Camp. Biochem.Physiol.Vol. 102A, No. 1, pp. 21 I-216, 1992

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YOUNG

DIETARY ARGININE REQUIREMENT RAINBOW TROUT (ONCORHYNCHUS C. YOUNG CHO,*~

SADASIVAM KAUSHIK~

and

OF MYKISS)

BILL WOODWARD*

*Department

of Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada NlG 2Wl. Telephone: 519-824-4120, ext. 3744; Fax: 519-763-5902; TFisheries Branch, Ontario Ministry of Natural Resources, Ontario, Canada; SLaboratoire de Nutrition des Poissons, I.N.R.A. 64310 Saint-PC-sur-Nivelle, France (Received 5 August 1991)

Abstract-l. Two growth trials were conducted with young rainbow trout (Oncorhynchus mykiss) to determine the dietary arginine requirement under conditions of rapid weight gain at 15°C. 2. The growth requirement does not exceed 4.2 g arginine/l6 g dietary nitrogen and, thus, is much lower than the value of 6.0 g arginine/l6 g dietary nitrogen presently listed by the NRC for Chinook salmon and widely applied to all Salmonids. 3. Comparison of the present results with the arginine requirement of the chick reveals remarkable similarity despite the phylogenetic distance between the Iwo species, and demonstrates the need to re-evaluate, as anomalously high, the presently-accepted value for Chinook salmon.

INTRODUCTION

Arginine (Arg) is an essential amino acid (EAA) for Salmonid fish (Halver and Shanks, 1960; Klein and Halver, 1970). The EAA levels in most animal proteins are not limiting for the purpose of fish diet formulation. Increasing use of plant proteins in fish diets, however, could lead to situations in which not only sulphur amino acids but also arginine and lysine might become limiting. Precise information regarding the dietary arginine requirement is therefore becoming important for formulation of practical fish diets. Presently available estimates of the arginine requirement of rainbow trout (Oncorhynchus mykiss) are widely divergent. A requirement level of 5.4-5.9% of dietary protein (5.4-5.9g Arg/l6g dietary N) was reported for maximal growth rate (Ketola, 1983). By contrast, Ogino (1980) reported a requirement of 3.5 g Arg/l6 g N on the basis of whole body arginine retention, and other authors (Kaushik, 1978; Kim et al., 1983; Chiu, 1984; Walton et al., 1986; Kaushik et al., 1988) have reported values ranging from 3.0 to 4.5 g Arg/l6 g N on the basis of a variety of biochemical criteria. Much of the disparity among published studies may be attributable to diverse diet formulations and culture conditions which have produced widely different feed intakes and growth rates. In particular, weight gains were extremely slow, despite consideration of water temperature, in the only available study providing an estimate of arginine requirement in the trout on the basis of growth parameters (Ketola, 1983). The primary objective of the present investigation, therefore, was to determine the dietary a&nine requirement of young rainbow trout according to growth parameters under conditions which permit rapid weight gain. In addition, some of the divergence in published results may be due to the use of numerous different assessment criteria varying in sensitivity to dietary arginine level. Consequently, a secondary objective of this investigation was to compare, under similar dietary and

environmental conditions, the usefulness of growth parameters and of post-prandial blood concentration parameters for determining the dietary arginine requirement of the trout. MATERIALS AND METHODS

Experimenral procedures Rearing system, basal diet, statistics and general experimental design. Two growth trials were conducted as well

as a study in which arginine requirement was determined on the basis of plasma concentration of the amino acid. In the growth experiments, fish were maintained in a flow-through system of 60 1tanks supplied with a mixture of well water and city water at a rate of 3 l/min. The water was continuously aerated, and temperature was controlled thermostatically at 15°C. Photoperiod was maintained on a 12 hr light: 12 hr dark schedule. The third experiment. conducted in SaintPte-sur-Nivelle, France, employed similar, but not identical conditions. Recycled city water was maintained at 16°C and supplied to 60 1 tanks at a flow rate of 2 l/min. Photoperiod in this study was 10 hr light: 14 hr darkness. The basal diet used throughout (designated “Experimental diet” in Table 1) was formulated to be limiting in arginine while being adequate, according to the NRC (1981), in all other EAA. This was achieved by setting the crude protein content of the diet at 30% with half of the protein supplied by skim milk and corn gluten meal and the other half by an amino acid premix devoid of arginine. All experimental diets contained 16.4 MJ digestible energy (DE) per kg and the dietary digestible protein (DP) to DE ratio was kept at 20 g/MJ. The apparent digestibilities of dietary energy and protein were calculated to be 81.9 and 97.9%, respectively, based on the assumption that the digestibility coefficients of nutrients in individual ingredients are additive (Cho et al., 1982; Cho and Kaushik, 1985). The level of arginine in the unsupplemented diet, by analysis, was 1.9 g/16 g N (0.55% by weight). Step-wise increases in arginine concentration were achieved by weight-for-weight replacement of L-glutamic acid by L-arginine-free base in the amino acid premix. Each amino acid premix was blended with dissolved agar (150 g agar in 2 kg water) before being mixed with other ingredients. This was intended to retard absorption of the free amino acids while digestion of the intact dietary proteins 211

