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Effect of dietary carbohydrate levels on growth, body composition and glycaemia in rainbow trout, Oncorhynchus mykiss, reared in seawater
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Aquaculture 123 (1994) 109-120
Effect of dietary carbohydrate levels on growth, body composition and glycaemia in rainbow trout, Oncorhynchus mykiss, reared in seawater C. Brauge, F. Medale*, G. Corraze INRA, Fish Nutrition Laboratory, Hydrobiology Station, BP3, 64310 Saint Pie-sur-Nivelle, France (Accepted 13 January 1994)
Abstract Three experimental diets were fed to near-satiation twice a day for 9 weeks to triplicate groups of 40 rainbow trout (mean initial weight 230 g) reared in seawater (36.5 ppt) at 9°C. The diets were formulated to have the same levels of nitrogen (crude protein 43% DM) and energy (digestible energy: around 15 kJ/g DM) but they contained different amounts of digestible carbohydrate (8-24.4%) and fat (5.7-l 1.1%). Digestibility was measured using chromic oxide as a marker. Plasma glucose concentrations were measured in 48-h fasted trout and at selected intervals after a single meal. Digestibility of starch and energy was lowest in trout fed the diet containing the highest level of crude starch. Apparent digestibility values of protein (86.0-87.4%) and fat (76.8-8 1.5%) were similar for the three diets. Fish growth and body composition were not affected significantly (P> 0.05) by the non-protein energy sources of the diets. However, the amounts of digestible protein and digestible energy required per kilogram production were higher for fish fed the highcarbohydrate diet (475 ? 7 g DP and 18.8 ?- 0.3 MJ DE) than for fish fed the high-fat diet (4 19 f 2 1 g DP and 16.0 5 0.8 MJ DE). Liver size and liver lipid content increased in direct relationship to the dietary level of digestible carbohydrate. The highest percentage of muscle lipid was found in fish fed the diet containing 25% carbohydrate and 10% lipid as non-protein energy sources. Glycaemia was increased by elevated digestible carbohydrate intake. In conclusion, 230 g rainbow trout were able to utilize carbohydrate efficiently for growth in seawater (up to 25% of digestible carbohydrate in diet) under the conditions of this study. However, dietary lipid had a slightly better protein-sparing effect than digestible carbohydrate at a similar level of digestible energy intake. High dietary levels of digestible carbohydrate induced lipogenesis and enhanced lipid deposition in the liver. *Corresponding author. 0044-8486/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SsDIOO44-8486(94)00018-J
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1. Introduction Many previous studies have been conducted with the aim of decreasing the level of protein in fish diets by adding energy from non-protein sources. Energy from dietary lipid has been shown to effectively spare protein energy in several fish species (Watanabe, 1982). The effect of dietary carbohydrate on fish growth seems to depend upon the source, dietary concentration and digestibility, the level of dietary intake, rearing conditions and fish species. Hilton and Atkinson ( 1982) reported that high dietary levels ( > 14%) of cerelose depressed growth of rainbow trout. However, it has been demonstrated that digestible carbohydrate in the form of gelatinized starch improves protein and energy utilization in the same species (Kaushik and Oliva Teles, 1985; Kaushik et al., 1989; Kim and Kaushik, 1992). Also, according to Pieper and Pfeffer ( 1980), gelatinized starch is just as effective as lipid as an energy source for rainbow trout. These studies were carried out with small juvenile trout ( 15-40 g) in freshwater, and at high temperature ( 15- 18 “C) . Sea-ranching of large rainbow trout has been extended for the past 10 years in France. This type of ranching needs lower water temperature than in freshwater since euryhalinity of rainbow trout is reduced at high temperature (Maxime et al., 1986). Little is known about the effect of dietary carbohydrate level on growth of salmonids reared in seawater. Mazur et al. ( 1992)) however, found that lipid was a better non-protein energy source than digestible carbohydrate for chinook salmon held in seawater. Further data about nutrient utilization of large rainbow trout held in seawater are needed to improve production efficiency of trout sea-ranching. The aim of this study was to assess the effect of increasing dietary levels of digestible carbohydrate on growth, feed efficiency and body composition of rainbow trout reared in seawater at low temperature. Since high dietary levels of digestible carbohydrate have led to prolonged hyperglycaemia in salmon (Mazur et al., 1992), the effect of feeding the experimental diets to trout on plasma glucose level was also examined.
