Methionine requirement of juvenile tiger shrimp Penaeus monodon Fabricius

Aquaculture Aquaculture 143 (1996) 403-410

Methionine requirement of juvenile tiger shrimp Penaeus monodon Fabricius Oseni M. Millamena av*, Myma N. Bautista-Temel A. Kanazawa b

a,

aAquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan, Iloilo 5021, Philippines b Faculty of Fisheries, Kagoshima University, 4-50-20, Shimoarata. Kagoshima, Japan

Accepted 18 January

1996

Abstract An 8-week feeding trial was conducted to determine the dietary requirement of postlarval Penaeus monodon for the sulfur-containing amino acid methionine. Shrimp postlarvae (mean weight 21 + 0.3 mg) were reared in 40-l fiberglass tanks in a flow-through seawater system. Test diets (37% protein and 360 kcal per 100 g diet) were formulated containing casein-gelatin as protein sources and supplemented with crystalline amino acids to simulate the amino acid pattern in shrimp tissue protein except methionine. The diets contained graded levels of methionine at a range of 0.72- 1.12% of the diet or 2.0-3.0% of protein. In diet preparation, the crystalline amino acids were pre-coated with carboxymethylcellulose (CMC) to reduce leaching. Diets were further coated with CMC, cornstarch, and K-carrageenan to improve water stability and the diet pH was kept at 7.0-7.2 by neutralization with 6N NaOH. Shrimp were fed the diets at 25-30% of their biomass thrice daily. At termination of the feeding experiment, various parameters including growth, survival, and feed conversion efficiency were determined and nutritional deficiency signs noted. The methionine requirement was determined from the relationship between weight gain and dietary methionine level using the broken-line regression method. The requirement of P. monodon postlarvae for methionine was 0.89% of the diet or 2.4% of protein. In a diet containing 0.41% cystine, the total sulfur amino acid requirement (methionine + cystine) would be 1.3% of the diet or 3.5% of protein. This requirement is slightly lower than the methionine level present in shrimp tissue protein. Keywords:

Tiger shrimp; Penaeus monadon Fabricius; Methionine requirement

* Corresponding author. Tel.: 63-33-271-009; 0044-8486/96/$15.00 PII SOO44-8486(96)0

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0 1996 Elsevier Science B.V. All rights reserved.

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1. Introduction Penaeus monodon, commonly known as the tiger shrimp, is a widely cultured species in Southeast Asia. Successful culture of this species in captivity requires a nutritionally adequate and economical diet. Shrimp diets are formulated to contain a high level of protein, an important but expensive nutrient, the nutritional quality of which is largely defined by its amino acid content. Thus, knowledge of amino acid requirements is crucial in optimizing growth and feed efficiency and for development of cost-effective diets for the tiger shrimp. The quantitative requirements for the ten essential amino acids of several fish species, such as common carp, Japanese eel, rainbow trout, channel catfish, Nile tilapia, and milkfish have been defined (National Research Council, 1983; Wilson, 1989). However, studies to quantify the requirements for shrimp have resulted in limited success. Deshimaru and Kuroki (1974) and Deshimaru (1981) showed that crystalline amino acids are poorly utilized by kuruma shrimp compared with a natural protein source. Akiyama (1986) and Akiyama et al. (1991) attributed the poor growth and survival of P. monodon fed moist amino acid test diets to leaching of the crystalline amino acids when the diets were immersed in seawater. Thus far, the only published amino acid information on shrimp species is the lysine requirement of Penaeus uannamei (Akiyama, 1986; Fox et al., 1992) and the arginine requirement of P. monodon (Chen et al., 1992). Success in these experiments was partly attributed to improvement in test diet preparation to prevent leaching losses of crystalline amino acids. Millamena et al. (1996a) and Millamena et al. (1996b) have developed a technique to quantify the amino acid requirement for shrimp and other species utilizing water-stable diets. This experiment was conducted to determine the methionine requirement of juvenile P. monodon. Methionine is one of the ten essential amino acids that have been found to be critical for optimal growth and survival of shrimp (Coloso and Cruz, 1980; Pascual and Kanazawa, 1986). This amino acid is deficient in plant proteins such as soybean meal, a common component of most aquaculture diets.

