Blackwell Science, LtdOxford, UK FISFisheries Science0919-92682003 Blackwell Science Asia Pty Ltd 696December 2003 744 EPA and

DHA on lipid metabolism AD OM et al. 10.1046/j.0919-9268.2003.00744.x Original Article11821193BEES SGML

FISHERIES SCIENCE

2003; 69: 11821193

Dietary effects of eicosapentaenoic acid and docosahexaenoic acid on lipid metabolism in black sea bream
AHMAD DAUD OM,1 HONG JI,1 Tetsuya UMINO,1 Heisuke NAKAGAWA,1* Toshiyuki SASAKI,2 Kenji OKADA,2 Masaya ASANO3 AND Atsushi NAKAGAWA3 Graduate School of Biosphere Science, Hiroshima University, Higashi-hiroshima, 739-8528, Hiroshima City Marine Products Promotion Association, Shok Center, Hiroshima, Hiroshima 733-0833 and 3Research Laboratories, Aquaculture Center, Kyowa Hakko Kogyo Co. Ltd, Ube, Yamaguchi 755-8501, Japan
2 1

ABSTRACT: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were fortied at a level of 1.5% in a composed diet. The effects were conrmed in 0-year black sea bream Acanthopagrus schlegeli in terms of lipid metabolism and physiological activity. The EPA group was high in EPA in muscle, liver, intraperitoneal fat body (IPF), eye and brain. The levels of DHA in liver, eye, brain and heart were also high in the EPA group, suggesting that conversion of EPA to DHA occurred in those organs. Fortication of DHA increased the levels of DHA in organs except the eye, but did not affect EPA levels. Both the EPA and DHA groups showed smaller adipocytes or lower levels of lipid content than the control group. The starvation followed by feeding experiment caused marked body weight loss in the control group by consumption of muscle protein and lipids in IPF. Fortications of EPA and DHA induced less mobilization of muscle protein and IPF lipids as energy. Liver function and resistance to air-dipping were improved by both EPA and DHA fortications. The present results implied conversion of EPA to DHA in the sh with regard to parameters, such as lipid metabolism and physiological vitality. KEY WORDS: Acanthopagrus schlegeli, black sea bream, eicosapentaenoic acid, lipid accumulation, lipolysis, vitality. INTRODUCTION Although sufcient amounts of eicosapentaenoic acids (EPA) and docosahexaenoic acids (DHA) are supplemented to diets, the levels of these fatty acids in the body of hatchery-reared juvenile black sea bream Acanthopagrus schlegeli are lower than in those of wild sh.1 However, sh released into the natural environment immediately increase their fatty acid proportion to the same level as wild sh within 5 days.1 Therefore, we examined the effects of incorporation of these fatty acids on the physiological condition of sh by fortifying ethyl esters of an EPA-DHA mixture in a composed diet. Accumulation of EPA and DHA in the same proportion as wild sh accompanied improvement of lipid metabolism and vitality.2 This phenomenon implied that the proportion of EPA and DHA required by the juveniles might be higher than the level of essential fatty acids.
*Corresponding author: Tel: 81-824-24-7989. Fax: 81-824-22-7059. Email: heisuke@hiroshima-u.ac.jp Received 14 November 2002. Accepted 5 August 2003.

docosahexaenoic

acid,

Our previous study showed that with the fortication of 3% DHA and EPA mixture in the commercial feed, muscle DHA and EPA levels in cultured juvenile black sea bream reached a similar level to those in wild sh.2 Therefore, we fortied 1.5% DHA and EPA in commercial feed to distinguish their effects on lipid metabolism and vitality. MATERIALS AND METHODS Fish and rearing conditions Juvenile black sea bream produced by Hiroshima City Marine Products Promotion Association were used for feeding experiments. A total of 900 sh (0.2 g, 0.13 cm average) were divided into six indoor berglass reinforced plastic (FRP) conical round tanks (400 L capacity) and each treatment was performed in a duplicate tank. The sh were fed for 60 days. Water temperature and dissolved oxygen were measured daily and ranged between 20.3 and 25.7C and 4.28.3 p.p.m., respectively. Water ow rate in the rearing tanks was maintained

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Table 1 Proximate composition of diets (wet basis) Dietary group Control Moisture (%) Ash (%) Crude protein (%) Lipid (%) 10.3 13.7 54.8 14.5 EPA 7.5 14.1 54.9 16.2 DHA 7.7 13.9 54.9 16.3

Table 2 Fatty acid composition of dietary lipid (%) Dietary group Fatty acid 14:0 15:0 16:0 18:0 20:0 21:0 23:0 16:1n-7 17:1n-8 17:1n-7 18:1n-9 20:1n-9 22:1n-11 22:1n-9 24:1n-9 16:4n-1 18:2n-6 18:3n-3 18:3n-6 18:4n-3 20:2n-6 20:3n-6 20:4n-6 20:3n-3 20:4n-3 20:5n-3 22:2n-6 22:5n-6 22:3n-3 22:5n-3 22:6n-3 Control 3.5 0.3 16.4 19.4 0.1 0.1 0.8 5.0 1.5 0.5 2.5 4.1 1.5 1.0 0.1 0.4 8.8 1.3 0.2 1.2 0.2 0.1 1.0 0.1 0.4 11.4 0.4 0.2 0.3 0.6 13.6 EPA 3.4 0.3 14.4 17.8 0.1 0.1 0.5 4.7 1.4 0.4 2.0 3.6 0.0 0.3 0.6 0.4 7.6 1.2 0.2 1.1 0.2 0.0 0.9 0.1 0.4 23.6 0.3 0.3 0.2 0.7 12.4 DHA 3.5 0.3 14.8 18.2 0.1 0.1 0.5 5.1 1.7 0.4 0.0 3.5 0.7 2.0 0.1 0.4 7.8 1.2 0.4 1.3 0.2 0.0 0.8 0.1 0.4 10.3 0.3 0.2 0.2 0.6 20.1

DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid.

