Different levels of dietary dl -α-tocopheryl acetate affect the vitamin E status of juvenile Korean rockfish, Sebastes schlegeli

Aquaculture 161 Ž1998. 405–414

Different levels of dietary DL-a-tocopheryl acetate affect the vitamin E status of juvenile Korean rockfish, Sebastes schlegeli Sungchul C. Bai ) , Kyeong-Jun Lee

1

Department of Aquaculture, Pukyong National UniÕersity, Pusan 608-737, South Korea

Abstract This experiment was conducted to study the effects of different levels of DL-a-tocopheryl acetate on vitamin E status of juvenile Korean rockfish and estimate the dietary vitamin E requirement. After 6 weeks of conditioning with a basal diet, fish were divided into six groups and fed semipurified diets for 20 weeks. The experimental diets were supplemented with 0, 20, 40, 60, 120 or 500 mg DL-a-tocopheryl acetaterkg diet ŽE 0 , E 20 , E 40 , E 60 , E 120 and E 500 .. These diets contained approximately 16 mg of endogenous a-tocopherolrkg. Fish averaging 12.3 g were fed twice a day during the first 8 weeks, and then once a day during the second 16 weeks. Fish were fed to satiation throughout the experimental periods. Fish fed diet E 0 began to show lower appetite and reduced growth rate after 16 weeks. Fish fed diet E 0 had lower Ž P - 0.05. weight gain, feed conversion ratio, and hematocrit compared to fish fed diets E 20 –E 120 . Vitamin E deficiency signs such as muscular dystrophy, shortened operculae and exophthalmia were observed only in fish fed diet E 0 . Fish fed diets E 0 and E 20 had lower Ž P - 0.05. liver a-tocopherol concentration than did fish feed diet E 120 . The highest level of DL-a-tocopheryl acetate supplementation Ždiet E 500 . did not result in greater growth performance or liver a-tocopherol concentration than the other diets. Fatty acid composition of muscle and liver were only affected by the diet E 0 Ž P - 0.05.. Based on broken line analysis of weight gain, the optimum level of dietary vitamin E was determined to be about 45 mgrkg diet for good growth and maximum tissue accretion in juvenile Korean rockfish. q 1998 Elsevier Science B.V. Keywords: Korean rockfish; Semipurified diet; a-Tocopherol; Deficiency signs

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Corresponding author. Fax: q82-51-628-7430. Fax: q82-51-628-7430.

0044-8486r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 4 4 - 8 4 8 6 Ž 9 7 . 0 0 2 8 8 - 3

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1. Introduction Vitamin E functions as a lipid-soluble antioxidant protecting biological membranes and lipoproteins against oxidation, and it has been demonstrated to be an essential dietary nutrient for all fish studied ŽNRC, 1993.. The level and state of oxidation of polyunsaturated fatty acids ŽPUFA. in the diet, as well as the presence of other antioxidants and selenium, may affect the dietary vitamin E requirement of fish ŽMurai and Andrews, 1974; Poston et al., 1976; Watanabe et al., 1977; Hung et al., 1981; Cowey et al., 1983; Lovell et al., 1984; Gatlin et al., 1986.. Numerous vitamin E deficiency signs have been induced in fish with some of the more pronounced signs being muscular dystrophy, erythrocyte fragility, anemia, and exudative diathesis ŽNRC, 1993.. These deficiency signs have been described for chinook salmon, ŽWoodall et al., 1964., Atlantic salmon, ŽPoston et al., 1976., channel catfish, ŽMurai and Andrews, 1974; Lovell et al., 1984; Wilson et al., 1984., common carp, ŽWatanabe et al., 1970, 1981a., rainbow trout, ŽCowey et al., 1981, 1983; Watanabe et al., 1981b. and yellowtail ŽToyoda, 1985.. The recommended levels of dietary a-tocopherol for growing fish determined with chemically defined diets under controlled conditions include the following: 30–50 mgrkg diet for salmon ŽWoodall et al., 1964; Halver, 1972; Lall et al., 1988., 25–100 mgrkg diet for rainbow trout ŽWoodall et al., 1964; Hung et al., 1980; Watanabe et al., 1981b; Cowey et al., 1983., 25–50 mgrkg diet for channel catfish ŽMurai and Andrews, 1974; Wilson et al., 1984., 100 mgrkg diet for common carp ŽWatanabe et al., 1970., 25 mgrkg diet for blue tilapia, ŽRoem et al., 1990., 50–100 mgrkg diet for Nile tilapia, ŽSatoh et al., 1987., and 119 mgrkg diet for yellowtail ŽShimeno, 1991.. Korean rockfish is a commercially important fish species ŽLee et al., 1994. in Korea. The development of an experimental model for investigating vitamin C requirements ŽBai et al., 1996. and the long-term effects of dietary vitamin C ŽBai and Lee, 1996. on Korean rockfish have been studied. Objectives of this study were to investigate the effects of different levels of vitamin E on growth performance and fatty acid composition of tissues, and to determine the dietary vitamin E requirement for juvenile Korean rockfish.

