et al. 2000). In
more recent work, Li et al. (2009) reported that methionine
deficiency in aquafeeds may reduce/exhaust reservoirs of an-
tioxidants such as ascorbic acid, glutathione and vitamin E in
various tissues of fish, which may result in irreversible oxida-
tive stress, further aggravating growth retardation and feeding
depression, which could also partly explain the preset results.
The present study showed significantly decreased PER at high
dietary methionine. It may be because of unbalance amino
acids composition in diets and diverting amino acids into
catabolic rather than anabolic processes (Cowey & Sargent
1979). However, values of nitrogen gain and PPV decreased
insignificantly at higher methionine level diets when compared
with fish fed optimal methionine level diet. Similar results were
observed in some previous studies (Ruchimat et al. 1997; Luo
et al. 2005; Yan et al. 2007). This is to be expected as growth is
largely driven by protein deposition (Sá et al. 2008). Indeed, it
was concluded that using maximum nitrogen gain as response
criteria results in higher EAAs requirement estimates than
WG in rainbow trout and Atlantic salmon (Rodehutscord
et al. 1997; Hauler & Carter 2001; Espe et al. 2007). Results
were similar to our observation; the optimal methionine for
PPV was estimated to be 17.2 g kg)1 diet (corresponding to
53.2 g kg)1 of dietary protein), which was slightly higher than
the optimum requirement for growth in the present study.
In Asian sea bass, HSI were unaffected by dietary methi-
onine level (Coloso et al. 1999), and in Atlantic salmon, low
methionine intake resulted in a significantly higher liver
weight relative to body weight (Espe et al. 2008). However,
HSI was reported to increase with increased dietary methi-
onine up to requirement level and kept constant at higher
methionine levels in yellowtail (Ruchimat et al. 1997) and
rockfish (Yan et al. 2007), similar in the present study. In
addition, Walton et al. (1982) pointed out that the methio-
nine/cystine ratio affected HSI, and HSI decreased when
methionine increased with the cystine level was 0.5 g kg)1
diet. However, when the cystine level was 20.5 g kg)1,
increasing methionine level resulted in a slight increase in
HSI. There were, however, no significant differences ob-
served in CF of black sea bream among dietary treatments.
Similar result was also found in Atlantic salmon (Sveier et al.
2001) and cobia (Zhou et al. 2006), but in contradiction with
the observation in grouper (Luo et al. 2005).
Methionine has three major metabolic functions: as an
EAA for protein synthesis; as a sulphur source for synthesis
of other sulphur-containing biochemicals; and as a methyl
donor for methylation reactions (Mehler 1986). In the pres-
ent study, protein contents in whole body and muscle of
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black sea bream both tended to increase with dietary
methionine level up to the requirement level, beyond which it
remained nearly unchanged, which is in agreement with other
reports (Kim et al. 1992; Ruchimat et al. 1997; Alam et al.
2000; Luo et al. 2005). De la Higuera et al. (1997) suggested
protein synthesis and accretion in fish required all amino
acids present simultaneously at the synthesis sites, or the
process is impaired or prevented. The variational tendency
on protein accretion was also consistent to nitrogen retention
values that obtained in the present study. Among amino
acids, the branched-chain amino acid leucine is clearly rec-
ognized as a most effective nutrient regulator of mRNA
translation and proteolysis (Kimball & Jefferson 2004; Yo-
shizawa 2004; Nakashima et al. 2005). However, the role of
methionine acting a nutrient signal to regulate protein syn-
thesis still needs extensive studies. It has demonstrated that
the first step of the initiation of mRNA translation consists
of the binding of initiator Met-tRNAi to the 40S ribosomal
subunit to form the 43S preinitiation complex. This initiation
step may be inhibited by methionine deficiency (Métayer
et al. 2008). With regard to the potential effect of methionine
on intracellular kinases, studies are sparse but indicate that
this sulphur amino acid may exert a signal function by
inducing 70-kDa ribosomal protein S6 kinase (p70S6K)
activation in mammals (Shigemitsu et al. 1999; Stubbs et al.
