Aquaculture 221 (2003) 427–438 www.elsevier.com/locate/aqua-online

Utilization of glucose, maltose, dextrin and cellulose by juvenile flounder (Paralichthys olivaceus)

Sang-Min Leea,*, Kyoung-Duck Kima, Santosh P. Lallb

a Faculty of Marine Bioscience and Technology, Kangnung National University, Kangnung, Gangneung 210-702, South Korea b National Research Council, Institute for Marine Biosciences, 1411 Oxford Street, Halifax, NS, Canada

Received 18 September 2002; received in revised form 6 January 2003; accepted 6 January 2003

Abstract

A study was conducted to determine the ability of juvenile flounder to utilize different sources of carbohydrate in their diets. Triplicate groups of fish (average weight, 4 g) were hand- fed visual satiety one of seven diets containing 15% cellulose, 15% glucose, 15% maltose, and 5–25% dextrin for 45 days in flush-out aquarium system. Weight gain, feed efficiency ratio, protein efficiency ratio (PER), energy retention efficiency, hepatosomatic index (HSI), and liver glycogen were measured. Fish fed the diet containing 15% cellulose had the lowest weight gain ( P < 0.05) among all groups. Weight gain of fish fed the diets containing 15% maltose and 15– 25% dextrin was higher than that of fish fed the diets containing 15% cellulose and 5% dextrin. The feed efficiency ratio and PER of fish fed the diets containing 15–25% dextrin were significantly higher ( P < 0.05) than those of the other groups. Growth and feed utilization increased with an increase in the dextrin level of the diet. Lipid content of the whole body and liver decreased with an increase in dietary dextrin level. Liver glycogen and HSI of fish fed the 15% glucose and 15% maltose diets were higher ( P < 0.05) than those of fish fed the other carbohydrates, however these values were not affected by an increase in dextrin intake. Flounder (average weight, 15 g) were fasted for 48 h for a glucose tolerance test and blood was collected after feeding at 0, 1, 3, 5, 8, 11, 16, 24 and 48 h. Plasma glucose concentrations of fish fed the diets containing 15% glucose and 15% maltose peaked at 5–8 h (200 mg/100 ml) and 5 h (148 mg/100 ml), respectively, then decreased at 24 and 16 h. Fish fed the diets containing 5–25% dextrin showed a lower glucose level (87–97 mg/100 ml) than fish fed the other diets and it peaked between 3 and 5 h. Flounder utilized dextrin more efficiently than glucose, and dextrin

* Corresponding author. Tel.: +82-33-640-2414; fax: +82-33-640-2410. E-mail address: [email protected] (S.-M. Lee).

0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(03)00061-9 428 S.-M. Lee et al. / Aquaculture 221 (2003) 427–438 was a better source of energy than lipid. The best growth and feed utilization were achieved with the diet containing 25% dextrin and 6% lipid. D 2003 Elsevier Science B.V. All rights reserved.

Keywords: Flounder; Carbohydrate; Plasma chemistry; Growth; Glucose tolerance

1. Introduction

Carbohydrates are the most economical source of dietary energy for terrestrial animals, however, the ability of fish to utilize carbohydrate varies among fish (NRC, 1993). The utilization of dietary carbohydrate by fish appears to be related to their digestive and metabolic systems adapted to the different aquatic environment (Walton and Cowey, 1982) and dietary carbohydrate level and complexity (Bergot, 1979; Hutchins et al., 1998). Generally, warmwater herbivorous or omnivorous fish utilize much higher levels of carbohydrate than carnivorous coldwater salmonids and marine fish (Wilson, 1994). The efficiency of dietary carbohydrate utilization is in order of common carp>gilthead seabream>rainbow trout (Furuichi and Yone, 1981,1982a; Wilson, 1994; Girad et al., 1997). In rainbow trout, either ingestion of a high carbohydrate diet or oral administration of glucose causes poor utilization of dietary glucose and prolonged hyperglycemia (Palmer and Ryman, 1972; Bergot, 1979; Brauge et al., 1995). An early work of Buhler and Halver (1961) demonstrated that glucose, maltose, and sucrose resulted in the best growth rates of chinook salmon, followed by dextrin and fructose, galactose and potato starch, and glucosamine. Studies on chum salmon fry indicated that dietary glucose, maltose, sucrose, dextrin, and gelatinized starch but not fructose, galactose, or lactose were good as energy sources (Akiyama et al., 1982). A review of more recent studies by Wilson (1994) showed that cooked starch and dextrin are utilized more efficiently than simple sugars by most fish. It is important to provide an adequate carbohydrate level in the diet in order to reduce catabolism of protein for energy and for synthesis of glucose, which reduces protein retention and increases the nitrogen release to the environment (Suarez and Mommsen, 1987; Cowey and Walton, 1989; Wilson, 1994). Several studies have shown that adequate levels of non-protein energy sources in the diet can minimize the use of protein as a source of energy (NRC, 1993). The protein-sparing effect obtained by increasing lipid or carbohydrate levels in the diet has been reported in several species of fish (Cho and Kaushik, 1990; Vergara et al., 1996). Flounder are one of the most important farmed marine fish in Korea and information on dietary carbohydrate utilization by this species is not available. Recently, Lee et al. (2002a) suggested that carbohydrate may be more efficiently utilized than lipid as a source of energy by flounder. The cost of flounder feed production could be reduced by using low value dietary sources of energy such as carbohydrate and optimum use of protein and lipid. The present study, therefore, was conducted to evaluate the ability of juvenile flounder to utilize several carbohydrates including glucose, maltose, dextrin and cellulose. S.-M. Lee et al. / Aquaculture 221 (2003) 427–438 429

