The influence of dietary protein sources on tissue free amino acid levels of fingerling rainbow trout

Transcription

1 FISHERIES SCIENCE 2000; 66: Original Article The influence of dietary protein sources on tissue free amino acid levels of fingerling rainbow trout Takeshi YAMAMOTO, 1, * Tatsuya UNUMA 2 AND Toshio AKIYAMA 3,a 1 Fish Feed Section, Inland Station, National Research Institute of Aquaculture, Tamaki, Mie , 2 Reproductive Technology Section, Coastal Station, National Research Institute of Aquaculture, Nansei, Mie and 3 Research Coordinating Section, National Research Institute of Fisheries Science, Yokohama, Kanagawa , Japan SUMMARY: To estimate the quantitative adequacy of amino acids in practical dietary proteins, free amino acid (FAA) levels in various tissues of rainbow trout Oncorhynchus mykiss fed different protein sources were compared. Four diets, the proteins of which were derived mainly from fish meal (FM diet), soybean meal (SBM diet), malt protein flour (MPF diet), and the combination of SBM and MPF (COMB diet), were fed to duplicate groups (30 fish/group, 10 g/fish) for 6 to 9 weeks at 15 C, until each treatment group reached the weight of the FM diet group attained in 6 weeks. The fish for FAA analysis were sampled 12 h after the last feeding. The body weight of the FM group was attained in 7 weeks by the COMB diet group, and in 9 weeks by the SBM and the MPF diet groups. Concentrations of free threonine, methionine, and lysine in most of the tissues of the COMB diet group, as well as the SBM and the MPF diet groups, were quite lower than those of the FM diet group. Levels of these amino acids between the four diets and most of the tissues examined were highly correlated. Although the threonine content in the COMB diet was slightly lower than the FM diet, these findings suggest that not only methionine and lysine but also threonine is still insufficient in the COMB diet. KEY WORDS: diet, fish meal, free amino acids, malt protein flour, Oncorhynchus mykiss, soybean meal, tissue. INTRODUCTION To determine the nutritional protein status of animals, it is a common practice to measure the tissue free amino acid (FAA) levels. 1 Plasma FAA levels of fish fed different proteins have sometimes been compared to determine the influence and utilization of the dietary amino acids. 2 5 Moreover, a relationship between tissue essential amino acid (EAA) levels and dietary requirements has been suggested. 6 9 Thus, tissue FAA levels in fish may be useful to know the sufficiency of amino acids in practical diets containing various protein sources. Studies of the influence of practical proteins on FAA levels in tissues other than plasma are, however, limited to the *Corresponding author: Tel: Fax: takejpn@nria-tmk.affrc.go.jp a Present address: Fukui Prefectural Fisheries Experimental Station, Tsuruga, Fukui , Japan. Received 2 August works of Kaushik and Luquet, 10 and of Dabrowska, 11 on rainbow trout Oncorhynchus mykiss muscle. It has been reported that muscle FAA levels of certain fish species also vary with fresh food organisms consumed. 12,13 In several fish species receiving a single feed, the FAA profiles and their levels vary widely among the tissues Since there is a slower rate of protein turnover in muscle than in the other organs, 19 the influence of dietary protein quality (amino acid profile) may be more responsive in other tissues than muscle. To evaluate the influence of dietary composition on tissue FAA, we examined in a previous study using semipurified diets, the influence of dietary protein and fat levels, and dietary amino acid profiles, on the FAA levels in several tissues of rainbow trout. 20 We demonstrated that not only dietary protein levels and dietary amino acid profiles, but also dietary fat levels affected the levels of certain FAA. We also found that the correlations between the proportions of dietary EAA levels and tissue free EAA concentrations were high in plasma, but very

