Arginine and threonine requirements of milkfish (Chanos chanos Forsskal) juveniles

.4quaculture, 93 ( 1991) 3 13-322 Elsevier Science Publishers B.V., Amsterdam

313

Arginine and threonine requirements of milkfish (Chanos chanos Forsskal) juveniles I.G. Borlongan Aquaculture Department, Southeast Asian Fisheries Development Center, P.O. Box 256, Iloilo City3 Philippines (Accepted 27 July 1990)

ABSTRACT Borlongan, I.G., 199 1. Arginine and threonine juveniles. Aquaculture, 93: 3 13-322.

requirements

of milkfish ( Chanos chanos Forsskal I

Growth studies were conducted with milkfish (Chanos chanos Forsskal) juveniles to determine the quantitative requirements for arginine and threonine. The amino-acid test diets ( 40°Yacrude protein I contained casein and gelatin supplemented with crystalline L-amino acids to provide an amino-acid profile similar to milktish protein except for the test amino acid. Each set of experimental diets consisted of six isonitrogenous and isocaloric diets containing graded levels of the essential amino acid to be tested. Break-points in the growth curves which represent the optimum dietary concentration of arginine and threonine for fish growth were determined by the broken-line regression method. Based on dry diet, the requirement of milkfish juveniles for arginine is 2.1 OI and for threonine, 1.80%. These values correspond to 5.25% arginine and 4.50% threonine when expressed as a percentage of dietary protein.

INTRODUCTION

The economic success of the controlled production of milklish depends mainly on the cost of feeds and particularly on that of proteins, as protein is the major determinant for growth and is the most expensive component of artificial diets. Information on the protein and amino-acid requirements is essential in the formulation of nutritionally adequate and low-cost artificial diets. More accurate diet formulation and the screening of potential dietary protein sources would be facilitated by knowledge of the quantitative essential amino-acid requirements of this species. So far, the complete quantitative amino-acid requirements have been established for only five finfish species, namely: chinook salmon, common carp, Japanese eel, Nile tilapia and channel catfish and significant differences between species have been found to exist (Tacon and Cowey, 1985; Wilson and Halver, 1986; Wilson, 1989 ) . For milklish, only the quantitative requirements for tryptophan (Coloso et 0044-8486/91/$03.50

0 199 1 -

Elsevier Science Publishers

B.V.

314

LG. BORLONGAN

al., 1986), lysine (Borlongan and Benitez, 1990) and methionine plus cystine (Sastrillo, 1990) have been determined. The objective of this study was to determine the quantitative requirements for arginine and threonine for growth of juvenile milkfish.

MATERIALS

AND METHODS

Experimental diets The experimental diets with graded levels of arginine and threonine were formulated as shown in Table 1 and Table 2, respectively. The range of levels was such that the lowest and highest levels were below and above the anticipated requirements, based on the essential amino-acid profile of milkfish protein. These assumptions were based on the observation of Cowey and Tacon ( 1983), and Wilson and Poe ( i985) that there is a close similarity or direct correlation between dietary EAA requirement of fishes and the EAA profile of fish muscle/carcass. The experimental diets contained vitamin-free casein and gelatin as natural protein sources. Crystalline L-amino acids were added TABLE 1 Composition

(g/kg dry diet) of the experimental Diet 1

Casein Gelatin EAA mix’ Non-EAA mix’ Arginine Dextrin Cod liver oil Vitamin mix3 Mineral mix3 DL-cu-tocopherol acetate BHA Carboxymethylcellulose Celutil Total Total arginine Computed I crude protein g Arginine/ 100 g protein Crude protein (Ohanalyzed)

