Methionine Requirement of Juvenile Japanese Flounder Paralichthys olivaceus

JOURNALOFTHE WORLD AQUACULTURE SOCIETY

Vol. 31, No. 4 December, 2000

Methionine Requirement of Juvenile Japanese Flounder Paralichthys olivaceus

SHUNSUKE KOSHIO MD. SHAH ALAM, SHIN-ICHI TESHIMA,~ MANABU ISHIKAWA, AND

laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, Shimoarata 4-50-20, Kagoshima 890-0056 Japan

Abstracr.-Growth studies were conducted to determine the dietary methionine requirement of juvenile Japanese flounder faralichthys olivaceus. A basal diet was formulated to contain 50% crude protein from casein and gelatin, as intact protein sources, supplemented with crystalline L-amino acids (CAA), to cor- respond to the amino acid pattern found in the whole body protein of the juvenile Japanese flounder, except methionine. Test diets contained six graded levels of L-methionine 0.53, 0.83, 1.13, 1.43, 1.73, and 2.03% of diet (dry matter basis) or 1.06, 1.66, 2.26,2.86, 3.46 and 4.06% of protein. To prevent leaching losses of water-soluble amino acids, CAA were pre-coated with carboxymethylcellulose (CMC), and diets were further bound with both CMC and K-carrageenan after addi- tion of the pre-coated CAA. Weighing about 2.80 g, each triplicate group of the juvenile flounder were fed test diets twice a day (5% of body weight) for 40 d. Survival rate, specific growth rate, feed conversion efficiency and apparent protein retention were significantly (P < 0.05) affected by dietary methionine con- centrations. The optimum dietary level of methionine in the presence of 0.06% of dietary cystine for Japa- nese flounder juvenile was estimated by using break point analysis. The values determined based on weight gain and feed efficiency were 1.49% of diet (or 2.98% of protein) and 1.44% of diet (or 2.88% of protein), respectively. These requirement values are close to the methionine level of flounder whole body protein.

Aquaculture of the Japanese flounder Paralicthys olivaceus is one of the rapidly increased industries in Japan due to the high market value of this species. Knowledge of the dietary essential amino acid (EAA) requirement of aquaculture species like the Japanese flounder is essential for develop- ing commercial feeds using low levels of marine proteins. The nutritive value of dietary protein for fish is influenced by their amino acid compositions (Wilson and Poe 1985). To formulate a cost-effective diet for

I Corresponding author.

Japanese flounder, the dietary requirement for essential amino acids should be defined.

Methionine is one EAA required for nor- mal growth of many fishes (Wilson 1989). This sulfur-containing EAA serves as a pre- cursor of many body components; the methyl group being a major contributor to the whole body pool of one-carbon units required for transmethylations, the biosyn- thesis of choline, thymidine, etc. A part of methionine is converted to cystine in ani- mals; therefore, the presence of cystine spares the requirement of methionine for maximum growth. The requirement for total sulfur amino acids can be met by either methionine alone or the proper mixture of methionine and cystine. In fact, the methionine-sparing effect of cystine is presented in fish, such as channel catfish Ictalurus punctatus (Harding et al. 1977), red drum Sciaenops ocellatus (Moon and Gatlin 1991), and rainbow trout Oncorhynchus mykiss (Kim et al. 1992).

Methionine was determined to be the most limiting amino acid in diets for warm- water fish (Lovell 1989) and some semi- purified diets for the red drum (Moon and Gatlin 1989). Quantitative requirements of all EAA have been reported for only a limited number of cultured fish species including common carp Cyprinus carpio (Nose 1979), rainbow trout (Ogino 1980), Nile tilapia Oreochromis niloticus (Santiago and Lovell 1988), catla Cutla catla (Ravi and Devaraj 1991), Japanese eel Anguillu ja- ponica, channel catfish, chinook salmon Oncorhynchus tshawytscha (NRC 19931, chum salmon Oncorhynchus ketu (Akiyama and Arai 1993). coho salmon Oncorhyn-

0 Copyright by the World Aquaculture Society ZOO0

METHIONINE REQUIREMENT OF PARALICHTHYS OLIVACEUS 619

chus kisutch (Arai and Ogata 1993), milk fish Chanos chanos (Borlongan and Coloso 1993), and white sturgeon Acipenser truns- montanus (Ng and Hung 1995).