2. Materials and methods

2.1. Experimental diets

Ingredients and proximate composition of the experimental diets are given in Table 1. The seven experimental diets were formulated to contain 15% cellulose, 15% glucose, 15% maltose, and 5–25% dextrin with isocaloric (19 MJ of gross energy/kg) except for 5% dextrin diet. Lipid levels in experimental diets containing 15% carbohydrates were maintained between 7% and 8% to achieve the optimum lipid level required for flounder (Lee et al., 2000). In order to determine the effects of various levels of dextrin, the amount of dextrin in the experimental diets increased at the expense of soybean oil and cellulose. All the diets were mixed in a mixer (Patterson-Kelley Blend, East Strouds- burg, PA, USA), water was added to the dry mix (40 g water/100 g diet) and extruded through a grinder to produce pellets. The experimental diets were stored in airtight containers at À 30 jC until use.

Table 1 Ingredient and proximate composition of the experimental diets Diets 15C 15G 15M 15D 5D 10D 25D Ingredients (g/100 g) Casein, vitamin-freea 7.0 7.0 7.0 7.0 7.0 7.0 4.0 White fish mealb 65.0 65.0 65.0 65.0 65.0 65.0 60.0 Alpha-cellulosec 15.0 5.0 2.5 Glucosec 15.0 Maltosec 15.0 Dextrinc 15.0 5.0 10.0 25.0 Squid liver oild 2.0 2.0 2.0 2.0 2.0 2.0 2.0 Soybean oil 5.0 2.5 Vitamin premixe 2.5 2.5 2.5 2.5 2.5 2.5 2.5 Mineral premixe 3.0 3.0 3.0 3.0 3.0 3.0 3.0 Carboxymethyl cellulosec 5.0 5.0 5.0 5.0 5.0 5.0 3.0 Choline saltc 0.5 0.5 0.5 0.5 0.5 0.5 0.5

Proximate analysis (%, dry matter basis) Crude protein 53.1 52.4 52.2 52.9 53.3 52.9 49.0 Crude lipid 7.8 8.0 7.5 6.6 13.3 9.5 6.3 Crude fiber 17.4 1.3 1.3 1.3 6.6 3.9 1.3 Ash 17.7 17.5 17.3 17.0 17.2 16.8 16.0 N-free extractf 4.0 20.8 21.8 22.2 9.6 16.8 27.4 Gross energy (MJ/kg) 19.0 19.5 19.3 19.2 20.6 19.3 19.2 a Serva, Feinbiochemica (Heidelberg, Germany). b Pollack fish meal, produced by steam dry method, Han Chang Fish Meal (Busan, Korea). c Sigma (St. Louis, MO, USA). d Provided by E-wha Oil & Fat Ind. (Busan, Korea). e Same as Lee et al. (2002a). f Calculated by difference (100 À crude protein À crude lipid À crude fiber À ash). 430 S.-M. Lee et al. / Aquaculture 221 (2003) 427–438

2.2. Fish and feeding trial

Juvenile flounder (Paralichthys olivaceus) were purchased from a private fish hatchery (Hang-Do Fisheries, Gangneung, Korea). They were acclimated to the experimental conditions for 2 weeks prior to the commencement of the study and fed a commercial feed during this period. Juvenile flounder (average body weight, 4.1 g) were randomly distributed into 21 green circular, fiberglass-reinforced plastic tanks (90 cm t, 100 cm depth) with 25 fish in each tank. Three replicate groups of fish were hand-fed to visual satiety twice daily at 0900 and 1700 h for 45 days. Pellet size was adjusted and appropriate sized pellet was fed as the fish grew. Filtered seawater (34 F 0.2x, mean F S.D.) was supplied at a flow rate of 5 l/min to each tank. Fish were held under natural photoperiod and water temperature was maintained at 18.8 F 2.0 jC during the feeding trail. Fish were starved for 36 h and anesthetized with MS222 (tricaine methane sulfonate, Sigma, USA) at the concentration of 100 mg/l prior to growth measurements. Each tank of fish was bulk weighed at the beginning and the end of the experiment. Records were kept for daily feed consumption, mortalities and feeding behavior. Fifty fish at the beginning of the feeding trial and 15 fish from each tank at the termination of experiment were killed by a blow to the head and were stored at À 25 jC in freezer for proximate analysis.

