2. Materials and methods
2.1. Diet The ingredients and proximate composition of the experimental diets are shown in
Ž .
Table 1. Six semi-purified diets were prepared using vitamin-free casein Wako, Japan Ž
. Ž
and gelatin Difco Laboratories, USA as the protein sources, pollock visceral oil Riken .
Ž .
Vitamin, Japan and soybean oil Wako as the fat sources, and gelatinized potato starch Ž
. Ž
. Matsuya Chemical Industry, Japan and dextrin Wako as the digestible carbohydrate
Ž Ž
. Ž
.. sources. Four diets contained combinations of two levels 35 LP and 50 HP
of a Ž
. Ž
Ž .
balanced amino acid protein casein:gelatin s 6:1 and two levels 10 LF and 20 Ž
.. Ž
. HF
of fat pollock oil:soybean oil s 2:3 . The ratio of casein to gelatin was in Ž
. accordance with an optimal value for carp Murai et al., 1984 . The other two diets
Ž .
contained 50 imbalanced amino acid protein IMB-HP, casein:gelatins 1:1 and two Ž
Ž .
Ž ..
levels of fat 10 LF and 20 HF . The ingredients were thoroughly mixed, Ž
. moistened by addition of hot water 508C , and made into pellets using a garlic squeezer.
The pellets were freeze-dried and stored at y208C until fed to the fish. The amino acid composition of the dietary protein sources and of the experimental
Ž .
diets are shown in Table 2. The EAA index EAAI based on the EAA composition of Ž
. trout eggs Suyama and Ogino, 1958 were estimated at 93 for the LP and HP diets, and
73 for the IMB-HP diets. Although the EAAI of the IMB-HP diets was lower than the
Table 1 Formulation and proximate composition of the experimental diets
a
Diet LPLF
LPHF HPLF
HPHF IMB-HPLF
IMB-HPHF Ingredients
Ž .
Casein vitamin free 32.29
32.29 46.13
46.13 26.91
26.91 Gelatin
5.74 5.74
8.20 8.20
28.70 28.70
Pollock visceral oil 4.00
8.00 4.00
8.00 4.00
8.00 Soybean oil
6.00 12.00
6.00 12.00
6.00 12.00
a-Starch 15.00
15.00 15.00
15.00 15.00
15.00 Dextrin
16.00 16.00
4.67 4.67
3.39 3.39
b
Vitamin mix 2.00
2.00 2.00
2.00 2.00
2.00
b
Mineral mix 4.00
4.00 4.00
4.00 4.00
4.00 Cellulose
14.97 4.97
10.00 –
10.00 –
Proximate composition dry matter basis Ž
. Crude protein N=6.25
36.5 36.7
52.8 52.2
55.5 55.0
Ž . Crude fat
9.7 19.9
9.4 19.9
10.3 20.9
Ž . Crude ash
3.0 3.0
3.4 3.4
3.2 3.2
c
Ž .
Gross energy KJrg 21.6
24.6 22.6
25.3 21.9
24.4
a
LP; low protein with balanced amino acid diet, HP; high protein with balanced amino acid diet, IMB-HP; high protein with imbalanced amino acid diet, LF; low fat diet, HF; high fat diet.
b
Ž .
Same mixtures as previously reported Yamamoto et al., 1998 .
c
Ž .
Analyzed value using Bomb Calorimeter CA-4PJ Shimadzu, Japan .
Table 2 Ž
. Amino acid composition gr100 g of the experimental diets
a
Amino acids Ingredient
Diet ArE ratio of diet
b
Casein Gelatin
LP HP
IMB-HP LP and HP
IMB-HP Arginine
3.62 7.65
1.61 2.30
3.17 86
149 Histidine
2.83 0.99
0.97 1.39
1.05 52
50 Isoleucine
4.95 1.26
1.67 2.39
1.69 89
80 Leucine
9.32 2.84
3.17 4.53
3.32 170
156 Lysine
7.84 3.56
2.74 3.91
3.13 147
147 Methionine
2.77 0.82
0.94 1.35
0.98 50
46
c
Cystine 0.41
ND 0.13
0.19 0.11
7 5
Phenylalanine 5.00
1.96 1.73
2.47 1.91
93 90
Tyrosine 5.57
0.66 1.84
2.62 1.69
98 80
Threonine 4.01
1.69 1.39
1.99 1.56
74 73
Tryptophan 1.09
ND 0.35
0.50 0.29
19 14
Valine 6.22
2.28 2.14
3.06 2.33
115 110
Alanine 3.14
10.18 1.60
2.28 3.77
Aspartic acid 6.60
5.45 2.44
3.49 3.34
Glutamic acid 21.73
9.55 7.56
10.81 8.59
Glycine 1.82
32.33 2.44
3.49 9.77
Proline 10.51
12.83 4.13
5.90 6.51
Hydroxyproline ND
10.11 0.58
0.83 2.90
Serine 5.44
3.16 1.94
2.77 2.37
d
EAA index 93.9
28.4 93.1
93.1 73.2
a
Amino acid levels of the diets were calculated based on the analytical values of casein and gelatin.