2 Influence of dietary proteins on trout tissue FAA FISHERIES SCIENCE 311 low in dorsal white muscle and brain. 20 In addition, levels of total EAA and certain amino compounds in the brain did not differ regardless of the dietary amino acid profiles. 20 Therefore, the influence of practical dietary protein sources on tissue FAA levels may also differ among the tissues. As the next step to estimate quantitative adequacy of amino acids in practical fish feeds by comparing tissue FAA levels, we conducted a feeding experiment with fingerling rainbow trout using practical-type diets containing different protein sources. Four diets were prepared, the main proteins were derived from white fish meal (FM), defatted soybean meal (SBM), malt protein flour (MPF), or a combination of SBM and MPF (COMB). We compared the growth performance parameters and FAA levels in various tissues of the fish. To estimate the relationship between levels of certain amino acids in the diets and concentrations of the amino acids in tissues, correlation coefficients between the levels of amino acids in the diets and the tissues were also evaluated. MATERIALS AND METHODS Diet The ingredients and the proximate composition of the experimental diets, and the amino acid composition of the diets, are shown in Tables 1 and 2, respectively. Four Table 1 Formulation and proximate composition of the experimental diets Diet FM SBM MPF COMB Ingredients (% wet weight) White fish meal Soybean meal Malt protein flour Pollock visceral oil Soybean oil a-starch Cellulose Vitamin mix Mineral mix Sodium alginate Proximate composition (dry matter basis) Crude protein (N 6.25%) Crude fat (%) Crude ash (%) Gross energy (MJ/kg) FM, white fish meal base diet; SBM, defatted soybean meal base diet; MPF, malt protein flour base diet; COMB, combination of soybean meal and malt protein flour base diet. 1 Same as previously reported Analyzed value using Bomb Calorimeter CA-4PJ (Shimadzu Co., Japan). Diet FM SBM MPF COMB % A/E ratio % A/E ratio % A/E ratio % A/E ratio Arginine Histidine Isoleucine Leucine Lysine Methionine Cystine Phenylalanine Tyrosine Threonine Tryptophan Valine Alanine Aspartic acid Glutamic acid Glycine Proline Hydroxyproline Serine Taurine Amino acid levels of the diets were calculated based on the analyzed values of the protein sources employed in this study. FM, white fish meal base diet; SBM, defatted soybean meal base diet; MPF, malt protein flour base diet; COMB, combination of soybean meal and malt protein flour base diet.

3 312 FISHERIES SCIENCE T Yamamoto et al. diets were formulated to provide a diet of approximately 35% crude protein and 15% crude fat. One diet (FM) contained white fish meal (Alaska pollock meal, produced by JSC Oceanrybflot, USSR) as the sole protein source, and two diets (SBM and MPF) each contained the fish meal and defatted soybean meal (Marubeni Shiryo Co. Ltd, Tokyo, Japan), or the fish meal and malt protein flour (Kirin Brewery Co. Ltd, Tokyo, Japan), at the inclusion ratio of 1:4 (crude protein basis). A diet (COMB) containing the FM, SBM, and MPF at the inclusion ratio of 1:2:2 was also prepared. The ratio of dietary fat from animal and vegetable origins was adjusted to 1:2 by the addition of pollock visceral oil (Riken Vitamin Co. Ltd, Tokyo, Japan) and soybean oil (Wako Pure Chemical Industries Ltd, Osaka, Japan). The ingredients were thoroughly mixed and made into dry pellets according to the method described in Yamamoto et al. 21 The pellets were stored at -20 C until being fed to the fish. Fish and feeding Fingerling rainbow trout (1.1 g/fish) were obtained from the Shiga Prefectural Samegai Trout Farm and transported to the Inland Station of the National Research Institute of Aquaculture. The fish were reared with a commercial dry pellet for trout (Nippon Formula Feed Mfg. Co. Ltd, Kanagawa, Japan) for 7 weeks prior to the feeding experiment, according to the same manner as previously reported. 20 The fish were then sorted by hand for a uniform size (approximately 8 g/fish) and 30 fish were allotted into each of eight polyvinyl chloride tanks ( cm, holding 20 L of water). The fish were acclimated with a commercial feed to the new rearing condition for 2 weeks prior to the feeding trial. The tanks were each supplied with well water (15.0 ± 0.1 C) at a flow rate of 1.5 L per min. All the fish were weighed initially and every 2 weeks. Each diet was fed to duplicate groups to satiation twice per day, 6 days per week for 6 weeks. To avoid the influence of fish size on chemical compositions of the fish, the fish size for subsequent chemical analyses were equalized. The fish group receiving the FM diet showed the best growth and this treatment was terminated at 6 weeks. The other treatment groups were reared for another 1 3 weeks until they attained the weight of the FM group. Sampling After the final body weight measurement, the fish in the tanks were fed their respective diets for another 2 days. In rainbow trout fed intact protein diets to satiation, Nose 25 and Walton and Wilson 6 reported that plasma FAA attained maximum levels at 12 h after feeding and declined to the fasting levels by 48 h. By contrast, FAA in trout liver did not show distinct postprandial changes. 