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Requirement for phenylalanine and replacement value of tyrosine for phenylalanine in rainbow trout (Oncorhynchus mykiss)
243
AquacuZture, 113 (1993) 243-250 Elsevier Science Publishers B.V., Amsterdam AQUA 50068
Requirement for phenylalanine and replacement val‘ue of tyrosine for phenylalanine in rainbow trout (Oncorhynchus mykiss) Kyu-11 Rim Aquaculture Program, Department of Food Science, University of Wisconsin, Madison, WI, USA (Accepted
13 October 1992)
ABSTRACT The requirement for phenylalanine (Phe) and replacement value of tyrosine (Tyr) for Phe in lingerling rainbow trout were determined by feeding diets containing various levels of Phe and Tyr. Four tanks of 30 trout each were assigned per diet and fed 3 times daily to satiation at 15°C. With diets containing excess Tyr ( 1.33%) and 35% crude protein, weight gain of 13-g trout over a 6-week period increased linearly with increasing levels of Phe up to 0.75% of dry diet but was not different (P> 0.05) at 0.75% and 1.75% Phe. Feed efficiency and nitrogen retention improved as Phe level in the diet increased up to 0.55%, but no significant effects were found at levels above 0.55%. Carcass protein content increased with Phe level up to 0.65% and fat content decreased concurrently. Increasing Tyr level up to 0.7% in a diet containing 0.6% Phe increased weight gain of 14-g trout over a 6-week period, but a further increase in Tyr level to 0.8% had no effect (P>O.O5). Analysis of data using a broken-line model showed that the Phe requirement of fingerling rainbow trout is 0.7&0.05% (mean k s.e.) of dry diet or 2.0% of dietary protein, when a diet contains excess Tyr and 35% protein, and that the replacement value of Tyr for Phe is approximately 53% on a weight basis or 48% on a molar basis. Thus the total aromatic amino acid requirement is 1.5% of dry diet or 4.3% of dietary protein.
INTRODUCTION
Total aromatic amino acid requirements of various tish species are summarized in recent U.S. National Research Council (NRC) ( 198 1, 1983) publications: 2.1% of diet (5.1% of dietary protein) for chinook salmon, 1.2(5.0)% for channel catfish, 2.5(6.5)% for common carp and 2.2(5.8)% for Japanese eel. More recently, a comprehensive review of the amino acid requirements of various tish species has been published (Wilson, 1989 ) . Ogino ( 1980) has also estimated the amino acid requirements of rainbow trout and carp on the basis of daily increase in body protein (amino acids) Correspondence to: Dr. K.I. Kim, Department of Animal Science, Cheju National Cheju 690-756, Republic of Korea. Tel. (64) 54-3335; Fax (64) 55-6130.
0044-8486/93/$06.00
0 1993 Elsevier Science Publishers
B.V. All rights reserved.
University,
244
K.I. KIM
and daily excretion of nitrogen by fish fed a protein-free diet. His calculated values for phenylalanine (Phe ) requirements of rainbow trout and common carp were 1.1(3.2)% and 1.O( 3.0)% of diet (protein), respectively. The respective values for tyrosine (Tyr) requirements were 0.73 (2.1)% and 0.7 (2.0)% of diet (protein). The replacement value of Tyr for Phe has been reported for channel cattish (50%) by Robinson et al. ( 1980) and for common carp (60%) by Nose ( 1979). However, no data on Phe requirement or the replacement value of Tyr for Phe as determined by feeding trials have been reported for rainbow trout. Therefore, the following experiments were performed to determine the Phe requirement and the replacement value of Tyr for Phe in fingerling rainbow trout. EXPERIMENTAL
PROCEDURES
Experiment 1 was conducted to determine the absolute Phe requirement, excluding any fraction that might be attributable to a need for Tyr. Diets used for Experiment 1 contained 1.33% Tyr, and 0.26,0.35,0.45,0.55, 0.65,0.75 or 1.75% Phe on a dry-matter basis. Of these levels, 0.26% Phe and 0.28% Tyr were supplied by casein and gelatin, and the remainder was supplied by crystalline amino acids. In Experiment 2, which was performed to determine the Tyr replacement value for Phe, diets contained varying levels of Tyr from 0.28 to 0.80% along TABLE
1
Composition
of the basal diet (% on an air-dry basis)
’
Ingredient
%
Ingredient
%
IDAA’ DAA’ Casein (92% CP)3,4 Gelatin (89% CP)4 Dextrin4 Dextrose4 cr-Cellulose’
17.74 12.51 5.00 2.00 27.80 5.00 8.20
Herring oil6 DL-a-tocophero14*7 Carboxymethyl cellulose4 Vitamin mixture’ Mineral mixture’ CaHP04 Choline chloride4
10.00 0.0455 1 .oo 2.00 5.06 2.94 0.70
‘Diets contained approximately 35% crude protein and were neutralized to give a final pH of 6.6. *Amounts of Phe and Tyr in IDAA (indispensable amino acids) were reduced while the amount of DAA (dispensable amino acids) was increased so as to vary the level of phenylalanine or tyrosine in the diet and to maintain the diets isonitrogenous (see column 3 of Table 2 ) 3Vitamin-free. 4Purchased from United States Biochemical Corp., Cleveland, OH, USA. 5Purchased from Sigma, St. Louis, MO, USA. 6Purchased from Glencoe Mills, Glencoe, MN, USA. 7Acetate form, 1 .O IU/mg added to herring oil and mixed. ‘Kimetal. (1991).
