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Requirements of juvenile marine shrimp, Penaeus monodon (Fabricius) for lysine and arginine
Aquaculture 164 Ž1998. 95–104
Requirements of juvenile marine shrimp, Penaeus monodon ž Fabricius/ for lysine and arginine O.M. Millamena a
a,)
, M.N. Bautista-Teruel a , O.S. Reyes a , A. Kanazawa b
Aquaculture Department, Southeast Asian Fisheries, DeÕelopment Center, 5021 Tigbauan, Iloilo, Philippines b Faculty of Fisheries, Kagoshima UniÕersity, 4-50-20, Shimoarata, Kagoshima, Japan
Abstract Feeding experiments were conducted using amino acid test diets to determine the dietary requirements of juvenile Penaeus monodon for lysine and arginine. Two sets of the test diets were prepared. The natural protein was supplied by casein and gelatin. Crystalline L-amino acids were added to provide an amino acid profile similar to shrimp muscle protein except for the test amino acid. One set of experimental diets contained graded levels of lysine at 1.18–3.28% of the diet and another set contained arginine at 0.6–3.0% of the diet. The amino acid mixture was pre-coated with carboxymethylcellulose ŽCMC. and diets were further bound with CMC, cornstarch, and K–carrageenan to prevent leaching losses of amino acids. Shrimp postlarvae, PL20, with mean weight of 21 " 0.5 mg, were randomly distributed at 10 shrimp per tank in 40-l fiberglass tanks and reared on the diets for 50–56 days. Growth, survival and feed conversion efficiency were determined at termination of feeding trials and signs of nutritional deficiency noted. Lysine and arginine requirements were determined from relationships between weight gains and dietary lysine and arginine levels as analyzed by the broken-line regression method. The requirement of juvenile P. monodon for lysine was estimated to be 2.08% of the diet or 5.2% of dietary protein while the requirement for arginine was 1.85% of the diet or 5.3% of dietary protein. This information is crucial in formulating cost-effective practical diets for juvenile tiger shrimp. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Penaeus monodon Fabricius; Amino acid requirement
)
C orresponding author. [email protected].
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O.M. Millamena et al.r Aquaculture 164 (1998) 95–104
1. Introduction The formulation of a nutritionally adequate and economical diet is most important for successful culture of the tiger shrimp, Penaeus monodon. Since feed constitutes the major item in shrimp production, its cost-effectiveness and nutritional adequacy must be well taken into consideration. High cost and less than optimal feeds are largely due to limited knowledge on shrimp nutritional requirements. Determination of essential amino acid requirements is considered to be the highest priority area in shrimp nutrition research ŽAkiyama, 1986.; however, information concerning amino acid nutrition is still limited. Studies to quantify the essential amino acid requirements using test diets supplemented with crystalline amino acids were generally unsuccessful due to poor shrimp growth and survival ŽDeshimaru and Kuroki, 1974; Deshimaru, 1981; Teshima et al., 1986; Pascual, 1990.. Among penaeid shrimp species, the lysine requirement of P. Õannamei ŽAkiyama, 1986. and arginine requirement of P. monodon ŽChen et al., 1992; Fox et al., 1992. had been reported. More recently, quantification of requirements for valine and methionine ŽMillamena et al., 1996a,b. and threonine ŽMillamena et al., 1997. of juvenile P. monodon have been achieved. Shrimps, due to their slow feeding behavior, require a water-stable diet. These reports described the coating technique and diet binders used for test diet preparation in order to minimize amino acid leaching and ensure availability of dietary nutrients to shrimp. The present experiments aimed to determine the dietary requirements of juvenile P. monodon for lysine and arginine using the same techniques as in the previous studies and further described in this paper.
2. Materials and methods 2.1. Experimental diet composition The composition of amino acid test diets used to determine lysine and arginine requirements are presented in Tables 1 and 2. The basal diet was based on a modified formula used by Pascual and Kanazawa Ž1986.. The amounts of casein and gelatin in the basal diets were adjusted such that the lowest level of test amino acid came solely from intact proteins. Experimental diet used for lysine study contained 40% protein and 1.18% lysine from 3:1 casein–gelatin, the natural sources of protein. Diets used for arginine study contained 35% protein with 0.6% arginine from 2:1 casein–gelatin. Crystalline L-amino acids were added to provide an amino acid pattern similar to P. monodon tissue protein except for the test amino acid. Graded levels of lysine were incorporated to obtain 1.18, 1.60, 2.02, 2.44, 2.86 and 3.28 and 3.0 g per 100 g diet and arginine at 0.6, 1.0, 1.4, 1.8, 2.2, 2.6 and 3.0 g per 100 g diet. These levels range below and above the lysine and arginine levels in shrimp muscle protein. Separate amino acid mixes were prepared for each set of test diets and the amount of amino acids used are presented in Tables 3 and 4.
O.M. Millamena et al.r Aquaculture 164 (1998) 95–104
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Table 1 Composition of experimental diets for lysine requirement study in g per 100 g dry diet Ingredients
Casein Žvitamin-free. Gelatin Amino acid mix Lysine Cornstarch Glucose Sucrose Cholesterol Lecithin Cod liver oil Soybean oil Vitamin mix a Mineral mix a Carboxymethyl cellulose ŽCMC. Celufil K–carrageenan % lysine diet protein a
Diet 1
2
3
4
5
6
15 5 20 0 15 5 10 2 2 3 3 8 3 4 2.5 2.5
15 5 19.58 0.42 15 5 10 2 2 3 3 8 3 4 2.5 2.5
15 5 19.16 0.84 15 5 10 2 2 3 3 8 3 4 2.5 2.5
15 5 18.74 1.26 15 5 10 2 2 3 3 8 3 4 2.5 2.5
15 5 18.32 1.68 15 5 10 2 2 3 3 8 3 4 2.5 2.5
15 5 17.90 2.10 15 5 10 2 2 3 3 8 3 4 2.5 2.5
1.60 4.0
2.02 5.05
2.44 6.10
2.86 7.15
3.28 8.20
1.18 2.95
Vitamin and mineral mixes after Kanazawa Ž1981..
