Aquaculture Nutrition
2012
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1,2 1 1
1
1 2
School of Life Science, Sun Yat-Sen University, Guangzhou, China;
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 28CP diets were formulated to contain 320 g kg 1,
300 g kg 1 and 280 g kg 1 crude protein without lysine
and methionine supplementation. In the supplementary
group, lysine and methionine were added to formulate
32AA, 30AA and 28AA diets with 320 g kg 1, 300 g kg 1
and 280 g kg 1 dietary crude protein, respectively, 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
(SGR, % day 1) of the fish fed 32CP diet were significantly
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
methionine supplementation (P < 0.05), and the interaction
between dietary protein level and amino acid supplementa-
tion was noted between WG and SGR (P < 0.05). Feed
intake (FI) was significantly increased with the increase in
dietary protein level and the supplementation of lysine and
methionine (P < 0.05), but feed conversion ratio (FCR)
showed a significant decreasing trend (P < 0.05). Two days
after total ammonia nitrogen (TAN) concentration test, the
values of TAN discharged by the fish 8 h after feeding
..............................................................................................
ª 2012 Blackwell Publishing Ltd
doi: 10.1111/j.1365-2095.2012.00937.x
1 1 1 1
Animal Science College, South China Agricultural
were 207.1, 187.5, 170.6, 157.3, 141.3 and 128.9 mg kg 1
body weight for fish fed 32CP, 32AA, 30CP, 30AA, 28CP
and 28AA diets, respectively. TAN excretion by grass carp
was reduced in plant-based practical diets with the increase
in dietary protein level and the supplementation of lysine
and methionine (P < 0.05). The results indicated that
lysine and methionine supplementation to the plant protein
sources-based practical diets can improve growth perfor-
mance and feed utilization of grass carp, and the dietary
crude protein can be reduced from 320 g kg 1 to
300 g kg 1 through balancing amino acids profile. The
positive effect was not observed at 280 g kg 1 crude
protein level.
KEY WORDS: Ctenopharyngodon idella, lysine, methionine,
protein reduction, total ammonia nitrogen
Received 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 second
largest aquaculture industry in the world inferior to silver
carp Hypophthalmichthys molitrix, constituting 14.7% of
the world aquaculture production, with an average annual
increase of 14% in China (FAO 1999). One of the major
reasons for the increase in production is the use of pelleted
feed, which enables the higher density or net–cage mono-
culture of this species to be achieved.
Protein is a major component in fish feeds, because it pro-
vides the essential and nonessential amino acids to synthesize
body protein and in part provides energy for maintenance.
Dabrowski (1977) found the protein requirement of grass
carp was between 410 g kg 1 and 430 g kg 1. Khan et al.
(2004) reported high WG in grass carp fed 300 g kg 1 and
350 g kg 1 crude protein diets. In China, grass carp are typi-
cally fed a commercial feed with 220–320 g kg 1 crude pro-
tein content. Protein, especially when derived from fishmeal
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
270 g kg 1 to 243 g kg 1) with crystalline amino acid had
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
can be reduced from 460 g kg 1 to 415 g kg 1 by 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 experimental
diets are shown in Table 2.
All dry ingredients were finely ground, weighed, mixed
manually for 5 min and then transferred to a Hobart mixer
(A-200T Mixer Bench Model unit, Resell Food Equipment
Ltd, Ottawa, ON, Canada) for another 15-min mixing.
Soya lecithin was added to a preweighed premix of soy oil
and mixed until homogenous. The oil mix was then added
to the Hobart mixer slowly while mixing was still contin-
uing. All ingredients were mixed for another 10 min. Then,
distilled water (about 300 g kg 1 of diets) was added to the
mixture-form dough. The wet dough was placed in a dual-
screw extruder (Institute of Chemical Engineering, South
China University of Technology, Guangzhou, China) and
extruded through an 1.25-mm die. The diets were dried
with forced air at 20 °C for 24 h, and the moisture was
reduced to about 100 g kg 1. The dry pellets were placed
in plastic bags and stored in a deep freezer at 20 °C until
used.
Grass carp (Ctenopharyngodon idella) juvenile from our facil-
ities were used in this experiment, and their initial wet
weights were 2.10 ± 0.01 g. Before the experiment, the fish
were acclimated to the experimental conditions for 2 weeks
and fed a commercial diet containing 300 g kg 1 protein and
40 g kg 1 lipid to satiation. Twenty-five healthy fish were
randomly distributed to each of the 18 experimental fibre-
glass tanks (98 L 9 48 W 9 42 H cm, water volume of
200 L) connected to a recirculation system. Water exchange
in each tank was maintained at 10 L min 1. The water was
oxygenated, passed through artificial sponge (3 cm thick-
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 quality
parameters monitored weekly as follows: temperature,
29.1 ± 2.4 °C; dissolved oxygen, 7.4 ± 0.36 mg L 1; TAN,
0.089 ± 0.006 mgL 1; pH, 7.9 ± 0.09, respectively. Faeces
were collected daily during the last 2 weeks as described by
Wang et al. (2005). Faeces tank 1 was dried at 105 °C and
stored at 70 °C for determination of digestibility with
Y2O3 as indicator.
In the growth experiment, the fish were fed three times
per day and 7 days per week to apparent satiation for
9 weeks. After the growth experiment, ten healthy fish
(average wet weight 65.0 ± 0.25 g) were randomly distrib-
uted to each of the 12 experimental fibreglass tanks
(98 L 9 48 W 9 42 H cm, water volume of 200 L) and
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Aquaculture Nutrition ª 2012 Blackwell Publishing Ltd
 Table 1 Formulation and approximate composition of practical diets for grass carp