2.3. Blood chemistry

After the 45-day feeding trial, another experiment was carried out to investigate the changes in plasma glucose, protein and triglyceride levels of flounder. For this experiment, juvenile flounder obtained from a private hatchery were acclimated to the laboratory conditions. After 45 days of acclimation period, the mean weight of 15 F 0.5 g fish were selected and distributed into 21 tanks used for feeding trial. Three replicate groups (60 fish per tank) of flounder were acclimated for 2 weeks to commercial feed containing 50% protein and 10% lipid. Following this period, fish were fasted for 48 h and hand-fed to visual satiety the experimental diets (Table 1). Blood samples were collected at 0, 1, 3, 5, 8, 11, 16, 24 and 48 h after the feeding. In order to properly determine the changes in plasma chemistry at the specified sampling time while maintaining the schedule, three separate teams were involved simultaneously in blood collection, centrifugation and separation of plasma. The fish were anesthetized with MS222 and blood samples collected from the caudal vein of six fish removed at each sampling time from each tank by using heparinized syringe. Blood was centrifuged at 3500 Â g for 5 min, plasma-separated and stored in À 75 jC freezer. Before freezing, plasma was divided into separate aliquots for protein, glucose and triglyceride analyses.

2.4. Chemical analysis

Chemical composition of the experimental diets and fish carcasses were determined using the following AOAC (1990) procedures: dry matter by drying in an oven at 105 jC for 24 h; crude protein (N Â 6.25) by the Kjeldahl method after an acid digestion method using an Auto Kjeldahl System (Buchi, Flawil, Switzerland); crude lipid by ether extraction after acid hydrolysis; ash by incineration in a muffle furnace at 550 jC for S.-M. Lee et al. / Aquaculture 221 (2003) 427–438 431

24 h and crude fiber by Fibertec automatic analyzer (Tecator, Hoganas, Sweden). Gross energy contents were analyzed using an adiabatic bomb calorimeter (Parr, Moline, IL, USA) and liver glycogen was measured by enzymatic method using amyloglucosidase (Fluka, EC 3.2.1.3) as described by Murat and Serfaty (1974). The plasma glucose, protein and triglyceride content were measured using commercial clinical investigation kits (Wako, Japan).

2.5. Statistical analysis

The data were subjected to one-way analysis of variance (ANOVA) using the SPSS program Version 7.5 (SPSS, Michigan Avenue, Chicago, IL, USA). Significant differences ( P < 0.05) among mean were determined by Duncan’s multiple range test (Duncan, 1955).

3. Results

3.1. Growth performance

The performance of juvenile flounder fed various experimental diets for 45 days is shown in Table 2. Survival of fish in each dietary group was over 83% and there was no significant difference among treatments. Weight gain, feed efficiency ratio, protein efficiency ratio (PER), protein retention efficiency and energy retention efficiency of fish

Table 2 Growth performance of juvenile flounder fed the diets containing various carbohydrate source and dextrin level for 45 days Diets 15C 15G 15M 15D 5D 10D 25D Initial wt. 4.0 F 0.10 4.0 F 0.04 4.1 F 0.07 4.1 F 0.09 4.0 F 0.07 4.1 F 0.03 4.0 F 0.10 (g/fish) Survival (%) 87 F 2.7 92 F 2.3 93 F 6.7 95 F 1.3 83 F 2.7 84 F 6.9 87 F 8.1 WG (%)1 153 F 14.3a 226 F 5.9bc 306 F 27.3de 272 F 6.4cde 209 F 16.3b 259 F 23.8bcd 325 F 18.7e FER (%)2 54 F 1.5a 83 F 0.8c 105 F 3.8d 117 F 5.1e 65 F 1.9b 97 F 1.1d 123 F 2.3e DFI (%)3 3.22 F 0.09e 2.70 F 0.05d 2.48 F 0.07cd 2.17 F 0.08ab 3.15 F 0.06e 2.38 F 0.09bc 2.13 F 0.09a PER4 1.02 F 0.03a 1.59 F 0.01c 2.02 F 0.07e 2.21 F 0.10f 1.23 F 0.04b 1.83 F 0.02d 2.52 F 0.05g PRE (%)5 14.7 F 1.42a 23.9 F 1.07b 32.8 F 1.27c 35.5 F 0.86cd 19.3 F 0.54ab 31.5 F 1.33c 39.9 F 3.51d ERE (%)6 14.3 F 1.01a 21.5 F 0.27c 27.8 F 0.38d 30.2 F 1.08de 18.2 F 0.92b 28.8 F 1.65d 32.2 F 0.98e CF7 1.01 F 0.02 1.08 F 0.01 1.09 F 0.03 1.04 F 0.02 1.04 F 0.03 1.03 F 0.04 1.04 F 0.02 Values (mean F S.E.M. of three replications) in the same row not sharing a common superscript are significantly different ( P < 0.05). 1 Weight gain=(final body weight À initial body weight)  100/initial body weight. 2 Feed efficiency ratio=(body wet weight gain  100)/feed intake (dry matter). 3 Daily feed intake = feed intake  100/[(initial fish wt. + final fish wt. + dead fish wt.)/2  days fed]. 4 Protein efficiency ratio = body wet weight gain/protein intake. 5 Protein retention efficiency = fish protein deposited  100/protein intake. 6 Energy retention efficiency = fish energy deposited  100/energy intake. 7 Condition factor=(body weight  100)/total body length (cm)3. 432 S.-M. Lee et al. / Aquaculture 221 (2003) 427–438