C. YOUNG CHO et al.

212

occurred. All diets were pelleted with steam (Laboratory Pellet Mill, California Pellet Mill, San Francisco, CA) and stored at - 10°C until used. All experiments were conducted according to a completely randomized design. Differences among means were determined using Tukey’s honestly significant difference procedure (Steel and Torrie, 1960) at the 5% level of significance. kor each parameter measured, except plasma f& arginine level, values were pooled on a per tank basis. This was not necessary for analysis of plasma arginine data, however, because each fish was fed individually. Live weight gain data from the two growth trials were also analysed vs dietary arginine level according to the four parameter model of non-linear regression described elsewhere (Mercer, 1982; Mercer et al., 1986). Arginine requirement for maximal growth rate was estimated, according to this procedure, as the dietary concentration corresponding to the point on the curve at which the slope (determined from the first derivative) was reduced to 5% of maximum. For regression analysis, gain data were first converted to a parameter termed the daily growth coefficient (DGC) (Cho and Woodward, 1989) using the cube root of body weight relationship discussed by Iwama and Tautz (1981) and by Kleiber (1975): DGC = (FBW’:’ - IBW”‘) x ,oo number of days

Table I. Composition

Ingredient Skim milk? (33% CP) Corn eluten meal? (60% Agar,‘bacteriolog$§ L-Arginine free baseI/ Casein, vitamin-free11 Gelatin11 o-Glucose (cerelose)T Dextrin, white11 Starch, raw, corn** Cellulose (Celufil)jl Amino acid premixtt Vitamin premixtf Mineral premix8 Fish oil, marine11II

of basal experimental reference diets’ Experimental (g/kg)

diet and purified Reference diets (g/kg)

250 I50 IO +

150 30 170(l) 30 60 150

300 30 40 70 250 30 20 (2) 30 80 150

400 40 50 90 110 :8 (3) 30 80 150

*Proximate analysis (% as-fed basis; basal, 30% protein and 40% nrotein reference diets. resoectivelv): drv matter. 92.2. 91.6. 89.6: crude protein (%N x 6.25;, 33.3,32.7,*38.3; ether extract; 15.5; 15.5, 14.8; crude fiber, 2.4, 2.6, 2.4; ash, 6.5, 6.6, 5.9; gross energy, 20. I, 19.8,20.7 kJ/g. tGay Lea Foods Co-operative, Guelph, Ontario. fMartin Feed Mills, Elmira, Ontario. @p&rum Diagnostics, Glenwood, IL. I\U.S. Biochemicals, Cleveland, OH. 7Corn Products, Englewood Cliffs, NJ. **St Lawrence Starch Co., Mississauga, Ontario. tt(l) g L-form/kg: met 7; cys 3; his 4; ile 2; lea 2; lys 15; phe 3; tyr 2; thr 3; trp 2; val4; asp IO; glu 33; gly 10; pro 25; ser 7; sc-cellulose 868. (2) g/kg: DL-met 4; L-arg I I; o!-cellulose 5. (3) g/kg: DL-met 5; L-arg 13; ol-cellulose 2. ff(lU/kg): retinyl acetate 7000; cholecalciferol 3000; all-rac-cc-tocopheryl acetate 200. (mg/kg): menadione sodium bisulfite 50; thiamine~HCI40; riboflavin 60, o-Ca-pantothenate 200; biotin 0.5; folic acid 20; cyanocobalamin 0.2; niacin 300; pyridoxine.HCI 40; myo-inositol 500; ascorbic acid 500; choline dihydrogen citrate II ,000; ~-cellulose. @(mg/kg): dibasic calcium phosphate (23% Ca, 18% P) 30,000; calcium carbonate (40% Ca) 3000; NaCl 15,000; potassium sulfate (45% K) 20,OW; magnesium sulfate (20% Mg) 10,000; ferrous sulfate (21% Fe) 700; manganous sulfate (33% Mn) 300; zinc sulfate (36% Zn) 550; cupric sulfate (25% Co) 160; cobalt chloride (25% Co) 26; potassium iodide (76% I) 15; sodium selenite (42% Se) 2.5; a-cellulose. //IIDeaerated with nitrogen and stabilized with 0.05% ethoxyquin.