2.2. Diet preparation The procedures for diet preparation were similar to those reported earlier ŽMillamena et al., 1996a,b. and are further described here. The dry ingredients, except crystalline amino acids, cholesterol and binders, were thoroughly mixed in a food mixer. Crystalline amino acids ŽCAA. were weighed individually for each of experimental diet. The crystalline amino acid mixture Ž20 g. was pre-coated with 1.5 g cooked carboxymethylcellulose ŽCMC.. The bound CAA was then mixed with dry ingredients and re-coated with 2.5 g cooked CMC. The vitamin premix was blended in the oils and lecithin and added to the coated mixture. Diet pH was maintained at 7.0–7.5 by gradually adding 6 N NaOH. The pH was determined by homogenizing 5 g of mixed ingredient with 50 ml distilled water. The diet binders Žcornstarch and K–carrageenan. were pre-gelatinized before adding to the coated mixture. Cornstarch was gelatinized at 1008C, cooled, and added to the mixture until a dough was formed. K–carrageenan was gelatinized at 858C using a water bath to form a homogeneous gel and quickly added as the final binder. The moist mixture was extruded in a Hobart food grinder and dried at 408C in an air-draft oven. Dry pellets Ž2 mm diameter, 3–5 mm long. were stored at y48C until fed. When tested for water stability, diets were water stable within the 4 h feeding interval. Diet samples were analyzed for proximate composition according to AOAC Ž1984..
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O.M. Millamena et al.r Aquaculture 164 (1998) 95–104
Table 2 Composition of experimental diets for arginine requirement study in g per 100 g dry diet Ingredients
Diet
Casein Žvitamin-free. Gelatin Amino acid mix Arginine Cornstarch Glucose Sucrose Cholesterol Lecithin Cod liver oil Soybean oil Vitamin mix a Mineral mix a Carboxymethyl cellulose ŽCMC. Celufil K–carrageenan % arginine diet protein a
1
2
3
4
5
6
7
10 5 20 0 15 5 10 2 2 3 3 8 3 4 7.5 2.5
10 5 19.6 0.4 15 5 10 2 2 3 3 8 3 4 7.5 2.5
10 5 19.2 0.8 15 5 10 2 2 3 3 8 3 4 7.5 2.5
10 5 18.8 1.2 15 5 10 2 2 3 3 8 3 4 7.5 2.5
10 5 18.4 1.6 15 5 10 2 2 3 3 8 3 4 7.5 2.5
10 5 18.0 2.0 15 5 10 2 2 3 3 8 3 4 7.5 2.5
10 5 17.6 2.4 15 5 10 2 2 3 3 8 3 4 7.5 2.5
0.6 1.7
1.0 2.9
1.4 4.0
1.8 5.1
2.2 6.3
2.6 7.4
3.0 8.6
Vitamin and mineral mixes after Kanazawa Ž1981..
Table 3 Amino acid composition of experimental diets for lysine requirement study Žg per 100 g dry diet. Amino acids Essential amino acids Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine
Amount in casein Ž15%. and gelatin Ž5%.
Crystalline amino acids
Amount in 40% shrimp tissue protein
0.73 0.60 0.74 1.34 1.18 0.38 0.87 0.64 0.06 1.00
2.43 0.38 1.02 1.57 variable 0.62 0.79 0.92 0.38 1.04
3.16 0.98 1.76 2.91 3.00 1.00 1.66 1.56 0.44 2.04
2.68 2.92 0.84 y 2.08 1.60 0.37 1.00
4.03 6.35 1.64 1.59 3.21 2.42 0.41 1.56
Non-essential amino acids Aspartic acid 1.35 Glutamic acid 3.43 Serine 0.86 Proline 1.72 Glycine 1.13 Alanine 0.82 Cystine 0.04 Tyrosine 0.56
O.M. Millamena et al.r Aquaculture 164 (1998) 95–104
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Table 4 Amino acid composition of experimental diets for arginine requirement study Žg per 100 g dry diet. Amino acids Essential amino acids Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine
Amount in casein Ž10%. and gelatin Ž5%.
Crystalline amino acids
Amount in 40% shrimp tissue protein
0.60 0.42 0.52 0.95 0.85 0.27 0.62 0.47 0.04 0.70
variable 0.56 1.24 1.96 2.15 0.73 1.04 1.09 0.40 1.34
3.16 0.98 1.76 2.91 3.00 1.00 1.66 1.56 0.44 2.04
3.0 3.91 1.0 0.31 2.16 1.72 0.38 1.18
4.03 6.35 1.64 1.59 3.21 2.42 0.41 1.56
Non-essential amino acids Aspartic acid 1.03 Glutamic acid 2.44 Serine 0.64 Proline 1.28 Glycine 1.05 Alanine 0.70 Cystine 0.03 Tyrosine 0.38
2.3. Feeding experiments Two feeding trials were conducted at the nutrition wet laboratory of SEAFDEC Aquaculture Department at Tigbauan, Iloilo, Philippines. P. monodon PL 20 , initial mean wt s 21 " 0.5 mg, were acclimated to the basal diets for one week before actual start of feeding trials. For each study, 10 postlarvae were randomly stocked in each of 36 units of 40 l oval fiberglass tanks. Sand-filtered sea water was supplied in a flow-through system and provided with continuous aeration. Dietary treatments were arranged in a completely randomized design each with six replicates. Shrimp were fed the experimental diets thrice daily at 0900, 1300, and 1700 h. Uneaten feeds and faeces were siphoned and 1r3 of the culture water was drained and replaced before the first feeding every morning. Daily feeding rate was 25–30% of shrimp biomass adjusted for mortalities. Shrimps were bulk-weighed every 15 days to determine weight gains. Survival was determined at termination of the feeding trial. Water quality indicators including pH, ammonia, and nitrite nitrogen were analyzed thrice weekly according to Strickland and Parsons Ž1972. while water temperature and salinity were monitored daily. These parameters remained within suitable levels throughout the culture period. 2.4. Statistical analysis Percent weight gain, specific growth rate, and percent survival were subjected to one-way analysis of variance ŽGomez and Gomez, 1976.. Computer software developed
O.M. Millamena et al.r Aquaculture 164 (1998) 95–104
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by Statistical Analysis Systems Ž1992. was used. The quantitative lysine and arginine requirements were derived using the broken-line regression analysis ŽZeitoun et al., 1976; Robbins et al., 1979. of the growth response curve of percent weight gains against dietary arginine and lysine levels. 3. Results 3.1. Lysine requirement Table 5 shows percent weight gain, specific growth rate, survival and feed conversion ratio of P. monodon fed the experimental diets containing graded levels of lysine for 56 days. Mean weight gains ranged from 672 to 1581%. Weight gain and specific growth rate were highest in shrimps fed diet 3 Ž2.02% lysine. and lowest in those fed diet 1 with no supplemental lysine Ždiet 1 s 1.18% lysine.. Survival rates varied from 57 to 80%. Although survival was not significantly different among the dietary treatments, highest survival was found on the diet with maximum weight gain and highest feed conversion efficiency. Results show that the quantitative dietary lysine requirements of juvenile P. monodon is 2.08% of a 40% protein diet or 5.2% of dietary protein ŽFig. 1a.. 3.2. Arginine requirement Table 6 shows the percent weight gain, specific growth rate, survival and feed conversion of P. monodon fed the experimental diets containing graded levels of arginine for 56 days.