Diet
1
Ingredients (g kg diet) 32AA 30AA 28AA 32CP 30CP 28CP

Soybean meal1 143.6 86.6 27.4 143.6 86.6 27.4

Canola meal1 250.0 250.0 250.0 250 250. 0 250.0
Cotton meal1 140.0 140.0 140.0 140 140.0 140.0
Rice bran meal1 50.0 50.0 50.0 50 50.0 50.0
Wheat flour1 284.8 284.8 284.8 284.8 284.8 284.8
Maize1 50.0 104.3 160.7 68.5 125.5 184.7
Mineral mix2 5.00 5.00 5.00 5 5.00 5.00
Vitamin mix3 5.00 5.00 5.00 5 5.00 5.00
Soy oil1 20.0 20.0 20.0 20 20.0 20.0
Choline chlorine (50%)1 2.00 2.00 2.00 2 2.00 2.00
Monocalcium phosphate1 20.0 20.0 20.0 20 20.0 20.0
51% L-Lysine SO45 12.0 14.5 17.0 0 0.00 0.00
84%MHA-Ca5 6.50 6.70 7.00 0 0.00 0.00
Phospholipid1 10.0 10.0 10.0 10 10.0 10.0
VC Ascorbic acid 1.00 1.00 1.00 1 1.00 1.00
Y2O36 0.10 0.10 0.10 0.1 0.10 0.10
Total 1000 1000 1000 1000 1000 1000
1
(DM) Approximate composition (g kg dry matter)
Moisture 105.7 97.6 101.1 113.3 106.5 108.1
Crude protein 333.5 311.2 285.5 319.7 293.9 273.5
Digestive Protein 264.0 258.9 231.4 261.7 249.2 234.1
Crude fat 44.8 44. 1 45. 4 45 45.0 44. 6
Lysine 19.906 19.558 19.502 13.509 12.404 12.173
Methionine 10.250 10.121 10.210 4.802 4.588 4.281
Ash 83.8 81.3 80.1 83.1 78.7 76.1
1
Digestive energy kcal kg 3397 3334 3429 3380 3456 3436
Gross energy kcal kg 1 4792 4728 4751 4841 4839 4783

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): MgSO4·7H2O,315; ZnSO4·7H2O,285; CaHPO4·2H2O,250; FeSO4·7H2O,200; MnSO4·H2O,25; CoSO4·7H2O,25;
CaIO3,25; CuSO4·5H2O,15; Na2SeO3,10. (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).
L-Lysine SO4 contained L-Lysine  51% (CJ Co., Ltd., Liaocheng, China).
4

5
MHA-Ca contained DL-HMTBA (2-hydroxy-4-methylthio butanoic acid)  84% (Novus International Inc., Zhibo, China).
6
Y2O3 (Yttrium oxide), analytical pure (Weibo Chemical Ltd, Guangzhou, China).