2. Materials and methods
Experimental diets Three experimental diets were formulated to contain constant levels of crude protein (Table 1) and digestible energy (Tables 1 and 2 ). Increasing amounts of fish oil were included in the diets to obtain graded levels of lipid. The amount of digestible energy supplied by carbohydrate was adjusted by using either gelatinized wheat starch (diet HC), crude wheat starch (diet LC) or a nearly equal mixture of both kinds of starch (diet MC). The diets HC and MC were coldpelleted using a laboratory pellet mill ( Simon-Heesen, Oslo). Because of its high fat level, diet LC was drawn into spaghetti-like strands using a meat grinder after
C. Brauge et al. /Aquaculture, 123 (1994) 109-120 Table 1 Ingredients and proximate compositions Ingredients
(%)
Fish meal’ Fish oil* Gelatinized wheat starch Crude wheat starch Vitamin mix3 Mineral mix4 Sodium alginate
of the experimental
111
diets
Diet HC
Diet MC
Diet LC
60.1 0.0 30.1 6.8 1.0 1.0 1.0
60.1 4.9 16.6 15.4 1.0 1.0 1.0
60.1 9.8 0.6 26.5 1.0 1.0 1.0
87.9 44.1 7.0 30.1 12.1 19.1
91.8 42.9 10.4 25.8 12.2 20.1
92.4 42.8 14.4 22.8 12.1 21.1
Proximate composition Dry matter (% WW) Crude protein (%DM) Crude lipid (% DM) Starch (% DM ) Ash (O/oDM) Gross energy (W/g DM)
‘Norwegian herring fish meal (CP = 69.8% DM), Descal, Donzacq, France. ?Soprop&che, Boulogne-sur-mer, France. Acidity index= 6%, stabilized with 200 ppm ethoxyquin, peroxide index < 10, iodine index between 140 and 160. 3Composition of vitamin mix (mg/g): vitamin A acetate, 1 (500 UI); nL-cholecakiferol, 2 (200 UI); DL-cf-tocopheryl acetate, 5 (2.5 UI); menadione, 0.30; thiamine hydrochloride, 0.75; riboflavin, 1; pyridoxine hydrochloride, 0.75; cyanocobalamin, 0.0025; nicotinic acid, 10; ascorbic acid, 25; folic acid, 0.25; calcium pantothenate, 2.5; choline chloride, 100; inositol, 50; biotin, 0.12. All ingredients were diluted with cellulose to 1 g. 4Composition of mineral mix (mg/g): calcium biphosphate*2H,O, 500; calcium carbonate, 2 15; sodium chloride, 40; potassium chloride, 90; magnesium hydroxide, 124; zinc sulfatee7H,O, 4; manganese sulfate.H,O, 3; cuprous sulfate*5H,O, 3; cobaltous sulfate, 0.02; potassium iodide, 0.04; ferric citrate- 7Hz0, 20; sodium fluoride, 1.
the addition of 20% water. Diet LC was then dried in an oven at 35°C for 48 h and subsequently cut into pellets. Chromic oxide was added to a portion of each diet at a level of 1% as an inert marker for digestibility measurement. The three experimental diets, kept in a cold room (4’ C) until use, were analysed for proximate composition according to the following procedures: dry matter by drying at 110 oC for 24 h, ash by combustion at 550 “C in a muffle furnace for 24 h, crude protein by the Kjeldahl method after acid digestion ( %Nx 6.25)) lipid according to Folch et al. ( 1957), starch using amylase and glucose oxidase (Thivend et al., 1972)) and gross energy using an IRA adiabatic bomb calorimeter. Feeding trial
Rainbow trout (Oncorhynchus mykiss) were transferred from freshwater to diluted seawater ( 17 ppt). After a 7-day acclimation period, 369 rainbow trout were alloted randomly and equally to 9 tanks (water volume: 450 litres) which were each supplied by running seawater (water flow rate: 750 l/h, salinity 36.5 ppt ). The photoperiod was 10 h light/ 14 h darkness. Initial weighing was carried
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out after another 7-day acclimation period to tanks and salinity. Nine fish ( l/ tank) were sampled for determination of initial whole body composition. Triplicate groups of 40 trout (initial mean body weight 230 g) were fed one of three experimental diets to near-satiation twice a day for 9 weeks. Fish were groupweighed and counted every 3 weeks, and feed intake was recorded daily. Water temperature varied between 7.9 and 10.7 ‘C during the experiment. At the end of the feeding trial, 10 fish per tank were withdrawn randomly and killed after anaesthesia (ethylene glycol monophenyl ether 1:2500). Composition of whole bodies was determined on 3 groups of 8 fish per diet, using the same analytical procedures as for proximate composition of diets. Whole bodies were freeze-dried, ground, and pooled for each tank before analyses. Liver, dorsal and ventral muscles were removed from 6 fish per diet, frozen in liquid nitrogen and stored at - 20” C. Liver and muscle samples were then analysed individually for lipid content by the Folch method.