Table 3 Proximate analysis (%) of whole body of juvenile flounder fed the diets containing various carbohydrate source and dextrin level for 45 days Diets 15C 15G 15M 15D 5D 10D 25D Moisture 76.2 F 0.1 76.6 F 0.1 76.6 F 0.3 76.6 F 0.2 75.4 F 0.5 74.8 F 0.9 76.0 F 0.2 Crude protein 15.7 F 0.5 15.8 F 0.5 16.6 F 0.8 16.4 F 0.2 16.4 F 0.2 17.2 F 0.4 16.2 F 0.8 Crude lipid 2.3 F 0.1a 2.8 F 0.2ab 2.8 F 0.1ab 2.6 F 0.1ab 3.4 F 0.3c 3.1 F 0.2bc 2.5 F 0.2ab Ash 3.3 F 0.3 3.5 F 0.2 3.4 F 0.1 3.6 F 0.3 3.2 F 0.5 3.6 F 0.4 4.1 F 0.1 Moisture, crude protein, crude lipid, and ash contents of initial fish were 75.0, 17.3, 1.8, and 4.2%, respectively. Values (mean F S.E.M. of three replications) in the same row not sharing a common superscript are significantly different ( P < 0.05). fed the diet containing 15% cellulose were the lowest among all groups. The best weight gain (272–325%) was observed in fish fed the diets containing 15% maltose and 15–25% dextrin. Feed efficiency ratios (117–123%) of fish fed the diets containing 15 and 25% dextrin were significantly higher than those (54–105%) of other groups. Daily feed intake of fish fed the diets with 15% cellulose and 5% dextrin was significantly higher than that of other groups. PER was highest in fish fed the 25% dextrin diet. Protein retention efficiency and energy retention efficiency of fish fed the diets containing 15% and 25% dextrin were significantly higher than those of fish fed the diets containing 15% cellulose, 15% glucose and 5% dextrin. Feed efficiency ratio, PER, protein retention efficiency and energy retention efficiency of fish fed the glucose were lower than those of fish fed the maltose and dextrin at the same carbohydrate level in the diets. Growth and feed utilization increased with increasing dietary dextrin level. Daily feed intake decreased with increasing dietary dextrin level. Condition factor was not significantly affected by dietary carbohy- drates.

3.2. Body composition

The proximate composition of whole body is shown in Table 3. Dietary carbohydrates had no significant effect on the moisture, crude protein and ash contents of whole body.

Table 4 Crude lipid and glycogen contents of liver and hepatosomatic index of juvenile flounder fed the diets containing various carbohydrate source and dextrin level for 45 days Diets 15C 15G 15M 15D 5D 10D 25D Moisture (%) 71.8 F 0.9bc 72.7 F 1.3bc 73.4 F 0.9c 72.6 F 0.2bc 67.8 F 0.6a 70.3 F 0.7ab 69.9 F 1.0ab Crude lipid (%) 9.0 F 0.9b 5.9 F 0.7a 8.9 F 0.6b 8.6 F 0.6b 15.5 F 0.8c 10.7 F 0.4b 9.5 F 1.2b Glycogen (%) 0.2 F 0.1a 7.2 F 0.5d 5.4 F 0.8c 3.4 F 0.4b 2.4 F 0.2b 3.2 F 0.5b 3.4 F 0.3b HSI1 0.85 F 0.07a 1.65 F 0.05c 1.99 F 0.12d 1.40 F 0.03b 1.36 F 0.06b 1.39 F 0.13b 1.40 F 0.01b Values (mean F S.E.M. of three replications) in the same row not sharing a common superscript are significantly different ( P < 0.05). 1 Hepatosomatic index=(liver wet weight  100)/body weight. S.-M. Lee et al. / Aquaculture 221 (2003) 427–438 433