diets for the arginine requirement

Diet 2

Diet 3

Diet 4

study

Diet 5

Diet 6

300.00 100.00 24.50 27.40 0 250.00 100.00 30.00 30.00 0.10 2.50 50.00 85.50

300.00 100.00 24.50 25.01 2.39 250.00 100.00 30.00 30.00 0.10 2.50 50.00 85.50

300.00 100.00 24.50 23.04 4.39 250.00 100.00 30.00 30.00 0.10 2.50 50.00 85.50

300.00 100.00 24.50 21.01 6.39 250.00 100.00 30.00 30.00 0.10 2.50 50.00 85.50

300.00 100.00 24.50 19.01 8.39 250.00 100.00 30.00 30.00 0.10 2.50 50.00 85.50

300.00 100.00 24.50 17.01 10.39 250.00 100.00 30.00 30.00 0.10 2.50 50.00 85.50

1000.0 14.61 40.0 3.65

1000.0 17.00 40.0 4.25

1000.0 19.00 40.0 4.75

1000.0 21.00 40.0 5.25

1000.0 23.00 40.0 5.75

1000.0 25.00 40.0 6.25

40.38

40.21

40.32

40.46

40.26

40.42

‘EAA mix (g/kg dry diet): Be, 2.70; Leu, 4.96; Lys, 7.95; Met, 1.63; Thr, 4.31; Trp, 2.985. ‘Non-EAA mix (g/kg dry diet): Asp, 17.42; Cys, 8.00; Tyr, 1.98. 3Vitamin and mineral mixes: Borlongan and Benitez ( 1990).

ARGININE

AND THREONINE

REQUIREMENTS

OF MILKFISH

315

JUVENILES

TABLE 2 Composition

(g/kg dry diet) of experimental

Ingredients Casein Gelatin EA.4 mix’ Non-EAA mix’ Threonine Dextrin Cod liver oil Vitamin mix3 Mineral mix3 DL-cr-tocopherol acetate BHA Carboxymethylcellulose Celufil Total Total threonine Computed % crude protein g Threonine/ 100 g protein Crude protein (% analyzed)

Diet 1

diets for the threonine requirement Diet 2

Diet 3

Diet 4

study Diet 5

Diet 6

200.00 200.00 43.50 16.40 0 250.00 100.00 30.00 30.00 0.10 2.50 50.00 71.50

200.00 200.00 43.50 13.75 2.65 250.00 100.00 30.00 30.00 0.10 2.50 50.00 77.50

200.00 200.00 43.50 11.75 4.65 250.00 100.00 30.00 30.00 0.10 2.50 50.00 77.50

200.00 200.00 43.50 9.75 6.65 250.00 100.00 30.00 30.00 0.10 2.50 50.00 77.50

200.00 200.00 43.50 7.75 8.65 250.00 l00.00 30.00 30.00 0.10 2.50 50.00 77.50

200.00 200.00 43.50 5.75 10.65 250.00 l00.00 30.00 30.00 0.10 2.50 50.00 77.50

1000.0 11.35 40.0 2.84

1000.0 14.00 40.0 3.50

1000.0 16.00 40.0 4.00

1000.0 18.00 40.0 4.50

1000.0 20.00 40.0 5.00

1000.0 22.00 40.0 5.50

40.18

40.37

40.41

40.34

40.36

40.28

‘EAA mix (g/kgdry diet): Arg, 5.72; Be, 5.66; Leu, 9.69: Lys. 10.87; Met. 3.19: Phe, 2.76: Trp, 3.38; Val. 2.26. ‘Non-EAA mix (g/kg dry diet): Asp, 7.36; Cys, 4.18; Ser, 0.55: Tyr, 4.34. ‘Vitamin and mineral mixes: Borlongan and Benitez ( 1990).

to the natural proteins to simulate the reference amino-acid profile of milkfish tissue protein except for the amino acid under investigation. The diets were made isonitrogenous by decreasing the non-essential amino acids as the level of arginine and threonine increased. Other dietary components were cod liver oil, which served as source of essential fatty acids and non-protein energy; dextrin as carbohydrate source, carboxymethylcellulose as binder; vitamin and mineral premixes, BHA (butylated hydroxyanisole ), DL-cr-tocopherol acetate as anti-oxidant and celufil, a non-nutritive tiller. The diets were prepared using a Hobart mixer. All dry ingredients except the CMC (carboxymethylcellulose) were mixed until homogeneous. DL-atocopherol and BHA were dissolved in cod liver oil, then the oil mix and the dry ingredient mix were blended together. The pH of the experimental diets was determined on the mixture obtained by homogenizing a 10-g portion of the dry ingredient mixture with 100 ml of distilled water. After the pH was determined, a measured amount of 6 N NaOH was blended into the mixture