AROMATIC AMINO ACID REQUIREMENTS OF TROUT
245
TABLE 2 Amino acid composition
of the basal diet (% on a dry-matter basis) From casein + gelatin’
From crystalline L-amino acids
Total’
Arginine Histidine Isoleucine Leucine Lysine Methionine Cystine Phenylalanine Tyrosine Threonine Tryptophan Valine
0.337 0.146 0.288 0.459 0.546 0.140 0.017 0.259 0.280 0.248 0.055 0.360
1.940 0.773 1.638 2.736 1.860” 1.042 0.174 1.754b 1.325b 1.574 0.472 1.989
2.277 0.919 1.926 3.195 2.406 1.182 0.191 2.013 1.605 1.822 0.527 2.349
DAA Alanine Aspartic acid Glycine Glutamic acid Proline Serine
0.290 0.437 0.625 1.198 0.845 0.278
1.787 3.326 0.671 3.716 0.513 2.494
2.086 3.763 1.296 4.914 1.358 2.772
Amino acid
ZDAA
‘Calculated from values provided by the United States Biochemical Corp., Cleveland, OH, USA. 2The amino acid profile simulated that of 35% whole chicken egg protein (Robinson et al., 1980). “Added as lysine HCl (2.324% for 1.860% lysine). bathe level of crystalline phenylalanine or tyrosine was reduced while the level of DAA was increased.
with 0.6% Phe on a dry-matter basis. The Phe level of 0.6% was chosen because the Tyr replacement value should be determined at the Phe level below the absolute requirement. [The result of Experiment 1 showed that the Phe requirement was about 0.7% of dry diet.] Another diet, which was used as a reference to ensure that 0.6% Phe was not in excess, contained 1.33% Tyr and 1.75% Phe. Ingredients and amino acid composition of the basal diet are shown in Tables 1 and 2, respectively. All experimental diets were formulated to contain 35% crude protein, the amino acid profile of which simulated that of 35% whole chicken egg protein. Four replicate groups, each consisting of 30 rainbow trout (Oncorhynchus m$ziss), were assigned to each diet. Osceola strain trout were provided by the U.S. Fish and Wildlife Service, National Fish Hatchery, Osceola, WI, and mean initial body weight + s.e.m. (for 28 tanks) was 12.7 2 0.06 g for fish used in Experiment 1 and 14.0 + 0.03 g for fish used in Experiment 2. Fish were fed the experimental diets 3 times daily to satiation in tanks containing 100 liter of water at 15 & 0.5 oC with a flow rate of 2 to 4 l/min. Other
K.I. KIM
246
procedures including experimental conditions, diet preparation, carcass analysis and statistical analysis were as described in Kim et al. ( 1987, 199 1). RESULTS
Elevating Phe from 0.26 to 0.75% in diets containing excess Tyr increased weight gain, feed efficiency and nitrogen retention (Table 3), but a further elevation to 1.75 had no effect (P> 0.05). A high mortality was observed with diets containing lower than 0.45% Phe. By applying a broken-line model (Robbins et al., 1977) to the analysis of weight gain versus dietary Phe level, the Phe requirement was estimated at approximately 0.7 20.05% (mean + s.e. ) of dry diet or 2% of dietary protein in the presence of excess Tyr. The carcass of trout fed diets deficient in Phe ( ~0.45%) tended to have lower protein but a higher fat and ash content than that of trout fed diets containing higher than 0.55% Phe (Table 4). No significant differences were found in dry matter content of the carcass, or carcass percentage (data not shown). In Experiment 2, weight gain increased as the Tyr level in the diet increased up to 0.8% (Table 5 ). But weight gain was not significantly different between trout fed diets containing 0.7% and 0.8% Tyr. The break point was found at the Tyr level of 0.68 + 0.01% (mean 2 se. ). Thus the replacement value of TABLE 3 Effects of dietary levels of phenylalanine (Experiment 1) ’
(Phe) on weight gain, feed efficiency and nitrogen retention
Level of Phe’ (%)
Weight gain (g/fish)
Feed efficiency3
Nitrogen retention4 (%)
Mortality (%)
0.26 0.35 0.45 0.55 0.65 0.75 1.75
7.7 * 0.4a~5 12.2i0.4b 16.0+0.8’ 21.71b0.7~ 22.0+0.4* 23.9 f 0.3’ 24.2kO.3”
0.43 * 0.05” 0.55 &0.03b 0.68 + 0.02’ 0.79 & 0.03* 0.81?0.02* 0.84+0.01* 0.82+0.01*
12.7f 1.5” 20.6? 1.4b 26.8 f 1.6’ 32.5 f0.7* 34.3 Ifrl.Od*” 36.7+0.9e 34.5 + 0.6*pe
44.0 25.0 9.2 0 0.8 0 0
‘Means Z!Is.e.m. of four tanks of 30 fish each over a 6-week period. Mean initial body weight + s.e.m. of 28 tanks of 30 fish each was 12.7 I!I0.06 g. *0.26% phenylalanine was supplied by casein and gelatin and all the experimental diets contained 1.33% tyrosine. 3g gain/g feed fed. “(Nitrogen gained in eviscerated carcass/nitrogen intake) x 100. ‘Values in the same column sharing the same superscript letters are not significantly different at the 5% level, when analyzed by the Neuman-Keuls test.