However, research about the marine fish requirement for essential amino acids is limited except for the studies on the requirements of a couple of amino acids in commonly cultured species. Kanazawa et al. (1989) reported that a microbound diet with an amino acid pattern similar to that of the larval fish whole body protein pro- duced good growth and survival of the larval Japanese flounder. Supplements of crystalline amino acids to the diets containing soybean meal, feather meal, and corn gluten meal as major protein sources improved the nutritive value of the diets of Japanese flounder juveniles (Kikuchi et al. 1994a, 1994b; Kikuchi 1999). Except for lysine (Forster and Ogata 1998), there have been no studies with regard to essential amino acid requirements of the juvenile Japanese flounder. The purpose of the present study was to quantify the optimum dietary methionine level for growth and survival of the juvenile Japanese flounder.

Materials and Methods Experimental Fish

Juvenile Japanese flounder (120 d after hatching) were obtained from a commercial hatchery, Matsumoto Suisan, Miyazaki, Ja- pan, transported to Kamoike Marine Pro- duction Laboratory, Faculty of Fisheries, Kagoshima University, and maintained on a commercial formulated diet (Higashimaru Foods, Kagoshima, Japan) for 2 wk.

Experimental Diets The composition of the test diets are

shown in Table 1. Casein and gelatin served as intact protein sources. Crystalline amino acids (CAA) were added to provide an amino acid pattern similar to that of the juvenile Japanese flounder whole body protein except for methionine. The basal diet (diet 1) contained the minimum level of methionine, 0.53% of diet or 1.06% of protein,

obtained from the casein and gelatin. Five additional test diets were prepared by adding incremental levels (0.3%) of methionine to the basal diet at 0.83, 1.13, 1.43, 1.73 and 2.03% of diet, corresponding to 1.66, 2.26, 2.86, 3.46 and 4.06% of dietary protein. These levels were below and above the methionine level found in the juvenile flounder whole body protein. Diets were kept isonitrogenous by decreasing gluta- mate while increasing the methionine levels. Diets were prepared according to Mil- lamena et d. (1996) with some modifica- tions as follows. The CAA mixture (25.28 g) was pre-coated with (1.90 g) cooked carboxymethylcellulose (CMC). Casein and gelatin were mixed well with water to make a paste form in a hot water bath. The bound CAA mixture and the other dry ingredients (vitamin mixture, mineral mixture, attractants, a-starch and a-cellulose) were added to the casein-gelatin paste. To this, blended oil and lecithin were added and mixed with a spoon by continuously stirring; sufficient water was added to produce a dough-like consistency. An additional 2.5 g CMC per 100 g diet was then mixed into the dough. To improve the water stability of the diets, 2.5 g K-carrageenan was gelatinized at 85 C in a water bath to form a homogenous gel and added to the mixture. The pH of the diets was adjusted to 7.0-7.5 by gradually adding 4 N sodium hydroxide. The dough was then passed through a pelletizer to ob- tain 2-mm diameter pellets and dried at 40 C in a constant temperature oven (DK 400, Yamato Scientific Co., Ltd., Japan) for 2 h. The dry pellets were ground, sieved and stored at -30 C until used. Table 2 indicates amino acid composition of dietary ingredients used for the test diets and flounder whole body obtained from high perfor- mance liquid chromatography (HPLC).

Feeding Experiment

Prior to the feeding experiment, all fish underwent a 2-wk conditioning period during which they adjusted to the pelleted diet and standardized environmental condition.