316

I.G. BORLONGAN

to establish a pH level of 7-8. The carboxymethylcellulose was gelatinized at 80°C in 450 ml water and was added to the pH-adjusted mixture. The semimoist mixture that resulted after thorough mixing was passed through a Hobart food grinder to form 2-mm diameter pellets. The pellets were dried in an air-convector oven at a temperature of 40’ C, ground, sieved to uniform sizes and stored at 4 oC until used. Experimental design and feeding Experimental fish underwent a 2-week conditioning period before the start of each experiment, during which they were fed a (40% CP) purified diet and reared under standardized environmental conditions. All experiments were conducted in 60-l flow-through fiberglass tanks with a flow rate of approximately 0.5 l/min. Airstones provided supplemental aeration and supplemental incandescent lighting provided a diurnal light : dark cycle of 12 : 12 h. At the start of the experimental period, milkfish fingerlings were sorted into groups of 20 fish/tank for the arginine study and 15 fish/tank for the threonine study. The initial mean weights of the milkfish were 0.70?0.04 and I .29 -+0.13, respectively for the arginine and threonine studies. Each of the experimental diets was fed to four replicate groups of fish in a completely randomized design. The fish were fed as much as they would consume three times a day at 8.00, 12.00 and 15.30 h for 12 weeks. All the fish were counted and weighed every 3 weeks. Tanks were cleaned daily by siphoning excess feed and fecal matter which had accumulated on the bottom. The tanks were scrubbed and thoroughly cleaned when the fish were removed during weighings. Water temperature and salinity were monitored daily, while pH, ammonia-N, nitrite-N, phosphate-P and dissolved oxygen were measured twice weekly. Water temperature and salinity ranged from 28 to 30°C and 27 to 32 ppt, respectively. Measurements of the other water parameters indicated that they were within favorable limits and not stressful to the fish. Chemical analyses At the termination of the experiments, 100 g of randomly selected fish were taken from each treatment for body composition analysis. Proximate analysis of casein, gelatin, all experimental diets and carcasses were conducted according to the standard Association of Official Analytical Chemists ( AOAC, 1980) methods. Amino-acid analyses on random samples of casein and gelatin were conducted as previously described by Borlongan and Benitez ( 1990). Statistical methods All data on growth, feed conversion and survival rates were subjected to one-way analysis of variance (Steel and Torrie, 1960) and Duncan’s multiple-range test (DMRT) to determine the significant differences among treat-

.ARGININE

AND

THREONINE

REQUIREMENTS

OF MILKFISH

317

JUVENILES

ments (Duncan, 1955 ). The data on percentage weight gains were analyzed to determine whether the regressions were linear, quadratic or cubic with increasing dietary amino-acid level. The broken-line regression model (Zeitoun et al., 1976; Robbins et al., 1979) was used to determine the break-points in the growth curves which represented the optimum dietary concentration of the amino acids for fish growth. This procedure assumes a linear relation between weight gain and dietary level of amino acid at or below the requirement and when the requirement is met the weight gains abruptly plateau. RESULTS AND DISCUSSION

Arginine requirement Weight gains, specific growth rates, food conversion ratios and survival rates of milkfish given the diets with graded arginine levels are shown in Table 3. The fish given diets 1 and 2 grew less well and had lower survival rates than the fish given the other four diets. When the weight gains were plotted against levels of arginine in the diet (Fig. 1) a break-point occurred at approximately 2.10% of the dry diet and this was taken as the dietary requirement level. This requirement, based on weight gain, corresponds also with the dietary level which gave the maximal food conversion ratio and highest survival rate. This value corresponds to 5.25% of the dietary protein, which is near the requirements reported for gilthead bream, 5.0% (Luquet and Sabaut, 1974); coho salmon, 5.8% and chinook salmon, 6.0% (Klein and Halver, 1970). However, the requirement figure found here is much higher than that reported for other species, such as 4.3% for common carp (Nose, 1979); 4.3% for channel TABLE 3 Growth. feed conversion and survival of milkfish given diets containing graded levels of arginine for I2 weeks’ Dietary arginine g/lOOg dry diet

g/lOOg protein

1.46 1.70 1.90 2.10 2.30 2.50

3.65 4.25 4.75 5.25 5.75 6.25

Mean initial weight (g)ts.e.m.

Mean final weight (g)is.e.m.

Mean weight gain (%)+_s.e.m.