6 Thus, the fish in this study were then starved for 48 h, fed the diets once again to satiation. 20 At 12 h after the last feeding, three fish from each tank were sampled for plasma and tissue FAA Table 3 Growth and feed performance of fingerling rainbow trout fed diets containing different protein sources 1 Diet FM SBM MPF COMB Pooled standard Initial Mean body weight (g) At 6 weeks Mean body weight (g) 28.7 c a 21.1 a 26.3 b 0.53 Weight gain (%) c a a b 2.39 Feed efficiency (%) d 69.4 a 77.5 b 85.2 c 1.74 Daily feed consumption (%) 2.19 a 2.39 a 2.17 a 2.60 b Mortality (%) At final Rearing period (weeks) Mean body weight (g) Weight gain (%) a b a a 2.76 Feed efficiency (%) c 76.1 a 74.4 a 84.4 b 1.48 Daily feed consumption (%) 2.19 a 2.10 a 2.06 a 2.31 b Mortality (%) Average values of duplicate groups containing 30 fish each reared at 15 C. 2 Values in the same row followed by the same superscript are not significantly different (P > 0.05). FM, white fish meal base diet; SBM, defatted soybean meal base diet; MPF, malt protein flour base diet; COMB, combination of soybean meal and malt protein flour base diet.

4 Influence of dietary proteins on trout tissue FAA FISHERIES SCIENCE 313 analysis according to the methods described in Yamamoto et al. 20 Analysis Proximate compositions of the experimental diets, amino acid compositions of the diets, and tissue FAA concentrations, were determined according to the same methods as described in Yamamoto et al. 20 Eye, gill, dorsal white muscle, and scaled skin samples were collected from the right side of the body. The digesta in stomach and intestine (excluding pyloric ceca) were washed out with distilled water. FAA concentrations of plasma, liver, and muscle were individually analyzed (six samples/ treatment). The other tissues (brain, eye, gill, heart, kidney, spleen, stomach, intestine, and skin) of fish from the same tank (three samples/tank) were pooled before extracting FAA from the tissues (two pooled samples/treatment). Significance of difference in means between treatments (P < 0.05) was calculated using oneway analysis of variance and Duncan s new multiple range test. 26 Correlation coefficients were calculated for levels of certain amino acid between the diets and each tissue of all the treatments combined. In the calculation, a unit of mm per 100 g diet was used for dietary amino acid levels. Table 4 Levels of free amino acids and related compounds in plasma (nm/ml) and brain (nm/g wet tissue) of fingerling rainbow trout fed diets containing different protein sources Plasma 1 Brain 2 Threonine 423 b3 195 a 203 a 271 a b 533 a 396 a 517 a 34.9 Valine 603 b 425 a 468 ab 510 ab Cystine 9 b 6 a 6 a 9 b 0.8 ND ND ND ND Methionine 309 c 73 a 141 b 147 b Isoleucine 277 b 209 ab 187 a 227 ab Leucine 496 b 345 a 405 ab 420 ab Tyrosine 75 a 96 a 143 b 134 b a 77 ab 99 b 94 b 6.1 Phenylalanine 140 a 144 a 193 b 182 b Tryptophan b 60 ab 55 a 66 b 2.5 Lysine 474 c 214 b 79 a 187 ab d 225 c 116 a 179 b 10.4 Histidine 136 a 156 a 160 a 205 b b 568 a b 687 a 76.8 Arginine 223 c 130 ab 75 a 146 b b 137 ab 108 a 135 ab 7.6 EAA + C + T 3209 b 2036 a 2100 a 2470 a b a a a Aspartic acid 8 ab 9 bc 6 a 12 c a b 699 a 776 a 47.2 Serine 288 b 129 a 139 a 173 a b 488 ab 397 a 415 a 40.4 Asparagine 176 b 153 ab 130 a 211 c 11.3 ND ND ND ND Glutamic acid 60 a 43 a 59 a 97 b Glutamine 237 ab 223 a 446 c 318 b Glycine 1325 c 495 a 540 ab 703 b c bc 888 ab 823 a 50.1 Alanine 478 b 392 ab 274 a 345 ab Hydroxyproline 233 b 48 a 54 a 58 a ND ND ND 15.9 Proline 141 a 92 a 331 c 230 b NEAA 2946 c 1585 a 1980 ab 2148 b ab b a ab Taurine 506 b 232 a 381 b 474 b b a a b Citrulline 37 a 39 a 47 a 43 a b 132 ab 139 ab 116 a 11.0 Cystathionine ND ND ND ND b-alanine 36 a 29 a 21 a 93 b g-aminobutyric acid 12 a 15 a 95 c 64 b Ethanolamine 10 b 7 a 5 a 5 a Ammonia 356 ab 323 a 328 a 428 b Ornithine 35 ab 81 c 14 a 48 b a 65 c 25 a 45 b 2.7 Anserine ND ND ND ND ND ND ND ND Carnosine ND ND ND ND a b ab ab Average values of six samples of duplicate groups (three fish/group) for each dietary treatment. 2 Average values of two pooled samples (three fish/sample) of duplicate groups for each dietary treatment. 3 Values in the same row followed by the same superscript are not significantly different (P > 0.05).