at 4.5 L/min. One hundred sh were used for biological and biochemical analyses. The remaining sh were then starved for an additional 51 days to compare energy consumption during starvation. Water temperature ranged from 23.1 to 25.7C during this period. The other conditions were same as those in feeding trial. Diet The control group was fed a commercial-based diet (Type C; Kyowa Hakko Kogyo Co. Ltd, Ube, Japan). The diets of the EPA group and DHA group were, respectively, fortied with 1.5% EPA and DHA ethyl esters (Bizen Kasei Co. Ltd, Okayama, Japan). The purities of EPA and DHA were 84.4% and 85.4%, respectively. The proximate composition and fatty acid composition of the diets are shown in Tables 1 and 2. Fish were hand-fed the diets two times daily to apparent satiation to minimize wastage. Biological measurements At the end of the experiment, 100 sh in each replicate treatment group were weighed. Fifteen sh chosen at random from each tank were used for biological and morphological measurements. Whole muscle (llet), liver and intraperitoneal fat body (IPF) were weighed. Intestinal length was dened as the length between the pyloric cecum and the anus. Biological parameters (wet basis) were dened as follows: Feed conversion efciency (%) = (body weight gain/diet given) 100 Protein efciency ratio = body weight gain/dietary protein gain Muscle ratio (%) = (muscle weight/body weight) 100 Hepatosomatic index (HSI; %) = (liver weight/body weight) 100 IPF ratio (%) = (IPF weight/body weight) 100 Relative intestine length = intestine length/body length

DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid.

Biochemical measurements Fish from the each treatment group were immediately frozen at - 20C after death until biochemical analyses were carried out. The muscle, liver and IPF were taken from 30 frozen sh from each group and analyzed separately. Crude protein was determined by the Kjeldhal method. The lipids were extracted with methanol-chloroform according to the method of Bligh and Dyer.3 The lipid content in liver was measured using the phospho-vanillin method. After saponication, the fatty acids were converted into their methyl esters by methanolHCl. A Hitachi Gas Chromatograph 263-30 with FID detector (Tokyo, Japan) was used to determine fatty acid composition. A 30-m capillary column packed with Omegawax TM 320 (Supelco Co., Bellefonte, PA, USA) was used. The oven temperature was 200C. Fatty acid methyl esters were identied using authentic standards (Sigma Co. St Louis, MO, USA).

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Table 3 Effect of EPA- and DHA-fortied diets on growth and biological parameters Dietary group n Total diet given (g) Biomass gain (g) Survival (%) Feed conversion efciency (%) Protein efciency ratio Mean body weight (g) Mean total length (mm) Muscle ratio (%) Hepatosomatic index (%) IPF ratio (%) Relative intestine length Adipocyte (mm) Control 906 1310 92.7 143 2.58 10.4 3.5 72 5 34.3 2.0 1.25 0.38a 1.13 0.50 1.52 0.21a 59.8 14.0a EPA 852 1149 94.4 131 2.37 9.3 2.9 70 5 33.9 1.8 0.80 0.16b 0.83 0.36 1.37 0.23b 39.1 11.5b DHA 817 1151 94.0 137 2.50 9.2 3.2 70 6 33.4 2.1 0.93 0.19b 0.93 0.39 1.49 0.20ab 42.5 11.3c

(100) (100) (30) (30) (30) (30) (5)

Initial body weight 0.2 0.0 g. Initial body length 13.1 1.3 mm. DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; IPF, intraperitoneal fat body. Values (mean SD) in the same line with different superscripts are signicantly different (P < 0.05).

Table 4 Effects of EPA- and DHA-fortied diets on the biochemical composition (%) in black sea bream juveniles Dietary group n Muscle Moisture Ash Crude protein Lipid Liver Lipid Glycogen (2) (2) (2) (2) (2) (2) Control 78.2 0.2 1.5 0.1 18.9 0.0 1.6 0.1 12.2 0.6a 0.17 0.10a EPA 78.4 0.2 1.4 0.0 18.9 0.0 1.4 0.1 5.5 0.3b 0.03 0.00b DHA 78.4 0.1 1.5 0.1 17.9 0.0 1.9 0.1 4.5 0.4b 0.02 0.00b

DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid. Values (mean SD) in the same line with different superscripts are signicantly different (P < 0.05).

Liver glycogen extracted by 30% KOH was puried by 95% ethanol and 10% trichloroacetic acid. The content was measured by the anthrone method. Measurement of adipose cell diameter Five sh from each group were xed in Bouins solution. The adipose tissue taken from IPF was embedded in parafn, cut into 10 mm-thick sections and stained with hematoxylin and eosin. The diameter of adipocytes was microscopically measured with 50 cells in ve sh taken from each tank. Liver function and air-dipping test Liver function was checked by the time required to recover from anesthesia with 2-phenoxyethanol according to Hilton and Dixon.4 Twenty sh from
Lipid (g/fish)

1.5 Muscle IPF Control 1

EPA DHA 0.5

0 F S F S F S

Fig. 1 Lipid accumulation in the muscle and intraperitoneal fat body (IPF) of black sea bream juveniles fed eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)-fortied diets (F) and in starved sh (S).