Lipid content was also not affected by dietary glucose, maltose, dextrin, and cellulose at the same level, however the increase in the dietary dextrin level cause a decreased in body lipid. Moisture, crude lipid and glycogen contents of liver and hepatosomatic index (HSI) of flounder are shown in Table 4. Moisture content of fish fed the diet containing 5% dextrin was lowest among the groups. Liver lipid content was lowest in flounder fed the glucose diet. An increase in dietary dextrin level caused a decrease in liver lipid content. Glycogen level was highest in flounder fed the glucose diet, followed by those given maltose, dextrin, and cellulose diets. HSI of flounder fed the diets containing glucose and maltose

Fig. 1. Postprandial plasma glucose, triglyceride and protein concentrations of juvenile flounder fed the diets containing different carbohydrate source and dextrin level. Values are the mean F S.E.M. of three replications. 434 S.-M. Lee et al. / Aquaculture 221 (2003) 427–438 was higher than that of fish fed the diets containing either dextrin or cellulose. However, HSI was not affected by increase in the dietary dextrin content.

3.3. Blood chemistry

Plasma glucose, triglyceride and protein values for juvenile flounder are shown in Fig. 1. Plasma glucose concentrations of fish fed the diets containing 15% glucose and 15% maltose peaked at 5–8 h (200 mg/100 ml) and 5 h (148 mg/100 ml), then decreased to 24 and 16 h, respectively, after feeding; whereas fish fed the diets containing 5–25% dextrin showed lower peak (87–97 mg/100 ml) at 3–5 h. Plasma triglyceride content of fish fed the diets containing 15% cellulose, 15% glucose, 15% maltose, and 5–15% dextrin peaked 11–16 h after feeding then decreased. However, fish fed the 25% dextrin diet exhibited a significantly delayed (24 h) response. Either dietary carbohydrate source or the level of their incorporation in the diet did not significantly affect plasma protein concentration.

4. Discussion

Dietary carbohydrate utilization by fish varies and appears to be related to the complexity of carbohydrate (Wilson, 1994). The relatively higher growth and protein utilization of flounder fed the diet containing polysaccharides in this study are similar to those reported for other fish (Furuichi et al., 1986; Shiau and Chuang, 1995; Hutchins et al., 1998). Growth and feed utilization of channel catfish fed diets containing 33% polysaccharides (dextrin and corn starch) were higher than those of fish fed diets containing mono- and disaccharides (Wilson and Poe, 1987). Although the mechanism responsible for the observed differences in carbohydrate utilization is not known, the lower weight gain, feed efficiency ratio and PER of flounder fed the glucose diet in this study is probably due to rapid absorption of glucose in the gut (Pieper and Pfeffer, 1980; Tung and Shiau, 1991), and the excess absorbed glucose may be cleared from blood before body cells can utilize it efficiently (Furuichi and Yone, 1982b; Hilton and Atkinson, 1982). However, the weight gain of red sea bream was not affected by different dietary carbohydrate sources such as glucose, dextrin and starch (Furuichi and Yone, 1982a). Growth and feed utilization of flounder fed the maltose diet in the present study were higher than those fed the glucose diet. Similarly, higher utilization of maltose than glucose has been reported for hybrid tilapia (Shiau and Chuang, 1995). However, growth and feed efficiency of white sturgeon fed diets containing 27% maltose were comparable to those fed glucose and significantly higher than those fed starch (Hung et al., 1989). The different ability to utilize dietary carbohydrate by fish may be due to differences in fish species, dietary composition of the experimental diets, culture conditions, etc. Incorporation of cellulose in the diet caused reduced growth and poor feed utilization by flounder. Generally, dietary fiber is not hydrolyzed by fish (NRC, 1993) and high fiber levels may reduce the utilization of other nutrients (Anderson et al., 1984). Other studies have observed a negative correlation between cellulose content of the diet and growth or nutrient digestibility in fish (Hilton et al., 1983; Fynn-Aikins et al., 1992). S.-M. Lee et al. / Aquaculture 221 (2003) 427–438 435