AROMATIC AMINO ACID REQUIREMENTSOF TROUT
247
TABLE 4 Effects of dietary levels of phenylalanine Carcass composition
Level of Phe3 (%)
Dry matter
0.26 0.35 0.45 0.55 0.65 0.75 1.75
25.2f0.2 25.5f0.2 25.2kO.l 25.2kO.l 25.3LO.l 25.4kO.l 25.2fO.O
(Phe) on carcass composition
(Experiment
1) ’
(o/o)’
55.3f2.0”,5 56.5* 1.3”~~ 58.3 +2.2asb,c 59.3 * 2.7b*G 60.5* 1.5” 60.7kO.9” 60.2+ 1.0’
Fat4
Ash4
32.0*0.9=
10.6 + 0.2”,b 10.7?0.3a 10.5f0.7”Tb 10.1 * 0.2aab%c 10.0 + 0.4”,b,c 9.7f0.4” 9.9 + 0.4b*’
30.8+2.1a~b 30.4 + 1.gaab 28.3 k 0.6b 28.2+ 1.8b 27.7+0.6b 29.4 a 2.8”ab
‘Means k s.e.m. of four tanks, each consisting of 10 fish (combined). ‘Initial carcass composition was 22.7% dry matter, 70.2% protein, 13.8% fat and 12.7% ash. 3See footnote 2 to Table 3. 40n a dry-matter basis. 5Values in the same column sharing the same superscript letters are not significantly different at the 5% level, when analyzed by the Neuman-Keuls test.
TABLE 5 Effects of dietary levels of tyrosine (Tyr) and phenylalanine (Experiment 2) ’ Level (%) TY~
Phe’
0.28 0.40 0.50 0.60 0.70 0.80 1.33
0.60 0.60 0.60 0.60 0.60 0.60 1.75
(Phe) on weight gain and feed efficiency
Weight gain (g/fish)
Feed efficiency’
Mortality (%)
6.9 + 0.7as4 8.550.9” 9.0f0.6” 11.3*0.9b*c 12.8&0.8c,d 14.9+0.5d 22.4f0.5”
0.37 * 0.03” 0.49 * o.04b 0.50+0.04b 0.53+0.03b 0.52 f 0.04b 0.59 f 0.02b 0.94 t- 0.02”
0.8 0.8 6.7 2.5 0 1.6 0
‘Means f s.e.m. of four tanks of 30 fish each over a 6-week period. Mean initial body weight * s.e.m. of 28 tanks of 30 fish each was 14.0+ 0.03 g. ‘0.28% tyrosine and 0.26% phenylalanine were supplied by casein and gelatin. 3g, gain/g, feed as fed. 4Values in the same column sharing the same superscript letters are not significantly different at the 5% level, when analyzed by the Neuman-Keuls test.
Tyr for Phe was determined at about 53% [100x0.68/(0.6+0.68)] on a weight basis or 48% (53 x 165.19/ 18 I. 19) on a molar basis and the total aromatic amino acid requirement was estimated at 1.5% [0.7/( l-0.53) ] of diet or 4.3% of dietary protein.
248
K.I. KIM
DISCUSSION
The requirement of rainbow trout for total aromatic amino acids ( 1.5% of diet or 4.3% of protein) estimated in the present study is similar to the value ( 1.83% of diet) reported for trout by Ogino ( 1980)) who calculated the amino acid requirements on the basis of the daily increase of protein (amino acids) in the body of fish fed diets containing about 35% protein, and the daily excretion of nitrogen by fish fed a protein-free diet. However, Ogino (1980) has probably overestimated the value, because of a low feed-intake level (assuming 3% of body weight) used to calculate the amino acid requirements, although the average daily gain was 3.44% of body weight. The total aromatic amino acid requirements of channel cat&h ( 1.2% of diet) reported by Robinson et al. ( 1980) and Nile tilapia ( 1.55%) reported by Santiago and Lovell ( 1988) appear similar to that (1.5%) estimated for rainbow trout in the present study. However, the total aromatic amino acid requirements recommended by the NRC ( 1983 ) for chinook salmon (2.1% of diet or 5.1% of protein), common carp (2.5% or 6.5%) and Japanese eel (2.2% or 5.8%) are much higher than that estimated in the present study for rainbow trout. The differences between the NRC (1983) values and the value found in the present study may be due not only to species differences, but also more probably to the laboratory variances, such as using various dietary ingredients, different experimental conditions and fish of different sizes. Our previous study (Kim et al., 199 1) showed that the dietary protein level required by fingerling rainbow trout to meet the need for indispensable amino acids is no more than 25% of the diet (although the protein requirement for maximal growth of fingerling rainbow trout is 35%). In the diet containing 25% intact protein (provided by casein + gelatin supplemented with arginine and methionine) and an additional 10% dispensable amino acid mixture, the levels of arginine, histidine, isoleucine, leucine, lysine, sulfur amino acids, aromatic amino acids, threonine, tryptophan and valine were 1.6, 0.67, 1.4, 2.1, 1.9, 0.98, 2.5, 1.1, 0.28 and 1.9%, respectively. The amino acid requirements of rainbow trout which have been determined at the University of Wisconsin-Aquaculture Laboratory do not exceed the level of the corresponding amino acids contained in the 25% intact protein diet supplemented with arginine and methionine. The amino acid requirements of rainbow trout (Kim et al., 1987, 1992a, b, present study) are also comparable to those of young pigs (NRC, 1988) except for arginine ( 1.4 vs. 0.6; an obvious reason for this is that trout are ammonotelic and thus produce little arginine via the urea cycle). The comparisons include lysine ( 1.3 vs. 1.4)) sulfur amino acids (0.8 vs. 0.68), tryptophan (0.2 vs. 0.2 ) and aromatic amino acids ( 1.5 vs. 1.1) . The replacement value (53% on a weight basis or 48% on a molar basis) of Tyr for Phe determined in the present study for rainbow trout appears lower