5 314 FISHERIES SCIENCE T Yamamoto et al. RESULTS Growth and feed performance At 6 weeks of feeding, the fish fed the FM diet showed the best growth (P < 0.05; Table 3). The fish fed the COMB diet were significantly smaller (P < 0.05) and the fish fed the SBM and the MPF diets were still smaller (P < 0.05). Thus, the rearing periods when the body weight of the FM group in 6 week feeding was attained were 7 weeks in the COMB group and 9 weeks in both the SBM and the MPF groups. Feed efficiency throughout the rearing periods was the highest in the FM groups (P < 0.05), intermediate in the COMB group (P < 0.05), and the lowest in both the SBM and the MPF groups (P < 0.05). Daily feed consumption rate was significantly higher in the COMB group (P < 0.05) than the other treatment groups. Free amino acid levels in tissues Levels of FAA and related compounds in various tissues are shown in Tables 4 to 9. In the spleen (Table 7) and the intestine (Table 8), although most of the individual FAA levels of the plantprotein diet (SBM, MPF and COMB) groups appeared lower than in the FM group, significant differences were not detected, due to large variances of the FAA levels within the FM group. Except these tissues, differences in individual amino acid levels between the FM diet group and the plant-protein diet groups in the brain Table 5 Levels of free amino acids and related compounds (nm/g wet tissue) in eye and gill of fingerling rainbow trout fed diets containing different protein sources Eye 1 Gill 1 Threonine 390 b2 237 a 203 a 244 a b 303 a 257 a 305 a 24.5 Valine 338 b 281 a 284 a 297 ab c 234 a 254 a 301 b 8.5 Cystine ND ND 25 ND 3.5 Methionine 375 c 90 a 189 b 181 b Isoleucine 152 b 133 ab 109 a 121 a Leucine 377 b 300 a 320 ab 315 a c 213 a 246 ab 255 b 10.1 Tyrosine 92 a 116 ab 158 c 137 bc ab 69 a 105 c 97 bc 5.8 Phenylalanine 151 a 231 b 326 c 256 b Tryptophan Lysine 355 c 191 b 105 a 162 b b 213 a 153 a 171 a 38.9 Histidine Arginine b 141 ab 101 a 139 ab 25.4 EAA + C + T b a a a b a a a Aspartic acid 218 a 270 b 214 a 234 ab a 516 c 339 a 453 b 13.8 Serine 332 b 203 a 189 a 209 a b 427 a 380 a 447 a 54.5 Asparagine 107 ab 132 bc 90 a 147 c a 359 b 235 a 377 b 15.9 Glutamic acid a ab ab b 87.6 Glutamine b a c b a 524 a 826 b 803 b 24.4 Glycine b 739 a 661 a 727 a b a a a Alanine 332 c 312 bc 234 a 251 ab c 748 bc 551 a 609 ab 36.4 Hydroxyproline 147 b 34 a 54 a 64 a b 138 a 142 a 120 a 14.2 Proline 105 a 86 a 229 c 173 b a 183 a 544 c 370 b 20.5 NEAA b a ab ab b a a ab Taurine b a b b ab a ab b Citrulline b 117 a 163 a 122 a 21.4 Cystathionine 175 b 80 a 109 a 106 a ND 23 ND 4.5 b-alanine g-aminobutyric acid a 184 ab 237 bc 286 c 22.9 Ethanolamine 66 a 65 a 58 a 77 b Ammonia c 927 a b d b a ab ab Ornithine 34 a 80 b 25 a 51 ab a 167 b 96 ab 134 ab 24.3 Anserine a 687 b 455 a 479 a 31.2 Carnosine ND ND ND ND ND ND ND ND 1 Average values of two pooled samples (three fish/sample) of duplicate groups for each dietary treatment. 2 Values in the same row followed by the same superscript are not significantly different (P > 0.05).