Growth and feed utilization of flounder increased with the increase in dietary dextrin level (0–25%) and also by decreasing the dietary lipid level (13–6%). Total lipid content of whole body and liver decreased with the increase in dietary dextrin level, probably as a result of the dietary lipid content. Negative effect of high dietary lipid on growth and body composition has also been reported for juvenile flounder (Lee et al., 2000) and they suggested that low dietary lipid (7%) was suitable for growth of this species. These results are similar to those reported for turbot (Caceres-Martinez et al., 1984; Regost et al., 2001), but opposite to other observations on the protein-sparing effect of high dietary lipid which results in better growth or feed efficiency of fish (De Silva et al., 1991; Lee et al., 2002b). Protein utilization may be improved by partially replacing dietary protein with lipid or carbohydrate to produce the protein-sparing effect. Growth and protein utilization of flounder fed the 25% dextrin diet with 49% protein and 6% lipid in this study were higher than those of fish fed the 0–10% dextrin diet with 53% protein and 8–13% lipid. These results indicate that flounder utilizes dietary carbohydrate more efficiently than lipid as an energy source, and carbohydrate may be used to spare a part of protein in the diet. Similar results have been observed in other marine fish such as European sea bass (Peres and Oliva-Teles, 2002), plaice (Cowey et al., 1975) and turbot (Adron et al., 1976). The feed efficiency ratio (73%) and PER (1.4) of flounder fed the 4% dextrin diet with 50% protein in the previous study (Lee et al., 2000) were lower than those (117–123% and 2.2–2.5, respectively) of 15–25% dextrin diets, but comparable to those (65% and 1.2) of 5% dextrin diet in this study. These differences in response to dietary carbohydrate levels could be the result of different ratios of carbohydrate to protein in the experimental diets, indicating that protein utilization in flounder can be improved by feeding diets containing adequate amount of carbohydrate (dextrin) as a source of energy as previously suggested by Lee et al. (2002a). Recently, Peres and Oliva-Teles (2002) observed that the dietary incorporation of 25% of starch had no negative effects on growth or feed efficiency ratio for European sea bass. Better growth and feed utilization have also been reported in rainbow trout fed 30% dextrin and 9% lipid diet as compare with diets containing 8% dextrin and 20% lipid diet (Yamamoto et al., 2000). Based on these results, more detail studies on proper ratio of carbohydrate, lipid and protein are needed to establish their protein-sparing effect in practical diets for flounder. Lipid content of whole body was not affected by various carbohydrates when incorporated at the same level in the diet. Moreover, body lipid of fish fed diet containing 25% dextrin diet was not different from that of fish fed diets containing other carbohydrates (15% of the diet). On the other hand, whole body and liver lipid contents of fish fed the diet containing 5% dextrin and 13% lipid were higher than those receiving 15–25% carbohy- drates with 6–8% lipids. This indicates that flounder may have limited metabolic ability to synthesize lipid from glucose and that body lipid is directly affected by dietary lipid intake. Liver glycogen content of flounder fed the glucose diet was significantly higher than fish fed the other carbohydrate sources, probably because absorbed excess glucose, which may not be utilized for energy, accumulated as glycogen but not lipid in the liver. The increase of HSI seems to be closely related to accumulation of liver glycogen, but not liver lipid content. HSI was positively correlated with liver glycogen (r = 0.88; P < 0.01). This trend is similar to results reported in other fish species (Hung et al., 1990; Fynn-Aikins et al., 1992; Hutchins et al., 1998), but different from other marine fish such as cod. In gadoid fishes, an 436 S.-M. Lee et al. / Aquaculture 221 (2003) 427–438 increase in liver lipid content is associated with an increase in the HSI (Hemre et al., 1989; Nanton et al., 2001). There was no relationship between dietary dextrin level and liver glycogen deposition in this study and HSI was not affected by dietary dextrin level. A similar trend was observed in other studies (Hemre et al., 1989; Hutchins et al., 1998). However, liver glycogen content has been shown to increase with an increase in dietary digestible carbohydrate level in rainbow trout (Cowey et al., 1975; Bergot, 1979). Blood glucose levels of flounder were variable depending on the dietary carbohydrates. Glucose level peaked 3–5 h after feeding the glucose diet, followed by maltose, dextrin and cellulose diets. Similar results have been also observed in other fish fed different carbohydrate source (Hutchins et al., 1998). The relatively higher and prolonged plasma glucose concentration of flounder fed the glucose diet compared to maltose and dextrin diets indicates an overload of glucose in blood, which was not utilized efficiently. On the other hand, blood triglyceride level reached a peak 11–24 h after feeding then decreased more slowly than glucose levels regardless of dietary carbohydrate source and dextrin level. This slower peak response and gradual decline in blood triglyceride compared to blood glucose indicates that flounder was unable to utilize dietary lipid efficiently compared to carbohydrate. This conclusion is supported by our results showing that growth and feed efficiency of flounder fed the 15–25% dextrin diets with 6–7% lipid were relatively high compared to those of fish fed the 5–10% dextrin diets with 10–13% lipid in this study. Although fish do not have a carbohydrate requirement, carbohydrate is the least expensive nutrient in feed formulating and can improve the physical properties of extruded and steam pelleted feeds (Wilson, 1994). Therefore, most studies have been focused on formulations to contain the maximum level of carbohydrate in the diet that fish can utilize efficiently. Considering improvement of growth and feed utilization of flounder fed the 25% dextrin diet without an effect on HSI and liver glycogen content, inclusion of up to 25% dextrin in the diet does produce any negative effects on the carbohydrate metabolism of juvenile flounder. These data indicate that juvenile flounder are able to efficiently utilize dextrin compared to glucose, and that dietary lipid could be partially replaced by dextrin without reducing growth and protein utilization. Moreover, an increase of dietary dextrin level has a protein-sparing effect. It appears that the diet containing 49% protein, 25% dextrin and 6% lipid (19 MJ/kg diet) was suitable for optimum growth and efficient protein utilization by juvenile flounder.