2002). Similar results have been found in an avian myoblast
cell line (QM7) of quail origin (Tesseraud et al. 2003), in
which the methionine or leucine regulates S6K1 phosphory-
lation and protein synthesis. It seems there may be also a
regulation of protein synthesis by methionine in fish; how-
ever, the mechanism was not clear and more investigations
are needed to confirm these aspects. Lipid content of dorsal
muscle in the present study was higher in fish fed methionine-
unsupplemented diet than those of fish fed the high-methio-
nine diets, which might be because of better utilization of
protein with reduced deposition of lipid in the presence of
methionine resulting lean growth of fish (Sardar et al. 2009).
The results were consistent with Kim et al. (1992) and Sch-
warz et al. (1998), but in contrast with the study in grouper
(Luo et al. 2005).
With regard to the effect of methionine levels on dietary
nutrients digestibility, studies are sparse. Digestibility data
are important to the fish nutritionist because the nutrients
contained in poorly digested ingredients are less available to
support growth and metabolism (Lin et al. 2004). ADCs of
crude protein in this study were higher than 95.32% and
averaging 96.64%, indicating that all the nitrogen sources
provided in the experimental diets were well absorbed and
utilized by the juvenile black sea bream. It has reported that
CAAs coated with CMC and j-carrageenan used in diets can
improve the utilization effectiveness by reducing its solubility
in water, and encapsulation or covalent binding may also
help to slow the rate of release of CAAs to transport sites in
the intestinal mucosa of fish (Millamena et al. 1996; Alam
et al. 2004), thus protecting free amino acids in the gut long
enough to improve their utilization efficiency (Segovia-
Quintero & Reigh 2004). ADCs of crude protein was
significantly higher in black sea bream fed the test diets
supplying methionine when compared to those fed the con-
trol diet, which was consistent with the findings by Espe et al.
(2008) and Chi et al. (in press). In the present study, ADCs
of gross energy showed a similar tendency among the groups
as the FER; it is according to the result observed in salmon,
in which the reduced feed efficiency was to a large extent
explained by reduced energy and protein digestibility
(Mundheim et al. 2004). The ADCs of crude of lipid in all
groups were relatively higher (more than 96.59%) in present
study, and independent of the dietary treatments, the values
compare favourably to data obtained in other sparids
(Santinha et al. 1996; Robaina et al. 1995; Biswas et al.
2007). Nevertheless, it should be noted that extraction of
faeces with petroleum ether does not extract the fatty acids
bound as calcium soaps, and this extraction will therefore to
some extent underestimate the amount of lipids in the faeces
and overestimate lipid digestibility (Santinha et al. 1996). In
general, few investigations with effects of methionine sup-
plemented on nutrients digestibility were reported. Di- and
tripeptides are absorbed into the enterocytes without any
hydrolysis by microvillous peptidases (Jose et al. 1997), and
c-glutamyltransferase (c-GT) is involved in peptide transport
(Griffith & Meister 1980), and in the study with Jian carp,
Tang et al. (2009) found that there was significant improve-
ment in intestine and hepatopancreas weight, as well as c-GT
activity by dietary methionine level. Also in the study by
Tang et al. (2009), methionine was reported to improve
intestinal creatine kinase activity, which plays a role in energy
transfer in tissue. Anyhow, it is necessary to conduct further
studies to make clear this aspect.
To support the requirement estimated by growth response,
whole fish body EAA contents of experimental black sea
bream were analysed (Table 7). Dietary amino acid profiles
are known to influence the postfeeding levels of free amino
acids in fish tissues, such as plasma, liver, muscle and whole
body (Twibell et al. 2000; Zhou et al. 2006; Espe et al. 2007).
Both the total EAA and NEAA content of whole fish body
showed marked increase with increasing dietary methionine
level, which was similar to other results which have demon-
strated that dietary methionine level affects tissue free amino
acids in fish (Griffin et al. 1994; Luo et al. 2005). It was
probably because dietary restriction of one EAA led to an
increase in oxidation of other essential and non-essential
amino acids present at normal levels in the diets (Ronnestad
et al. 2000; Ozório et al. 2002). Mai et al. (2006) suggested
methionine deficiency in diet could inhibit methionine par-
ticipating in protein synthesis and reduce its level in amino
acid pool in tissues; however, in the present study, methio-
nine concentration in fish body increased significantly with
dietary methionine levels, similar result was also observed in
group (Luo et al. 2005). According to Espe et al. (2007), the
P P
ratio of EAA to NEAA was held close to 1 to maximize
amino acid utilization; however, in the present trial, the
P P
EAA/ NEAA ratio was observed to increase significantly
with dietary methionine level. It has been demonstrated that
excessive intake of one amino acid or disproportionate
amounts of one amino acid affect the utilization of the other
amino acids (Choo et al. 1991; Coloso et al. 1999), but the
P P
maximum value of EAA/ NEAA ratio in whole fish
body occured in fish fed the highest methionine level diet.