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Table 5 Effect of EPA- and DHA-fortied diets on the fatty acid composition (area %) of the muscle at the end of feeding (Feeding)and after starvation (Starvation) Control Fatty acid 14:0 16:0 18:0 16:1n-7 17:1n-7 18:1n-9 18:1n-7 20:1n-11 20:1n-9 20:1n-7 22:1n-11 22:1n-9 24:1n-9 18:2n-6 18:3n-6 20:2n-6 20:3n-6 20:4n-6 22:2n-6 18:3n-3 18:4n-3 20:3n-3 20:4n-3 20:5n-3 21:5n-3 22:3n-3 22:5n-3 22:6n-3 Saturates Monoenes n-6 n-3 n-3/n-6 Feeding 3.3 0.2 18.5 0.8 4.5 0.2 5.0 0.5 0.4 0.0 11.9 1.1 7.2 2.1 3.2 0.4 0.2 0.0 Tr. 1.2 0.2 0.4 0.5 0.1 0.0 6.7 0.2 0.2 0.0 0.2 0.0 0.1 0.2 0.3 0.0 0.2 0.0 0.7 0.2 0.5 0.1 1.4 0.1 0.4 0.0 7.4 0.3 0.3 0.0 0.6 0.5 2.0 0.1 18.7 2.5 26.5 0.6 29.5 0.4 7.4 5.1 31.9 0.1 4.3 Starvation 1.2 0.2* 19.3 0.4 7.1 0.1* 2.9 0.9 0.4 0.0 11.4 3.0 2.1 2.1* 1.8 0.4 0.1 0.1 Tr. 0.4 0.2* 0.0 0.1 0.5 0.6 6.6 0.3 0.3 0.0 0.3 0.0 0.4 0.0 0.2 0.0 0.1 0.0 0.2 0.0 0.1 0.0 4.3 0.1* 0.4 0.0 7.6 0.2 0.8 0.0 0.4 0.5 2.0 0.2 25.9 0.5* 27.8 1.7 19.5 0.4* 7.6 5.1 41.8 0.7* 5.5 Feeding 3.5 0.2 17.3 0.1 4.1 0.3 5.2 1.1 0.4 0.1 11.8 2.9 6.6 2.3 2.8 0.3 0.4 0.3 0.2 0.0 1.4 0.1 0.2 0.0 0.6 0.5 6.4 0.1 0.3 0.0 0.2 0.0 1.2 0.0 0.2 0.0 0.2 0.0 0.7 0.1 0.6 0.1 0.1 0.0 0.3 0.1 10.7 0.2 0.4 0.1 0.3 0.0 2.4 0.3 16.3 0.8 25.1 0.2 29.5 0.1 8.2 0.2 31.9 0.6 3.9 EPA Starvation 2.7 0.9 18.4 0.4 5.5 0.8 4.4 0.2 0.4 0.1 13.7 0.2 3.2 0.5 2.9 0.7 0.1 0.1 0.2 0.1 0.2 0.3 0.1 0.1 Tr. 5.8 0.1 0.2 0.0 1.4 2.0 0.6 0.8 0.2 0.0 Tr. 0.4 0.3 0.3 0.2 0.3 0.0 0.9 0.1 4.1 1.5* 1.4 0.0 0.3 0.1 1.0 1.3 24.9 1.0* 26.7 0.9 25.0 1.5* 8.0 1.7 33.5 3.0 4.2 Feeding 2.8 1.1 16.5 0.6 4.5 1.6 4.7 1.6 0.4 0.1 9.2 0.2 7.7 2.6 2.7 0.5 0.2 0.0 0.2 0.0 1.0 0.4 0.4 0.2 0.9 0.0 6.2 0.5 0.2 0.0 0.2 0.1 0.9 0.0 0.2 0.0 0.1 0.1 0.5 0.2 0.4 0.2 1.3 1.6 0.3 0.0 5.6 0.2 0.3 0.2 0.3 0.3 1.5 0.1 27.1 2.3 23.9 0.9 27.4 1.5 7.6 1.7 37.4 3.0 4.9 DHA Starvation 2.7 0.9 18.4 0.4* 5.5 0.8 4.4 0.2 0.4 0.1 13.7 0.2* 3.2 0.5 3.0 0.7 0.1 0.1 0.2 0.0 0.2 0.3 0.1 0.1 Tr. 5.8 0.1 0.2 0.0 1.4 2.0 0.6 0.8 0.1 0.0 Tr. 0.4 0.3 0.3 0.2 0.3 0.0 0.9 1.5 4.1 1.5 0.4 0.1 0.3 0.1 1.5 1.3 24.8 1.0 26.7 0.9 25.0 1.5 8.0 1.7 33.5 3.0 4.2

n = 2. *Signicantly different from the value after feeding (P < 0.05). Signicantly different from the value of the control group (P < 0.05). DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; Tr., trace.

each group were dipped for 50 s in water containing the anesthetic at a concentration of 0.1%. The sh were transferred to oxygen-saturated fresh seawater and recovery time of individual sh from anesthetic condition was recorded. For the air-dipping test, 20 sh from each group were exposed to air-dipping for 5 min and returned to oxygen-saturated seawater. The recovery time from the succumbed condition was recorded. The water temperature during the treatment was 25.3C. Statistical analysis The data for growth, feed utilization, biological and biochemical parameters were examined by one-

way ANOVA and Fishers Protected Least Signicant Difference test for multiple range tests. Probabilities of 0.05 or less were considered statistically signicant. RESULTS Fortication of EPA and DHA elevated the lipid content and, consequently, decreased the moisture content in diets (Table 1). Fortication with EPA and DHA did not affect feeding activity. The effects of dietary EPA and DHA fortication on growth and biological parameters are shown in Table 3. The amounts of food given to the EPA group and DHA group were not signicantly different among the groups. The EPA- and DHA-forti-