6 Influence of dietary proteins on trout tissue FAA FISHERIES SCIENCE 315 Table 6 Levels of free amino acids and related compounds (nm/g wet tissue) in heart and liver of fingerling rainbow trout fed diets containing different protein sources Heart 1 Liver 2 Threonine 521 b3 358 ab 326 a 338 a b b 541 a 632 a Valine b 551 ab 515 a 529 a 37.3 Cystine ND ND ND ND Methionine 60 b ND 52 b 20 a Isoleucine b 330 a 297 a 324 a 22.2 Leucine b 680 a 721 ab 710 a 50.9 Tyrosine 80 a 90 a 159 b 145 b Phenylalanine 143 a 151 a 206 b 187 b Tryptophan 48 ab 41 a 56 b 49 ab Lysine 292 c 155 b 76 a 133 ab c 615 b 338 a 468 ab 62.9 Histidine 730 a b b b c ab a bc 58.3 Arginine 148 b 99 a 57 a 95 a EAA + C + T c bc a ab Aspartic acid a b a a Serine 476 b 341 a 380 ab 328 a c bc 733 a 940 ab Asparagine b 187 a 232 ab 345 b 43.5 Glutamic acid a ab b ab a a a b Glutamine 984 a a b ab ab a c bc Glycine b a a a b a a a Alanine 817 b 963 c 845 b 729 a Hydroxyproline 294 b ND ND 76 a b 174 a 122 a 135 a 33.2 Proline 176 ab 108 a 288 b 196 ab a 976 a b b NEAA ab a b ab Taurine a ab b ab b a b b Citrulline a 737 ab b b Cystathionine 20 ND ND ND b 73 a 67 a 61 a 12.5 b-alanine 62 a 119 b 60 a 68 a g-aminobutyric acid 24 a 26 a ND 61 b a 35 ab 116 c 65 b 10.4 Ethanolamine 54 a 111 b 117 b 95 ab a 118 ab 112 ab 130 b 15.9 Ammonia a ab b ab b b a ab Ornithine b 351 c 132 a 235 b 27.5 Anserine 14 a 81 b 21 a ND 7.0 ND ND ND ND Carnosine ND ND ND ND ND ND ND ND 1 Average values of two pooled samples (three fish/sample) of duplicate groups for each dietary treatment. 2 Average values of six samples of duplicate groups (three fish/group) for each dietary treatment. 3 Values in the same row followed by the same superscript are not significantly different (P > 0.05). (Table 4), kidney (Table 7), and skin (Table 9) seemed small. Among free EAA, the levels of threonine, valine, methionine, leucine, and lysine in most of the tissues of the SBM group were lower than in the FM group. In the MPF group, the levels of threonine, methionine, lysine, and arginine were generally lower than in the FM group. Among these EAA, the levels of methionine of the SBM group and those of lysine of the MPF group, in several tissues such as plasma (Table 4), eye (Table 5), heart (Table 6), and dorsal white muscle (Table 9), were markedly lower than the FM group. Although the levels of these EAA of the COMB group tended to be higher compared with the SBM or the MPF group, the threonine, methionine, and lysine levels were generally still lower than the FM group. The levels of aromatic amino acids (phenylalanine and tyrosine) in most of the tissues of the MPF group were higher than the FM group. However, since most of the individual EAA levels of the plant-protein diet groups were lower than the FM group, the total EAA levels of the plant-protein groups were generally lower than the FM group. In the eye, relatively high levels of sulfur-containing compounds such as methionine, cystine, and cystathionine were present compared with the other tissues. Among free NEAA, serine, glycine, and hydroxyproline levels of the plant-protein diet groups were generally lower than the FM group. In contrast, the levels of aspartic acid and asparagine of the SBM group, and those of glutamine and proline of the MPF group, tended to be higher than the FM group. Therefore, differences in the total NEAA levels between the FM group and the plantprotein groups appeared small compared with the case in the total EAA levels.