620 ALAM ET AL

TABLE 1. Composition of the test diets (g per 100 g dry diet) used for the methionine requirements study. ~~

Test diet

Ingredient 1 2 3 4 5 6

Casein Gelatin Amino acid mixture' Squid liver oilb Soybean lecithin' Vitamin mixtured Mineral mixtureC

Carboxymethyl cellulose (CMC) K-Carrageenan a-Cellulose Attractants' L-methionine

Total methionine % of diet % of protein Crude protein % (dry matter basis) Crude lipid Crude ash

a-Stxch

17.00 8.00

25.28 5.00 5.00 6.00 5.00

12.00 4.40 2.50 8.82 1 .00 0

0.53 1.06

50.2 9.7 5.4

17.00 8.00

24.98 5 .00 5 .OO 6.00 5 .OO

12.00 4.40 2.50 8.82 1 .oo 0.30

0.83 1.66

50.3 9.7 5.5

17.00 8.00

24.68 5.00 5.00 6.00 5.00

12.00 4.40 2.50 8.82 1 .00 0.60

1.13 2.26

49.5 9.8 5.5

17.00 8.00

24.38 5.00 5.00 6.00 5.00

12.00 4.40 2.50 8.82 1 .oo 0.90

1.43 2.86

50.4 9.4 5.6

17.00 8.00

24.08 5.00 5.00 6.00 5.00

12.00 4.40 2.50 8.82 1 .oo 1.20

1.73 3.46

50.7 9.6 5.3

17.00 8.00

23.78 5.00 5.00 6.00 5.00

12.00 4.40 2.50 8.82 1 .OO 1 S O

2.03 4.06

50.0 9.6 5.5

a See Table 2. Feed oil ika, Riken Vitamin, Tokyo, Japan. Kanto Chemical Co., Inc., Tokyo, Japan. (gkg) p-amino benzoic acid, 1.60; biotin, 0.02; inositol. 16.02; nicotinic acid, 3.20; ca-pantothenate, 1.12;

pyridoxine-HC1, 0.19; riboflovin, 0.80; thiamin-HCL 0.24; menadione, 0.19; vitamin A-palmitate, 0.77; a-to- copherol. 1.60; cyanocoalamine, 1.10; calciferol, 0.04; ascorbyl-2-phosphate-Mg, 0.28; folic acid, 0.06 and cholin chloride, 32.75.

(gkg) NaC1, 1.838; MgS04.7H,0, 6.850; NaH,P04.2H,0, 4.360; KH,P04, 1 1.990; Ca(H,P04),.2H,0, 6.790; Fe-citrate, 1.485; Ca-lactate, 16.350; AICl,.BH,O, 0.009; ZnS04.7H,0, 0.179; CuCI,, 0.005, MnS04.4H,0, 0.040; KI, 0.008 and CoCI,, 0.050. ' Taurine 0.5, betaine 0.4 and inosine-5-monophosphate 0.1.

The growth study using test diets was conducted in 18 100-L polypropylene tanks (filled with 80 L water), and 22 juveniles with a mean weight of 2.8 g were stocked randomly in each tank. Each test diet was fed to three replicate groups of the juveniles. The juveniles were fed the respective diets at a rate equaling 5% of their body weight per day. Daily ration size was divid- ed into two equal feedings at 0830 and 1630 h. The juveniles were weighed every 10 d and the ration size was adjusted ac- cordingly. Uneaten feed was removed 1 h after feeding and dried to quantify feed intake. Fecal matter was removed by siphon- ing the water from the bottom of each tank 1 h before giving the diet. The water flow

of the tank was 1.2 Wmin and a 12:12 h light-dark photoperiod was maintained. The water temperature was 21.6 ? 0.98 C (mean t SD) during the feeding period. The other parameters were dissolved oxy- gen 5.92 t 0.18 mg/L, pH 8.02 k 0.10, and salinity 33.5 t 0.31 ppt. The growth study was conducted for a period of 40 d. After termination of the feeding experiment, the eyes of four or five fish from each tank were inspected to evaluate cataract or eye opucity as described by EstCvez et al. ( 1997).

Chemical and Statistical Analysis

Amino acid analysis was performed by HPLC according to Teshima et al. (1986).

METHIONINE REQUIREMENT OF PARAWCHTHYS OLIVACEUS 62 1

TABLE 2. Amino crcid composition (g per 100 g dry diet) of dietav ingredients for determining the methionine requirement.