AROMATIC AMINO ACID REQUIREMENTSOF TROUT
249
than the value of 60% (on a weight basis) found for common carp (Nose, 1979), but similar to the value of 50% (on a molar basis) reported for channel catfish (Robinson et al., 1980). The Tyr replacement value reported in the present study was obtained with a diet containing Phe at a level lower than the requirement, assuming that the minimum ratio of Phe to the total aromatic amino acids required for maintenance and growth is the same. If the Phe level in the test diet is higher than that required, the Tyr replacement value may be underestimated. The low weight gain obtained with the diets containing 0.6% Phe and either 0.7 or 0.8% Tyr, as compared to that obtained with the diet containing 1.75% Phe and 1.33% Tyr or with the diet containing 0.55% Phe and 1.33% Tyr in Experiment 1, was unexpected. However, such variations have been seen in feeding experiments performed with fish (see Robinson et al., 1980). The findings in the present study suggest that the total aromatic amino acid requirement of fingerling rainbow trout (about 1.5% of diet ) is similar to that recommended for young pigs-l .4% by Kim et al. ( 1983 ), assuming that the replacement value of Tyr for Phe is 50%, or 1.1% by NRC ( 1988 )-and for broiler chicks ( 1.34% by NRC, 1984). This similarity supports our contention that the individual amino acid requirements of rainbow trout are similar to those of rapidly growing farm animals (Kim et al., 1987, 1992a, b), and that the higher protein requirement of fish is due to use of protein by fish to meet their energy requirements (Kim et al., 199 1). This conclusion is well explained by the fact that trout are carnivorous and have a limited ability to utilize carbohydrate, which is a main energy source for farm animals. ACKNOWLEDGEMENTS
I thank Terry Kayes for making this study possible. I also thank Penny Swanson, Tom Kuczynski, Jean Grassman, Thorn Grimshaw and Larry Zehner for their technical help and the U.S. Fish and Wildlife Service for providing fish. This work was supported in part by the University of WisconsinMadison, College of Agricultural and Life Sciences and Graduate School, and by the University of Wisconsin Sea Grant College Program under grants from the National Oceanic and Atmospheric Administration, the U.S. Department of Commerce, and from the State of Wisconsin (Federal grant NA80AA-D00086, projects R/AQ-8 and R/AQ-9).
REFERENCES Kim, K.I., McMillan, I. and Bayley, H.S., 1983. Determination of amino acid requirements of young pigs using an indicator amino acid. Br. J. Nutr., 50: 369-382. Kim, K.I., Kayes, T.B. and Amundson, C.H., 1987. Effects of dietary tryptophan levels on growth,
250
K.I. KIM
feed/gain, carcass composition and liver glutamate dehydrogenase activity in rainbow trout (Sulmogairdneri). Comp. Biochem. Physiol., MB: 737-741. Kim, K.I., Kayes, T.B. and Amundson, C.H., 199 1. Purified diet development and re-evaluation of the dietary protein requirement of fingerling rainbow trout (Oncorhynchus mykiss). Aquaculture, 97: 57-67. Kim, K.I., Kayes, T.B. and Amundson, C.H., 1992a. Requirements for sulfur amino acids and utilization of D-methionine by rainbow trout (Oncorhynchus mykiss). Aquaculture, 10 1: 95-103. Kim, K.I., Kayes, T.B. and Amundson, C.H., 1992b. Requirements for lysine and arginine by rainbow trout (Oncorhynchus mykiss). Aquaculture, 106: 333-344. National Research Council, 198 1. Nutrient Requirements of Coldwater Fishes. National Academy Press, Washington, DC, 63 pp. National Research Council, 1983. Nutrient Requirements of Warmwater Fishes and Shellfishes. National Academy Press, Washington, DC, 102 pp. National Research Council, 1984. Nutrient Requirements of Poultry, 8th ed. National Academy Press, Washington, DC, 77 pp. National Research Council, 1988. Nutrient Requirements of Swine, 9th ed. National Academy Press, Washington, DC, 93 pp. Nose, T., 1979. Summary report on the requirements of essential amino acids for carp. In: J.E. Halver and K. Tews (Editors), Finfish Nutrition and Fishfeed Technology I. Heeneman, Berlin, pp. 145-156. Ogino, C., 1980. Requirements of carp and rainbow trout for essential amino acids. Bull. Jpn. Sot. Sci. Fish., 46: 171-174. Robbins, K.P., Baker, D.H. and Norton, H.W., 1977. Histidine status of the chick as measured by growth rate, plasma free histidine and breast muscle carnosine. J. Nutr., 107: 2055-206 1. Robinson, E.H., Wilson, R.P. and Poe, W.E., 1980. Total aromatic amino acid requirement, phenylalanine requirement and tyrosine replacement value for fingerling channel catfish. J. Nutr., 110: 1805-1812. Santiago, C.B. and Lovell, R.T., 1988. Amino acid requirements for growth of Nile tilapia. J. Nutr., 118: 1540-l 546. Wilson, R.P., 1989. Amino acids and proteins. In: J.E. Halver (Editor), Fish Nutrition, 2nd ed., Academic Press, New York, NY, pp. 112- 15 1.