2. Materials and methods 2.1. Experimental diets Composition of the basal diet is shown in Table 1. Six experimental diets supplemented with 0, 20, 40, 60, 120, or 500 mg DL-a-tocopheryl acetaterkg dry diet ŽE 0 , E 20 , E 40 , E 60 , E 120 , and E 500 . were prepared by adding appropriate amounts of DL-a-tocopheryl acetate pre-mixture Ž10 mg DL-a-tocopheryl acetaterg cellulose. at the expense of cellulose. The experimental diets were formulated to contain 48% crude protein and 18 kJ available energyrg. Estimated available energy ŽLee and Putnam, 1973; Garling and Wilson, 1977. of experimental diets was 18 kJrg Ž16.7, 16.7 and 37.7 kJrg for protein, carbohydrate and lipid, respectively.. Fish meal Ž10%. was added

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Table 1 Composition of the basal diet Ždry-matter basis. Ingredients

%

Ingredients

%

Casein, vitamin free a Gelatina White fish meal Ždefatted. b Dextrina c L-Arginine b L-Lysine Methionineb

30.0 10.0 10.0 27.0 0.5 0.5 0.5

Fish oil Žpollack. Corn oil Carboxymethylcellulose Cellulose Vitamin premix d Mineral premix e DHAqEPA mixture Ž25%. f

5.0 4.9 2.0 2.5 3.0 4.0 0.1

a

United States Biochemical, Cleveland, OH 44122. Kum Sung Feed, Pusan, Korea. c Myeng Sung Science, Pusan, Korea. d Contains Žas gr100 g premix.: DL-calcium pantothenate, 0.5; choline bitartrate, 10; inositol, 0.5; menadione, 0.02; niacin, 0.5; pyridoxine. HCl, 0.05; riboflavin, 0.1; thiamin mononitrate, 0.05; L-ascorbic acid, 5.0; retinyl acetate, 0.02; biotin, 0.005; folic acid, 0.018; B12 , 0.0002; cholecalciferol, 0.008; alpha-cellulose, 82.26. e H-440 premix No. 5 Žmineral. ŽNRC, 1973.. f E-HWA OIL and FAT Ind., Pusan, Korea. b

to the experimental diets to enhance palatability to Korean rockfish because the purified casein–gelatin based diet was not readily accepted by the fish in a previous feeding trial. Fish meal was extracted three times with a chloroformrmethanol mixture Ž2:1, vrv. for 1 day and air dried before incorporating into the experimental diets ŽFolch et al., 1957.. At the end of the feeding trial, diets were analyzed for a-tocopherol by HPLC. Based on this analysis, levels of 15.9, 37.7, 50.3, 63.5, 114 and 471 mg a-tocopherolrkg diet were determined for E 0 , E 20 , E 40 , E 60 , E 120 , and E 500 diets, respectively. The pelleted experimental diets were stored at y358C before feeding to fish. 2.2. Experimental fish and feeding trial Juvenile Korean rockfish Ž Sebastes schlegeli . were produced at the Wando hatchery ŽResearch station branch of the National Fisheries Research and Development Agency. in Korea. During a 6-week conditioning period, fish were fed the basal diet to adjust to the experimental conditions and to deplete body reserves of vitamin E. The feeding trial was conducted for 20 weeks in 40 l flow-through aquaria receiving filtered seawater at a rate of 2 lrmin. Supplemental aeration also was provided to maintain dissolved oxygen near saturation. Water temperature ranged from 17 " 0.58C at the beginning to 13 " 0.28C at the end of the experiment due to natural fluctuation in temperature of the seawater. Experimental fish averaging 12.3 g were randomly distributed in each aquarium as groups of 30 fish. The average total weight of 30 fish was 371 " 4.3 g Žmean " sd.. The diets were fed to triplicate groups of fish to satiation Žapproximately 2% of wet body weight. per day on a dry-matter basis. Fish were fed twice a day at 0900 and 1600 during the first 8 weeks, and then once a day at 1600 after 8 weeks until the end of the experiment. Total fish weight in each aquarium was determined every 4 weeks following anesthesia with 100 mg MS 222 Žtricaine.rl, and the amount of diet fed adjusted accordingly.