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-
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sition and 8 were anaesthetized with tricaine methane sul-
phonate (MS222) (50 mg L 1) to obtain weights of
individual whole body, viscera, liver and mesenteric fat.
White muscle from both sides of the fillets without skin
and liver was dissected and frozen immediately in liquid
nitrogen and stored at 70 °C until used.
Diets and fish samples (including white muscle and liver)
were analysed in triplicate for proximate composition. Crude
protein, crude lipid, moisture, crude ash and gross energy
(GE) were determined following standard methods (AOAC
1984). Crude protein (N 9 6.25) was determined by the Kjel-
dahl method after acid digestion using an Auto Kjeldahl Sys-
tem (1030-Auto-analyzer, Tecator, Sweden). Crude lipid was
determined by the ether extraction method using a Soxtec

Aquaculture Nutrition ª 2012 Blackwell Publishing Ltd

 Table 2 Amino acid composition of
Diet
experimental diets for grass carp (g kg 1
Amino acids 32AA 30AA 28AA 32CP 30CP 28CP dry diets)

Essential amino acids

Lysine 19.9 19.6 19.5 13.5 12.4 12.2
Methionine 10.3 10.1 10.2 4.8 4.6 4.3
Phenylalanine 12.8 11.7 10.8 12.5 11.9 11.1
Histidine 6.1 5.7 5.2 5.9 5.7 5.2
Tryptophan 3.9 3.5 3.1 3.9 3.5 3.1
Arginine 18.7 17.7 15.3 18.4 17.3 15.6
Threonine 8.4 8.3 7.4 8.6 8.4 7.5
Isoleucine 11.0 10.2 9.3 11.1 10.7 9.6
Leucine 19.2 17.9 16.4 19.3 18.7 17.0
Valine 15.0 13.9 12.9 14.8 14.4 13.2

Non-essential amino acids

Serine 7.0 7.3 6.3 7.4 7.5 6.6
Proline 16.7 15.6 14.9 16.9 19.1 15.8
Cystine 1.8 1.8 1.7 1.8 1.7 1.8
Tyrosine 4.7 4.3 3.5 4.9 4.7 3.9
Aspartic acid 22.6 20.0 17.2 22.3 20.3 17.4
Glutamic acid 59.8 56.3 52.4 59.1 57.5 53.0
Glycine 13.2 12.1 11.2 12.9 12.4 11.3
Alanine 12.5 11.9 11.1 12.5 12.5 11.6

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.

System HT (Soxtec System HT6, Tecator, Sweden). Moisture
was determined by oven-drying at 105 °C for 24 h. Crude
ash was determined by incineration in a muffle furnace at
550 °C for 24 h. GE was determined using an adiabatic
bomb calorimeter. Amino acids were analysed following acid
hydrolysis using high-pressure liquid chromatography
(HPLC; Hewlett Packard 1090, Palo Alto, CA, USA). The
concentrations of dietary and faecal Y2O3 were determined
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.
All data are presented as means ± SEM The SPSS software
ver 13.0 for Windows of GLM procedure (SPSS Inc.,
Chicago, IL, USA) was used to conduct factorial ANOVA to
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 par-
ticular response, differences among protein levels within
each diet type were determined using Tukey’s mean separa-
tion. Treatment effects and interactions were considered
significant at P < 0.05.
Fish readily accepted the experimental diets. At the end of
the growth trial, the survival were high (>94.67%), and there
were no significant differences in survival among fish fed all
the 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 trial
are presented in Table 3. FI and NR were significantly
increased with the increase in dietary protein level and the
supplementation of lysine and methionine (P < 0.05), and
WG and SGR also showed the same trends, but there was
interaction found between dietary protein level and the
supplementation of lysine and methionine (P < 0.05). FCR
significantly decreased with the increase in dietary protein
level and the supplementation of lysine and methionine
(P < 0.05). LR was not significantly affected by the diet
treatments (P > 0.05).
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Aquaculture Nutrition ª 2012 Blackwell Publishing Ltd

 Table 3 Effect of with or without lysine and methionion supplementation in different protein level on growth and feed utilization of grass carp