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Table 6 Effect of EPA- and DHA-fortied diets on the fatty acid composition (area %) of the liver at the end of feeding (Feeding)and after starvation (Starvation) Control Fatty acid 14:0 16:0 18:0 16:1n-7 17:1n-7 18:1n-9 18:1n-7 20:1n-11 20:1n-9 20:1n-7 22:1n-11 22:1n-9 24:1n-9 18:2n-6 18:3n-6 20:2n-6 20:3n-6 20:4n-6 22:2n-6 18:3n-3 18:4n-3 20:4n-3 20:5n-3 21:5n-3 22:3n-3 22:5n-3 22:6n-3 Saturates Monoenes n-6 n-3 n-3/n-6 Feeding 4.1 0.6 25.5 1.0 9.0 0.2 0.3 0.1 5.3 0.7 0.3 0.1 10.5 0.5 11.1 1.1 3.8 0.2 0.4 0.0 0.4 0.0 1.4 0.6 0.3 0.1 4.3 0.4 0.3 0.1 0.3 0.0 0.7 0.1 0.3 0.0 0.1 0.0 0.4 0.0 0.2 0.0 0.4 0.0 2.6 0.2 0.1 0.1 0.3 0.1 1.8 0.8 8.3 2.4 38.9 1.7 34.8 0.3 5.6 2.1 14.2 0.1 2.5 Starvation 2.8 1.8* 21.7 1.8* 9.3 0.3 0.4 0.0 3.1 0.7* 0.4 0.0 8.2 2.7 4.2 0.3* 2.1 1.1 0.1 0.0 0.4 0.0 0.7 0.5 Tr. 3.2 0.4 0.2 0.1 0.2 0.1 2.6 1.6 0.2 0.0 0.1 0.0 0.2 0.1 0.2 0.0 0.2 0.0 2.8 1.4 0.4 0.2 1.1 0.5 1.8 0.1 29.3 0.3* 34.2 1.7 19.9 0.3* 6.4 0.9 36.0 0.1* 5.6 Feeding 3.8 0.4 20.7 0.3 5.2 0.2 0.4 0.1 4.9 0.5 0.3 0.0 12.6 0.3 2.9 0.0 2.9 0.2 0.5 0.5 0.3 0.1 1.8 0.6 Tr. 5.7 1.1 0.3 0.0 0.1 0.1 1.1 0.1 0.2 0.0 0.1 0.1 0.6 0.1 0.4 0.1 0.4 0.0 7.9 2.2 0.2 0.0 0.2 0.1 3.5 0.0 17.0 1.5 29.8 0.1 27.3 0.2 7.3 0.2 30.0 0.2 4.1 EPA Starvation 2.3 0.9 19.3 3.6 10.0 2.0* 0.4 0.0 2.9 0.1 0.7 0.1 7.2 1.7* 5.3 0.6 1.7 0.8 Tr. 0.3 0.1 0.5 0.4 0.2 0.1 3.3 0.1 0.3 0.1 0.2 0.0 3.0 1.3 0.2 0.0 Tr. 0.2 0.0 0.1 0.2 0.4 0.0 4.7 2.6* 0.4 0.0 1.5 0.3 2.4 0.3 29.7 0.7* 32.7 1.1 19.4 0.9* 6.7 1.3 39.3 0.7* 5.9 Feeding 4.5 0.3 17.9 0.4 4.6 0.2 0.4 0.0 4.8 0.2 0.4 0.1 11.3 1.2 4.5 0.5 2.8 0.3 0.1 0.4 0.3 0.0 1.4 0.1 0.4 0.4 5.3 0.3 0.3 0.0 0.2 0.0 1.0 0.1 0.3 0.0 Tr. 0.8 0.3 0.4 0.1 0.4 0.0 4.5 0.3 0.2 0.0 0.3 0.1 2.0 0.3 25.2 2.6 27.2 0.2 26.8 1.1 6.9 0.9 33.9 2.3 4.9 DHA Starvation 4.0 1.8 19.1 1.6 6.2 1.4 0.4 0.0 3.8 0.6 0.4 0.0 10.3 2.1 4.4 1.0 2.5 0.3 Tr. 0.3 0.0 0.8 0.4 0.6 0.0. 4.1 0.8 0.2 0.0 0.1 0.1 1.8 0.6 0.1 0.0 0.1 0.1 0.3 0.1 0.2 0.2 0.3 0.2 3.0 0.8 0.2 0.1 0.5 0.3 1.2 0.6 32.4 2.1* 29.4 0.9 23.3 1.1 6.4 1.0 28.1 1.3* 6.0

n = 2. *Signicantly different from the value after feeding (P < 0.05). Signicantly different from the value of the control group (P < 0.05). DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; Tr., trace.

ed diets given did not affect sh size. Relative intestine length was signicantly lower in the EPA group than in the control group. Hepatosomatic index was signicantly lower in the EPA and DHA groups. The fortication of EPA and DHA slightly depressed the IPF ratio and markedly reduced adipocyte diameter. Although the proximate composition of muscle was not signicantly different among the groups, liver lipid and glycogen were signicantly lower in both the EPA and DHA groups (Table 4). Reserved lipids in muscle and IPF of fed and starved sh were depressed both in EPA and DHA groups as shown in Fig. 1. Fortication of EPA and DHA markedly depressed lipid accumulation in muscle, liver and IPF. Liver glycogen was also depressed by the fortication (Table 4).

Tables 510 show the fatty acid composition of various organs after feeding. While dietary EPA and DHA were incorporated in the muscle and liver, the fatty acid composition of the brain was affected by the dietary fatty acids. In addition, fortication of EPA enhanced DHA accumulation in the liver, eye and brain, but dietary DHA was scarcely incorporated in liver. Effect of starvation was compared on mortality, body weight and proximate composition (Fig. 2). The mortalities of the control, EPA and DHA groups after 51 days starvation were 40%, 30% and 22%, respectively, showing that EPA- and DHA-fortied diets could suppress the mortality during starvation. The starved sh showed a resultant loss in liver weight and intestine length and IPF, but the muscle ratio was almost con-