where FBW and IBW are final and initial live weights, respectively. DGC is little affected by differences in initial body weight (Cho, 1990) so that, using this parameter, data from the two growth trials of the present study could be pooled for regression analysis. Each growth trial was conducted over a period of 12 weeks, and fish were fed near satiety three times daily. The animals were weighed every 4 weeks and food intake was recorded biweekly. Daily mortality and behavioural records were kept. Experiment 1. Rainbow trout fry (Ontario Ministry of Natural Resources, Normandale Hatchery) weighing 2.5 g each, were randomly distributed among 24 tanks. Each tank initially contained 80 fish. Six diets were formulated from the basal (Table 1) so as to contain 1.9, 5.2, 6.6, 8.0, 9.2 or 10.6e Arei16e N. i.e. from 0.55 to 3.35% of the diet bv weight. In addition, two purified reference diets containing 30 and 40% crude protein, respectively, were formulated (Table 1) as positive controls. Each diet was fed to three tanks of fish. Experiment 2. A second growth trial was conducted with rainbow trout fry (Ontario Ministry of Natural Resources, Normandale Hatchery) initially weighing 1.3 g each. The fish were randomly distributed, 100 per tank, among 21 tanks. Seven diets were formulated from the basal (Table 1) so as to contain 1.9, 2.6, 3.2, 4.2, 5.2, 6.4 or 8.3 g Arg/l6 g N, i.e. from 0.55 to 2.75% of the diet by weight. Each diet was fed to three tanks of fish. Experiment 3. This experiment was conducted on the basis of previous work (Kaushik, 1978) which demonstrated the responsiveness of the blood free arginine concentration of the rainbow trout to intake of a single meal. Rainbow trout (INRA, Donzacq Experimental Station) initially weighing IOOg each, were randomly distributed between two tanks, 20 animals per tank. The fish were fed a commercial diet (Aqualim, France; 42% crude protein, 10% ether extract) for 3 weeks and were then fasted for 3 days. Fish were then given a single oral administration, by stomach tube, of one of seven experimental diets containing I .9, 3.3, 5.2,6.6, 8.0,9.2 or 10.6 g Arg/l6 g N. Each diet was given to three fish at an intake level of 1% of body weight, and fish were anaesthetized with phenoxy-ethanol monophenyl ether (4 ml in 10 1 of water) during the procedure. Blood samples were taken 6 hr later. Analytical procedures. Analyses of dry matter, crude protein, crude fiber, ether extract, ash and gross energy were carried out on samples from each diet according to the methods of the AOAC (1970). Dietary arginine levels in Experiments 1 and 2 were confirmed by HPLC analysis (following hydrolysis in 6 N HCI) in the Centre de Recherches en Nutrition, University of Laval, PQ, Canada. Carcass nitrogen and lipid concentrations were analysed by methods described elsewhere (AOAC, 1970; Bligh and Dyer, 1959, respectively). In each growth trial, an initial pooled sample of 30 fish was analysed, whereas final samples consisted of five fish pooled per tank. Daily retained nitrogen and lipid was determined per unit of “average live weight” calculated as the mean of initial and final live weights. Serum urea concentration (Experiment 1) was determined by autoanalyser (American Monitor Parallel) according to a calorimetric method (Marsh et al., 1965). Blood was collected by severing the caudal fin from fish anaesthetized with tricaine methanesulfonate, and serum was stored at -20” for up to 3 days before analysis. A preliminary experiment was conducted to determine the time pattern of post-prandial changes in serum urea levels. One tank of fish fed the 40% digestible orotein reference diet (Table 1) was used for this pu-&se at-the end of the growth study. Six fish were withdrawn for sampling 1,2,3,4,6, 8, 12, 14 and 24 hr after the first morning meal. Peak urea level was found 6 hr post-prandial. Consequently, all other blood samples for urea measurement were taken 6 hr after the first morning I

_I

213

Arginine requirement of rainbow trout Table 2.