Supplied by Supplied by Supplied by 50% whole Amino acidsA 17% casein 8% gelatin CAA Total body protein

EAAh Arginine Histidine Isoleucine Leucine Lysine' Methionine Phenylalanine Threonine Tryptophan W i n e

NEAA' Asp'artic acid Glutamic acid Serine Proline Glycine Alanine Tyrosine Cystine Hydroxyproline

0.60 0.49 0.8 1 1 S O 1.60 0.48 0.84 0.65 ND 0.99

1.06 3.84 0.7 1 1.76 0.24 0.43 1.01 0.05' -

0.65 0.08 0.12 0.24 0.24 0.05 0.16 0.17 ND 0.20

0.48 0.87 0.20 I .oo 1.76 0.83 0.02 0.01' 0.92

1.88 0.69 1.28 2.14 3.29

variable 1.10 1.31 0.36 1.37

3.22 3.47 1.02 0 1.14 2.04 0.97 -

-

3.13 1.26 2.21 3.88 5.13

variable 2.10 2.13 0.36 2.56

4.76 8.18 1.93 2.76 3.14 3.30 2.0 0.06 0.92

3.13 1.26 2.21 3.88 5.13 I .42 2.10 2.13 0.36d 2.56

4.76 8.18 1.93 2.34 3.14 3.30 2.0 ND -

a Supplied as L-form (Ajinomoto Co.. Inc., Japan). Essential amino acids. Supplied as L-Lysine HCI. Kanazawa et al. (1989). Non-essential amino acids.

' NRC (1993). ND = Not detected.

Approximately 2 mg of dry sample was weighed and hydrolyzed with N-methane- sulfonic acid for 22 h at 110 C. The pH of the hydrolysate was adjusted to pH 2.2 and injected into a HPLC unit with an ion ex- change resin column. Norleucine was used as an internal standard. The crude protein and lipid contents of the test diets were determined by Kjeldahl method and Bligh and Dyer (1 959) method, respectively. Ash and moisture contents were analysed by Asso- ciation of Official Analytical Chemists (AOAC 1990) method. Weight gain data were tested using one-way analysis of var- iance (package super-ANOVA, Abacus Concepts, Berkeley, California, USA). Sig- nificant differences between means were evaluated by Tukey Kramer test (Kramer 1956). Probabilities of P < 0.05 were con-

sidered significant. The optimum dietary methionine level was determined using the broken-line regression method (Zeitoun et al. 1976; Robbins et al. 1979). The 95% confidence interval of the breakpoint was estimated as described by Jones and Moli- toris (1984). Regression analysis was performed using the software package Stat- Vie+ (Abacus Concepts, Berkeley, Cali- fornia, USA).

Results Mean weight gain (WG), survival, feed

conversion efficiency (FCE), feed intake, specific growth rate (SGR) and apparent protein retention (APR) of the Japanese flounder juveniles fed graded levels of methionine are presented in Table 3. There was a trend that WG and SGR increased with

622 ALAM ET AL

TABLE 3. Final body weight (FEW), weight gain, feed conversion eficiency (FCE), feed intake (FI), percentage survival, specific growth rate (SGR) and apparent protein retention (APR) of juvenile Japanese flounder fed diets graded levels of methionine .for 40 d. Values are means of three replicate groups. Means with different letter in the same column differ significantly (P < 0.05).

Methionine (%) FBW Weight gain FI Survival SGRZ APR' Diet (protein) ($9 (%) FCE' (glfishld) (%) (%)/d (%)

0.53 (1.06) 4.28a 46.67a 0.28a 0.14a 77.67a 0.83 (1.66) 6.55b 133.81b 0.48b 0.20ab 78.67a 1.13 (2.26) 8 . 5 1 ~ 196.90~ 0 . 6 7 ~ 0.22b 77.67b 1.43 (2.86) 12.45d 344.52d 0.74cd 0 . 3 3 ~ 87.67ab 1.73 (3.46) 12.42d 345.24d 0.78cd 0 . 3 1 ~ 98.00b 2.03 (4.06) 12.27d 338.104 0.82d 0 . 2 9 ~ 88.33ab Pooled SEM 0.60 21.85 0.04 0.02 3.20

I Feed conversion efficiency = weight gain &)/total feed intake in dry basis (g). Specific growth rate = [In (mean final weight) -In (mean initial weight)/40] X Apparent protein retention = protein gain X 100/protein intake.