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2.3. Sample collection and analysis At the end of the feeding trial, all fish were weighed and counted to calculate weight gain, feed conversion ratio, specific growth rate, protein efficiency ratio, and survival. Hematocrit was determined on five fish randomly selected per aquarium by the microhematocrit method ŽBrown, 1980., and hemoglobin was measured in the same five fish by the cyanmethemoglobin procedure using Drabkin’s solution. Hemoglobin standard prepared from human blood ŽSigma Chemical, St. Louis, MO. was used. Blood was obtained from the caudal vasculature of five fish per aquarium and pooled prior to centrifugation. Plasma was then separated and stored at y758C until analyzed. Diet and tissue a-tocopherol concentrations were determined by HPLC. Liver, muscle and plasma samples were prepared from five additional fish randomly selected per aquarium for the analyses of a-tocopherol and fatty acid composition. The HPLC system ŽShimadzu. consisted of a model RF-551 Spectro Fluorometric detector, a Waters model 510 HPLC Pump, and a model LC 13 PVDF ŽGelman Science. Aerodisc. The HPLC was operated by conditions of Lichropher 100RP-18Ž5 m m. column, 95% methanol mobile phase, 1.4 mlrm flow rate, 408C column temperature and 20 m l injection size. Lipid in liver and muscle were extracted ŽFolch et al., 1957. and then fatty acid composition determined after methylation with 5% sulfuric acidrmethanol. Fatty acid analysis was conducted using a Hewlett Packard Gas–Liquid Chromatograph 5890 with flame ionization detector. The column Ž30 m = 0.25 mm ID., 0.25 m m, film thickness. was operated isothermally at 2308C. Injector and detector temperatures were 2508C. Helium served as carrier gas at a flow rate 30 mlrmin. Crude protein, moisture and ash of the experimental diets were analyzed by AOAC Ž1995. methods and crude lipid was determined by Folch et al. Ž1957.. All data were subjected to ANOVA test using Statistix 3.1 ŽAnalytical Software, St. Paul, MN, USA.. When a significant treatment effect was observed, a Least Significant Difference ŽLSD. test was used to compare means. Treatment effects were considered significant at P - 0.05. The a-tocopherol requirement was determined by the broken line model ŽRobbins et al., 1979..

3. Results and discussion 3.1. Growth performance Growth and other responses of fish in the 20-week feeding trial are summarized in Table 2. Fish fed the basal diet had significantly lower weight gain and feed conversion ratio ŽFCR. than those fed diets E 20 –E 120 Ž P - 0.05.. On the basis of the broken line regression analysis, the optimum level of dietary DL-a-tocopheryl acetate leading to optimum weight gain was established to be about 45 mg DL-a-tocopheryl acetaterkg diet ŽFig. 1.. Fish fed diet E 500 with the highest concentration of a-tocopherol showed adverse responses in terms of growth and hemoglobin concentration. Weight gain and FCR of fish fed diet E 500 were significantly lower Ž P - 0.05. than those of fish fed diets E 20 –E 120 . Poston and Livingston Ž1969. reported that poor growth and lower hematocrit

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Table 2 Percent weight gain, feed conversion ratio ŽFCR., protein efficiency ratio ŽPER., hematocrit, and hemoglobin of Korean rockfish fed the experimental diets for 20 weeks1 Pooled SEM 3

Diets

Initial weight Žg. Final weight Žg. Weight gain Ž%. 4 FCR5 PER6 Hematocrit Ž%. Hemoglobin Žgrdl.