2. Material and methods 2.1. Experimental

diet composition

and preparation

Six experimental diets were formulated to contain 37% protein and 360 kcal per 100 g diet using purified ingredients (Table 1). The diet formula was derived from Pascual and Kanazawa (1986) but modified based on current information on nutrient requirements of tiger shrimp. Casein and gelatin (2:l) served as natural protein sources. Crystalline amino acids were added to provide an amino acid pattern similar to that of P. monodon juveniles except for methionine. The basal diet contained 0.72% methionine from the casein and gelatin (Table 2). Five additional diets were prepared by adding incremental levels of methionine at 0.80, 0.88, 0.96, 1.04 and 1.12% of the diet. These diets contained methionine at levels of 2.0, 2.2, 2.4, 2.6, 2.8 and 3.0% of dietary protein. These levels were below and above the methionine level found in shrimp muscle

O.M. Millamena et al./Aquaculture Table 1 Composition

of experimental

diets (g per 100 g dry diet)

Ingredient

Diet

Casein Gelatin Ammo acid mix Methionine Cornstarch Glucose Sucrose Cholesterol Lecithin Cod liver oil Soybean oil Vitamin mix a Mineral mix a Carboxymethyl cellulose (CMC) K-carrageenan Cellulose Total methionine % diet % protein

1

2

3

4

5

6

10 5 20 0 15 5 10 2 2 3 3 8 3 4 2.5 7.5

10 5 19.92 0.08 15 5 10 2 2 3 3 8 3 4 2.5 7.5

10 5 19.84 0.16 15 5 10 2 2 3 3 8 3 4 2.5 7.5

10 5 19.76 0.24 15 5 10 2 2 3 3 8 3 4 2.5 7.5

10 5 19.68 0.32 15 5 10 2 2 3 3 8 3 4 2.5 7.5

10 5 19.60 0.40 15 5 10 2 2 3 3 8 3 4 2.5 7.5

0.80 2.2

0.88 2.4

0.96 2.6

1.04 2.8

1.12 3.0

0.72 2.0

a Vitamin and mineral mixes after Kanazawa

Table 2 Amino acid composition Amino acids

143 (1996) 403-410

(1981).

of the basal diet (g per 100 g dry diet)

Amount in casein (10%) and gelatin (5%)

Crystalline

amino acids

Amount in 40% shrimp tissue orotein

Essential amino 1 acids Arginine 0.60 Histidine 0.42 lsoleucine 0.52 Leucine 0.95 Lysine 0.85 Methionine 0.72 Phenylalanine 0.62 Threonine 0.47 Tryptophan 0.04 Valine 0.70

2.56 0.56 1.24 l.% 2.15 variable 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

Non-essential amino acids Aspartic acid 1.03 Glutamic acid 2.46 Serine 0.64 Proline I .26 Glycine 1.07 Alanine 0.72 Cystine 0.03 Tyrosine 0.38