Table 5 Mean weight gain, specific growth rate ŽSGR., survival and feed conversion ratio ŽFCR. of P. monodon fed diets with graded levels of lysine for 50 days Weight gain Ž%."SEM
Dietary lysine % diet 1.18 1.6 2.02 2.44 2.86 3.28
b
Survival Ž%.
FCRb
4.62 4.80 5.59 4.68 5.12 4.71
73 65 80 67 72 57
2.12 2.07 1.66 2.00 1.96 2.31
Mean square 412143.27 87745.01
F value 4.70
P)F 0.0035
% protein 2.95 4.0 5.05 6.1 7.15 8.2
Analysis of Õariance Source df Model 5 Error 26 Corrected total 31 R 2 s 0.474 a
SGRa
672"137 959"50 1581"142 902"61 1147"84 984"164
Sum of squares 2060716.37 2281370.38 4342086.75 CVs 28.95714
Root MSEs 296.218
Specific growth rates lnŽmean final wt.ylnŽmean initial wt.r50 days=100. Feed conversion rations g dry weight feedrg wet weight gain.
O.M. Millamena et al.r Aquaculture 164 (1998) 95–104
101
Fig. 1. Ža. Growth response of P. monodon fed graded levels of lysine for 50 days. Žb. Growth response of P. monodon fed graded levels of arginine for 56 days.
O.M. Millamena et al.r Aquaculture 164 (1998) 95–104
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Table 6 Mean weight gain, specific growth rate ŽSGR., survival, and feed conversion ratio ŽFCR. of P. monodon fed diets with graded levels of arginine for 56 days Weight gain Ž%."SEM
Dietary arginine % diet 0.6 1.0 1.4 1.8 2.2 2.6 3.0
b
Survival Ž%.
FCRb
4.09 4.40 4.64 4.82 4.55 4.48 4.32
80 78 74 87 93 80 70
1.70 1.88 2.21 1.65 1.88 2.03 2.18
Mean square 48049.136 18531.765
F-value 2.59
P)F 0.0470
% protein 1.7 2.9 4.0 5.1 6.3 7.4 8.6
Analysis of Õariance Source df Model 6 Error 22 Corrected Total 28 R 2 s 0.414 a
SGRa
678"35 812"78 926"59 1017"25 881"64 846"53 778"69
Sum of squares 288294.814 407698.814 695993.638 CVs16.21824
Root MSEs136.131
Specific growth rates lnŽmean final wt.ylnŽmean initial wt.r56 days=100. Feed conversion ratios g dry weight feedrg wet weight gain.
Mean weight gain and specific growth rate of shrimp were improved by supplementation with arginine. Highest mean weight gain Ž1017%. was obtained for shrimp fed diet 4 Ž1.80% arginine. and lowest for Ž678%. in those fed the unsupplemented diet Ždiet 1 s 0.60% arginine.. Survival rate ranged from 70 to 93%. High survival and feed conversion efficiency were recorded in diet 4 which was near the requirement level. Based on the broken-line analysis of percent weight gain and dietary arginine levels, the arginine requirement of juvenile P. monodon is 1.85% of a 37.5% protein diet or 5.3% of protein ŽFig. 1a..
4. Discussion The dietary lysine requirement for P. monodon of 5.2% protein obtained in this study is considerably higher than the reported lysine requirement of another penaeid species, P. Õannamei, at 3.1% of protein ŽAkiyama, 1986.; however, Fox et al. Ž1992. reported closer requirement values P. Õannamei using covalent and crystalline lysine supplementation at 4.67% and 5.19% respectively. Requirement values among fish species also exhibit a wide variation. Fishes with requirement levels closer to that of P. monodon, when expressed in percent of dietary, are chinock salmom, 5.0% ŽHalver et al., 1958., gilthead sea bream, 5.0%, ŽLuquet and Sabaut, 1974., channel catfish, 5.0%, ŽRobinson et al., 1980., Nile tilapia, 5.1%, Santiago and Lovel, 1988. and Japanese eel, 5.3%, ŽNose, 1979.. The requirement obtained for arginine Ž5.3% of protein. agrees well with that reported for the same species, P. monodon, at 5.45% arginine ŽChen et al., 1992.. This
O.M. Millamena et al.r Aquaculture 164 (1998) 95–104
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value is similar to that of milkfish, 5.2%,ŽBorlongan and Coloso, 1993. and is generally higher than for some other fish species such as Nile tilapia, 4.2%, ŽSantiago and Lovel, 1988.; channel catfish, 4.3%, Robinson et al., 1981.; common carp, 4.3%, and Japanese eel, 4.5%, ŽNose, 1979.; but lower than that reported for chinook salmon, 5.75%, ŽKlein and Halver, 1970.. This observed variation is attributed to inherent differences among species, differences in levels and quality of proteins used in the test diets, and other dietary and environmental factors. In both studies, growth responses of shrimp improved with lysine and arginine-supplementation beyond those coming from intact proteins suggesting the ability of juvenile shrimp to utilize crystalline forms of lysine and arginine. There is a marked decline in growth rates beyond the optimum requirement levels for both lysine and arginine. The growth depression was also observed in studies on valine and methionine of P. monodon ŽMillamena et al., 1996a,b. and in a study by Recodo Ž1991. on histidine requirement of the tiger shrimp. The basis for growth-depression has not been fully established; however, these observations may suggest toxicity of excessive amino acid levels beyond that found in the shrimp tissue protein. Mertz Ž1972. suggested that high dietary amino acid levels can cause accumulation and toxicity in tissues. Further, Recodo Ž1991., through electron microscopy technique, noted extensive necrosis in the shrimp hepatopancreas when fed high levels of histidine. In this study, visual observations showed no gross pathological signs in P. monodon juveniles fed diets low or high in either of the test amino acids except for depressed growth rate. Lysine and arginine are found in high quantities in the muscle protein of penaeid shrimp and are considered as most limiting amino acids in most animals, particularly in P. monodon ŽAkiyama, 1986; Pascual, 1990.. These information on dietary lysine and arginine requirements, along with data obtained on requirements for other essential amino acids, will provide valuable knowledge in protein and amino acid nutrition of P. monodon and thus will lead to developing cost-effective diets for the tiger shrimp. Acknowledgements The authors wish to thank Mr. Vicente Balinas and Mr. Dolfus Miciano for their assistance in the statistical analysis and computer work and Ms. Mae Fernando for her assistance during the conduct of these studies. References Akiyama, D., 1986. The development of purified diet and nutritional requirement of lysine in penaeid shrimp. PhD dissertation. Texas A&M University, College Station, 78 pp. AOAC, 1984. Williams, S. ŽEd.., Official Methods of Analysis of the Association of Official Analytical Chemists, 14th edn. Arlington, VA, 114 pp. Borlongan, I.G., Coloso, R.M., 1993. Requirements of juvenile milkfish Chanos chanos for essential amino acids. J. Nutr. 123, 125–132. Chen, H.Y., Len, Y.T., Roelants, I., 1992. Quantification of arginine requirements of juvenile marine shrimp Penaeus monodon using microencapsulated arginine. Mar. Biol. 114, 229–233.