E 20

E 20

E 40

E 60

E 120

E 500

12.4 35.8 c 188 d 1.04 b 1.17 ab 52.0 c 9.6 c

12.4 38.3 ab 210 bc 0.97 a 1.20 ab 59.7 a 10.6 ab

12.3 39.8 ab 224 ab 0.94 a 1.22 ab 58.8 ab 11.1ab

12.4 39.5ab 218 ab 0.97 a 1.24 a 58.2 ab 10.2 bc

12.2 40.4 a 231a 0.93 a 1.16 ab 57.7 ab 11.4 a

12.4 37.1bc 199 cd 1.07 b 1.09 b 56.2 b 9.7 c

0.03 0.46 3.98 0.03 0.19 0.10 0.25

1

Values are means from triplicate groups of fish. Means in each row with a different superscript are significantly different Ž P - 0.05.. 2 a-Tocopherol concentrations of diets E 0 , E 20 , E 40 , E 60 , E 120 and E 500 were 15.9, 37.7, 50.3, 63.5, 114 and 471 mgrkg dry diet. 3 Pooled standard error of mean. 4 Percent weight gain: Žfinal wt.yinitial wt..=100rinitial wt. 5 Feed conversion ratio: dry feed intake Žg.rweight gain Žg.. 6 Protein efficiency ratio: wet weight gainrprotein intake.

were observed in brook trout fry fed a diet containing a high level of vitamin E Ž5000 mgrkg.. Baker and Davies Ž1996. also reported that African catfish, Clarias gariepinus, fed high a-tocopheryl acetate dose Ž500 mgrkg dry feed. were observed to have significantly lower Ž P - 0.05. hematocrit than fish fed the basal diet Ž0 mgrkg.. Similar results were also found in this study. Fish fed diets E 0 and E 500 had lower hematocrit

Fig. 1. Broken line regression analysis of percent weight gain in Korean rockfish fed different six levels of dietary DL-a-tocopheryl acetate during 20 weeks. Values of the X-axis are the total vitamin E levels in experimental diets. a-Tocopherol concentrations of diets E 0 , E 20 , E 40 , E 60 , E 120 and E 500 were 15.9, 37.7, 50.3, 63.5, 114 and 471 mgrkg dry diet. Values are means"SEM, ns 3 tanks.

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than those fed the other diets Ž P - 0.05.. Hematocrit of fish fed E 0 was lower than that of fish fed E 500 diet Ž P - 0.05.. These findings suggest that the adverse effects of high levels of dietary vitamin E may need to be studied further. The anemia in fish fed low amounts of a-tocopherol was characterized by a reduced number of erythrocytes, with less volume and lower hemoglobin concentration, and decreased hematocrit has been observed in vitamin E-deficient rainbow trout and Atlantic salmon ŽPoston et al., 1976; Cowey et al., 1984; Hamre et al., 1994.. Hematocrit reduction observed in this experiment may have been due to a combination of aforementioned factors. Hamre et al. Ž1994. suggested that the low hemoglobin was due to either an inhibition of hemoglobin synthesis, or an increased rate of destruction of hemoglobin in fish fed the lower levels of vitamin E. In the present study, the vitamin E deficiency signs such as exophthalmia, shortened operculae and muscular dystrophy were observed in fish fed the basal diet after 16 weeks. Exophthalmia and shortened operculae have been reported as vitamin E deficiency signs in chinook salmon by Woodall et al. Ž1964.. Survival in the current experiment was 100%. 3.2. Tissue a-tocopherol concentrations Liver, muscle and plasma a-tocopherol concentrations after the 20-week feeding trial are shown in Table 3. Liver a-tocopherol concentrations of fish fed diets E 0 and E 20 were significantly lower than that of fish fed diet E 120 Ž P - 0.05.. However, there were no significant differences among fish fed diets E 40 –E 500 Ž P ) 0.05.. The highest level of a-tocopherol supplementation Ždiet E 500 . did not result in an elevated level of liver a-tocopherol compared to the other groups, but did result in higher muscle and plasma a-tocopherol concentrations than those of fish fed the other diets. Muscle a-tocopherol concentration of fish fed diet E 0 was lower than that of fish fed diets E 60 and E 500 Ž P - 0.05.. Baker and Davies Ž1996. observed linear tissue accretion of a-tocopherol in muscle Ž r 2 s 0.83., blood plasma Ž r 2 s 0.93. and liver Ž r 2 s 0.82. of African catfish in response to dietary a-tocopheryl acetate concentrations of 0, 80, 200 and 500 mgrkg