SGR’ (%/day)

FCR’ (g/g)

Survival (O/o)

0.69 k 0.02 0.71 kO.04 0.68 f 0.02 0.69 f 0.03 0.71 kO.05 0.7oio.04

1.73kO.14 2.15kO.08 2.6420.16 3.23kO.16 3.lOf0.30 2.82+0.12

151.27? 6.48” 202.60? 13.38“ 288.76 + 7.99’ 368.lOk 7.76” 336.76+ 5.52b 302.41 & 4.32‘

1.10* 1.31’ l.62b 1.84” 1.76” 1.66b

4.02a 3.30d 2.79’ 1.94” 2.16” 2.48b

62d 72’ 84’ 97” 90b 85’

‘Mean values within a column not sharing the same superscript are significantly different at F’< 0.05. In( mean final weight) - In( mean initial weight) x loo, ‘Specific growth rate= 84 days ‘Feed conversion ratio=dry weight feed(g)/wet weight gain (g).

LG. BORLONGAN

318 430 Y = -36&K)+ Y3

400

369.67

346.13X

for X62.10

for X 7 2.10

330

: ..

E L

300

. : .

z (3 cI

230

(3 3 z

200

130

100

L

-

I.30

1.30

I.70

ARGININE

1.90

LEVEL

2.10

2.30

2.30

2.70

1% dry diet)

Fig. 1. Relationship between weight gain of milkfish and dietary arginine level as described the broken-line model which allows derivation of the optimum arginine level.

by

cattish (Robinsonetal., 1981);3.9%foreel (Nose, 1979);4.0%,3.6%, 3.5% and 3.3% for rainbow trout as reported by Kim et al. ( 1983), Walton et al. ( 1986), Ogino ( 1980) and Kaushik ( 1979), respectively; 2.82% for Oreochromis mossambicus (Jauncey et al., 1983); and 4.2% for Oreochromis niloticus (Santiago and Lovell, 1988). Weight gain was significantly depressed at dietary arginine levels exceeding 2.10%. High dietary arginine level may have caused accumulation and hence toxicity in the tissue, although the basis for the growth-depressing or toxic effect of excess arginine has not been established for fish. Threonine requirement The responses of milkfish juveniles to varying threonine levels are shown in Table 4. Mean weight gains and specific growth rates increased significantly with increases in threonine level of the diets. Feed efficiency expressed

.ARGININE

AND

THREONINE

REQUIREMENTS

OF MILKFISH

319

JUVENILES

TABLE 4 Growth, feed conversion nine for 12 weeks’ Dietary threonine g/lOOg dry diet

g/lOOg protein

1.13 1.40 I .60 1.80 2.00 _.3 ‘0

2.84 3.50 4.00 4.50 5.00 5.50

ratio and survival of milk&h given diets containing graded levels of threo-

Mean initial weight (g)+s.e.m.

Mean final weight (g) *s.e.m.

Mean weight gain (%) fs.e.m.

SGR2 (%/day)

FCR3

Survival

k/P)

(O/o)

1.37f0.06 1.32f0.04 1.34f0.11 1.30*0.03 1.33kO.06 1.3oio.13

3.04?0.11 3.47?0.10 4.38kO.86 5.52kO.54 6.02f0.19 5.68kO.17

122.80* 2.89’ 162.62rf- 1.75’ 226.48?26.94b 324.70+20.99” 352.925 8.37” 336.01 k28.58”

0.95d l.lY l.40b 1.72a 1.80a 1.75”

4.77’ 4.48’ 3.2S’ 2.44” 2.04” 2.39”

50d 67d 80’ 90h 91” 80

‘Mean values within a column not sharing the same superscript are significantly different at Pi 0.05. In (mean final weight ) - ln( mean initial weight) x ,oo. 84 days ‘Feed conversion ratio=dry weight feed (g)/wet weight gain (g).