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Deshimaru, O., 1981. Protein and amino acid nutrition of the prawn Penaeus japonicus. In: Pruder, G.D., Langdon, C., Conklin, D. ŽEds.., Proceedings of the 2nd International Conference on Aquaculture Nutrition, Biochemical and Physiological Approaches to Shellfish Nutrition. Rehoboth Beach, DE, U.S.A., pp. 106–123. Deshimaru, O., Kuroki, K., 1974. Studies on a purified diet for prawn: III. A feeding experiment with amino acid test diets. Bull. Jpn. Soc. Sci. Fish. 41, 101–103. Fox, J., Lawrence, A.L., Li-Chan, E., 1992. Apparent lysine requirement of Penaeus Õannamei ŽBoone. using covalent and crystalline lysine supplementation. Abstracts of Aquaculture ’92. Marriott’s Orlando World Center, Orlando, FL, May 21–25, pp. 96–97. Gomez, K.A., Gomez, A.A., 1976. Statistical Procedures for Agricultural Research with Emphasis on Rice. The Inernational Rice Research Institute, Los Banos, ˜ Laguna, The Philippines, 294 pp. Halver, J.E., DeLong, D.C., Mertz, E.T., 1958. Threonine and lysine requirements of chinook salmon. Fed. Proc., Fed. Am. Soc. Exp. Biol. 17, 478. Kanazawa, A., 1981. Penaeid nutrition. In: Pruder, G.D., Langdon, C., Conklin, D. ŽEds.., Proceedings of the 2nd International Conference on Aquaculture Nutrition: Biochemical and Physiological Approaches to Shellfish Nutrition. Louisiana State University, Baton Rouge, LA, U.S.A., pp. 87–105. Klein, R.G., Halver, J.E., 1970. Nutrition of salmonid fishes: arginine and histidine requirements of chinook and coho salmon. J. Nutr. 100, 1105–1110. Luquet, P., Sabaut, J.J., 1974. Nutrition azotee et croissance chez la daurade et la truite. Actes des colloques. Colloques sur l’Aquacult. 1, 243–253. Mertz, E.T., 1972. The protein and amino acid needs. In: Halver, J.E. ŽEd.., Fish Nutrition. Academic Press, New York, pp. 105–143. Millamena, O.M., Bautista-Teruel, M.N., Kanazawa, A., 1996a. Valine requirement of postlarval tiger shrimp Penaeus monodon Fabricius. Aquacult. Nutr. 2 Ž3., 129–132. Millamena, O.M., Bautista-Teruel, M.N., Kanazawa, A., 1996b. Methionine requirement of juvenile tiger shrimp Penaeus monodon Fabricius. Aquaculture 143, 403–410. Millamena, O.M., Bautista, M.N. Reyes, O.S., Kanazawa, A., 1997. Threonine requirement of juvenile marine shrimp Penaeus monodon. Aquaculture Žin press.. Nose, T., 1979. Summary report on the requirements for essential amino acids for carp. In: Trews, K., Halner, J.E. ŽEds.., Finfish Nutrition and Fishfeed Technology. Heinemann, Berlin, Germany, pp. 145–156. Pascual, F.P., 1990. Terminal report submitted to SEAFDEC Aquaculture Department. Tigbauan, Iloilo, Philippines, 84 pp. Pascual, F.P., Kanazawa, A., 1986. Specific amino acid-free semi-purified diets for Penaeus monodon juveniles. Mem. Kagoshima Univ. Res. Center South Pacific 7, 65–72. Recodo, A.G., 1991. Histophysiological effects of various histidine levels in the diet on the hepatopancreas of Penaeus monodon Fabricius. Masters thesis. Institute of Biology, College of Science, U.P. Diliman, Quezon City, Philippines, 104 pp. Robbins, K.R., Norton, H.W., Baker, D.H., 1979. Estimation of nutrient requirements from growth data. J. Nutr. 109, 1710–1714. Robinson, E.H., Wilson, R.P., Poe, W.E., 1980. Re-evaluation of the lysine requirement and lysine utilization by fingerling channel catfish. J. Nutr. 110, 2313–2316. Robinson, E.H., Wilson, R.P., Poe, W.E., 1981. Arginine requirement and apparent absence of lysine–arginine antagonist in fingerling channel catfish. J. Nutr. 111, 46–52. Santiago, C.B., Lovel, R.T., 1988. Amino acid requirements of Nile tilapia. J. Nutr. 118, 1540–1546. Statistical Analysis Systems, 1992. Manual on statistical analysis of fisheries data using SAS. SEAFDEC Aquaculture Department and Division of Statistics and Statistical Laboratory, University of the Philippines at Los Banos, ˜ College, Laguna, 70 pp. Strickland, J.D.H., Parsons, T.R., 1972. Inorganic micronutrients in sea water. In: Stevenson, J. ŽEd.., A Practical Handbook of Seawater Analysis. Fish. Res. Board Can., Canada Dept. Fish Oceans, Ottawa, Canada. Teshima, S., Kanazawa, A., Yamashita, M., 1986. Dietary value of several proteins and supplemental amino acids for larvae of the prawn Penaeus japonicus. Aquacult. Amsterdam 51, 225–235. Zeitoun, I.H., Ulrey, D.E., Magee, W.T., Gill, J.L., Bergen, W.G., 1976. Quantifying nutrient requirements of fish. J. Fish. Res. Board Can. 33, 167–172.