3.00 3.89 1.00 0.33 2.14 1.70 0.38 1.18

4.03 6.35 1.64 1.59 3.21 2.42 0.41 1.56

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protein. As the methionine level was increased, aspartate and glutamate levels were proportionately decreased, on a nitrogen basis, to keep the diets isonitrogenous. Separate amino acid pre-mixes were made for each of diets l-6. Other dietary components were cod liver oil, soybean oil, cholesterol, and lecithin as lipid sources; glucose, sucrose, and cornstarch as carbohydrate sources; vitamins and mineral premixes; carboxymethylcellulose (CMC) and k-carrageenan as diet binders; and cellulose, a non-nutritive filler. The following procedure for diet preparation is similar to that described by Millamena et al. (1996a) and Millamena et al. (1996b) in earlier reports on threonine and valine. All dry ingredients, except the amino acids and binders, were throughly mixed in a food mixer. Crystalline amino acids (CAA) were individually weighed, mixed and pre-coated with 1.5 g cooked CMC per 20 g amino acid mix. The bound CAA mixture was then mixed with the dry ingredients and re-coated with 2.5 g cooked CMC per 100 g diet. The oils, lecithin, and vitamins were blended and added to the coated mixture. Cornstarch at 15 g per 20 ml water was gelatinized at 100°C then added to produce a dough-like consistency. The pH of the diets was adjusted to 7.0-7.2 by gradually adding 6N NaOH. To further improve diet stability, 2.5 g k-carrageenan was gelatinized at 85°C in a water bath to form a homogeneous gel and added to the coated mixture. The semi-moist mixture was pressure pelleted in a food grinder using a 2 mm die and dried at 40°C in an air-draft oven. The dry pellets were uniformly cut to an approximate length of 5 mm and stored at - 4°C until use. When tested for water stability, the diets were water stable within the feeding time interval of 4 h. 2.2. Feeding experiment Feeding trials were conducted at the nutrition wet laboratory of the SEAFDEC Aquaculture Department in Tigbauan, Iloilo, Philippines. Penaeus monodon postlarvae (mean weight 21 + 0.3 mg) were obtained from a private shrimp hatchery and acclimated to the basal diet for 1 week. Postlarvae were randomly distributed in 36 units of 40-l oval fiberglass tanks at 12 shrimp per tank and reared for 8 weeks. Each tank was supplied with filtered seawater in a flow-through system and adequate aeration. Six dietary treatments representing the graded levels of methionine were arranged in a completely randomized design with six replicates. Experimental animals were fed thrice daily at 09:00, 13:00, and 17:00 h. Feeding rate was 30% of shrimp biomass during the first 4 weeks and reduced to 25% thereafter. Tanks were cleaned of excess diet and feces daily and one-third of the culture water replaced. Every 2 weeks, shrimp were group-weighed and the feed ration adjusted accordingly. Weight gain, survival, specific growth rate (SGR) and feed conversion ratio (FCR) were determined at the termination of the feeding experiment. The water quality parameters temperature and salinity were monitored daily, while pH, ammonia and nitrite nitrogen were measured thrice weekly according to Strickland and Parsons (1972). 2.3. Chemical analyses Amino University,

acid analysis was performed at the chemistry laboratory of Kagoshima Japan using an automated amino acid analyzer SHIMADZU LC system with

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4 N methane sulfonic acid as the hydrolyzing agent. Proximate analysis of the diets was conducted according to methods of the AOAC (1984) at the Centralized Analytical Laboratory of SEAFDEC Aquaculture Department. 2.4. Statistical analysis Data were statistically analyzed using the procedures of the Statistical Analysis Systems Institute Inc. (1992). One-way analysis of variance was performed with weight gain, survival, specific growth rate and feed conversion efficiency of shrimp fed the test diets. The methionine requirement was determined using the broken-line regression method (Zeitoun et al., 1976; Robbins et al., 1979).

3. Results Table 3 shows the mean values of percent weight gain, survival, specific growth rate (SGR), and feed conversion ratio (FCR) of P. mono&m postlarvae fed the test diets. After 8 weeks, growth rates ranged from 578 to 897% while survival varied from 63 to 88%. Poorest weight gain, survival, and feed conversion (FCR) were observed in shrimp fed the basal diet (diet 1 at 0.72% methionine from intact protein). Highest weight gain, SGR, and best FCR were observed in shrimp fed diet 3 containing 0.88% methionine. The growth curve (Fig. 1) showed an increase in weight gain with increasing methionine level up to the breakpoint. Analysis by the broken-line regression method

Table 3 Mean percentage diets with graded

weight gain, survival, specific growth rate, and feed conversion ratio of P. monodon levels of methionine. Initial mean weight, 2 1f 0.3 mg; cultum period, 56 days Weight gain

Survival

%diet

%protein

(%)

(%)

0.72 0.80 0.88 0.96

2.0 2.2 2.4 2.6 2.8 3.0

578 f 54 732 f 38 897 f 56 817k39 800+34 779 + 36

Source

d.f.