AquacuZture, 113 (1993) 243-250 Elsevier Science Publishers B.V., Amsterdam AQUA 50068
Requirement for phenylalanine and replacement val‘ue of tyrosine for phenylalanine in rainbow trout (Oncorhynchus mykiss) Kyu-11 Rim Aquaculture Program, Department of Food Science, University of Wisconsin, Madison, WI, USA (Accepted
13 October 1992)
ABSTRACT The requirement for phenylalanine (Phe) and replacement value of tyrosine (Tyr) for Phe in lingerling rainbow trout were determined by feeding diets containing various levels of Phe and Tyr. Four tanks of 30 trout each were assigned per diet and fed 3 times daily to satiation at 15°C. With diets containing excess Tyr ( 1.33%) and 35% crude protein, weight gain of 13-g trout over a 6-week period increased linearly with increasing levels of Phe up to 0.75% of dry diet but was not different (P> 0.05) at 0.75% and 1.75% Phe. Feed efficiency and nitrogen retention improved as Phe level in the diet increased up to 0.55%, but no significant effects were found at levels above 0.55%. Carcass protein content increased with Phe level up to 0.65% and fat content decreased concurrently. Increasing Tyr level up to 0.7% in a diet containing 0.6% Phe increased weight gain of 14-g trout over a 6-week period, but a further increase in Tyr level to 0.8% had no effect (P>O.O5). Analysis of data using a broken-line model showed that the Phe requirement of fingerling rainbow trout is 0.7&0.05% (mean k s.e.) of dry diet or 2.0% of dietary protein, when a diet contains excess Tyr and 35% protein, and that the replacement value of Tyr for Phe is approximately 53% on a weight basis or 48% on a molar basis. Thus the total aromatic amino acid requirement is 1.5% of dry diet or 4.3% of dietary protein.
INTRODUCTION
Total aromatic amino acid requirements of various tish species are summarized in recent U.S. National Research Council (NRC) ( 198 1, 1983) publications: 2.1% of diet (5.1% of dietary protein) for chinook salmon, 1.2(5.0)% for channel catfish, 2.5(6.5)% for common carp and 2.2(5.8)% for Japanese eel. More recently, a comprehensive review of the amino acid requirements of various tish species has been published (Wilson, 1989 ) . Ogino ( 1980) has also estimated the amino acid requirements of rainbow trout and carp on the basis of daily increase in body protein (amino acids) Correspondence to: Dr. K.I. Kim, Department of Animal Science, Cheju National Cheju 690-756, Republic of Korea. Tel. (64) 54-3335; Fax (64) 55-6130.
0044-8486/93/$06.00
0 1993 Elsevier Science Publishers
B.V. All rights reserved.
University,
244
K.I. KIM
and daily excretion of nitrogen by fish fed a protein-free diet. His calculated values for phenylalanine (Phe ) requirements of rainbow trout and common carp were 1.1(3.2)% and 1.O( 3.0)% of diet (protein), respectively. The respective values for tyrosine (Tyr) requirements were 0.73 (2.1)% and 0.7 (2.0)% of diet (protein). The replacement value of Tyr for Phe has been reported for channel cattish (50%) by Robinson et al. ( 1980) and for common carp (60%) by Nose ( 1979). However, no data on Phe requirement or the replacement value of Tyr for Phe as determined by feeding trials have been reported for rainbow trout. Therefore, the following experiments were performed to determine the Phe requirement and the replacement value of Tyr for Phe in fingerling rainbow trout. EXPERIMENTAL
PROCEDURES
Experiment 1 was conducted to determine the absolute Phe requirement, excluding any fraction that might be attributable to a need for Tyr. Diets used for Experiment 1 contained 1.33% Tyr, and 0.26,0.35,0.45,0.55, 0.65,0.75 or 1.75% Phe on a dry-matter basis. Of these levels, 0.26% Phe and 0.28% Tyr were supplied by casein and gelatin, and the remainder was supplied by crystalline amino acids. In Experiment 2, which was performed to determine the Tyr replacement value for Phe, diets contained varying levels of Tyr from 0.28 to 0.80% along TABLE
1
Composition
of the basal diet (% on an air-dry basis)
’
Ingredient
%
Ingredient
%
IDAA’ DAA’ Casein (92% CP)3,4 Gelatin (89% CP)4 Dextrin4 Dextrose4 cr-Cellulose’
17.74 12.51 5.00 2.00 27.80 5.00 8.20
Herring oil6 DL-a-tocophero14*7 Carboxymethyl cellulose4 Vitamin mixture’ Mineral mixture’ CaHP04 Choline chloride4
10.00 0.0455 1 .oo 2.00 5.06 2.94 0.70
‘Diets contained approximately 35% crude protein and were neutralized to give a final pH of 6.6. *Amounts of Phe and Tyr in IDAA (indispensable amino acids) were reduced while the amount of DAA (dispensable amino acids) was increased so as to vary the level of phenylalanine or tyrosine in the diet and to maintain the diets isonitrogenous (see column 3 of Table 2 ) 3Vitamin-free. 4Purchased from United States Biochemical Corp., Cleveland, OH, USA. 5Purchased from Sigma, St. Louis, MO, USA. 6Purchased from Glencoe Mills, Glencoe, MN, USA. 7Acetate form, 1 .O IU/mg added to herring oil and mixed. ‘Kimetal. (1991).