Acknowledgements

This work was supported by the funds of the Ministry of Marine Affairs in Korea.

References

Adron, J.W., Blair, A., Cowey, C.B., Shanks, A.M., 1976. Effects of dietary energy level and dietary energy source on growth, feed conversion and body composition of turbot (Scophthalmus maximus L.). Aquaculture 7, 125–132. Akiyama, T., Murai, T., Nose, T., 1982. Effects of various dietary carbohydrates on growth, feed efficiency, and body composition of chum salmon fry. Bull. Jpn. Soc. Sci. Fish. 33, 112–116. S.-M. Lee et al. / Aquaculture 221 (2003) 427–438 437

Anderson, J.S., Jackson, A.J., Matty, A.J., Capper, B.S., 1984. Effects of dietary carbohydrate and fiber on the tilapia, Orechromis niloticus (Linn.). Aquaculture 37, 303–314. AOAC, 1990. Official Methods of Analysis, 15th ed. Association of Official Analytical Chemists, Arlington, VA. 1298 pp. Bergot, F., 1979. Carbohydrate in rainbow trout diets: effects of the level and source of carbohydrate and the number of meals on growth and body composition. Aquaculture 18, 157–167. Brauge, C., Corraze, G., Me´dale, F., 1995. Effect of dietary levels of lipid and carbohydrate on growth perform- ance, body composition, nitrogen excretion and plasma glucose levels in rainbow trout reared at 8 or 18 jC. Reprod. Nutr. Dev. 35, 277–290. Buhler, D.R., Halver, J.E., 1961. Nutrition of salmonid fishes: IX. Carbohydrate requirements of chinook salmon. J. Nutr. 74, 307–318. Caceres-Martinez, C., Cadena-Roa, M., Metailler, R., 1984. Nutritional requirements of turbot (Scophthalmus maximus): I. A preliminary study of protein and lipid utilization. J. World Maricult. Soc. 15, 191–202. Cho, C.Y., Kaushik, S.J., 1990. Nutritional energetics in fish: energy and protein utilization in rainbow trout (Salmo gairdneri). World Rev. Nutr. Diet 61, 132–172. Cowey, C.B., Walton, M.J., 1989. Intermediary metabolism. In: Halver, J.E. (Ed.), Fish Nutrition, 2nd ed. Academic Press, New York, pp. 259–329. Cowey, C.B., Adron, J.W., Brown, D.A., 1975. Studies on the nutrition of marine flatfish. The metabolism of glucose by plaice (Pleuronectes platessa) and the effects of dietary energy source on protein utilization in plaice. Br. J. Nutr. 33, 219–231. De Silva, S.S., Gunasekera, R.M., Shim, K.F., 1991. Interactions of varying dietary protein and lipid levels in young red tilapia: evidence of protein sparing. Aquaculture 95, 305–318. Duncan, D.B., 1955. Multiple-range and multiple F tests. Biometrics 11, 1–42. Furuichi, M., Yone, Y., 1981. Changes in blood sugar and plasma insulin levels fishes in glucose tolerance tests. Bull. Jpn. Soc. Sci. Fish. 47, 761–764. Furuichi, M., Yone, Y., 1982a. Availability of carbohydrate in nutrition of carp and red sea bream. Bull. Jpn. Soc. Sci. Fish. 48, 945–948. Furuichi, M., Yone, Y., 1982b. Change in activities of hepatic enzymes related to carbohydrate metabolism of fishes in glucose and insulin–glucose tolerance tests. Bull. Jpn. Soc. Sci. Fish. 48, 463–466. Furuichi, M., Taira, H., Yone, Y., 1986. Availability of carbohydrate in nutrition of yellowtail. Bull. Jpn. Soc. Sci. Fish. 52, 99–102. Fynn-Aikins, K., Hung, S.S.O., Liu, W., Li, H., 1992. Growth, lipogenesis and liver composition of juvenile white sturgeon fed different levels of D-glucose. Aquaculture 105, 61–72. Girad, J., Ferre´, P., Foufelle, F., 1997. Mechanisms by which carbohydrates regulate expression of genes for glycolytic and lipogenic enzymes. Annu. Rev. Nutr. 17, 325–352. Hemre, G.I., Lie, O., Lied, E., Lambertsen, G., 1989. Starch as an energy source in feed for cod (Gadus morhua): digestibility and retention. Aquaculture 80, 261–270. Hilton, J.W., Atkinson, J.L., 1982. Response of rainbow trout (Salmo gairdneri) to increased levels of available carbohydrate in practical trout diets. Br. J. Nutr. 47, 597–607. Hilton, J.W., Atkinson, J.I., Slinger, S.J., 1983. Effect of increased dietary fiber on the growth of rainbow trout (Salmo gairdneri). Can. J. Fish. Aquat. Sci. 40, 81–85. Hung, S.S.O., Fynn-Aikins, K.F., Lutes, P.B., Xu, R.P., 1989. Ability of juvenile white sturgeon (Acipenser transmontanus) to utilize different carbohydrate source. J. Nutr. 119, 727–733. Hung, S.S.O., Groff, J.M., Lutes, P.B., Fynn-Aikins, K.F., 1990. Hepatic and intestinal histology of juvenile white sturgeon fed different carbohydrates. Aquaculture 87, 149–160. Hutchins, C.G., Rawles, S.D., Gatlin III, D.M., 1998. Effects of dietary carbohydrate kind and level on growth, body composition and glycemic response of juvenile sunshine bass (Morone chrysops U Â M. saxatilis h). Aquaculture 161, 187–199. Lee, S.-M., Cho, S.H., Kim, K.-D., 2000. Effects of dietary protein and energy levels on growth and body composition of juvenile flounder (Paralichthys olivaceus). J. World Aquacult. Soc. 31, 306–315. Lee, S.-M., Park, C.S., Bang, I.C., 2002a. Dietary protein requirement of young Japanese flounder Paralichthys olivaceus fed isocaloric diets. Fish. Sci. 68, 158–164. Lee, S.-M., Jeon, I.G., Lee, J.Y., 2002b. Effects of digestible protein and lipid levels in practical diets on 438 S.-M. Lee et al. / Aquaculture 221 (2003) 427–438