Model Error Corrected total Adj. RZ = 0.368

5 30 35

Metbionine

level

1.04 1.12

SGR a

FCR b

63 77 83 83 88 88

3.79 4.22 4.58 4.43 4.38 4.37

1.4 1.2 1.1 1.2 1.2 1.3

Sum of Squares

Mean square

F value

Pr.>F

346340.455 440783.422 787 123.876 CV = 15.79270

69268.091 14692.781

4.71

0.0027

Analysis of variance

a SGR = hi(mean

final wt.)-m(mean

initial wt.)

56 days b Feed conversion

ratio = g dry weight feed/g

Xl00 wet weight gain.

Root MSE = 121.214

fed

408

O.M. Millamena y = -859.72 y = 895.5

et al./Aquaculture + 1994.38% for

for

143 (1996) 403-410

x < or = 0.89

x > 0.89

900

850 ‘;;

ar

800 -

2

750 -

.F s

700 -

f 650 iii 600 550 500 I

O 0.72

I

I

0.8

0.88

I

0.96

I

1.04

I

1

1.12

Dietary methionine level (OX,of diet) Fig. 1. Relationship between weight gain by tiger shrimp and dietary broken-line regression model.

methionine

level as described

by the

showed that the breakpoint (0.89% of diet or 2.4% of protein) occurred at a dietary methionine level which is slightly lower than that present (1.00%) in shrimp muscle (Table 2). Beyond the breakpoint, weight gain of P. monodon tended to decrease as the methionine concentration in the diet increased.

4. Discussion The quantitative methionine requirement for optimal growth of Penaeus monoabn is 0.89% of the diet or 2.4% of a 37% protein diet. In the presence of cystine, this corresponds to 1.3% of the diet or 3.5% of dietary protein. The methionine requirement for P. monodon appears to be lower than values reported (in percent of total protein) for fish species such as gilthead seabream (Luquet and Sabaut, 1974) and chinook salmon (Chance et al., 1964) at 4.0%; but agrees well with those of Japanese eel (Nose, 1979), Nile tilapia (Santiago and Lovell, 1988), and milkfish (Borlongan and Coloso, 1993) at 3.2% of total protein. The methionine requirements obtained for rainbow trout (3.0%,

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143 (1996) 403-410

409

et al., 1983), carp (2.7%, Nose, 19791, and channel catfish (2.3%, Harding et 1977) are all below that determined for P. monodon in the present study. The results showed that shrimp weight gain and survival of P. monodon improved beyond that observed in shrimp fed the basal diet indicating that juvenile P. monodon are able to utilize methionine from crystalline amino acids. Increasing methionine supplementation beyond the requirement did not enhance growth. However, survival tended to improve as the dietary methionine level was increased. Feed conversion ratios in all dietary treatments were not significantly different from each other. In this experiment, no gross pathological signs were observed in postlarvae fed diets deficient in methionine. Success in this experiment may be partly attributed to the diet preparation technique and use of efficient dietary binders to increase the water stability of the experimental diets. Pellet stability and concurrently nutrient leaching are of particular importance in nutritional studies on shrimp. Shrimp by nature of their feeding habits are slow continious feeders; hence, if feeds are not water stable, crystalline amino acids are quickly leached out and may no longer be present when the feed is consumed. Excessive nutrient leaching in crustacean diets and substantial losses of crystalline amino acids from poorly bound diets have been reported (Meyers and Z&n-Eldin, 1972; Goldblatt et al., 1980). In this study, the amino acid test diets supported good growth, survival, and efficient feed conversion suggesting the availability of crystalline amino acids and other dietary nutrients to shrimp. This is the first report published on the quantitative methionine requirement for P. monodon and hence provides valuable knowledge of amino acid nutrition of the tiger shrimp. The data provide a useful basis for the assessment of methionine sufficiency in shrimp diet formulations and may thus facilitate an improvement in the nutritional quality and help to lower the cost of shrimp feeds. Rumsey al.,

Acknowledgements

The authors acknowledge with gratitude the assistance of Mae Fernando in culture aspects, Lami Espada in statistical and computer work and the Centralized Analytical Laboratory staff of SEAFDEC Aquaculture Department in feed and water analyses.

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