Lys + met supplementation No Lys + met supplementation Pr > F

Group
Protein*
Diet 32AA 30AA 28AA 32CP 30CP 28CP Protein Lys + met (Lys + met)

IBW 2.10 ± 0.01 2.09 ± 0.00 2.10 ± 0.01 2.10 ± 0.01 2.10 ± 0.01 2.09 ± 0.01
FBW 13.7 ± 0.42C 10.6 ± 1.01B 6.11 ± 0.38A 9.25 ± 1.13b 6.19 ± 0.14ab 5.05 ± 0.16a 0.043 AA supply > No AA supply

Aquaculture Nutrition ª 2012 Blackwell Publishing Ltd

<0.001 <0.001
Survival 96.0 ± 2.31 97.3 ± 2.67 97.3 ± 2.67 94.7 ± 3.53 100 ± 0.00 98.67 ± 1.33 0.560 0.855 0.290
WG% 554 ± 22.0C 405 ± 48.2B 192 ± 18.6A 340 ± 56.1b 194 ± 6.19ab 142 ± 8.17a <0.001 <0.001 0.047 AA supply > No AA supply
SGR 2.98 ± 0.05C 2.56 ± 0.15B 1.69 ± 0.10A 2.34 ± 0.20b 1.71 ± 0.03ab 1.40 ± 0.05a <0.001 <0.001 0.049 AA supply > No AA supply
FCR 1.31 ± 0.03 1.39 ± 0.01 1.56 ± 0.02 1.40 ± 0.02 1.53 ± 0.02 1.58 ± 0.04 <0.001 0.003 0.096 AA supply < No AA supply

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FI 1.75 ± 0.02 1.75 ± 0.08 1.60 ± 0.06 1.66 ± 0.07 1.60 ± 0.03 1.44 ± 0.03 0.028 0.025 0.806 AA supply > No AA supply
NR 28.0 ± 0.91 27.2 ± 0.30 26.9 ± 0.29 26.8 ± 0.59 26.1 ± 0.41 23.9 ± 1.04 0.030 0.009 0.256 AA supply > No AA supply
LR 184 ± 8.48 179 ± 23.4 202 ± 13.2 211 ± 24.8 222 ± 3.69 222 ± 19. 6 0.801 0.066 0.801

Means ± SEM of three replicates. Probability associated with the F statistic for the factorial ANOVA. Within a row, capital letters indicated differences within diets with lys + met
supplementation and lowercase letters indicate differences within diets without Lys + met supplementation at P < 0.05 when interactions occurred for the full model.
IBW (g fish 1), initial body weight.
FBW (g fish 1), final body weight.
Survival (%) = 100 9 (final fish number)/(initial fish number).
Weight gain (WG, %) = 100 9 (final body weight-initial body weight)/initial body weight.
Specific growth rate (SGR, % day 1) = 100 9 (ln final wt ln initial wt)/63 days.
Feed conversion ratio (FCR) = Feed consumed/(FBW IBW).
Feed intake (FI) = grams of dry feed consumed 9 100/100 g body mass/63 days.
Nitrogen retention (NR) = 100 9 retained nitrogen (g)/nitrogen fed (g).
Lipid retention (LR) = 100 9 retained lipid (g)/lipid fed (g).
 Table 4 Body composition and morphometry index of grass carp fed experimental diets for 63 days

Lys + met supplementation No Lys + met supplementation Pr > F

Group
Protein*
Composition (g kg 1)1 32AA 30AA 28AA 32CP 30CP 28CP Protein Lys + met (Lys + met)