7 316 FISHERIES SCIENCE T Yamamoto et al. Table 7 Levels of free amino acids and related compounds (nm/g wet tissue) in kidney and spleen of fingerling rainbow trout fed diets containing different protein sources Kidney 1 Spleen 1 Threonine Valine Cystine ND ND ND 1.6 Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Lysine Histidine Arginine EAA + C + T Aspartic acid ab b a a 94.8 Serine Asparagine Glutamic acid Glutamine 577 a2 599 a b 798 ab a 802 a b ab Glycine b a a a b a a a Alanine Hydroxyproline 540 b 146 a 167 a 175 a b 112 a 146 a 159 a 21.9 Proline ab 290 a 921 b 508 ab NEAA Taurine b a b b Citrulline Cystathionine 63 b 44 a 39 a 39 a b-alanine 332 a 512 b 237 a 304 a g-aminobutyric acid Ethanolamine Ammonia Ornithine Anserine Carnosine ND ND ND ND 1 Average values of two pooled samples (three fish/sample) of duplicate groups for each dietary treatment. 2 Values in the same row followed by the same superscript are not significantly different (P > 0.05). In the SBM group, tissue taurine levels were generally lower, and those of ornithine were higher, than in the FM group. The levels of g-aminobutyric acid (GABA) and carnosine in the brain were relatively high compared with the other tissues, however, the levels of these compounds in the plant-protein groups were not lower than in the FM group. In the brain, kidney, and stomach (Table 8), the levels of carnosine were always higher than those of anserine in all treatments. The levels of anserine in the muscle were very high compared with the other tissues, but did not differ among the treatments. Relationship in amino acid levels between diets and tissues Correlations between the levels of certain amino acids in the diets and in each tissue for all the treatments combined are given in Table 10. In general, the correlations were highly positive for lysine and threonine. In several tissues, high positive correlations were also found for methionine, glycine, and proline. DISCUSSION In the present study, the levels of lysine and threonine in all four experimental diets had strong positive correlations with their levels in most of the tissues examined (Table 10). To a lesser extent, high correlations were also found for methionine. Dietary contents of these three EAA are commonly lower in plant-protein diets than in the FM diet (Table 2). Compared with the differences in the dietary levels, the free methionine level of the SBM group, and free lysine level of the MPF group in several tissues, such as plasma, heart, and muscle, were much

8 Influence of dietary proteins on trout tissue FAA FISHERIES SCIENCE 317 Table 8 Levels of free amino acids and related compounds (nm/g wet tissue) in stomach and intestine of fingerling rainbow trout fed diets containing different protein sources Stomach 1 Intestine 1 Threonine 500 b2 325 a 274 a 335 a Valine Cystine ND 17 a 31 b 14 a b 31 a 66 b 65 b 7.0 Methionine 229 c 99 a 115 ab 136 b Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Lysine 440 b 257 ab 120 a 192 a Histidine Arginine 276 b 225 ab 141 a 211 ab EAA + C + T b ab a ab Aspartic acid Serine 741 b 487 a 424 a 491 a Asparagine 185 ab 236 bc 171 a 254 c b 249 a 274 ab 335 ab 51.3 Glutamic acid b ab a ab Glutamine 498 a 453 a 755 b 603 ab Glycine b 884 a 758 a 851 a b a a a Alanine b 936 ab 762 a 811 ab Hydroxyproline 224 b 68 a 79 a 90 a ND ND ND 35.3 Proline 284 a 268 a 576 b 447 ab NEAA Taurine Citrulline Cystathionine 18 b ND 9 a ND ND ND ND 4.3 b-alanine b 252 ab 200 ab 93 a 66.2 g-aminobutyric acid Ethanolamine Ammonia b a ab ab Ornithine Anserine ND Carnosine a b ab ab ND Average values of two pooled samples (three fish/sample) of duplicate groups for each dietary treatment. 2 Values in the same row followed by the same superscript are not significantly different (P > 0.05). lower than in the FM group (Tables 4 9). Moreover, while dietary threonine levels of the plant-protein diets were slightly (< 10%) lower than in the FM diet, free threonine levels in most of the tissues of the SBM and the MPF groups attained only 50 60% of the levels in the FM group. In general, individual free EAA levels in the tissues of the COMB diet group were equal to, or slightly higher than, the higher levels of either the SBM group or the MPF group. However, the tissue EAA levels of the COMB group were generally still lower than the FM group, especially the levels of threonine, methionine, and lysine. As