AROMATIC AMINO ACID REQUIREMENTS OF TROUT
245
TABLE 2 Amino acid composition
of the basal diet (% on a dry-matter basis) From casein + gelatin’
From crystalline L-amino acids
Total’
Arginine Histidine Isoleucine Leucine Lysine Methionine Cystine Phenylalanine Tyrosine Threonine Tryptophan Valine
0.337 0.146 0.288 0.459 0.546 0.140 0.017 0.259 0.280 0.248 0.055 0.360
1.940 0.773 1.638 2.736 1.860” 1.042 0.174 1.754b 1.325b 1.574 0.472 1.989
2.277 0.919 1.926 3.195 2.406 1.182 0.191 2.013 1.605 1.822 0.527 2.349
DAA Alanine Aspartic acid Glycine Glutamic acid Proline Serine
0.290 0.437 0.625 1.198 0.845 0.278
1.787 3.326 0.671 3.716 0.513 2.494
2.086 3.763 1.296 4.914 1.358 2.772
Amino acid
ZDAA
‘Calculated from values provided by the United States Biochemical Corp., Cleveland, OH, USA. 2The amino acid profile simulated that of 35% whole chicken egg protein (Robinson et al., 1980). “Added as lysine HCl (2.324% for 1.860% lysine). bathe level of crystalline phenylalanine or tyrosine was reduced while the level of DAA was increased.
with 0.6% Phe on a dry-matter basis. The Phe level of 0.6% was chosen because the Tyr replacement value should be determined at the Phe level below the absolute requirement. [The result of Experiment 1 showed that the Phe requirement was about 0.7% of dry diet.] Another diet, which was used as a reference to ensure that 0.6% Phe was not in excess, contained 1.33% Tyr and 1.75% Phe. Ingredients and amino acid composition of the basal diet are shown in Tables 1 and 2, respectively. All experimental diets were formulated to contain 35% crude protein, the amino acid profile of which simulated that of 35% whole chicken egg protein. Four replicate groups, each consisting of 30 rainbow trout (Oncorhynchus m$ziss), were assigned to each diet. Osceola strain trout were provided by the U.S. Fish and Wildlife Service, National Fish Hatchery, Osceola, WI, and mean initial body weight + s.e.m. (for 28 tanks) was 12.7 2 0.06 g for fish used in Experiment 1 and 14.0 + 0.03 g for fish used in Experiment 2. Fish were fed the experimental diets 3 times daily to satiation in tanks containing 100 liter of water at 15 & 0.5 oC with a flow rate of 2 to 4 l/min. Other
K.I. KIM
246
procedures including experimental conditions, diet preparation, carcass analysis and statistical analysis were as described in Kim et al. ( 1987, 199 1). RESULTS
Elevating Phe from 0.26 to 0.75% in diets containing excess Tyr increased weight gain, feed efficiency and nitrogen retention (Table 3), but a further elevation to 1.75 had no effect (P> 0.05). A high mortality was observed with diets containing lower than 0.45% Phe. By applying a broken-line model (Robbins et al., 1977) to the analysis of weight gain versus dietary Phe level, the Phe requirement was estimated at approximately 0.7 20.05% (mean + s.e. ) of dry diet or 2% of dietary protein in the presence of excess Tyr. The carcass of trout fed diets deficient in Phe ( ~0.45%) tended to have lower protein but a higher fat and ash content than that of trout fed diets containing higher than 0.55% Phe (Table 4). No significant differences were found in dry matter content of the carcass, or carcass percentage (data not shown). In Experiment 2, weight gain increased as the Tyr level in the diet increased up to 0.8% (Table 5 ). But weight gain was not significantly different between trout fed diets containing 0.7% and 0.8% Tyr. The break point was found at the Tyr level of 0.68 + 0.01% (mean 2 se. ). Thus the replacement value of TABLE 3 Effects of dietary levels of phenylalanine (Experiment 1) ’
(Phe) on weight gain, feed efficiency and nitrogen retention
Level of Phe’ (%)
Weight gain (g/fish)
Feed efficiency3
Nitrogen retention4 (%)
Mortality (%)
0.26 0.35 0.45 0.55 0.65 0.75 1.75
7.7 * 0.4a~5 12.2i0.4b 16.0+0.8’ 21.71b0.7~ 22.0+0.4* 23.9 f 0.3’ 24.2kO.3”
0.43 * 0.05” 0.55 &0.03b 0.68 + 0.02’ 0.79 & 0.03* 0.81?0.02* 0.84+0.01* 0.82+0.01*
12.7f 1.5” 20.6? 1.4b 26.8 f 1.6’ 32.5 f0.7* 34.3 Ifrl.Od*” 36.7+0.9e 34.5 + 0.6*pe
44.0 25.0 9.2 0 0.8 0 0
‘Means Z!Is.e.m. of four tanks of 30 fish each over a 6-week period. Mean initial body weight + s.e.m. of 28 tanks of 30 fish each was 12.7 I!I0.06 g. *0.26% phenylalanine was supplied by casein and gelatin and all the experimental diets contained 1.33% tyrosine. 3g gain/g feed fed. “(Nitrogen gained in eviscerated carcass/nitrogen intake) x 100. ‘Values in the same column sharing the same superscript letters are not significantly different at the 5% level, when analyzed by the Neuman-Keuls test.