b
LP; low protein with balanced amino acid diet, HP; high protein with balanced amino acid diet, IMB-HP; high protein with imbalanced amino acid diet.
c
Not detected.
d
Ž Essential amino acid index based on amino acid composition of rainbow trout egg Suyama and Ogino,
. 1958 .
balanced amino acid diets, the IMB-HP diets contained similar levels of individual EAA to those of the LP diets except for arginine.
2.2. Fish and feeding Ž
. Fingerling rainbow trout Oncorhynchus mykiss with a mean weight of 3.1 grfish
were transported from the Shiga Prefectural Samegai Trout Farm, Shiga, Japan, to the Inland Station of the National Research Institute of Aquaculture, Mie, Japan. The fish
Ž .
were reared for 6 weeks with a commercial trout feed Nippon Formula Feed, Japan at Ž
. 158C. The fish were then sorted by hand to an uniform size approximately 8 grfish ,
Ž and 30 fish were allotted into each of 12 polyvinyl chloride tanks 50 = 20 = 30 cm,
. holding 20 l of water , and fed a commercial feed for two weeks for acclimation to the
Ž new rearing condition. The tanks were continuously supplied with well water 15
. 0.38C at a flow rate of 1.5 lrmin. After the acclimation, all the fish were weighed
Ž .
initial weight; 9.6 0.7 grfish and duplicate groups of fish were fed one of the
experimental diets to satiation twice per day, 6 days per week for 6 weeks. The fish were weighed every 2 weeks to follow the overall growth.
2.3. Sampling Ž
After the 6-week feeding trial, the fish were weighed and four fish from each tank 8 .
fishrtreatment were sampled and stored at y208C for subsequent analyses of the proximate and the amino acid composition of the whole body. The remaining fish were
fed their respective diets for another 2 days. In rainbow trout fed intact protein diets to Ž
. Ž
. satiation, Nose 1973 and Walton and Wilson 1986 reported that plasma FAA attained
maximum levels at 12 h after feeding and declined to the fasting levels by 48 h. By Ž
contrast, FAA in trout liver did not show distinct postprandial changes Walton and .
Wilson, 1986 . Thus, the fish in this study were then starved for 48 h, fed once again the diets to satiation, and blood and tissue samples for FAA analysis were collected at 12 h
Ž .
after the last feeding. Three fish from each tank 6 fishrtreatment were anesthetized in Ž
. 0.01 ethyl 3-aminobenzoate methanesulfonic acid Aldrich Chemical, USA . Blood
was immediately withdrawn from the caudal vein–artery complex and plasma samples for determination of FAA were prepared according to the procedures described in
Ž .
Yamamoto et al. 1998 . The fish were then placed into liquid nitrogen and stored at y808C for up to 1 week until extraction of FAA from tissues.
2.4. Analysis Proximate analysis of the diets and the whole bodies were carried out using the
Ž .
AOAC method slightly modified by Akiyama et al. 1984 . Amino acid composition of the protein sources and the whole bodies was determined after acid hydrolysis in 6 N
Ž .
HCl for 22 h at 1108C, as described in Yamamoto et al. 1997 . Cystine and tryptophan Ž
. in the protein sources were analyzed according to the methods of Moore 1963 and
Ž .
Penke et al. 1974 , respectively. The partially thawed–frozen fish were dissected and Ž
. whole brain and liver, and a portion of dorsal white muscle approximately 500 mg
were taken for FAA analysis. FAA in tissue samples were extracted with a perchloric Ž
. acid solution according to the method of Ogata and Murai 1994 . FAA levels were
Ž .
determined individually by an automatic amino acid analyzer L-8500 Hitachi, Japan Ž
. with a ninhydrin reagent and lithium buffer system Yamamoto et al., 1998 .
Ž .
Significance P - 0.05
of treatment means of growth performance parameters, whole body proximate and amino acid compositions, and tissue FAA levels, were
compared using one-way ANOVA followed by Duncan’s new multiple range test Ž
. Wakabayashi, 1984 .
3. Results
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