we had already demonstrated, the nutritive value of the COMB diet protein is higher than the SBM diet or the MPF diet. 27,28 The COMB group showed better growth and feed efficiency than the SBM and the MPF groups, however, they were still inferior in performance to the FM group (Table 3). The above findings in this study may mean that the dietary contents of these three EAA would still have been insufficient to meet the requirement levels. Although the threonine level in the COMB diet is slightly lower than the FM diet, supplementation of this amino acid to the COMB diet, as well as methionine and lysine, is thought to be essential. In this study, we found several dietary protein-specific or tissue-specific phenomena in certain FAA levels. Changes in certain FAA levels in tissues reflect the dietary levels, such as aromatic amino acids, glutamine, and proline in malt protein flour. In the eye, relatively high amounts of methionine, cystine and cystathionine were present compared with the other tissues examined (Table 5). For example, ratios of methionine to total EAA level of the FM group are 0.12 in the eye, 0.1 in the plasma, and only 0.02 to 0.07 in the other tissues. Cowey et al. demonstrated that methionine deficiency in rainbow trout caused lens pathologies such as cataract

9 318 FISHERIES SCIENCE T Yamamoto et al. Table 9 Levels of free amino acids and related compounds (nm/g wet tissue) in dorsal white muscle and skin of fingerling rainbow trout fed diets containing different protein sources Dorsal white muscle 1 Skin 2 Threonine b3 890 ab ab 741 a b 761 b 500 a 515 a 34.5 Valine 327 ab 286 a 357 b 303 ab Cystine 25 b 10 a 24 b 12 a a 25 b ND 40 c 3.4 Methionine 196 c 38 a 125 b 86 b Isoleucine Leucine 271 a 211 a 335 b 247 a Tyrosine 49 a 73 ab 152 c 98 b Phenylalanine 102 a 130 b 202 d 157 c Tryptophan Lysine 503 c 283 b 12 a 66 a Histidine a b a b a b ab b Arginine EAA + C + T a c ab b Aspartic acid 145 a 187 a 293 b 144 a ab 654 b 406 a 466 a 45.0 Serine b 580 a 872 ab 722 a b 978 ab 737 a 777 a 64.6 Asparagine 982 a b b b a 861 b 615 a 861 b 53.4 Glutamic acid 627 a 730 a b 736 a a b ab a Glutamine 706 a 700 a c 967 b a 836 a b 973 ab 94.8 Glycine b ab a a Alanine a b b a a b a 856 a Hydroxyproline c 358 a 566 b 332 a b 218 a 255 ab 384 ab 92.5 Proline 387 a 411 a c 989 b a 490 a c 691 b 50.0 NEAA b ab ab a a b ab ab Taurine c 857 a b b Citrulline 182 a 116 a 259 b 167 a a 362 b 169 a 134 a 32.8 Cystathionine 77 c 2 a 34 b 8 a ab 12 a 26 ab 40 b 5.4 b-alanine 212 a 332 b 195 a 244 ab a 579 b 430 ab 406 ab 51.8 g-aminobutyric acid 11 a 33 b 65 c 30 b a 290 b 195 ab 291 b 29.1 Ethanolamine Ammonia b a c c Ornithine 42 a 754 c 33 a 221 b a 273 b 66 a 152 a 24.7 Anserine Carnosine a 116 a 49 a 236 b Average values of six samples of duplicate groups (three fish/group) for each dietary treatment. 2 Average values of two pooled samples (three fish/sample) of duplicate groups for each dietary treatment. 3 Values in the same row followed by the same superscript are not significantly different (P > 0.05). formation and focal length variability. 29 Although no pathological symptoms were observed in the eyes in any treatment in the present study, the levels of methionine and cystathionine in the eyes of the plant-protein diet groups were markedly lower than in the FM group. It may be necessary in further long-term feeding to add sulfur amino acids to a diet containing a high proportion of soybean meal. In several tissues of the SBM group in the present study, levels of taurine were lower than in the FM group, as we had found previously in trout fed high fat diets. 20 Several reports have shown that tissue taurine levels increase with the addition of sulfur amino acids to diets Thus, the lower taurine levels of the SBM group may have been caused by the lower levels of sulfur amino acids in this diet. It was also observed that the ornithine levels in several tissues, especially in muscle, of the SBM group were higher than in the other groups. Arginine, ornithine, and citrulline are the substrates of the urea cycle. 32 This cycle exists in rainbow trout but is not sufficiently functional to satisfy the arginine requirement, possibly because conversion of ornithine to citrulline is limited. 33 In a previous study, higher levels of ornithine were also found in trout fed diets containing high proportions of gelatin, which has an imbalanced amino acid profile but is rich in arginine. The soybean meal used in the present study contained slightly higher levels of arginine than the fish meal (Table 2). However, tissue free arginine levels of the SBM group were generally equal to or lower than that in the FM group. The levels of citrulline in the tissues were rather uniform among the treatments. Thus, the considerably higher level of arginine in the SBM diet compared with other diets might have been catabolized into