Table 3 Effects of different dietary levels of DL-a-tocopheryl acetate on liver Ž m grg., muscle Ž m grg. and plasma a-tocopherol concentrations Ž m grml. in Korean rockfish for 20 weeks1 Pooled S.E.M.3

Diets

Liver Muscle Plasma 1

E 20

E 20

E 40

E 60

E 120

E 500

9.7 b 3.9 c 2.0 b

11.2 b 5.3 bc 4.4 b

44.0 ab 6.4 bc 2.6 b

59.9 ab 8.0 b 3.5 b

103 a 5.3 bc 3.8 b

93.1ab 27.2 a 14.0 a

12.68 1.94 1.07

Values are means of pooled samples of five fish from each of three replicate groups. Means in each row with a different superscript are significantly different Ž P - 0.05.. 2 a-Tocopherol concentrations of diets E 0 , E 20 , E 40 , E 60 , E 120 and E 500 were 15.9, 37.7, 50.3, 63.5, 114 and 471 mgrkg dry diet. 3 Pooled standard error of mean.

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diet. Similar results were found in this study. Our results showed that tissue accretion of a-tocopherol in muscle Ž r 2 s 0.96. and plasma Ž r 2 s 0.96. increased linearly in response to elevated dietary supplementation of a-tocopheryl acetate within the range of the concentrations fed in this study. In liver tissue, a-tocopherol concentration increased Ž P - 0.05. linearly Ž r 2 s 0.94. as vitamin E intake increased from 0 to 120 mgrkg diet, but then reached a plateau. This finding may suggest that the high level of 500 mg a-tocopheryl acetaterkg diet could have a negative effects on liver a-tocopherol concentration in juvenile Korean rockfish. Liver a-tocopherol concentrations in the present study were similar to those reported for channel catfish ŽBai and Gatlin, 1993. and rainbow trout ŽCowey et al., 1983.. However, muscle and plasma a-tocopherol levels of fish in this study were lower than those of channel catfish ŽBai and Gatlin, 1993.. 3.3. Fatty acid composition Liver and muscle fatty acid compositions are summarized in Table 4. For liver fatty acid composition, monoene and saturated fatty acids were higher Ž P - 0.05. in fish fed diet E 0 than in fish fed diets E 20 –E 500 Ž P - 0.05.. Unsaturated fatty acids, polyunsaturated fatty acids ŽPUFA. and PUFArsaturated fatty acid ratio of fish fed E 0 diet also were lower than those of fish fed the other diets Ž P - 0.05.. For muscle fatty acid composition, fish fed diet E 0 had higher monoene and lower PUFA and PUFArsaturated fatty acid ratio than fish fed the other diets. Watanabe et al. Ž1977. found that the fatty acid composition of muscle and hepatopancreas was the same in carp fed a-tocopherol at more than 300 mg DL-atocopheryl acetaterkg diet. They suggested that a tocopherol deficiency exerts some effects on fatty acid composition, but the tocopherol supplements exceeding a level required by fish had little effect on fatty acid composition. In the present study, similar results were found in Korean rockfish after the 20-week feeding trial. Fish fed diets E 20 –E 500 showed the same monoene, saturated fatty acid, unsaturated fatty acid and PUFA concentrations as well as PUFArsaturated fatty acid ratio. However, fish fed diet E 0 showed significantly lower unsaturated fatty acid, PUFA and PUFArsaturated fatty acid ratio, and higher monoene and saturated fatty acids. These results showed that vitamin E deficiency affected fatty acid composition of Korean rockfish, and fatty acid composition of tissues Žliver and muscle. were not affected by dietary a-tocopherol content once fish had satisfied its minimum requirement. Hamre and Lie Ž1995. observed a tendency toward an increased percentage of polyenes and a decreased percentage of monoenes in the liver of Atlantic salmon in response to lower dietary vitamin E. They indicated that this result was opposite to that found in vitamin E-deficient carp by Watanabe and Takashima Ž1977., and the reason for the change in fatty acid composition was probably due to a reduced feed intake, rather than being a direct effect of vitamin E deficiency. Watanabe and Takashima Ž1977. reported that in most of the tissues of vitamin E deficient carp, the percentage of monoene increased, whereas the percentage of polyene decreased. They found that the most significant change observed was the marked decrease of 18:2 n y 6 level in all the tissues, especially in the triglyceride fraction of plasma, ovaries and muscle. In those tissues, the