Requirements of juvenile marine shrimp, Penaeus monodon ž Fabricius/ for lysine and arginine O.M. Millamena a
a,)
, M.N. Bautista-Teruel a , O.S. Reyes a , A. Kanazawa b
Aquaculture Department, Southeast Asian Fisheries, DeÕelopment Center, 5021 Tigbauan, Iloilo, Philippines b Faculty of Fisheries, Kagoshima UniÕersity, 4-50-20, Shimoarata, Kagoshima, Japan
Abstract Feeding experiments were conducted using amino acid test diets to determine the dietary requirements of juvenile Penaeus monodon for lysine and arginine. Two sets of the test diets were prepared. The natural protein was supplied by casein and gelatin. Crystalline L-amino acids were added to provide an amino acid profile similar to shrimp muscle protein except for the test amino acid. One set of experimental diets contained graded levels of lysine at 1.18–3.28% of the diet and another set contained arginine at 0.6–3.0% of the diet. The amino acid mixture was pre-coated with carboxymethylcellulose ŽCMC. and diets were further bound with CMC, cornstarch, and K–carrageenan to prevent leaching losses of amino acids. Shrimp postlarvae, PL20, with mean weight of 21 " 0.5 mg, were randomly distributed at 10 shrimp per tank in 40-l fiberglass tanks and reared on the diets for 50–56 days. Growth, survival and feed conversion efficiency were determined at termination of feeding trials and signs of nutritional deficiency noted. Lysine and arginine requirements were determined from relationships between weight gains and dietary lysine and arginine levels as analyzed by the broken-line regression method. The requirement of juvenile P. monodon for lysine was estimated to be 2.08% of the diet or 5.2% of dietary protein while the requirement for arginine was 1.85% of the diet or 5.3% of dietary protein. This information is crucial in formulating cost-effective practical diets for juvenile tiger shrimp. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Penaeus monodon Fabricius; Amino acid requirement
)
C orresponding author. [email protected].
T el.:
q 63-33-3351-009;
fax:
0044-8486r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 4 4 - 8 4 8 6 Ž 9 8 . 0 0 1 7 9 - 3
q 63-33-3351-008;
e-m ail:
96
O.M. Millamena et al.r Aquaculture 164 (1998) 95–104
1. Introduction The formulation of a nutritionally adequate and economical diet is most important for successful culture of the tiger shrimp, Penaeus monodon. Since feed constitutes the major item in shrimp production, its cost-effectiveness and nutritional adequacy must be well taken into consideration. High cost and less than optimal feeds are largely due to limited knowledge on shrimp nutritional requirements. Determination of essential amino acid requirements is considered to be the highest priority area in shrimp nutrition research ŽAkiyama, 1986.; however, information concerning amino acid nutrition is still limited. Studies to quantify the essential amino acid requirements using test diets supplemented with crystalline amino acids were generally unsuccessful due to poor shrimp growth and survival ŽDeshimaru and Kuroki, 1974; Deshimaru, 1981; Teshima et al., 1986; Pascual, 1990.. Among penaeid shrimp species, the lysine requirement of P. Õannamei ŽAkiyama, 1986. and arginine requirement of P. monodon ŽChen et al., 1992; Fox et al., 1992. had been reported. More recently, quantification of requirements for valine and methionine ŽMillamena et al., 1996a,b. and threonine ŽMillamena et al., 1997. of juvenile P. monodon have been achieved. Shrimps, due to their slow feeding behavior, require a water-stable diet. These reports described the coating technique and diet binders used for test diet preparation in order to minimize amino acid leaching and ensure availability of dietary nutrients to shrimp. The present experiments aimed to determine the dietary requirements of juvenile P. monodon for lysine and arginine using the same techniques as in the previous studies and further described in this paper.
2. Materials and methods 2.1. Experimental diet composition The composition of amino acid test diets used to determine lysine and arginine requirements are presented in Tables 1 and 2. The basal diet was based on a modified formula used by Pascual and Kanazawa Ž1986.. The amounts of casein and gelatin in the basal diets were adjusted such that the lowest level of test amino acid came solely from intact proteins. Experimental diet used for lysine study contained 40% protein and 1.18% lysine from 3:1 casein–gelatin, the natural sources of protein. Diets used for arginine study contained 35% protein with 0.6% arginine from 2:1 casein–gelatin. Crystalline L-amino acids were added to provide an amino acid pattern similar to P. monodon tissue protein except for the test amino acid. Graded levels of lysine were incorporated to obtain 1.18, 1.60, 2.02, 2.44, 2.86 and 3.28 and 3.0 g per 100 g diet and arginine at 0.6, 1.0, 1.4, 1.8, 2.2, 2.6 and 3.0 g per 100 g diet. These levels range below and above the lysine and arginine levels in shrimp muscle protein. Separate amino acid mixes were prepared for each set of test diets and the amount of amino acids used are presented in Tables 3 and 4.
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Table 1 Composition of experimental diets for lysine requirement study in g per 100 g dry diet Ingredients
Casein Žvitamin-free. Gelatin Amino acid mix Lysine Cornstarch Glucose Sucrose Cholesterol Lecithin Cod liver oil Soybean oil Vitamin mix a Mineral mix a Carboxymethyl cellulose ŽCMC. Celufil K–carrageenan % lysine diet protein a
Diet 1
2
3
4
5
6
15 5 20 0 15 5 10 2 2 3 3 8 3 4 2.5 2.5
15 5 19.58 0.42 15 5 10 2 2 3 3 8 3 4 2.5 2.5
15 5 19.16 0.84 15 5 10 2 2 3 3 8 3 4 2.5 2.5
15 5 18.74 1.26 15 5 10 2 2 3 3 8 3 4 2.5 2.5
15 5 18.32 1.68 15 5 10 2 2 3 3 8 3 4 2.5 2.5
15 5 17.90 2.10 15 5 10 2 2 3 3 8 3 4 2.5 2.5
1.60 4.0
2.02 5.05
2.44 6.10
2.86 7.15
3.28 8.20
1.18 2.95
Vitamin and mineral mixes after Kanazawa Ž1981..