1.05a 7.28a 2.13b 14.58b 2 . 7 8 ~ 22.07~ 3.72d 25.33cd 3.72d 25.74d 3.68d 25.77d 0.19 1.52

100.

the increment of methionine supplementation to the basal diet. These indices significantly improved as the dietary methionine level increased up to 1.43% of diet, beyond which those indices tended to plateau or slightly decrease. Although the maximum WG and SGR were recorded from the diet containing methionine level 1.73% of diet, no statistical differences were observed among 1.43%, 1.73% and 2.03% of diet. The poorest WG and SGR were obtained in the group fed the diet without supplemental methionine (diet 1 containing 0.53% methionine from intact protein), and were significantly lower than other groups. Due to

TABLE 4. Effects of dietary levels of methionine on bodv composition (% wet basis) of Japanese Jloun- der. Values are means of triplicate groups. Initial bodv cornposition was 80% moisture, 12.82% protein, 1.92% lipid and 3.60% ash. Means with different letter in the same column differ significantly (P < 0.05).

~~

Methionine level

(% diet) Moisture Protein Lipid Ash

0.53 78.89a 13.21a 1.96a 3.68d 0.83 77.72ab 14.44b 2.89b 3 . 2 3 ~ 1.13 76.57b 15.36bcd 3.28bc 2.89b 1.43 76.44b 16.35d 3.09bc 2.64ab 1.73 76.87b 15.96cd 3 . 5 6 ~ 2.58a 2.03 76.93b 15.12bc 3.37bc 2.69ab Pooled SEM 0.39 0.39 0.16 0.08

supplementation of methionine to the basal diet, FCE significantly increased together with increased feed intake. FCEs among the diets containing 1.13%, 1.43% and 1.73% methionine were not statistically significant, and those among the diets containing 1.43%, 1.73% and 2.03% were also not significant. However, a statistical significance of FCE was detected between the diets containing 1.13% and 2.03% of methionine. Juveniles fed the diets containing more than 1.43% of methionine showed significantly higher consumption than other lower methionine groups. Juveniles fed the basal diet exhibited significantly poorer consumption compared to those fed on the diets containing 1.13% methionine and up. Survival rate was significantly affected by methionine levels in the diets and the highest (98%) survival rate was recorded from the diet containing 1.73% of methionine. There was a trend that the survival of fish improved with increased dietary methionine supple- ment. The lowest APR was obtained in the group fed the basal diet. Increasing the methionine level up to 1.43% significantly increased APR, and further increase did not improve the APR significantly. Effects of dietary levels of methionine on proximate composition of the whole body are presented in Table 4. Protein, lipid, moisture

METHIONINE REQUIREMENT OF PARAWCHTHYS OLIVACEUS 623

400 i Y = - 131.81 t 318.59X for X5 1.49

Y = 342.62 for X>1.49

I

0 0.0 0.5 1.0 1.5 2.0 2.5

I I I I

Dietary methionine level (% of diet)

FIGURE I . Relationship between weight gain of Jap- anese flounder juvenile and dietary methionine level as described b y the broken-line regression model.

and ash contents were affected by the methionine level of the diet. The lowest protein content was observed in the group fed the diet containing the basal diet, whereas the highest value was obtained for the group fed the diet containing 1.43% of methionine. Increasing the methionine level from 1.43% to 2.03% of diet did not show any significant influences on body composition.

The optimum dietary level of methionine in the presence of 0.06% of dietary cystine for Japanese flounder juvenile was estimated by using break point analysis. Based on weight gain data, the requirement for methionine by juvenile Japanese flounder was estimated to be 1.49% of the diet (95% confidence interval: 1.45 to 1.53). This level is equivalent to 2.98 g/100 g protein (Fig. 1) . When feed efficiency was plotted against dietary methionine, the break-point oc- curred at 1 . 4 4 % of the diet (95% confidence interval: 1.41 to 1.47) or 2.88% of protein. This value was a little lower than that obtained from weight gain but agreed well.