Final live weights, cumulative

Dietary Arg level (g/l6gN)

Final live weightt (g/fish)

weight gains and feed Weight gaint (% initial weight)

1.9 5.2 6.6 8.0 9.2 10.6 33% DPf 40% DPf SEM§

22.ga 33.0b 35.2bs 35.oh 35.lk 35.lk 37.4d 40.0d 0.828

843’ 1220b 1335k I 324b5 1339h 1329” 1420cd 1513d 33.2

1.9 2.6 3.2 4.2 5.2 6.4 8.3 SEMG

18.2a 20.2”b 21.4b 25.3’ 24.3’ 24.6’ 24.9’ 0.439

1293” 142gPb 1523” 1695cd 1179d 1812d 1829” 38.3

conversion efficiencies,and daily nitrogen and lipid retentions and growth coefficients*

Experiment 0.7w 0.84b 0.87b 0.86b 0.92b 0.87b 0.90b I .03’ 0.021 Experiment 0.64’ 0.71b 0.75” 0.7v 0.8od’ 0.81dc 0.82’ 0.009

Daily lipid retention (g lipid/kg/day)

Daily N retention

Daily growth coefficient (g”‘/day)

Feed efficiencyt (g gain/g f-d)

(g N/kg/day)

1 1.78’ 2.20b 2.29” 2.29bc 2.29bc 2.29” 2.3p 2.46d 0.03 1

0.370 0.43b 0.43b 0.44* 0.44k 0.43b 0.47d 0.49d 0.0061

2.99 2.46’ 2.60b 2.46b 2.6Ob 2.5gb 2.45’ 2.57b 0.067

1.83’

0.46’ 0.4gPb 0.49b 0.535 0.53c 0.53c 0.54c 0.004

3.578 3.47Pb 3.36b 3.04c 2.91’ 3.03c 2.98’ 0.030

2

1.93Pb 2.OOb 2.llC 2.15’ 2.17’ 2.18’ 0.022

*Mean values. Within the same column and experiment, values not sharing a common superscript honestly significant difference procedure. tAfter 12 weeks. Initial live weight of 2.5 and 1.3 g/fish in Experiments I and 2, respectively. fReference diets containing either 30 or 40% digestible protein (DP). $Pooled standard error of mean, df = 16 (Experiment 1) or df = 15 (Experiment 2).

letter differ (P < 0.05) according

meal (designated “post-prandial”). Three fish were sampled per tank and time period. Plasma free arginine concentration was determined by means of a Technicon autoanalyser as described by Bacchus and London (1971). Blood was withdrawn into heparinized syringes from the caudal vein of fish under phenoxy-ethanol monophenyl ether anaesthesia. Heparin (sodium salt) was used as anticoagulant and plasma was recovered after centrifugation at 3000 g for 20 min. Plasma was stored at -2O”, and was analysed within 48 hr after withdrawal.

a

R

a

to Tukey’s

R

“,



“II

b = -0.26

R,,,

= 2.51

krJ5 = 0.66 n= 1.02

go.ou

RESULTS

1

Serum urea level (mM)

1.9

5.2

1.2a

I .6b

I

I

I

I

I

I

!

I

4

5

6

7

6

9

10

11

Dletary Arginine Level (g/16 g N)

Fig. 1. Daily growth coefficient as a function of dietary arginine level (reference diets excluded). Curve combines results from Experiment 1 (0) and Experiment 2 (X), and was derived using the four parameter model of non-linear regression (Mercer, 1982; Mercer et al., 1986). Dietary arginine requirement is indicated by the vertical dotted line, and corresponds to the arginine level at the point at which the maximum slope of the curve is reduced by 95%. The mathematical formula for the curve is as follows: Y = WG,)” + R,,(W (&,)“+(W where Y is daily growth coefficient, X is dietary arginine level, b is the intercept on the Y-axis, F&axis the maximal value of Y, K,,, is the value of X for gR_ + b), and n is the apparent kinetic order.