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Table 4 Fatty acid composition of liver and muscle lipid in Korean rockfish fed different dietary levels of tocopheryl acetate for 20 weeks Žarea %.1

DL-a-

Pooled SEM 3

Diets E 20

E 20

E 40

E 60

E 120

E 500

LiÕer 18:2 ny6 18:3ny6 18:3ny3 18:4ny3 20:2 ny6 20:3ny6 20:4ny6 20:5ny3 22:6ny3 Monoene SFA4 USFA5 PUFA6 PUFArSFA

1.37 0.11 0.39 0.57 0.25 0.14 0.18 2.43 1.22 68.2 a 25.2 a 74.9 b 6.7 b 0.27 b

14.99 0.45 0.40 0.28 0.42 0.19 0.10 1.14 0.77 59.2 b 22.1b 77.9 a 18.8 a 0.85a

14.19 0.49 0.44 0.40 0.63 0.31 0.19 1.17 0.81 58.8 b 22.6 b 77.4 a 18.6 a 0.82 a

15.17 0.43 0.38 0.26 0.38 0.19 0.11 1.16 0.73 58.6 b 22.6 b 77.4 a 18.8 a 0.84 a

15.11 0.47 0.44 0.31 0.41 0.20 0.13 1.24 0.81 58.5 b 22.4 b 77.6 a 19.1a 0.86 a

13.72 0.44 0.37 0.31 0.49 0.24 0.16 1.08 0.72 59.5 b 23.0 b 77.0 a 17.5a 0.76 a

0.19 0.03 0.02 0.03 0.04 0.02 0.02 0.13 0.04 0.83 0.29 0.29 1.22 0.05

Muscle 18:2 ny6 18:3ny6 18:3ny3 18:4ny3 20:2 ny6 20:3ny6 20:4ny6 20:5ny3 22:6ny3 Monoene SFA4 USFA5 PUFA6 PUFArSFA

2.32 0.13 0.95 1.25 0.20 trace 0.44 8.41 7.43 52.6 a 26.3 ab 73.7 ab 21.1b 0.80 c

18.24 0.30 1.56 0.73 0.67 trace 0.50 5.04 6.40 39.1b 27.5ab 72.5ab 33.4 a 1.22 ab

17.79 0.71 1.53 0.75 1.30 trace 0.60 5.15 6.32 38.0 b 27.8 ab 72.2 ab 34.2 a 1.23 ab

18.41 0.36 1.22 0.64 0.72 0.09 0.44 5.08 5.83 39.1b 28.1ab 71.9 ab 32.8 a 1.20 ab

15.24 0.55 1.58 0.65 1.70 trace 0.65 5.08 6.68 38.1b 29.8 a 70.2 b 32.1a 1.08 b

18.67 0.38 1.06 0.71 0.87 trace 0.36 5.20 6.37 41.9 b 24.5 b 75.5a 33.6 a 1.39 a

1.40 0.08 0.08 0.05 0.23 0.02 0.04 0.29 0.26 1.40 0.69 0.69 1.11 0.05

1

Values are means of pooled samples of five fish from each of three replicate groups. Means in each row with a different superscript are significantly different Ž P - 0.05.. 2 a-Tocopherol concentrations of diets E 0 , E 20 , E 40 , E 60 , E 120 and E 500 were 15.9, 37.7, 50.3, 63.5, 114 and 471 mgrkg dry diet. 3 Pooled standard error of mean. 4 Saturated fatty acids. 5 Unsaturated fatty acids. 6 Polyunsaturated fatty acids.