2.2. Diet preparation The procedures for diet preparation were similar to those reported earlier ŽMillamena et al., 1996a,b. and are further described here. The dry ingredients, except crystalline amino acids, cholesterol and binders, were thoroughly mixed in a food mixer. Crystalline amino acids ŽCAA. were weighed individually for each of experimental diet. The crystalline amino acid mixture Ž20 g. was pre-coated with 1.5 g cooked carboxymethylcellulose ŽCMC.. The bound CAA was then mixed with dry ingredients and re-coated with 2.5 g cooked CMC. The vitamin premix was blended in the oils and lecithin and added to the coated mixture. Diet pH was maintained at 7.0–7.5 by gradually adding 6 N NaOH. The pH was determined by homogenizing 5 g of mixed ingredient with 50 ml distilled water. The diet binders Žcornstarch and K–carrageenan. were pre-gelatinized before adding to the coated mixture. Cornstarch was gelatinized at 1008C, cooled, and added to the mixture until a dough was formed. K–carrageenan was gelatinized at 858C using a water bath to form a homogeneous gel and quickly added as the final binder. The moist mixture was extruded in a Hobart food grinder and dried at 408C in an air-draft oven. Dry pellets Ž2 mm diameter, 3–5 mm long. were stored at y48C until fed. When tested for water stability, diets were water stable within the 4 h feeding interval. Diet samples were analyzed for proximate composition according to AOAC Ž1984..
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Table 2 Composition of experimental diets for arginine requirement study in g per 100 g dry diet Ingredients
Diet
Casein Žvitamin-free. Gelatin Amino acid mix Arginine Cornstarch Glucose Sucrose Cholesterol Lecithin Cod liver oil Soybean oil Vitamin mix a Mineral mix a Carboxymethyl cellulose ŽCMC. Celufil K–carrageenan % arginine diet protein a
1
2
3
4
5
6
7
10 5 20 0 15 5 10 2 2 3 3 8 3 4 7.5 2.5
10 5 19.6 0.4 15 5 10 2 2 3 3 8 3 4 7.5 2.5
10 5 19.2 0.8 15 5 10 2 2 3 3 8 3 4 7.5 2.5
10 5 18.8 1.2 15 5 10 2 2 3 3 8 3 4 7.5 2.5
10 5 18.4 1.6 15 5 10 2 2 3 3 8 3 4 7.5 2.5
10 5 18.0 2.0 15 5 10 2 2 3 3 8 3 4 7.5 2.5
10 5 17.6 2.4 15 5 10 2 2 3 3 8 3 4 7.5 2.5
0.6 1.7
1.0 2.9
1.4 4.0
1.8 5.1
2.2 6.3
2.6 7.4
3.0 8.6
Vitamin and mineral mixes after Kanazawa Ž1981..
Table 3 Amino acid composition of experimental diets for lysine requirement study Žg per 100 g dry diet. Amino acids Essential amino acids Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine
Amount in casein Ž15%. and gelatin Ž5%.
Crystalline amino acids
Amount in 40% shrimp tissue protein
0.73 0.60 0.74 1.34 1.18 0.38 0.87 0.64 0.06 1.00
2.43 0.38 1.02 1.57 variable 0.62 0.79 0.92 0.38 1.04
3.16 0.98 1.76 2.91 3.00 1.00 1.66 1.56 0.44 2.04
2.68 2.92 0.84 y 2.08 1.60 0.37 1.00
4.03 6.35 1.64 1.59 3.21 2.42 0.41 1.56
Non-essential amino acids Aspartic acid 1.35 Glutamic acid 3.43 Serine 0.86 Proline 1.72 Glycine 1.13 Alanine 0.82 Cystine 0.04 Tyrosine 0.56
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Table 4 Amino acid composition of experimental diets for arginine requirement study Žg per 100 g dry diet. Amino acids Essential amino acids Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine
Amount in casein Ž10%. and gelatin Ž5%.
Crystalline amino acids
Amount in 40% shrimp tissue protein
0.60 0.42 0.52 0.95 0.85 0.27 0.62 0.47 0.04 0.70
variable 0.56 1.24 1.96 2.15 0.73 1.04 1.09 0.40 1.34
3.16 0.98 1.76 2.91 3.00 1.00 1.66 1.56 0.44 2.04
3.0 3.91 1.0 0.31 2.16 1.72 0.38 1.18
4.03 6.35 1.64 1.59 3.21 2.42 0.41 1.56
Non-essential amino acids Aspartic acid 1.03 Glutamic acid 2.44 Serine 0.64 Proline 1.28 Glycine 1.05 Alanine 0.70 Cystine 0.03 Tyrosine 0.38
2.3. Feeding experiments Two feeding trials were conducted at the nutrition wet laboratory of SEAFDEC Aquaculture Department at Tigbauan, Iloilo, Philippines. P. monodon PL 20 , initial mean wt s 21 " 0.5 mg, were acclimated to the basal diets for one week before actual start of feeding trials. For each study, 10 postlarvae were randomly stocked in each of 36 units of 40 l oval fiberglass tanks. Sand-filtered sea water was supplied in a flow-through system and provided with continuous aeration. Dietary treatments were arranged in a completely randomized design each with six replicates. Shrimp were fed the experimental diets thrice daily at 0900, 1300, and 1700 h. Uneaten feeds and faeces were siphoned and 1r3 of the culture water was drained and replaced before the first feeding every morning. Daily feeding rate was 25–30% of shrimp biomass adjusted for mortalities. Shrimps were bulk-weighed every 15 days to determine weight gains. Survival was determined at termination of the feeding trial. Water quality indicators including pH, ammonia, and nitrite nitrogen were analyzed thrice weekly according to Strickland and Parsons Ž1972. while water temperature and salinity were monitored daily. These parameters remained within suitable levels throughout the culture period. 2.4. Statistical analysis Percent weight gain, specific growth rate, and percent survival were subjected to one-way analysis of variance ŽGomez and Gomez, 1976.. Computer software developed
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by Statistical Analysis Systems Ž1992. was used. The quantitative lysine and arginine requirements were derived using the broken-line regression analysis ŽZeitoun et al., 1976; Robbins et al., 1979. of the growth response curve of percent weight gains against dietary arginine and lysine levels. 3. Results 3.1. Lysine requirement Table 5 shows percent weight gain, specific growth rate, survival and feed conversion ratio of P. monodon fed the experimental diets containing graded levels of lysine for 56 days. Mean weight gains ranged from 672 to 1581%. Weight gain and specific growth rate were highest in shrimps fed diet 3 Ž2.02% lysine. and lowest in those fed diet 1 with no supplemental lysine Ždiet 1 s 1.18% lysine.. Survival rates varied from 57 to 80%. Although survival was not significantly different among the dietary treatments, highest survival was found on the diet with maximum weight gain and highest feed conversion efficiency. Results show that the quantitative dietary lysine requirements of juvenile P. monodon is 2.08% of a 40% protein diet or 5.2% of dietary protein ŽFig. 1a.. 3.2. Arginine requirement Table 6 shows the percent weight gain, specific growth rate, survival and feed conversion of P. monodon fed the experimental diets containing graded levels of arginine for 56 days.