The 6 hr post-prandial serum urea concentrations (Experiment 1) are shown in Table 3. The values

serum urea concentrations*

Dietary Arg level (g/ 16 g N) 6.6 8.0 I .6b

I 3

-0.5

Growth parameters from Experiments 1 and 2 are shown in Table 2. In the two experimental diet series, final live weight, cumulative weight gain, daily growth coefficient and daily nitrogen and lipid retention varied with dietary arginine content up to the level of 5.2 g/l6 g N (Experiment 1) and 4.2 g/16 g N (Experiment 2). Feed conversion efficiency was maximized by 5.2 g Arg/l6 g N in both growth trials. Regression of daily growth coefficients vs dietary arginine level is presented in Fig. 1. This analysis indicated a requirement of 3.0 g Arg/l6 g N. The fish fed supplemented test diets exhibited good growth rates as underscored by direct comparison, in Experiment 1, to fish fed the reference diets. Apart from low food intake which was apparent by 4 or 8 weeks (Experiments 1 and 2, respectively, results not shown) fish fed the unsupplemented experimental diet (1.9 g Arg/l6 g N) displayed no outward signs or symptoms of deficiency. Table 3. Post-prandial

I 2

l.gb

*Mean values of serum levels 6 hr after first morning meal (Experiment (P < 0.05) by Tukey’s honestly significant difference procedure. tReference diets containing 30 or 40% digestible protein (DP). fPooled standard error of mean, df = 16.

9.2

10.6

I .gb

1.9b

1). Values not sharing

Reference dietst 30% DP 40% DP

a common

I .6b superscript

1.6b

SEMI 0.075

letter are different

214

C. YOUNGCHO et al. Table 4. Post-prandial plasma free @nine

Plasma Arg level (mM)

1.9

3.3

0.45”

0.44’

concentrations*

Dietary Arg level (g/l 6 g N) 5.2 6.6 8.0 0.45”

0.6Oab

0.611b

9.2

10.6

SEMt

0.65”

0.71b

0.036

*Mean values of plasma levels 6 hr after a single meal (I % live weight) in fish previously fasted 3 days (Experiment 3). Values not sharing a common superscript letter are different (P -c 0.05) by Tukey’s honestly significant difference procedure. tPooled standard error of mean, df = 14.

measured were maximized at and above the dietary arginine level of 5.2 g/16g N. Fasting serum urea levels (means + SD: 1.1 f 0.2 mM, N = 24) did not differ among the eight groups of this experiment (results not shown for individual dietary groups). Finally, 6 hr post-prandial free arginine concentration increased between dietary arginine levels of 5.2 and 9.2 g/16 g N (Table 4).

DISCUSSION

An unambiguous estimate of the dietary arginine requirement according to growth parameters has not been available, prior to this investigation, for young rainbow trout. The previous growth study (Ketola, 1983) is difficult to interpret because of low rates of weight gain. This is readily apparent on the basis of the daily growth coefficient, a criterion which permits comparison of growth results independently of factors such as fish size, water temperature and duration of feeding period (Cho, 1990). By contrast, the present results demonstrate that the dietary arginine requirement for maximal weight gain and N retention by the rainbow trout does not exceed 4.2 g/l6 g N (Table 2; Experiment 2) when this species is in rapid, exponential growth. The requirement predicted using regression analysis (Fig. 1) of the theoretical growth response curve was 3 g Arg/l6 g N based on a reduction of the maximum slope of 95%. Regardless of the growth criterion or the method of statistical analysis, therefore, the requirement derived from this work is substantially lower than the estimate from the growth trial of Ketola (1983) as well as the value of 6.0 g Arg/l6 g N which is listed for Chinook salmon (Oncorhynchus tshawytscha) by the NRC (1981) and generally applied to all salmonid fish. The critical factor in the present investigation was the formulation of an experimental test diet which had a low basal level of arginine (1.9 g/l6 g N) but which, if supplemented with arginine, could support good growth rates at the standard environmental temperature, 15”. According to the results of Experiment 1, the supplemented experimental diet permitted weight and nitrogen gains which exceeded 85% of those achieved with the reference diet containing 40% crude (intact) protein. This is important insofar as the crude protein level of the experimental diets (33%) was low relative to the presently recommended level (40%) for trout diets (NRC, 1981). The calculated DP level of our experimental diets (32%), however, was comparable to that of a commercial diet (35%) formulated by the Ontario Ministry of Natural Resources (Cho, 1986). Two particularly important considerations in formulating the experimental diet for this work were the avoidance of plant proteins such as zein and the