percentage of 18:2 n y 6 was approximately 14% in all fish fed the complete diet, but 0.2, 1.4, and 1.3% in fish fed the vitamin E-deficient diet. In the present study, similar results were observed. The most significant change observed was the marked decrease in 18:2 n y 6 level in liver and muscle of fish fed diet E 0 . The percentage of 18:2 n y 6 was

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approximately 15 and 18% in liver and muscle of fish fed diets E 20 –E 500 , but 1.4 and 2.3% in liver and muscle of fish fed diet E 0 . In conclusion, broken line regression analysis of percent weight gain indicated that the optimum level of a-tocopherol was 45 mgrkg diet for adequate growth and tissue levels of a-tocopherol in juvenile Korean rockfish.

Acknowledgements This research was supported in part by the funds of Woo Sung Feed and Research Center for Ocean Industrial Development ŽERC designated by KOSEF. at Pukyong National University.

References AOAC ŽAssociation of Official Analytical Chemists., 1995. Official Methods of Analysis, 16th edn., AOAC International, Arlington, VA. Bai, S.C., Gatlin, D.M. III, 1993. Dietary vitamin E concentration and duration of feeding affect tissue a-tocopherol concentrations of channel catfish Ž Ictalurus punctatus .. Aquaculture 113, 129–135. Bai, S.C., Lee, K.J., 1996. Long-term feeding effects of different dietary L-ascorbic acid levels on growth and tissue vitamin C concentrations in juvenile Korean rockfish. Bull. Korean Fish. Soc. 29, 643–650, Žin Korean with English abstract.. Bai, S.C., Lee, K.J., Jang, H.K., 1996. Development of an experimental model for vitamin C requirement study in Korean rockfish, Sebastes schlegeli. J. Aquacult. 9, 169–178, Žin Korean with English abstract.. Baker, R.T.M., Davies, S.J., 1996. Changes in tissue a-tocopherol status and degree of lipid peroxidation with varying a-tocopheryl acetate inclusion in diets for the African catfish. Aquacult. Nutr. 2, 71–79. Brown, B.A., 1980. Routine hematology procedures. In: Hematology: Principles and Procedures. Lea and Febiger, Philadelphia, pp. 71–112. Cowey, C.B., Adron, J.W., Walton, M.J., Murray, J., Youngson, A., Knox, D., 1981. Tissue distribution, uptake, and requirement for a-tocopherol of rainbow trout Ž Salmo gairdneri . fed diets with a minimal content of unsaturated fatty acids. J. Nutr. 111, 1556–1567. Cowey, C.B., Adron, J.W., Youngson, A., 1983. The vitamin E requirement of rainbow trout Ž Salmo gairdneri . given diets containing polyunsaturated fatty acids derived from fish oil. Aquaculture 30, 85–93. Cowey, C.B., Degener, E., Tacon, A.G.J., Youngson, A., Bell, J.G., 1984. The effect of vitamin E and oxidized fish oil on the nutrition of rainbow trout Ž Salmo gairdneri . grown at natural varying water temperature. Br. J. Nutr. 51, 443–451. Folch, J., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509. Garling, D.L. Jr., Wilson, R.P., 1977. Effects of dietary carbohydrate-to-lipid ratios on growth and body composition of fingerling channel catfish. Prog. Fish-Cult. 39, 43–47. Gatlin, D.M. III, Poe, W.E., Wilson, R.P., 1986. Effects of singular and combined dietary deficiencies of selenium and vitamin E on fingerling channel catfish. J. Nutr. 104, 1416–1431. Halver, J.E., 1972. The vitamins. In: Halver, J.E. ŽEd.., Fish Nutrition. Academic Press, New York, pp. 29–103. Hamre, K., Lie, Ø., 1995. Minimum requirement of vitamin E for Atlantic salmon, Salmo salar L., at first feeding. Aquacult. Res. 26, 175–184. Hamre, K., Hjeltnes, B., Kryvi, H., Sandberg, S., Lorentzen, M., Lie, Ø., 1994. Decreased concentration of hemoglobin accumulation of lipid oxidation products and unchanged skeletal muscle in Atlantic salmon Ž Salmo salar . fed low dietary vitamin E. Fish Physiol. Biochem. 12, 421–429.

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