10 Influence of dietary proteins on trout tissue FAA FISHERIES SCIENCE 319 Table 10 Correlations in the levels of certain amino acids between the diets and each tissue for all the treatments combined Amino acids Plasma Brain Eye Gill Heart Liver Kidney Spleen Stomach Intestine Muscle Skin Arginine Histidine Isoleucine Leucine Lysine Methionine Cystine NC NC Phenylalanine Tyrosine Threonine Tryptophan Valine Alanine Aspartic acid Glutamic acid Glycine Proline Serine P < P < P < Calculation was not carried out because free cystine was not detected in the tissue. 5 Tissue free asparagine and glutamine levels were included in free aspartic acid and glutamic acid levels, respectively. ornithine in the tissues, due to the amino acid imbalance of the diet. The distributions of anserine and carnosine were found to be variable among the tissues examined. In the gill, spleen, muscle, and skin, the anserine levels were higher than the levels of carnosine. By contrast, carnosine levels were higher than anserine in brain (hcarnosine), 34 kidney, and stomach. It is well known that the distribution of these imidazole dipeptides in muscle differs among aquatic animals, for example, tunas and salmonids mainly possess anserine, eels carnosine, and whales balenine. 35 Arai reported similar phenomena to the present findings that kidney contained only anserine although muscle and gill contained only carnosine in eel Anguilla anguilla. 18 The reason why the distribution of the two imidazole dipeptides in fish depends on the tissue is unclear. In contrast, muscle anserine levels did not differ, irrespective of the dietary amino acid profiles (Table 9). In addition, levels of carnosine and GABA in the brain of the plant-protein diet groups were also similar to the levels in the FM group (Table 4). These observations are consistent with our previous results, and have already been discussed. 20 The findings of the present study demonstrate that levels of threonine, methionine, and lysine among diets containing different protein sources with different amino acid profiles are strongly reflected by their tissue levels. The growth performance and the tissue levels of these EAA in the fish fed the combination of soybean meal and malt protein flour were still lower than the fish fed the fish meal base diet. Therefore, although the threonine level of the COMB diet was slightly lower than the FM diet, threonine, as well as methionine and lysine, is thought to still be insufficient in the COMB diet. The levels of carnosine and GABA in brain, and anserine in muscle, which may have important physiological roles in the tissues, seem unaffected by dietary amino acid profiles. ACKNOWLEDGMENTS We wish to thank the staff of the Shiga Prefectural Samegai Trout Farm for providing the test fish, and to the Kirin Brewery Co. Ltd, for providing malt protein flour. We also thank Dr K. D. Shearer, Northwest Fisheries Science Center, NMFS, USA, for reviewing the manuscript. REFERENCES 1. Pion R. Dietary effects and amino acids in tissues. In: Cole DJA, Boorman KN, Buttery PJ, Lewis D, Neale RJ, Swan H (eds). Protein Metabolism and Nutrition. Butterworths, London, 1976, Shimeno S, Seki S, Masumoto T, Hosokawa H. Post-feeding changes in digestion and serum constituent in juvenile yellowtail force-fed with raw and heated soybean meals. Nippon Suisan Gakkaishi 1994; 60: Shimeno S, Mima T, Masumoto T, Hosokawa H, Matsushita I, Watanabe K. Digestion and post-feeding changes in serum constituents of juvenile yellowtail fed moist pellet diet containing soybean meal. Suisanzoshoku 1995; 43: Yamamoto T, Unuma T, Akiyama T. Postprandial changes in plasma free amino acid concentrations of rainbow trout fed diets containing different protein sources. Fisheries Sci. 1998; 64: Watanabe T, Aoki H, Shimamoto K et al. A trial to culture yellowtail with non-fishmeal diets. Fisheries Sci. 1998; 64:

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