Discussion

The optimum dietary methionine level for the juvenile Japanese flounder based on weight gain and feed efficiency data was found to be 1.49% of diet (2.98% of protein) and 1.44% of diet (2.88% of protein), respectively. These values are less than that reported previously for gilthead sea bream (Luquet and Sabaut 1974), but are close to those for most of the commercially impor- tant finfish species, namely, rainbow trout (3%, Rumsey et al. 1983), common carp (3.1%, Nose 1979), Japanese eel (3.2%, NRC 1993), Nile tilapia (3.2%, Santiago and Love11 1988), coho salmon (2.7%, Arai and Ogata 1993), yellowtail (2.56%, Ruch- imat et al. 1997), red drum (2.69%, Moon and Gatlin 1991). Lower dietary requirements of methionine have been reported for channel catfish (2.3%, Harding et al. 1977) as compared with that for flounder in this present study. Forster and Ogata (1998) reported that methionine plus cystine requirement for the juvenile Japanese flounder was 1.9% of dietary protein, based on lysine requirement (calculated from nitrogen retention data), which was low compare to other fishes. Our results showed that the methionine requirement value is within the range of most of the commercially cultured fish species, and the value obtained is nearer to the whole body protein of the juvenile flounder. The value calculated for methionine by Forster and Ogata (1 998) is different from our result, perhaps due to different protein sources and different techniques to determine the optimum level; we used purified intact protein sources. There are many factors which may affect amino acid requirements, including species and age, dietary protein sources, crystalline amino acids, environmental conditions and experimental design (Tacon and Cowey 1985; Moon and Gatlin 1991).

Cataracts were observed in rainbow trout fed in methionine-deficient diets by Walton et al. (1982) and Rumsey et al. (1983), but not in other finfish, such as channel catfish

624 ALAM ET AL.

(Robinson et al. 1978). No cataracts (evi- dent from visual inspection of the lenses) or any other methionine deficiency signs, except reduced growth, were observed in the Japanese flounder fed the methionine- deficient diet in the present study. Estkvez et al. (1997) also reported that no cataracts were formed in the eyes of Japanese flounder fed methionine-deficient diets. The poor growth of the methionine-unsupplemented groups may be due to loss of appetite, which resulted in low feed intake, hence depressed growth. Rollin et al. (1994) reported that Atlantic salmon Sulmo sulur juveniles fed the methionine-deficient diet resulted in low feed intake and depressed growth. In the case of rats, Aguilar et al. (1974) found that the loss of weight was associated with low feed intake, when fed the diet devoid of methionine. Variable re- sponses in survival observed in our experiment were also related to various fish species fed amino acid-deficient diets (Arai et al. 1972; Santiago and Love11 1988). The APR as well as the whole body protein tended to increase up to the requirement level. This may be due to an increase in nitrogen retention with increasing dietary methionine, which was observed for rainbow trout (Kim et al. 1992) and yellow tail (Ruchimat et al. 1997). The results in the present study showed that weight gain of the juveniles fed methionine-unsupplemented diet was significantly lower than that of the juveniles fed methionine-supplemented diets. This indicates that Japanese flounder juveniles are able to utilize supplemental methionine from CAA and also indicates that methionine was essential for growth of Japanese flounder. The total sulfur-containing amino acids (methionine + cystine) requirement for Japanese flounder calculated based on the weight gain and feed efficiency data were 1.55% of diet (3.10% of protein) and 1.50% of diet (3.00% of protein), respectively. The cystine replacement value was not studied by conducting a separate experiment. Cho et al. (1992), when quantifying the arginine requirement of rainbow

trout, added agar-coated free amino acids to the diet and gave high rates of growth com- parable the complete test diet. In our experiment, the overall good growth of the flounder proved the availability of CAA by this fish. This may be due to the successful adaptation of the diet preparation technique (Millamena et al. 1996) to reduce leaching losses of CAA in amino acid test diets, through both pre-coating the CAA mixture and use of the efficient binders.

Acknowledgments The authors wish to acknowledge Aji-

nomoto Co., Inc., Japan for donation of crystalline amino acids. The financial sup- port received by the first author for this research from the Ministry of Education, Cul- ture and Sports (Monbusho) of Japan is gratefully acknowledged.

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Documents

Methionine Requirement of Juvenile Japanese Flounder Paralichthys olivaceus