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Table 7 Effect of EPA- and DHA-fortied diets on the fatty acid composition (area %) of the intraperitoneal fat at the end of feeding (Feeding) and after starvation (Starvation) Control Fatty acid 14:0 16:0 18:0 16:1n-7 17:1n-7 18:1n-9 18:1n-7 20:1n-11 20:1n-9 20:1n-7 22:1n-11 22:1n-9 24:1n-9 18:2n-6 18:3n-6 20:2n-6 20:3n-6 20:4n-6 22:2n-6 18:3n-3 18:4n-3 20:3n-3 20:4n-3 20:5n-3 21:5n-3 22:3n-3 22:5n-3 22:6n-3 Saturates Monoenes n-6 n-3 n-3/n-6 Feeding 5.6 0.2 18.5 0.7 3.4 0.1 0.5 0.0 7.4 0.3 0.5 0.0 16.8 0.8 6.5 0.9 4.1 0.9 Tr. 0.2 0.0 1.9 0.7 0.3 0.0 8.5 0.0 0.3 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.3 0.0 1.0 0.0 0.8 0.1 0.6 0.1 0.4 0.0 5.0 0.2 0.2 0.0 0.2 0.0 1.4 0.0 10.7 0.6 27.6 1.7 39.0 0.3 9.3 1.1 20.2 0.1 2.2 Starvation 1.7 0.1* 19.3 0.7 11.7 0.8* 0.3 0.0 3.4 0.1* 0.3 0.0 13.6 1.2 2.4 0.0* 1.7 0.2 Tr. 0.2 0.0 0.6 0.1 Tr. 4.6 0.3* 0.3 0.0 0.3 0.0 4.6 0.3 0.2 0.0 Tr. 0.1 0.1 0.1 0.0 Tr 0.1 0.0 5.0 1.0 0.7 0.1 0.4 0.1 2.0 0.6 22.9 0.5* 33.0 1.7* 22.7 0.3* 9.8 0.6 31.3 0.1* 3.2 Feeding 5.1 0.0 16.3 0.2 1.9 1.0 0.4 0.0 6.6 0.4 0.5 0.0 18.5 1.0 3.0 0.5 4.0 0.1 Tr. 0.4 0.2 2.1 0.1 0.4 0.1 8.4 0.3 0.3 0.0 0.2 0.0 0.1 0.0 0.3 0.0 0.3 0.0 1.0 0.1 0.9 0.1 0.6 0.0 0.5 0.0 1.2 0.2 0.2 0.0 0.1 0.1 1.8 0.3 10.5 0.6 23.4 0.8 36.2 0.5 9.3 0.2 26.7 0.6 2.9 EPA Starvation 1.9 0.0* 19.2 0.0 10.9 0.2* 0.3 0.0 3.4 0.0* 0.3 0.0 10.9 0.4* 5.1 0.8 1.8 0.0* Tr. 0.2 0.0 0.6 0.0 0.3 0.3 4.5 0.1* 0.3 0.0 0.2 0.0 4.2 0.1* 0.2 0.0 Tr. 0.1 0.0 0.1 0.0 Tr. 0.2 0.0 5.6 0.1* 0.7 0.0 0.4 0.1 2.6 0.1 23.3 0.2* 32.1 1.5* 23.3 0.9* 9.1 0.2 32.4 1.6* 3.6 Feeding 5.1 0.4 16.5 0.7 3.4 0.0 0.4 0.1 6.3 0.1 0.5 0.0 17.4 0.1 3.4 0.3 4.5 0.6 Tr. 0.2 0.0 3.5 0.8 0.3 0.1 8.5 0.2 0.3 0.0 0.2 0.0 0.5 0.0 0.4 0.0 0.3 0.0 1.1 0.0 0.8 0.1 0.1 0.0 0.4 0.0 4.5 0.5 0.2 0.0 0.1 0.1 1.4 0.1 14.2 0.2 25.1 0.5 38.8 1.3 9.6 1.2 22.8 0.5 2.4 DHA Starvation 3.6 0.1* 10.7 0.3* 6.4 0.1* 8.4 0.3* 2.6 2.2* 0.4 0.0 14.0 0.1* 2.5 0.2* 4.1 0.0 Tr. 0.2 0.0 1.8 0.0* 0.1 0.0. 3.9 1.8* 0.3 0.0 0.2 0.0 2.6 0.1 0.2 0.0 0.1 0.0 0.4 0.0* 0.4 0.0 Tr. 0.1 0.1 3.6 0.0 0.4 0.0 0.2 0.0 1.4 0.3 26.2 1.8* 20.9 0.2* 34.3 1.2 7.1 2.0 32.7 3.0* 4.6

n = 2. *Signicantly different from the value after feeding (P < 0.05). Signicantly different from the value of the control group (P < 0.05). DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; Tr., trace.

stant. Whole muscle protein loss calculated from the muscle ratio and protein percentage was higher in the control group (0.68 g/sh) than in the EPA (0.57 g/sh) and DHA groups (0.58 g/ sh). Nevertheless, starvation resulted in relatively high muscle protein loss in the control group rather than in the EPA and DHA groups. Decreasing rate of muscle protein and IPF lipid during starvation was lower in the EPA and DHA groups in comparison with the control group. Consequently, both EPA- and DHA-fortications suppressed body weight loss during starvation. Fortication of DHA signicantly suppressed shrinkage of the intestine length by starvation in the DHA group, compared with the EPA and control groups.

Liver function evaluated from the recovery time from the anesthetic condition is shown in Fig. 3. The DHA group and the EPA group seemed recover from anesthesia earlier than the control group. The 50% recovery time of the DHA group and EPA group were, respectively, 85 and 95 s, whereas the control group recovered in 120 s. Figure 4 shows recovery in the air-dipping test. The dietary EPA and DHA markedly reduced the impact of airdipping. During treatment, 30% of the sh died in the control group, while the other groups recovered completely. Tables 510 show the composition of fatty acid after starvation for 51 days. Starvation did not affect EPA in the control group, but signicantly reduced the value in muscle and liver of the EPA

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Table 8 Effect of EPA- and DHA-fortied diets on the fatty acid composition (area %) of the heart at the end of feeding (Feeding) and after starvation (Starvation) Control Fatty acid 14:0 16:0 18:0 16:1n-7 17:1n-7 18:1n-9 18:1n-7 20:1n-11 20:1n-9 20:1n-7 22:1n-11 22:1n-9 24:1n-9 18:2n-6 18:3n-6 20:2n-6 20:3n-6 20:4n-6 22:2n-6 18:3n-3 18:4n-3 20:3n-3 20:4n-3 20:5n-3 21:5n-3 22:3n-3 22:5n-3 22:6n-3 Saturates Monoenes n-6 n-3 n-3/n-6 Feeding 3.4 0.4 28.6 0.2 9.5 1.5 0.4 0.1 5.2 0.5 0.4 0.0 14.3 1.2 6.4 1.0 2.6 0.2 Tr. 0.1 0.0 0.9 0.1 Tr. 5.0 0.8 0.2 0.1 0.3 0.1 0.2 0.1 0.4 0.0 Tr. 0.3 0.0 0.1 0.0 2.0 0.1 0.1 0.0 4.0 1.0 0.2 0.0 Tr. 0.7 0.1 9.4 2.4 27.6 1.7 39.0 0.3 9.3 2.1 20.2 0.1 2.2 Starvation 1.8 0.7* 20.0 0.6* 8.9 0.3 0.3 0.1 3.2 0.9* 0.3 0.1 10.5 0.7 5.3 0.2 2.5 0.6 Tr. 0.1 0.0 0.7 0.1 0.1 0.0 4.5 0.4 0.3 0.1 0.3 0.0 0.3 0.1 0.2 0.0 0.5 0.0 0.3 0.0 0.2 0.0 5.0 1.8* 0.2 0.0 4.3 0.4 0.6 0.4 1.0 0.0 1.7 0.2 26.8 0.8* 30.9 1.7 23.2 0.3* 5.7 0.3* 39.9 0.1* 7.0 Feeding 3.4 0.7 26.6 2.2 9.0 0.4 0.4 0.0 4.8 0.4 0.3 0.0 13.3 1.8 4.5 1.0 1.9 0.2 Tr. 0.1 0.0 1.0 0.5 Tr. 4.7 0.4 0.2 0.0 0.2 0.0 0.1 0.0 0.3 0.0 0.1 0.0 0.3 0.0 0.2 0.1 2.1 0.7 0.1 0.0 5.9 1.6 0.3 0.1 Tr 1.2 0.0 12.0 0.7 39.1 0.1* 26.7 0.5 5.4 0.8 27.4 0.4 5.1 EPA Starvation 1.3 0.1* 20.2 2.5* 10.1 1.0* 0.3 0.0 2.9 0.2 0.2 0.1 9.1 1.3 5.1 0.3 1.4 0.3 Tr. 0.2 0.1 0.2 0.0 0.3 0.0 4.0 0.2 0.3 0.1 0.2 0.0 0.2 0.1 0.3 0.0 0.4 0.0 0.2 0.0 0.2 0.0 4.6 0.2* 0.2 0.0 6.3 0.5 0.6 0.5 Tr. 1.7 0.1 29.0 5.2* 31.8 0.6* 19.8 0.9* 5.0 0.3 42.2 2.3* 8.4 Feeding 3.6 0.8 24.4 1.3 7.5 1.3 0.4 0.0 5.3 0.9 0.4 0.0 14.3 3.7 5.4 2.0 2.9 1.1 Tr. 0.1 0.0 0.8 0.3 0.4 0.4 5.9 0.9 0.2 0.0 0.2 0.0 0.1 0.0 0.4 0.0 0.1 0.0 0.5 0.3 0.4 0.2 1.9 0.7 0.2 0.1 4.6 0.0 0.2 0.1 Tr. 0.8 0.3 13.8 0.3 35.1 0.4* 30.3 1.5 6.5 1.2 22.3 1.8* 3.4 DHA Starvation 1.6 0.2* 23.1 1.9 10.9 0.2* 0.4 0.1* 3.5 1.1* 0.3 0.1 10.5 0.5* 5.6 0.6 1.9 0.1 Tr. 0.1 0.0 0.3 0.0 0.3 0.0 4.2 0.1 0.3 0.0 0.2 0.1 0.2 0.0 0.6 0.0 0.2 0.0 0.1 0.0 0.1 0.1 4.3 0.6* 0.2 0.0 3.2 0.4* 0.3 0.3 0.4 0.0 0.7 0.2 24.0 3.3* 35.9 1.0 23.0 1.8* 5.2 1.2 33.3 3.1* 6.4