AROMATIC AMINO ACID REQUIREMENTSOF TROUT
247
TABLE 4 Effects of dietary levels of phenylalanine Carcass composition
Level of Phe3 (%)
Dry matter
0.26 0.35 0.45 0.55 0.65 0.75 1.75
25.2f0.2 25.5f0.2 25.2kO.l 25.2kO.l 25.3LO.l 25.4kO.l 25.2fO.O
(Phe) on carcass composition
(Experiment
1) ’
(o/o)’
55.3f2.0”,5 56.5* 1.3”~~ 58.3 +2.2asb,c 59.3 * 2.7b*G 60.5* 1.5” 60.7kO.9” 60.2+ 1.0’
Fat4
Ash4
32.0*0.9=
10.6 + 0.2”,b 10.7?0.3a 10.5f0.7”Tb 10.1 * 0.2aab%c 10.0 + 0.4”,b,c 9.7f0.4” 9.9 + 0.4b*’
30.8+2.1a~b 30.4 + 1.gaab 28.3 k 0.6b 28.2+ 1.8b 27.7+0.6b 29.4 a 2.8”ab
‘Means k s.e.m. of four tanks, each consisting of 10 fish (combined). ‘Initial carcass composition was 22.7% dry matter, 70.2% protein, 13.8% fat and 12.7% ash. 3See footnote 2 to Table 3. 40n a dry-matter basis. 5Values in the same column sharing the same superscript letters are not significantly different at the 5% level, when analyzed by the Neuman-Keuls test.
TABLE 5 Effects of dietary levels of tyrosine (Tyr) and phenylalanine (Experiment 2) ’ Level (%) TY~
Phe’
0.28 0.40 0.50 0.60 0.70 0.80 1.33
0.60 0.60 0.60 0.60 0.60 0.60 1.75
(Phe) on weight gain and feed efficiency
Weight gain (g/fish)
Feed efficiency’
Mortality (%)
6.9 + 0.7as4 8.550.9” 9.0f0.6” 11.3*0.9b*c 12.8&0.8c,d 14.9+0.5d 22.4f0.5”
0.37 * 0.03” 0.49 * o.04b 0.50+0.04b 0.53+0.03b 0.52 f 0.04b 0.59 f 0.02b 0.94 t- 0.02”
0.8 0.8 6.7 2.5 0 1.6 0
‘Means f s.e.m. of four tanks of 30 fish each over a 6-week period. Mean initial body weight * s.e.m. of 28 tanks of 30 fish each was 14.0+ 0.03 g. ‘0.28% tyrosine and 0.26% phenylalanine were supplied by casein and gelatin. 3g, gain/g, feed as fed. 4Values in the same column sharing the same superscript letters are not significantly different at the 5% level, when analyzed by the Neuman-Keuls test.
Tyr for Phe was determined at about 53% [100x0.68/(0.6+0.68)] on a weight basis or 48% (53 x 165.19/ 18 I. 19) on a molar basis and the total aromatic amino acid requirement was estimated at 1.5% [0.7/( l-0.53) ] of diet or 4.3% of dietary protein.
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K.I. KIM
DISCUSSION
The requirement of rainbow trout for total aromatic amino acids ( 1.5% of diet or 4.3% of protein) estimated in the present study is similar to the value ( 1.83% of diet) reported for trout by Ogino ( 1980)) who calculated the amino acid requirements on the basis of the daily increase of protein (amino acids) in the body of fish fed diets containing about 35% protein, and the daily excretion of nitrogen by fish fed a protein-free diet. However, Ogino (1980) has probably overestimated the value, because of a low feed-intake level (assuming 3% of body weight) used to calculate the amino acid requirements, although the average daily gain was 3.44% of body weight. The total aromatic amino acid requirements of channel cat&h ( 1.2% of diet) reported by Robinson et al. ( 1980) and Nile tilapia ( 1.55%) reported by Santiago and Lovell ( 1988) appear similar to that (1.5%) estimated for rainbow trout in the present study. However, the total aromatic amino acid requirements recommended by the NRC ( 1983 ) for chinook salmon (2.1% of diet or 5.1% of protein), common carp (2.5% or 6.5%) and Japanese eel (2.2% or 5.8%) are much higher than that estimated in the present study for rainbow trout. The differences between the NRC (1983) values and the value found in the present study may be due not only to species differences, but also more probably to the laboratory variances, such as using various dietary ingredients, different experimental conditions and fish of different sizes. Our previous study (Kim et al., 199 1) showed that the dietary protein level required by fingerling rainbow trout to meet the need for indispensable amino acids is no more than 25% of the diet (although the protein requirement for maximal growth of fingerling rainbow trout is 35%). In the diet containing 25% intact protein (provided by casein + gelatin supplemented with arginine and methionine) and an additional 10% dispensable amino acid mixture, the levels of arginine, histidine, isoleucine, leucine, lysine, sulfur amino acids, aromatic amino acids, threonine, tryptophan and valine were 1.6, 0.67, 1.4, 2.1, 1.9, 0.98, 2.5, 1.1, 0.28 and 1.9%, respectively. The amino acid requirements of rainbow trout which have been determined at the University of Wisconsin-Aquaculture Laboratory do not exceed the level of the corresponding amino acids contained in the 25% intact protein diet supplemented with arginine and methionine. The amino acid requirements of rainbow trout (Kim et al., 1987, 1992a, b, present study) are also comparable to those of young pigs (NRC, 1988) except for arginine ( 1.4 vs. 0.6; an obvious reason for this is that trout are ammonotelic and thus produce little arginine via the urea cycle). The comparisons include lysine ( 1.3 vs. 1.4)) sulfur amino acids (0.8 vs. 0.68), tryptophan (0.2 vs. 0.2 ) and aromatic amino acids ( 1.5 vs. 1.1) . The replacement value (53% on a weight basis or 48% on a molar basis) of Tyr for Phe determined in the present study for rainbow trout appears lower