provision of amino acids, in part as intact proteins and in part as a mixture of crystalline amino acids in agar. Zein-based diets have been used in previous studies of the arginine requirement of rainbow trout (Kaushik, 1978; Ketola, 1983; Walton et al., 1986), but give rise to poor feed intakes and growth rates, even in juvenile trout. Similarly, a mixture of free amino acids as either the sole source or a major source of dietary nitrogen generally fails to promote weight gains in rainbow trout comparable to those achieved with diets supplying amino acids mainly as intact proteins (Kaushik, 1978; Walton et al., 1986). This is presumably because of differing rates of intestinal absorption among the various amino acids (Murai et al., 1987). In the present investigations, however, an experimental diet in which crystalline amino acids supplied 50% of dietary nitrogen nevertheless supported weight gains of a similar magnitude to those obtained with a reference diet based on intact proteins. The agar coating of the amino acid premix may have overcome major differences in absorption rate among the amino acids included in free form within the experimental diets. A number of additional dietary factors may influence the arginine requirement, and merit attention in estimates of this value. In the first place, antagonism between lysine and arginine is widely recognized in rats and chicks (Austic and Calvert, 1981) and is reported, also, in rainbow trout (Kaushik and Fauconneau, 1984). The lysine concentration of the experimental diets in the present investigation exceeded the requirement listed by the NRC (1981) for rainbow trout. If the NRC value is excessive for this species, then an overestimate of arginine requirement could have resulted in the present study. Secondly, dietary electrolytes affect basic amino acid metabolism in poultry and swine (Austic and Calvert, 1981). No clear influence of electrolytes on arginine requirement of rainbow trout was apparent, however, over a range of dietary electrolyte contents (Na + K - Cl) from -200 to +200 meq/kg (Chiu, 1984). The experimental diets of the present study contained, by calculation, +220 meq/kg so that a major impact of electrolytes on the conclusions from this work should not be anticipated. Finally, upward adjustment of pH can be important in promoting good growth rates and feed conversion efficiencies in at least some fish species fed diets containing large quantities of crystalline amino acids (Murai et al., 1983). Good food intakes and growth rates, however, were obtained in the present studies without attention being given to dietary pH, so that this factor may also be of little significance to the interpretation of the results of this investigation. The present results are consistent with the viewpoint expressed elsewhere (Baker, 1983) that amino acid requirements I’. . are generally best defined in

Arginine requirement of rainbow trout growing animals by growth data”. Perhaps the most meaningful single criterion of assessment used in this work was daily nitrogen retention, although final live weight, cumulative weight gain, daily growth coefficient and daily lipid retention each appeared equally sensitive for the purpose of determining the dietary arginine requirement (Table 2). The parameter of post-prandial serum urea concentration provided results which were supportive of the conclusion that the arginine requirement listed for Chinook salmon by the NRC (198 1) is an overestimate for rainbow trout. Others have reported that increasing increments of dietary arginine resulted in an increased rate of urinary urea excretion (Kaushik and Fauconneau, 1984). In the latter study, plasma urea level was not affected by dietary arginine level, but the present results show the importance of postprandial timing in the use of this parameter. Responsiveness of urea production to excesses of dietary arginine in rainbow trout is predictable on the basis of the high level of arginase activity in this species (Portugal and Aksnese, 1983). The rate of urea synthesis or excretion might, therefore, be as sensitive as growth parameters in the measurement of dietary arginine requirement. In teleost fish, however, any such measurements must be made against a high baseline level of urea synthesis from purines (Walton and Cowey, 1982). Moreover, measurement of urea excretion requires invasive procedures in fish. Post-prandial plasma free arginine concentration proved to be insensitive relative to growth parameters for the purpose of this work. According to this criterion, the arginine requirement exceeds 5.2 g/16 g dietary N. As for the serum urea concentration, the transient nature of blood free arginine concentration renders this parameter of limited value in studies of dietary requirement. Measurement of arginine oxidation appears to be superior to measurement of tissue free arginine level for assessing the arginine requirement of the trout (Walton et al., 1986; Kaushik et al., 1988). This method produced an estimate of 4.0-4.5 g/16 g N and, thus, may be comparable to growth parameters in sensitivity to dietary arginine level. The dietary arginine requirement of young rainbow trout does not exceed that of another non-ureotelic animal, the broiler chicken. Expressed in several different ways, the growth requirements of the chick between three and six weeks of age (NRC, 1984) and of the trout (this study) are, respectively, as follows: 6.0 vs 4.2 g Arg/l6 g dietary N, 0.86 vs 0.84 g Arg/MJ DE, and 1.2 vs 1.4% of the diet. Each method of expressing requirement possesses unique merits either scientifically or from a practical standpoint. It is interesting to note, however, that two phylogenetically distant, non-ureotelic species exhibit similar dietary arginine requirements despite their divergent dietary protein needs, namely 20% of the diet by weight in the case of the chick (NRC, 1984) and 40% in the case of the trout (NRC, 1981). The apparent difference in requirement expressed as g Arg/l6 g N is artificial in that it reflects the differing dietary protein needs of these two species. From this perspective, the very high requirement of 6.0 g Arg/l6 g N (2.4% of the diet by weight) presently accepted for Chinook salmon (NRC, 1981), and generally applied to all