Table 5 Mean weight gain, specific growth rate ŽSGR., survival and feed conversion ratio ŽFCR. of P. monodon fed diets with graded levels of lysine for 50 days Weight gain Ž%."SEM
Dietary lysine % diet 1.18 1.6 2.02 2.44 2.86 3.28
b
Survival Ž%.
FCRb
4.62 4.80 5.59 4.68 5.12 4.71
73 65 80 67 72 57
2.12 2.07 1.66 2.00 1.96 2.31
Mean square 412143.27 87745.01
F value 4.70
P)F 0.0035
% protein 2.95 4.0 5.05 6.1 7.15 8.2
Analysis of Õariance Source df Model 5 Error 26 Corrected total 31 R 2 s 0.474 a
SGRa
672"137 959"50 1581"142 902"61 1147"84 984"164
Sum of squares 2060716.37 2281370.38 4342086.75 CVs 28.95714
Root MSEs 296.218
Specific growth rates lnŽmean final wt.ylnŽmean initial wt.r50 days=100. Feed conversion rations g dry weight feedrg wet weight gain.
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Fig. 1. Ža. Growth response of P. monodon fed graded levels of lysine for 50 days. Žb. Growth response of P. monodon fed graded levels of arginine for 56 days.
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Table 6 Mean weight gain, specific growth rate ŽSGR., survival, and feed conversion ratio ŽFCR. of P. monodon fed diets with graded levels of arginine for 56 days Weight gain Ž%."SEM
Dietary arginine % diet 0.6 1.0 1.4 1.8 2.2 2.6 3.0
b
Survival Ž%.
FCRb
4.09 4.40 4.64 4.82 4.55 4.48 4.32
80 78 74 87 93 80 70
1.70 1.88 2.21 1.65 1.88 2.03 2.18
Mean square 48049.136 18531.765
F-value 2.59
P)F 0.0470
% protein 1.7 2.9 4.0 5.1 6.3 7.4 8.6
Analysis of Õariance Source df Model 6 Error 22 Corrected Total 28 R 2 s 0.414 a
SGRa
678"35 812"78 926"59 1017"25 881"64 846"53 778"69
Sum of squares 288294.814 407698.814 695993.638 CVs16.21824
Root MSEs136.131
Specific growth rates lnŽmean final wt.ylnŽmean initial wt.r56 days=100. Feed conversion ratios g dry weight feedrg wet weight gain.
Mean weight gain and specific growth rate of shrimp were improved by supplementation with arginine. Highest mean weight gain Ž1017%. was obtained for shrimp fed diet 4 Ž1.80% arginine. and lowest for Ž678%. in those fed the unsupplemented diet Ždiet 1 s 0.60% arginine.. Survival rate ranged from 70 to 93%. High survival and feed conversion efficiency were recorded in diet 4 which was near the requirement level. Based on the broken-line analysis of percent weight gain and dietary arginine levels, the arginine requirement of juvenile P. monodon is 1.85% of a 37.5% protein diet or 5.3% of protein ŽFig. 1a..
4. Discussion The dietary lysine requirement for P. monodon of 5.2% protein obtained in this study is considerably higher than the reported lysine requirement of another penaeid species, P. Õannamei, at 3.1% of protein ŽAkiyama, 1986.; however, Fox et al. Ž1992. reported closer requirement values P. Õannamei using covalent and crystalline lysine supplementation at 4.67% and 5.19% respectively. Requirement values among fish species also exhibit a wide variation. Fishes with requirement levels closer to that of P. monodon, when expressed in percent of dietary, are chinock salmom, 5.0% ŽHalver et al., 1958., gilthead sea bream, 5.0%, ŽLuquet and Sabaut, 1974., channel catfish, 5.0%, ŽRobinson et al., 1980., Nile tilapia, 5.1%, Santiago and Lovel, 1988. and Japanese eel, 5.3%, ŽNose, 1979.. The requirement obtained for arginine Ž5.3% of protein. agrees well with that reported for the same species, P. monodon, at 5.45% arginine ŽChen et al., 1992.. This
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value is similar to that of milkfish, 5.2%,ŽBorlongan and Coloso, 1993. and is generally higher than for some other fish species such as Nile tilapia, 4.2%, ŽSantiago and Lovel, 1988.; channel catfish, 4.3%, Robinson et al., 1981.; common carp, 4.3%, and Japanese eel, 4.5%, ŽNose, 1979.; but lower than that reported for chinook salmon, 5.75%, ŽKlein and Halver, 1970.. This observed variation is attributed to inherent differences among species, differences in levels and quality of proteins used in the test diets, and other dietary and environmental factors. In both studies, growth responses of shrimp improved with lysine and arginine-supplementation beyond those coming from intact proteins suggesting the ability of juvenile shrimp to utilize crystalline forms of lysine and arginine. There is a marked decline in growth rates beyond the optimum requirement levels for both lysine and arginine. The growth depression was also observed in studies on valine and methionine of P. monodon ŽMillamena et al., 1996a,b. and in a study by Recodo Ž1991. on histidine requirement of the tiger shrimp. The basis for growth-depression has not been fully established; however, these observations may suggest toxicity of excessive amino acid levels beyond that found in the shrimp tissue protein. Mertz Ž1972. suggested that high dietary amino acid levels can cause accumulation and toxicity in tissues. Further, Recodo Ž1991., through electron microscopy technique, noted extensive necrosis in the shrimp hepatopancreas when fed high levels of histidine. In this study, visual observations showed no gross pathological signs in P. monodon juveniles fed diets low or high in either of the test amino acids except for depressed growth rate. Lysine and arginine are found in high quantities in the muscle protein of penaeid shrimp and are considered as most limiting amino acids in most animals, particularly in P. monodon ŽAkiyama, 1986; Pascual, 1990.. These information on dietary lysine and arginine requirements, along with data obtained on requirements for other essential amino acids, will provide valuable knowledge in protein and amino acid nutrition of P. monodon and thus will lead to developing cost-effective diets for the tiger shrimp. Acknowledgements The authors wish to thank Mr. Vicente Balinas and Mr. Dolfus Miciano for their assistance in the statistical analysis and computer work and Ms. Mae Fernando for her assistance during the conduct of these studies. References Akiyama, D., 1986. The development of purified diet and nutritional requirement of lysine in penaeid shrimp. PhD dissertation. Texas A&M University, College Station, 78 pp. AOAC, 1984. Williams, S. ŽEd.., Official Methods of Analysis of the Association of Official Analytical Chemists, 14th edn. Arlington, VA, 114 pp. Borlongan, I.G., Coloso, R.M., 1993. Requirements of juvenile milkfish Chanos chanos for essential amino acids. J. Nutr. 123, 125–132. Chen, H.Y., Len, Y.T., Roelants, I., 1992. Quantification of arginine requirements of juvenile marine shrimp Penaeus monodon using microencapsulated arginine. Mar. Biol. 114, 229–233.