AROMATIC AMINO ACID REQUIREMENTSOF TROUT
249
than the value of 60% (on a weight basis) found for common carp (Nose, 1979), but similar to the value of 50% (on a molar basis) reported for channel catfish (Robinson et al., 1980). The Tyr replacement value reported in the present study was obtained with a diet containing Phe at a level lower than the requirement, assuming that the minimum ratio of Phe to the total aromatic amino acids required for maintenance and growth is the same. If the Phe level in the test diet is higher than that required, the Tyr replacement value may be underestimated. The low weight gain obtained with the diets containing 0.6% Phe and either 0.7 or 0.8% Tyr, as compared to that obtained with the diet containing 1.75% Phe and 1.33% Tyr or with the diet containing 0.55% Phe and 1.33% Tyr in Experiment 1, was unexpected. However, such variations have been seen in feeding experiments performed with fish (see Robinson et al., 1980). The findings in the present study suggest that the total aromatic amino acid requirement of fingerling rainbow trout (about 1.5% of diet ) is similar to that recommended for young pigs-l .4% by Kim et al. ( 1983 ), assuming that the replacement value of Tyr for Phe is 50%, or 1.1% by NRC ( 1988 )-and for broiler chicks ( 1.34% by NRC, 1984). This similarity supports our contention that the individual amino acid requirements of rainbow trout are similar to those of rapidly growing farm animals (Kim et al., 1987, 1992a, b), and that the higher protein requirement of fish is due to use of protein by fish to meet their energy requirements (Kim et al., 199 1). This conclusion is well explained by the fact that trout are carnivorous and have a limited ability to utilize carbohydrate, which is a main energy source for farm animals. ACKNOWLEDGEMENTS
I thank Terry Kayes for making this study possible. I also thank Penny Swanson, Tom Kuczynski, Jean Grassman, Thorn Grimshaw and Larry Zehner for their technical help and the U.S. Fish and Wildlife Service for providing fish. This work was supported in part by the University of WisconsinMadison, College of Agricultural and Life Sciences and Graduate School, and by the University of Wisconsin Sea Grant College Program under grants from the National Oceanic and Atmospheric Administration, the U.S. Department of Commerce, and from the State of Wisconsin (Federal grant NA80AA-D00086, projects R/AQ-8 and R/AQ-9).
REFERENCES Kim, K.I., McMillan, I. and Bayley, H.S., 1983. Determination of amino acid requirements of young pigs using an indicator amino acid. Br. J. Nutr., 50: 369-382. Kim, K.I., Kayes, T.B. and Amundson, C.H., 1987. Effects of dietary tryptophan levels on growth,
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feed/gain, carcass composition and liver glutamate dehydrogenase activity in rainbow trout (Sulmogairdneri). Comp. Biochem. Physiol., MB: 737-741. Kim, K.I., Kayes, T.B. and Amundson, C.H., 199 1. Purified diet development and re-evaluation of the dietary protein requirement of fingerling rainbow trout (Oncorhynchus mykiss). Aquaculture, 97: 57-67. Kim, K.I., Kayes, T.B. and Amundson, C.H., 1992a. Requirements for sulfur amino acids and utilization of D-methionine by rainbow trout (Oncorhynchus mykiss). Aquaculture, 10 1: 95-103. Kim, K.I., Kayes, T.B. and Amundson, C.H., 1992b. Requirements for lysine and arginine by rainbow trout (Oncorhynchus mykiss). Aquaculture, 106: 333-344. National Research Council, 198 1. Nutrient Requirements of Coldwater Fishes. National Academy Press, Washington, DC, 63 pp. National Research Council, 1983. Nutrient Requirements of Warmwater Fishes and Shellfishes. National Academy Press, Washington, DC, 102 pp. National Research Council, 1984. Nutrient Requirements of Poultry, 8th ed. National Academy Press, Washington, DC, 77 pp. National Research Council, 1988. Nutrient Requirements of Swine, 9th ed. National Academy Press, Washington, DC, 93 pp. Nose, T., 1979. Summary report on the requirements of essential amino acids for carp. In: J.E. Halver and K. Tews (Editors), Finfish Nutrition and Fishfeed Technology I. Heeneman, Berlin, pp. 145-156. Ogino, C., 1980. Requirements of carp and rainbow trout for essential amino acids. Bull. Jpn. Sot. Sci. Fish., 46: 171-174. Robbins, K.P., Baker, D.H. and Norton, H.W., 1977. Histidine status of the chick as measured by growth rate, plasma free histidine and breast muscle carnosine. J. Nutr., 107: 2055-206 1. Robinson, E.H., Wilson, R.P. and Poe, W.E., 1980. Total aromatic amino acid requirement, phenylalanine requirement and tyrosine replacement value for fingerling channel catfish. J. Nutr., 110: 1805-1812. Santiago, C.B. and Lovell, R.T., 1988. Amino acid requirements for growth of Nile tilapia. J. Nutr., 118: 1540-l 546. Wilson, R.P., 1989. Amino acids and proteins. In: J.E. Halver (Editor), Fish Nutrition, 2nd ed., Academic Press, New York, NY, pp. 112- 15 1.