215

Salmonid fish, is unlikely to be attributable differences and requires re-evaluation.

to species

SUMMARY

1. Two growth trials were conducted with rainbow trout fry (0ncorhynchu.r mykiss), initially 1.3 or 2.5 g/ fish, in order to determine the dietary arginine requirement of this species under conditions permitting rapid weight gain. Each experiment was 12 weeks in duration, and was conducted at a water temperature of 15°C. 2. Skim milk, corn gluten meal and crystalline amino acids served as nitrogen sources for the test diets which were formulated to contain 30% crude protein. One half of the nitrogen of each test diet was supplied by free amino acids coated with agar. Two positive control diets were formulated with a mixture of casein and gelatin to contain 30 and 40% crude protein, respectively, as intact proteins. All diets contained 15% lipid supplied by fish oil and 16.4 MJ digestible energy per kg. 3. Weight gain as well as nitrogen and lipid retention of fish fed the test diets were maximized by an arginine concentration of 4.2 g/l6 g dietary N. Two biochemical parameters related to the dietary arginine requirement, namely post-prandial serum concentrations of free arginine and of urea, provided no insight beyond that achieved through the use of growth parameters 4. The dietary arginine requirement of young, rapidly growing rainbow trout is much lower than the value of 6.0 g/16 g dietary N which is presently listed by the NRC for Chinook salmon and widely applied to all Salmonid fish. Comparison of the present results with the arginine requirement of the chick, another non-ureotelic species, revealed remarkable similarity between this animal and the trout despite their phylogenetic distance, and suggested a need to re-evaluate the presently accepted value for Chinook salmon as anomalously high. Acknowledgemenrs-The authors gratefully acknowledge the skilled technical assistance of Mr Martin Hodgson

and Mr Jim Elliott (diet mixing, Pelleting and feeding), Mrs Vera Peacock and Mrs Paulette Peyrotte (diet and carcass analyses), Mr Evert Grift, A.R.T. (urea determinations) and Mrs Denise Blanc (blood free arginine measurements). This work was supported by the Ontario Ministry of Natural Resources and the Ontario Ministry of Agriculture and Food.

REFERENCES

Association of Official Analytical Chemists (1970) 0giciar Metho& of Analysis (Edited by Horwitz W., Chichilo P. and Reynolds H.), lith edition. Washington, DC. Austic R. E. and Calvert C. C. (1981) Nutritional inter. relationships of electrolytes and amino acids. Fed. Proc. 40, 63-67. Bacchus R. A. and London D. R. (1971) The measurement of arginine in plasma. Clin. Chik A& 33, 479-482. Baker D. H. (1983) Problems and uitfalls in animal experiments designed to establish dietary requirements for essential nutrients. J. Nutr. 116, 2339-2349. Bligh E. G. and Dyer W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917. I

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Dietary arginine requirement of young rainbow trout (Oncorhynchus mykiss).

1. Two growth trials were conducted with young rainbow trout (Oncorhynchus mykiss) to determine the dietary arginine requirement under conditions of r...
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