n = 2. *Signicantly different from the value after feeding (P < 0.05). Signicantly different from the value of the control group (P < 0.05). DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; Tr., trace.

and the DHA groups. The proportion of DHA became greater than 20% in the organs other than brain after starvation in all treatment groups. Monoenes in the muscle, liver and IPF were preferentially consumed. The saturated and n-3 fatty acids were relatively increased in the liver and IPF by the starvation. DISCUSSION The requirement of n-3 highly unsaturated fatty acids (HUFA) is about 0.4% for larval red sea bream Pagrus major5 and around 1.8% for juvenile striped jack Longirostris delicatissimus.6 Takeuchi et al.7 estimated the requirements of EPA and DHA were

1% and 0.5% in the diet of juvenile red sea bream, respectively. The fortication of 1.5% EPA and DHA increased their levels in muscle, which were similar to those of wild sh.2 Not only EPA, but also DHA fortication signicantly depressed reserved lipids in the present study. Low reserved lipids were mainly due to reduction of IPF lipids that was accompanied by a reduction in the adipocyte diameter. The growth of adipose tissue is explained from the increase in both the size and number of adipose cells.8 Adipocyte diameter might be a useful indicator to evaluate lipolysis activity.9,10 The present results showed that adipocyte diameter in the EPA group was signicantly smaller than in the DHA group, which was also signicantly smaller than in the control

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Table 9 Effect of EPA- and DHA-fortied diets on the fatty acid composition (area %) of the brain at the end of feeding (Feeding) and after starvation (Starvation) Control Fatty acid 14:0 16:0 18:0 16:1n-7 17:1n-7 18:1n-9 18:1n-7 20:1n-11 20:1n-9 22:1n-11 22:1n-9 24:1n-9 18:2n-6 20:2n-6 20:3n-6 20:4n-6 22:2n-6 18:3n-3 18:4n-3 20:3n-3 20:4n-3 20:5n-3 21:5n-3 22:3n-3 22:5n-3 22:6n-3 Saturates Monoenes n-6 n-3 n-3/n-6 Feeding 2.4 0.7 22.9 1.5 10.1 1.1 0.3 0.1 7.0 1.3 0.6 0.2 22.4 1.5 2.6 0.0 2.3 0.2 0.2 0.0 0.1 0.1 0.8 0.2 2.2 1.9 0.1 0.0 0.1 0.0 0.5 0.0 0.2 0.0 0.2 0.1 0.3 0.2 0.8 0.2 0.2 0.1 3.0 1.3 0.1 0.0 2.5 0.7 1.0 0.3 14.1 4.3 35.9 1.7 36.6 0.3 2.5 2.0 21.9 0.1 8.7 Starvation 0.8 0.0* 19.3 1.4 12.7 1.2 0.2 0.0 4.4 0.8* 0.4 0.0 24.5 0.2 1.8 0.0 1.1 0.1 0.1 0.2 0.2 0.0 0.1 0.0 1.1 0.9 0.1 0.0 0.2 0.0 0.2 0.0 Tr. 0.1 0.0 0.1 0.0 1.6 0.1 0.3 0.0 3.0 0.2 0.1 0.0 1.9 0.0 1.1 0.1 19.8 1.2* 32.9 1.7 34.0 0.3 1.4 0.2 27.9 0.1* 20.0 Feeding 2.1 0.6 17.6 0.2 11.0 0.9 0.3 0.0 6.1 1.5 0.4 0.2 19.2 0.7 4.3 0.0 1.3 0.9 0.2 0.0 0.4 0.0 1.1 0.0 1.1 0.7 0.2 0.1 0.4 0.4 0.5 0.0 Tr. 0.3 0.1 0.1 0.1 1.0 0.0 0.1 0.0 5.1 2.3 0.6 0.3 1.4 0.0 1.2 0.3 25.4 0.5 30.7 1.2 33.8 0.9 1.7 0.5 35.1 2.1 20.7 EPA Starvation 0.5 0.4 20.1 1.1* 9.1 1.8 0.3 0.0 4.5 1.1 0.3 0.1 26.4 0.3* 1.4 0.0 0.8 0.2 0.2 0.1 0.1 0.0 0.1 0.2* 0.8 0.2 0.1 0.0 0.2 0.2 0.3 0.0 0.6 0.6 0.1 0.0 0.1 0.0 1.6 0.0 0.5 0.2 3.7 0.3* 0.3 0.0 2.0 0.0 1.2 0.2 18.1 0.8* 29.8 0.4* 35.0 0.4 1.7 0.1 27.6 0.9* 16.2 Feeding 2.8 1.3 16.2 1.0 8.9 0.3 0.3 0.1 10.0 0.1 0.6 0.4 23.0 4.4 4.5 0.0 2.6 0.8 0.1 0.0 0.3 0.2 1.7 0.0 0.7 1.6 0.1 0.0 Tr. 0.6 0.0 0.2 0.1 0.2 0.1 0.1 0.1 0.6 0.3 0.2 0.1 1.8 0.5 0.1 0.0 1.3 0.0 0.5 0.1 22.3 0.1 28.0 1.1 43.7 1.0 1.9 0.9 27.1 1.1 14.2 DHA Starvation 1.8 0.5 21.0 1.8* 9.8 0.2 0.3 0.0 6.3 0.6* 0.4 0.1 28.1 1.0* 3.2 0.0 1.1 0.1* 0.1 0.0 0.2 0.1 Tr. 1.4 1.3 0.1 0.0 0.2 0.0 0.3 0.0 1.3 0.0 0.2 0.1 0.5 0.4 1.5 0.5 0.2 0.0 2.8 0.8 0.2 0.0 0.7 0.0 1.0 0.2 16.8 1.0* 32.7 0.8 40.6 1.1* 3.0 1.1 24.1 1.3* 8.0