‘Specific growth rate =

as feed conversion ratio was a reflection of weight gain; the best feed conversion ratios were obtained with the diets which gave the best growth rates. The poorer efficiency of feed utilization by fish fed a threonine-deficient diet was evident. Survival rates ranged from 50 to 97%, with highest survival obtained in the diet which gave the best growth rate and lowest survival in the threonine-deficient diet. Broken-line regression analysis of the growth data revealed a break-point in the growth-response curve at 1.80% dietary threonine (Fig. 2 ), indicating that increasing threonine beyond this level would not provide significant additional growth. This value corresponds to 4.50% of the dietary protein, which is higher than that reported for other fish. Threonine requirements as percentage of the protein are: 3.9% for common carp (Nose, 1979); 3.6% for Japanese eel (Arai et al., 1972); 3.75 for Oreochromis niloticus (Santiago and Lovell, 1988); 3.4% for rainbow trout (Ogino, 1980); 2.93% for Oreochromis mossabicus (Jauncey et al., 1983); 2.21% for channel catfish (Wilson et al., 1978); 2.25% for chinook salmon (DeLong et al., 1962) and 3.0% for chum salmon (Akiyama et al., 1985). The wide variations observed in the requirement levels for both arginine and threonine among species, may be due to differences in the methodologies used such as the nature of the dietary protein sources in the test diets, the reference protein whose amino-acid pattern is being mimicked and the culture conditions. These variations may also be due to real species differences. Cowey and Tacon ( 1983) and Wilson and Poe (1985) found a direct relationship between essential amino-acid requirements of certain fish and the essential amino-acid pattern of the whole body tissue of that fish. The threo-

LGBORLONGAN

320

450 Y= -192.7l+ 288.6lXfarX~ 1.80 Y= 326.00 for X 7 1.80

400

. :

.

350

. 2 2

.

. .

300

ii g

230

w 3 200

150

too

1 0

1

so THREONINE

LEVEL

(% dry diet

2.00

1

2.20

1

2.40

1

Fig. 2. Relationship between weight gain of milkfish and dietary threonine level as described the broken-line model which allows derivation of the optimum threonine level.

by

nine requirement value obtained in this study is very near the threonine level found by Coloso et al. ( 1988) in whole milkfish protein which is 4.69% but the arginine requirement value of 5.25% is lower than the arginine level of 6.23% found in the milkfish protein. Except for the loss of appetite and low feed efficiency which resulted in depressed growth, no signs of pathologies were observed in milk&h juveniles fed the arginine-deficient and threonine-deficient diets. ACKNOWLEDGEMENT

The author acknowledges with thanks the statistical and computer assistance of Ms. Larni Espada.