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Deshimaru, O., 1981. Protein and amino acid nutrition of the prawn Penaeus japonicus. In: Pruder, G.D., Langdon, C., Conklin, D. ŽEds.., Proceedings of the 2nd International Conference on Aquaculture Nutrition, Biochemical and Physiological Approaches to Shellfish Nutrition. Rehoboth Beach, DE, U.S.A., pp. 106–123. Deshimaru, O., Kuroki, K., 1974. Studies on a purified diet for prawn: III. A feeding experiment with amino acid test diets. Bull. Jpn. Soc. Sci. Fish. 41, 101–103. Fox, J., Lawrence, A.L., Li-Chan, E., 1992. Apparent lysine requirement of Penaeus Õannamei ŽBoone. using covalent and crystalline lysine supplementation. Abstracts of Aquaculture ’92. Marriott’s Orlando World Center, Orlando, FL, May 21–25, pp. 96–97. Gomez, K.A., Gomez, A.A., 1976. Statistical Procedures for Agricultural Research with Emphasis on Rice. The Inernational Rice Research Institute, Los Banos, ˜ Laguna, The Philippines, 294 pp. Halver, J.E., DeLong, D.C., Mertz, E.T., 1958. Threonine and lysine requirements of chinook salmon. Fed. Proc., Fed. Am. Soc. Exp. Biol. 17, 478. Kanazawa, A., 1981. Penaeid nutrition. In: Pruder, G.D., Langdon, C., Conklin, D. ŽEds.., Proceedings of the 2nd International Conference on Aquaculture Nutrition: Biochemical and Physiological Approaches to Shellfish Nutrition. Louisiana State University, Baton Rouge, LA, U.S.A., pp. 87–105. Klein, R.G., Halver, J.E., 1970. Nutrition of salmonid fishes: arginine and histidine requirements of chinook and coho salmon. J. Nutr. 100, 1105–1110. Luquet, P., Sabaut, J.J., 1974. Nutrition azotee et croissance chez la daurade et la truite. Actes des colloques. Colloques sur l’Aquacult. 1, 243–253. Mertz, E.T., 1972. The protein and amino acid needs. In: Halver, J.E. ŽEd.., Fish Nutrition. Academic Press, New York, pp. 105–143. Millamena, O.M., Bautista-Teruel, M.N., Kanazawa, A., 1996a. Valine requirement of postlarval tiger shrimp Penaeus monodon Fabricius. Aquacult. Nutr. 2 Ž3., 129–132. Millamena, O.M., Bautista-Teruel, M.N., Kanazawa, A., 1996b. Methionine requirement of juvenile tiger shrimp Penaeus monodon Fabricius. Aquaculture 143, 403–410. Millamena, O.M., Bautista, M.N. Reyes, O.S., Kanazawa, A., 1997. Threonine requirement of juvenile marine shrimp Penaeus monodon. Aquaculture Žin press.. Nose, T., 1979. Summary report on the requirements for essential amino acids for carp. In: Trews, K., Halner, J.E. ŽEds.., Finfish Nutrition and Fishfeed Technology. Heinemann, Berlin, Germany, pp. 145–156. Pascual, F.P., 1990. Terminal report submitted to SEAFDEC Aquaculture Department. Tigbauan, Iloilo, Philippines, 84 pp. Pascual, F.P., Kanazawa, A., 1986. Specific amino acid-free semi-purified diets for Penaeus monodon juveniles. Mem. Kagoshima Univ. Res. Center South Pacific 7, 65–72. Recodo, A.G., 1991. Histophysiological effects of various histidine levels in the diet on the hepatopancreas of Penaeus monodon Fabricius. Masters thesis. Institute of Biology, College of Science, U.P. Diliman, Quezon City, Philippines, 104 pp. Robbins, K.R., Norton, H.W., Baker, D.H., 1979. Estimation of nutrient requirements from growth data. J. Nutr. 109, 1710–1714. Robinson, E.H., Wilson, R.P., Poe, W.E., 1980. Re-evaluation of the lysine requirement and lysine utilization by fingerling channel catfish. J. Nutr. 110, 2313–2316. Robinson, E.H., Wilson, R.P., Poe, W.E., 1981. Arginine requirement and apparent absence of lysine–arginine antagonist in fingerling channel catfish. J. Nutr. 111, 46–52. Santiago, C.B., Lovel, R.T., 1988. Amino acid requirements of Nile tilapia. J. Nutr. 118, 1540–1546. Statistical Analysis Systems, 1992. Manual on statistical analysis of fisheries data using SAS. SEAFDEC Aquaculture Department and Division of Statistics and Statistical Laboratory, University of the Philippines at Los Banos, ˜ College, Laguna, 70 pp. Strickland, J.D.H., Parsons, T.R., 1972. Inorganic micronutrients in sea water. In: Stevenson, J. ŽEd.., A Practical Handbook of Seawater Analysis. Fish. Res. Board Can., Canada Dept. Fish Oceans, Ottawa, Canada. Teshima, S., Kanazawa, A., Yamashita, M., 1986. Dietary value of several proteins and supplemental amino acids for larvae of the prawn Penaeus japonicus. Aquacult. Amsterdam 51, 225–235. Zeitoun, I.H., Ulrey, D.E., Magee, W.T., Gill, J.L., Bergen, W.G., 1976. Quantifying nutrient requirements of fish. J. Fish. Res. Board Can. 33, 167–172.