n = 2. *Signicantly different from the value after feeding (P < 0.05). Signicantly different from the value of the control group (P < 0.05). DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; Tr., trace.

group, suggesting that dietary EPA and DHA suppressed adipocyte formation and lipid accumulation. In mammals, n-3 HUFA accelerates lipolysis activity.11 Suppression of body weight loss in EPA and DHA groups would be due to sparing the consumption of muscle protein and lipid reserves. While the body weight loss of the control group was 24% by starvation, the values of EPA and DHA groups were no greater than 15%. As body weight loss is generally caused by preferential consumption of muscle protein as an energy source during starvation, the body weight loss could be suppressed by effective consumption of reserved lipids and a sparing effect of muscle protein, as seen in the EPA and DHA groups. Nevertheless, the effects could not be differentiated with regard to lipid metabolism between the EPA

and DHA groups. Lipolysis activity might be closely connected with conformation of the triglyceride molecule and fatty acid composition of reserved lipids.12 The lipolysis might generally change the fatty acid composition because of preferential consumption of specic fatty acids. Monoenes appeared to be preferentially consumed as an energy source during dietary energy shortage. Investigations have suggested that DHA is related to the development of the nervous system including brain and retina in larval and juvenile sh.1315 The nding of the present study that dietary DHA was primarily incorporated in the brain was conrmed elsewhere.13 Accumulation of DHA in the brain is very important in the function of the nervous system in sh. In yellowtail Seriola quinqueradiata, the brain was involved in the

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Table 10 Effect of EPA- and DHA-fortied diets on the fatty acid composition (area %) of the eye at the end of feeding (Feeding) and after starvation (Starvation) Control Fatty acid 14:0 16:0 18:0 16:1n-7 17:1n-7 18:1n-9 18:1n-7 20:1n-11 20:1n-9 20:1n-7 22:1n-11 22:1n-9 24:1n-9 18:2n-6 18:3n-6 20:2n-6 20:3n-6 20:4n-6 22:2n-6 18:3n-3 18:4n-3 20:3n-3 20:4n-3 20:5n-3 21:5n-3 22:3n-3 22:5n-3 22:6n-3 Saturates Monoenes n-6 n-3 n-3/n-6 Feeding 4.2 1.1 23.0 2.7 6.7 0.1 0.4 0.1 7.4 0.4 0.5 0.0 20.2 3.6 5.1 0.2 2.8 1.6 1.1 0.7 0.1 0.0 1.4 0.2 0.6 0.0 5.8 0.8 0.5 0.1 0.1 0.0 0.1 0.0 0.4 0.2 0.3 0.0 0.1 0.1 0.3 0.1 0.7 0.0 0.1 0.0 2.9 1.6 0.1 0.0 1.6 1.5 0.7 0.4 15.3 2.1 34.1 1.7 39.8 0.4 6.9 1.1 21.7 0.1 3.1 Starvation 0.8 0.1* 15.0 0.6* 9.8 0.5 0.1 0.0 3.3 0.3* 0.3 0.1 13.3 0.0* 3.2 0.0* 0.9 0.1 0.4 0.0 0.2 0.1 0.1 0.1 0.1 0.0 1.5 0.2* 0.1 0.0 0.1 0.0 0.1 0.0 0.5 0.1 0.1 0.0 0.1 0.0 0.1 0.0 5.1 0.2* 0.1 0.1 1.7 0.0 0.1 0.0 1.0 0.6 0.7 0.7 37.6 3.7* 25.9 1.7* 22.7 0.4* 2.1 0.2* 46.4 0.1* 22.1 Feeding 3.1 0.5 16.9 1.2 6.2 0.6 0.3 0.0 6.3 1.4 0.4 0.0 16.8 3.1 3.3 0.0 2.8 0.8 0.3 0.0 0.2 0.0 1.0 0.2 0.4 0.0 4.9 1.3 0.3 0.1 0.1 0.1 0.1 0.1 0.1 0.5 0.2 0.0 0.5 0.3 0.4 0.4 0.8 0.1 0.2 0.2 8.9 2.0 0.1 0.1 0.8 0.7 1.6 0.2 20.3 1.4 26.3 0.2 32.1 0.1 5.8 0.2 33.7 0.6 5.8 EPA Starvation 1.8 0.4* 15.2 1.0 8.8 1.8 0.2 0.0 4.3 0.1 0.3 0.1 15.4 1.3 3.1 0.4 1.8 0.3 0.4 0.0 0.3 0.1 0.3 0.2 0.3 0.0 2.9 0.9* 0.2 0.0 0.1 0.0 0.2 0.1 0.2 0.0 1.0 1.0 0.3 0.1 0.3 0.1 0.8 0.0 0.2 0.2 4.3 1.3* 0.2 0.1 0.5 0.1 1.7 0.7 30.1 1.0* 25.9 0.2 27.0 0.1* 5.1 0.2 38.1 0.6* 7.5 DHA Feeding 4.4 0.6 19.6 3.4 3.3 0.8 0.4 0.1 6.2 0.7 0.5 0.0 20.2 2.1 3.2 1.5 3.5 0.3 0.4 0.0 0.1 0.0 1.6 0.3 0.6 0.0 5.5 0.8 0.5 0.3 0.2 0.0 0.1 0.2 0.3 0.0 0.4 0.0 0.6 0.0 0.2 0.2 0.6 0.7 0.1 0.0 4.2 2.0 0.1 0.0 1.7 0.1 0.9 0.5 13.9 0.3 27.4 0.9 36.7 1.5 6.8 1.7 22.3 3.0 3.3 Starvation 1.1 0.4* 20.0 1.4 9.3 0.3* 0.2 0.0 3.8 0.5* 0.3 0.0 17.0 0.7* 2.6 0.0 1.2 0.4 0.4 0.1 0.2 0.1 0.5 0.3 0.4 0.0 2.0 0.7* 0.2 0.1 0.1 0.0 0.1 0.0 0.1 0.1 0.1 0.0 0.1 0.0 0.1 0.0 0.6 0.4 Tr. 2.1 0.2* 0.1 0.0 0.4 0.1 0.5 0.4 40.0 4.0* 24.5 0.9 26.8 1.5* 3.1 1.7* 43.9 2.1* 14.2