ARGININE

4ND

THREONINE

REQUIREMENTS

OF MILKFISH

JUVENILES

321

REFERENCES of Akiyama, T., Arai, S. and Murai, T., 1985. Threonine, histidine and lysine requirements chum salmon fry. Bull. Jpn. Sot. Sci. Fish., 5 1: 635-639. AOAC (Association of Official Analytical Chemists), 1980. Official Methods of Analysis, I 3th edn. Association of Offtcial Analytical Chemists, Washington, DC, 1 14 1 pp. Arai, S., Nose, T. and Hashimoto, Y., 1972. Amino acids essential for the growth of eels, .-lnguilla anguilla and A. japonica. Bull. Jpn. Sot. Sci. Fish., 38: 753-759. Borlongan, LG. and Benitez, L.V., 1990. Quantitative lysine requirement of milktish (Charros clranos) juveniles. Aquaculture, 87: 341-347. Coloso. R.M., Tiro, L.B. and Benitez, L.V., 1986. Tryptophan requirement of milkfish, Chanos chanos Forsskal, juveniles. The Asian Fisheries Forum, Manila, The Philippines. 26-3 1 May 1986, p. 54 (abstract ). Coloso, R.M., Benitez, L.V. and Tiro, L.B., 1988. The effects ofdietary protein-energy levels on growth and metabolism of milkfish (Chanos chanos Forsskal). Comp. Biochem. Physiol.. 89(l): 11-17. Cowey. C.B. and Tacon. A.G.J., 1983. Fish nutrition - relevance to invertebrates. In: G.D. Pruder. C.J. Langdon and D.E. Conklin (Editors), Proc. 2nd Int. Conf. Aquaculture Nutrition: Biochemical and Physiological Approaches to Shellfish Nutrition. Louisiana State Univ. Continuing Educ., Baton Rouge, LA, pp. 13-30. DeLong, D.C.. Halver, J.E. and Mertz, E.T.. 1962. Nutrition of salmonoid fishes. X. Quantitative threonine requirements of chinook salmon at two water temperatures. J. Nutr., 76: I74178. Duncan, D.B.. 1955. Multiple-range and multiple F-tests. Biometrics, 11: l-42. Jauncey, K., Tacon, A.G.J. and Jackson, A.J., 1983. The quantitative essential amino acid requirements of Oreochrornis (Sarotherodon) mossarrzbicus. In: L. Fishelson and Z. Yaron (Compilers), Proc. First International Symposium on Tilapia in Aquaculture, 8- 13 May 1983. Nazareth, Israel. Tel Aviv University, Israel, pp. 328-337. Kaushik, S.J., 1979. Application of a biochemical method for the estimation of amino acid needs in fish: quantitative arginine requirements of rainbow trout in different salinities. In: J.E. Halver and K. Tiews (Editors), Fintish Nutrition and Fishfeed Technology. Vol. 1. Heenemann, Berlin, pp. 197-207. Kim, K.I., Kayes, T.B. and Amundson, C.H., 1983. Protein and arginine requirements of rainbow trout. Fed. Proc.. 42: 2 198. Klein. R.G. and Halver, J.E., 1970. Nutrition of salmonid fishes: arginine and histidine requirements of chinook and coho salmon. J. Nun., 100: 1105-l 109. Luquet, P. and Sabaut, J.J., 1974. Nutrition azotee et croissance chez la daurade et la truite. Actes Colloq., Colloq. I’Aquaculture. Brest, 1: 243-253. Nose, T.. 1979. Summary report on the requirements of essential amino acids for carp. In: J.E. Halver and K. Tiews (Editors), Fintish Nutrition and Fishfeed Technology, Vol. I. Heenemann, Berlin, pp. 145-156. Ogino. C., 1980. Requirements of carp and rainbow trout for essential amino acids. Bull. Jpn. Sot. Sci. Fish., 46: 17 l-l 74. Robbins, K.R., Norton, H.W. and Baker, D.H., 1979. Estimation ofnutrient requirements from growth data. J. Nutr.. 109: 1710-1714. Robinson, E.H., Wilson, R.P. and Poe, W.E., 198 1. Arginine requirement and apparent absence of a lysine-arginine antagonist in fingerling channel cattish. J. Nutr., 111: 46-52. Santiago, C.B. and Lovell, R.T., 1988. Amino acid requirements for growth of Nile tilapia. J. Nun.. 118: 1540-I 546. Sastrillo, A.S., 1990. Methionine requirement of milkfish (Chanos chanos Forsskal ) juveniles. MS Thesis. University of the Philippines in the Visayas, Iloilo. Philippines. 50 pp.

322

I.G.BORLONGAN

Steel, R.G.D. and Torrie. J.H., 1960. Principles and Procedures of Statistics. McGraw Hill, New York, NY, 48 1 pp. Tacon, A.G.J. and Cowey, C.B., 1985. Protein and amino acid requirements. In: P. Tytler and P. Calow (Editors), Fish Energetics, New Perspectives. Croom Helm, London, pp. 155-183. Walton, M.J., Cowey, C.B., Coloso, R.M. and Adron, J.W., 1986. Dietary requirements of rainbow trout for tryptophan, lysine and arginine determined by growth and biochemical measurements. Fish Physiol. Biochem., 2: 16 l-l 69. Wilson, R.P., 1989. Protein and amino acid requirements of fishes. In: Shi-Yen Shiau (Editor), Progress in Fish Nutrition. Proceedings of the Fish Nutrition Symposium, Keelung, Taiwan, 6-7 Sept. 1989. Marine Food Science Series No. 9, pp. 51-76. Wilson, R.P. and Halver, J.E., 1986. Protein and amino acid requirements offishes. Annu. Rev. Nutr., 6: 225-244. Wilson, R.P. and Poe, W.E., 1985. Relationship of whole body and egg essential amino acid patterns to amino acid requirement patterns in channel catfish, Zctalurus punctatus. Comp. Biochem. Physiol., 80B: 385-388. Wilson, R.P., Allen, O.W., Jr., Robinson, E.H. and Poe, W.E., 1978. Tryptophan and threonine requirements of fingerling channel catfish. J. Nutr., 108: 1595-l 599. Zeitoun, I.H., Ullrey, D.E., Magee, W.T., Gill, J.L. and Bergen, W.G., 1976. Quantifying nutrient requirements of fish. J. Fish. Res. Board Can., 33: 167-l 72.