The reason for this discrepancy is uncertain, and black sea
bream might be able to assimilate excess dietary amino acids
into tissues within a short period.
Currently, little information is available on the effect of
dietary methionine on serum characteristics. The free amino
acid content in the blood initially reflects the content of
amino acids in the dietary protein following absorption
(Yamada et al. 1981; Simmons et al. 1999), and methionine
may accumulate in blood when the total dietary methionine
supply exceeds the requirement (Mambrini et al. 1999). In
corroboration with the present findings, Harding et al.
(1977), Schwarz et al. (1998) and Zhou et al. (2006) all
observed that free methionine concentration in blood
increased with increasing dietary methionine. Plasma or
serum methionine levels have been used to confirm dietary
TSAA requirements derived from WG and FER data in
some fish species (Harding et al. 1977; Griffin et al. 1994);
however, Cowey (1995) considers the free amino acids in
plasma to be relatively unsuitable for the determination of
amino acid requirements, because no close relationship
exists between intake and concentration in animal tissues.
In the current study, serum methionine concentrations were
positively related to the dietary supply; consequently, it
could not be used evaluating methionine requirement for
black sea bream. There was an increasing tendency in serum
protein concentration with dietary methionine level, which
was similar with Luo et al. (2005). The observation of
higher serum cholesterol content for fish fed low-methionine
diets compared with high-methionine diets was also
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Aquaculture Nutrition 17; 469–481  2010 Blackwell Publishing Ltd
 reported by Yan et al. (2007), but inconsistent with Luo
et al. (2005). Contrary to the study in cobia (Zhou et al.
2006), serum glucose content in black sea bream signifi-
cantly decreased while dietary methionine levels increased;
however, it increased significantly at the maximum dietary
methionine level. Ruchimat et al. (1997) suggested that
plasma triacylglycerol in yellowtail significantly increased
with dietary methionine levels, while triacylglycerol con-
centrations in serum of black sea bream were more variable
and could not be related to dietary treatments. Zhou et al.
(2007) pointed out the effects on blood parameters were
more likely because of the nutritional stress of the fish
instead of a specific effect of the deficient amino acid. The
mechanism of methionine for those observations was not
clear and needs further investigation.
In conclusion, the current results on feeding methionine to
juvenile black sea bream (initial average weight: 14.21 ±
0.24 g) showed the improvement effects on growth perfor-
mance and feed utilization. Based on SGR and PPV, the
optimum requirement of dietary methionine for juvenile
black sea bream was estimated to be 17.1 g kg)1 of diet
(45.0 g kg)1 methionine of protein) and 17.2 g kg)1 of diet
(45.3 g kg)1 methionine of protein), in the presence of
3.1 g kg)1 of dietary cystine. Hence, under the present
experimental conditions, the corresponding TSAA require-
ments of this fish were calculated to be 20.2 g kg)1 of diet
(53.2 g kg)1 of dietary protein) and 20.3 g kg)1 of diet
(53.4 g kg)1 of dietary protein), respectively. The potential
for cystine sparing methionine and regulation mechanisms of
methionine on growth and protein synthesis warrant further
investigation.
The study was supported by Oceanic public-beneficial scien-
tific fund (State Oceanic Administration Peoples Republic Of
China, 201005013-08) and Scientific support program for
mariculture and demonstration in the East China Sea (The
Ministry of Science and Technology of the Peoples Republic
Of China, 2011).
We thank the Key Lab of Mariculture and Enhancement
of Zhejiang Province, and China-Norwegian Joint Labora-
tory of Nutrition and Feed for Marine Fish (Zhoushan,
ZheJiang province) for supplying the experimental black sea
bream, allowing us to use the experimental base and their
logistic support during the feeding experiment. We also
thank J.Z. Xu, Z. Sun, Y.J. Xu, F.P. Yu, G.Y. Zhong, W.X.
Song and W. Xiong for their assistance with caring for the
fish and sampling.
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Aquaculture Nutrition 17; 469–481  2010 Blackwell Publishing Ltd
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