Whole body
Moisture 741 ± 1.90 732 ± 10.3 728 ± 7.20 729 ± 5.70 721 ± 5.80 724 ± 4.71 0.214 0.290 0.287
Protein 123 ± 1.71 121 ± 1.01 123 ± 0.92 122 ± 3.93 122 ± 2.14 114 ± 0.93 0.161 0.098 0.112
Lipid 106 ± 2.83 117 ± 10.9 122 ± 0.13 124 ± 11.9 135 ± 0.53 132 ± 8.33 0.408 0.068 0.902
Ash 21.9 ± 0.02 21.9 ± 0.21 23.2 ± 0.64 23.0 ± 0.33 22.6 ± 0.64 21.7 ± 1.74 0.948 0.863 0.241
Muscle
Moisture 808 ± 2.14 793 ± 3.62 781 ± 6.13 803 ± 21.1 787 ± 2.92 779 ± 8.83 0.159 0.895 0.854
Protein 170 ± 0.83 169 ± 1.72 172 ± 1.51 157 ± 12.4 174 ± 2.41 168 ± 2.61 0.379 0.412 0.295
Lipid 17.1 ± 2.00 21.7 ± 1.81 31.4 ± 5.42 22.8 ± 6.81 28.6 ± 2.53 38.6 ± 11.1 0.073 0.200 0.990
Liver
Moisture 553 ± 16.7 518 ± 32.3 478 ± 3.41 524 ± 27.0 496 ± 16.6 475 ± 24.2 0.023 0.537 0.544
Protein 104 ± 2.72 102 ± 2.51 102 ± 3.62 102 ± 2.51 106 ± 6.10 99.2 ± 2.61 0.568 0.879 0.585
Lipid 214 ± 23.6 310 ± 29.3 311 ± 19.2 294 ± 25.1 314 ± 23.2 330 ± 31.6 0.019 0.442 0.157
Morphometry2
VSI 10.1 ± 0.43 11.3 ± 0.45 12.9 ± 0.52 11.4 ± 0.55 13.1 ± 0.40 13.4 ± 0.67 0.000 0.005 0.403 AA supply < No AA supply
HSI 1.79 ± 0.10 2.11 ± 0.11 2.37 ± 0.17 2.42 ± 0.18 2.61 ± 0. 22 2.51 ± 0.17 0.109 0.002 0.237 AA supply < No AA supply
IPF 3.58 ± 0.27 4.54 ± 0.40 6.02 ± 0.42 4.83 ± 0.39 5.83 ± 0.47 6.50 ± 0.45 0.000 0.003 0.535 AA supply < No AA supply
CF 2.09 ± 0.07 2.13 ± 0.06 2.30 ± 0.08 2.20 ± 0.07 2.24 ± 0.04 2.40 ± 0.08 0.007 0.053 0.986
1
Means ± SEM of three replicates. Probability associated with the F statistic for the factorial ANOVA. Within a row, capital letters indicated differences within diets with lys
+ met supplementation and lowercase letters indicate differences within diets without Lys + met supplementation at P < 0.05 when interactions occurred for the full model.
2
Means ± SEM of 24 replicates.
Viscerasomatic index (VSI) = 100 9 viscerasomatic weight (g)/body weight (g).
Hepatopancreasomatic index (HSI) = 100 9 liver weight (g)/body weight (g).
Intraperitoneal fat ratio (IPF) = 100 9 intraperitoneal fat weight (g)/body weight (g).
Condition factor (CF) = 100 9 body weight (g)/body length (cm)3.