growth, protein utilization and body composition of juvenile rockfish (Sebastes schlegeli). Aquaculture 211, 227–239. Murat, J.C., Serfaty, A., 1974. Simple enzymatic determination of polysaccharide (glycogen) content of animal tissues. Clin. Chem. 20, 1576–1577. Nanton, D.A., Lall, S.P., McNiven, M.A., 2001. Effects of dietary lipid level on liver and muscle lipid deposition in juvenile haddock, Melanogrammus aeglefinus. Aquacult. Res. 32 (Suppl. 1), 225–234. NRC (National Research Council), 1993. Nutrient Requirements of Fish. National Acad. Press, Washington, DC. 114 pp. Palmer, T.N., Ryman, B.E., 1972. Studies on glucose intolerance in fish. J. Fish Biol. 4, 311–319. Peres, H., Oliva-Teles, A., 2002. Utilization of raw and gelatinized starch by European sea bass (Dicentrarchus labrax) juveniles. Aquaculture 205, 287–299. Pieper, A., Pfeffer, E., 1980. Studies on the comparative efficiency of utilization of gross energy from some carbohydrate, protein and fats by rainbow trout (Salmo gairdneri R). Aquaculture 20, 323–332. Regost, C., Arzel, J., Cardinal, M., Robin, J., Laroche, M., Kaushik, S.J., 2001. Dietary lipid level, hepatic lipogenesis and flesh quality in turbot (Psetta maxima). Aquaculture 193, 291–309. Shiau, S.-Y., Chuang, J.-C., 1995. Utilization of disaccharides by juvenile tilapia (Oreochromis niloticus  O. aureus). Aquaculture 133, 249–256. Suarez, P.K., Mommsen, T.P., 1987. Gluconeogenesis in teleost fishes. Can. J. Zool. 65, 1869–1882. Tung, P.-H., Shiau, S.-Y., 1991. Effects of meal frequency on growth performance of hybrid tilapia, Oreochromis niloticus  O. aureus, fed different carbohydrate diets. Aquaculture 92, 343–350. Vergara, J.M., Fernandez-Palacios, H., Robaina, L., Jauncey, K., De La Higuera, M., Izquierdo, M., 1996. The effects of varying dietary protein level on the growth, feed efficiency, protein utilization and body compo- sition of gilthead sea bream. Fish. Sci. 62, 620–623. Walton, M.J., Cowey, C.B., 1982. Aspects of intermediary metabolism in fish. Comp. Biochem. Physiol. 73B, 59–79. Wilson, R.P., 1994. Utilization of dietary carbohydrate by fish. Aquaculture 124, 67–80. Wilson, R.P., Poe, W.E., 1987. Apparent inability of channel catfish to utilize dietary mono- and disaccharides as energy source. J. Nutr. 117, 280–285. Yamamoto, T., Shima, T., Unuma, T., Shiraishi, M., Akiyama, T., Tabata, M., 2000. Voluntary intake of diets with varying digestible energy contents and energy sources, by juvenile rainbow trout Oncorhynchus mykiss, using self-feeders. Fish. Sci. 66, 528–534.