n = 2. *Signicantly different from the value after feeding (P < 0.05). Signicantly different from the value of the control group (P < 0.05). DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; Tr., trace.

myelination of neurocytes and the construction of synapses.15 High dietary n-3 HUFA improved stress response and survival in red sea bream Pagrus major,16 Japanese ounder Paralichthys olivaceus,17 yellowtail,18 Pacic threadn Polydactylus sexlis19 and striped jack Pseudcaranx dentax.20 The fatty acid prole showed that EPA fortication increased DHA in the liver, eye and brain. Furthermore, it was noteworthy that DHA in the brain and eye of the EPA group was superior to that of the organs of the DHA group. Nevertheless, DHA fortication did not affect the EPA levels in the organs except for the liver. Vitality parameters, such as liver function and airdipping resistance, were also improved by DHA

fortication, rather than EPA fortication. While EPA incorporated in the sh body appeared to be converted to DHA, preferential absorption of dietary DHA could not be disregarded. Fortication of either EPA or DHA gave the same tendency in lipid metabolism and vitality. These phenomena implied a bioconversion of EPA to DHA in black sea bream. Some shes (plaice larvae and Japanese ounder) do not require DHA if an adequate quantity of EPA is present.14,21 Dietary DHA is superior to EPA in improving sh vitality.1418 Watanabe et al.16 found partial conversion of EPA to 22:5n-3 and no retroconversion from DHA to 22:5n-3 in red sea bream. The improvement in the liver function and airdipping resistance by dietary EPA and DHA

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80
Control EPA DHA a

100

DHA

EPA

60
b a b b b b a a b b b b b

% 40

75

Control

20

Recovery (%)

0
Mortality Body weight loss HSI loss RIL loss Muscle IPF ratio loss protein loss

50

Fig. 2 Inuence of starvation on biological parameters of black sea bream juveniles fed eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)-fortied diets. Loss by starvation was calculated from the values before and after starvation in an individual. HSI, hepatosomatic index; IPF, intraperitoneal fat body; RIL, relative intestine length. Different letters on the bar represent signicant differences (P < 0.05).

25

0 0 50 100 Time (s) 150 200

100

EPA Control

Fig. 4 Effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) fortication on recovery time after the air-dipping test. Different letters on the graph represent signicant differences (P < 0.05).

75 DHA Recovery (%)

50

25

0 0 50 100 150 200 250 Time (s)

Fig. 3 Effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) fortication on recovery after anesthesia with 2-phenoxyethanol.

might be partly explained by the phenomena such as bioconversion of EPA to DHA and/or superiority of DHA to EPA. While liver is highly sensitive to nutritional status, the fortication of EPA and DHA decreased HSI, hepatic triglycerides and glycogen. Takeuchi et al.22 showed higher HSI as a gen-

eral symptom in dietary essential fatty aciddecient sh, which was related to high liver lipids levels. The present study showed low HSI and low liver lipid levels by fortication of EPA and DHA. Liver glycogen was also lowered by fortication of EPA and DHA. Rustan et al.23 found low glycogen accumulation in the livers of rats fed with n-3 HUFA. Gilthead seabream larvae fed n-3 HUFA-decient diets were reported to have smaller hepatocytes and higher lipid content than those fed a control diet.24 Fujii and Yone25 also reported that dietary n-3 HUFA brought about a decrease in liver lipid content and an increase in the liver glycogen in red sea bream. Low glycogen and low triglycerides in the liver in the EPA group and the DHA group suggested a specic interaction between lipid metabolism and glycogen metabolism. The amounts of EPA and DHA in hatcheryproduced juveniles were inferior to those of wild juveniles.1 In the present study, the lack of growth performance by fortication of EPA and DHA might suggest that EPA and DHA contents in the control diet would be adequate to ll fatty acid requirements. Nevertheless, it is essential to consider not only growth and feed utilization, but also physiological conditions, such as metabolism and vitality. The levels of EPA and DHA in

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the fortied diets were equivalent to the values of wild juveniles. This level would be needed to stimulate physiological functions. The fatty acids incorporated from the articial diet in cultured sh were fairly low.1,2 Wild sh effectively absorbed EPA and DHA from feed organisms (Om et al., unpublished data). The sh could effectively absorb EPA and DHA from feed organisms in the natural environment.2 Adequate EPA and DHA levels in diets based on current results would be, respectively, 1.8% and 2.3%. However, the effect of further fortication of these fatty acids is impossible to determine because of excessive production cost. An adequate level of essential fatty acids might not be recommended by the dietary level only, but the value incorporated in sh should be considered. There might be certain unknown factors, which accelerate to incorporate dietary EPA and DHA from natural feed organisms. ACKNOWLEDGMENT We are grateful to Bizen Kasei Co. Ltd for supplying EPA and DHA esters.

REFERENCES
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