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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
treatments (P > 0.05). And the whole body lipid content
showed decreasing trends when fish was fed diets with
lysine and methionine supplementation (P = 0.068).
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
treatments (P > 0.05).
Liver moisture significantly increased with the increase in
the dietary protein level (P < 0.05), and liver lipid content
showed an opposite trend (P = 0.073). No significant dif-
ferences were found in liver protein contents of fish among
all the diet treatments (P > 0.05).
Condition factor (CF), hepatopancreasomatic index
(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
crude protein levels (P < 0.05). At the same dietary crude
protein level, fish fed lysine and methionine supplemented
diets discharged less TAN, demonstrating that lysine and
methionine supplementation reduced TAN discharge (P <
0.05), but the interaction was not found (P = 0.463).
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
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Aquaculture Nutrition ª 2012 Blackwell Publishing Ltd
250
207.08
Ammonia nitrogen aŌer fed 8 h
200 187.53
(mg kg–1 body weight)
170.64
157.31
141.31
150 128.87
100
50
0
32AA 32CP 30AA 30CP 28AA 28CP
Diets
Figure 1 Total ammonia nitrogen discharged into water (mg kg 1
body weight, mean ± SD) after fed 8 h.
content, was also observed by Kim et al. (2001) for the had-
dock (Melanogrammus aeglefinus L), and by Luo et al.
(2004) for the grouper Epinephelus coioides. Compared with
most aquaculture fish species, grass carp has a low energy
requirement, grass carp preferentially adjusted intake to
protein before energy (Du et al. 2005). FI of grass carp was
also significantly increased with addition of lysine and
methionine. Amino acids deficiency causes loss of appetite,
resulting in low FI as shown in Chanos chanos (Borlongan
& Benitez 1990), Labeo rohita (Khan & Jafri 1993),
Oncorhynchus mykiss (Yamamoto et al. 2000) and Cirrhinus
mrigala (Ahmed & Khan 2004). Yamamoto et al. (2000)
indicated that Oncorhynchus mykiss showed a preference
for the balanced amino acid diet over the imbalanced amino
acid, and Oncorhynchus mykiss can also discriminate
deficiency of lysine in diets and show a rapid reduction in the
consumption of the amino acid imbalanced diet (Yamamoto
et al. 2001).
Dabrowski (1977) found there were a linear relationship
between dietary protein level and body protein and growth
and up to optimum at dietary protein levels of 410 g kg 1
and 430 g kg 1, respectively. Our results also indicated that
low-protein diets induced adverse effects on growth perfor-
mance of grass carp, there were significant differences in
growth performance of fish fed 32CP, 30CP and 28CP
diets, which indicated that grass carp need to be fed diet
with higher protein content.
In the present experiment, the growth performance of fish
fed 32AA and 30AA diets were significantly higher than
that of fish fed 32CP and 30CP diets. Results of the present
investigation demonstrate significant improvement of
growth and feed utilization of grass carp can be achieved by
L-Lysine sulphate and MHA-Ca supplementation. Some
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
nile tilapia can be reduced from 270 g kg 1 to 243 g kg 1
(Botaro et al. 2007). Gaylord & Barrows (2009) found that
plant-based dietary protein content for rainbow trout could
be reduced from 460 g kg 1 to 415 g kg 1 by supplement-
ing lysine, methionine and threonine without growth reduc-
tion. The dietary protein content for common carp also
could be reduced from 300 g kg 1 to 250 g kg 1 by 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
et al. 2000). But essential amino acid supplementation to
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 proportion 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 morphological
measurements of grass carp (Table 4), VSI, IPF and HSI
were 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 & Barrows
2009). 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 and
IPF of rainbow trout (Oncorhynchus mykiss) were signifi-
cantly reduced with multiple amino acid supplementation
in plant-based feeds. Brown et al. (1992) suggested that
IPF and HSI reflect the proportional accumulation of
energy in both the abdominal cavity and the liver, and it
has been widely acknowledged that feeding diets deficient
in amino acid results in excess energy deposition as fat in
the liver, fillet or abdominal cavity. The results indicated
that muscle, liver and whole body lipid contents of fish fed
32CP diet were slightly lower than that of fish fed 30CP
and 28CP diets, and muscle, liver and whole body lipid
contents showed a reducing trend with increasing lysine
and methionine supplementation. Several studies have
demonstrated 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 of
channel catfish fed diet with supplemental lysine and
methionine (Li & Robinson 1998). The reduction in carcass
lipid content of fish may be related to enhanced protein
synthesis as a result of lysine and methionine supplementa-
tion. The NR was increased with lysine and methionine
supplementation.
Total ammonia nitrogen was directly related to dietary
nitrogen 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 carp
increased 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 die-
tary protein levels had lower dietary carbohydrate levels.
Some researchers found that increasing the dietary level
of non-protein digestible energy could increase NR by
decreasing nitrogen losses (Kaushik & Oliva Teles 1985;
Médale et al. 1995). In the present study, high crude
protein diets resulted in higher excretion of ammonia,
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Aquaculture Nutrition 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
a diet containing a 250 g kg 1 CP supplemented with
5 g kg 1 lysine could reduce the amount of nitrogen excre-
tion per unit WG by 20%. Viola et al.(1992b) further
reported that common carp could reduce nitrogen excretion
by about 20% by reducing dietary CP from 300 g kg 1 to
250 g kg 1 along with supplementing lysine (0.5%) and
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,
and we can reduce the dietary crude protein from 320 g kg 1
to 300 g kg 1 in the practical diets through balancing amino
acids profile. TAN excretion of grass carp was also reduced
with lysine and methionine supplementation.
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Aquaculture Nutrition ª 2012 Blackwell Publishing Ltd