Digestibility trial Apparent digestibility of nutrients and energy was determined using chromic oxide as an indigestible marker after a progressive acclimation period to salinity (36.5 ppt) of 14 days. One hundred and eighty trout (initial mean weight 230 g) were randomly and equally alloted to 6 tanks (water volume 260 litres) which were each supplied by running seawater (water flow rate 420 l/h, salinity 36.5 ppt ). Trout, in duplicate tanks per diet, were adapted to the chromic-oxide diets for 7 days. Faeces were then continously collected for 9 days by the faecal settling column method (Guelph system) according to Cho et al. ( 1982). Trout were fed twice a day a daily ration of 1.4% of body weight. Temperature ranged from 7.9 to 8.7”C during the trial. Faeces were freeze-dried, ground, and pooled for each tank before analyses. Chromic oxide was measured after perchloric acid digestion (Bolin et al., 1952). Apparent digestibility coefficients (ADC) were calculated according to Maynard and Loosli ( 1969).
Plasma glucose levels After the digestibility trial, trout were maintained on their respective experimental diets for another week. They were then fasted for 48 h. Blood samples were collected first on 5 fasted trout from each group (O-sampling time). These trout were then discarded while the others were fed one meal of the experimental diets at a ration level of 1.7% BW. Blood was sampled at each selected time interval (2,4,6, 8, 12, 16,24 and 30 h after feeding) from 5 fish per diet. These fish were removed randomly from the duplicate tanks. Fish were discarded after blood sampling in order to sample each trout once only. Blood samples were taken by caudal vessel puncture on anaesthetized fish (mean body weight 290 g). A mixture of potassium oxalate 4% and sodium fluoride 4% was used to prevent coagulation and glycolysis. Blood was immediately centrifuged and plasma glucose was analysed using a glucose analyser (Beckman II, USA). Water temperature was 8.2 oC during this study.
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Statistical analyses One-way analysis of variance was performed to assess the effect of the dietary treatment on the different parameters. The effect of sampling time on plasma glucose levels was also tested by one-way analysis of variance since each sample was taken from a different fish. When a significant effect was found, means were compared using the Neuman-Keuls multiple range test. Significance was tested at a 0.05 Type I error level. Statistical analyses were performed using the SAS package (1987).
3. Results The apparent digestibility coefficients for protein and lipid were not influenced by dietary treatment (Table 2). As expected, ADC of starch decreased significantly (P< 0.05 ) with increasing dietary levels of crude starch. Consequently, the ADC value of energy was the lowest for diet LC. The digestible energy provided by carbohydrate was 28,21 and 10% of total digestible energy for diets HC, MC and LC, respectively, and ratios of digestible carbohydrate to digestible lipid were 4.3,2.2 and 0.7, respectively (Table 2). Voluntary feed intake of fish fed diet LC, i.e. the diet containing the highest level of crude starch, was slightly but not significantly (P> 0.05) lower than found for fish fed the other diets (Table 3 ). Digestible protein intake was not changed, but digestible energy intake was significantly depressed (PC 0.05 ) in fish fed diet LC. Final body weights, specific growth rates and feed efficiencies tended to be lower for trout fed diet HC, but these differences were not significant (P> 0.05 ) (Table 3). Table 2 Apparent digestibility coefficients of the dietary components diets as digestible nutrients or energy Diet HC
and composition
of the experimental
Diet MC
Diet LC
Apparent digestibility coefficients (%) Protein Lipid Starch Energy
86.7 80.8 81.1” 78.3”
86.0 81.5 73.1b 76.9a
87.4 76.8 35.4” 67.2b
36.9 8.Sb 18.9b 1546”
37.4 11.1a 8.1’ 1418b
Digestible nutrients (%) or energy (W/100 g DM) in the diets Digestible Digestible Digestible Digestible
protein lipid carbohydrate energy
38.2 5.7” 24.4a 1496ab
Values (means of 2 tanks) with different (PiO.05).
superscripts
in the same row are significantly
different
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Table 3 Feed intake, growth performance diets for 9 weeks
and nutrient retention
efficiencies
in trout fed the experimental
Diet HC
Diet MC
Diet LC
12.1 5.4” 4.7 231.7 181.0”
12.3 5.3” 4.5 247.7 190.3a
11.5 4.9b 4.3 242.0 162.7b
230.5 f2.4 429.0 +5.1 1.04t0.02 0.79kO.02
227.6 i5.4 451.0 57.9 1.14f0.04 0.84kO.01
227.3 k9.8 444.1 k3.5 1.12+0.09 0.88 I!I0.05
33.9 f2.5 43.5 f2.5
36.6 k2.5 46.6 k2.5
Intake Feed (g DM/kg BW/d) Crude protein (g/kg BW/d) Digestible protein (g/kg BW/d) Gross energy (W/kg BW/d) Digestible energy (kJ/kg BW/d)
Growth performance Initial body weight (g) Final body weight (g ) Specific growth rate’ (o/o/d) Feed efficiency2
Retention ef$ciency as % of digestible intake Protein Energy
31.5 AI1.7 41.0 +2.6
Values (mean I!Istandard error of mean, n = 3) with different superscript letter in the same row are significantly different (PC 0.05 ) . ‘Specific growth rate: [ (In BWt end - In BWt start) /62 ] x 100. ‘Feed efficiency: wet weight gain/dry food intake. 3Retention efficiency coeficient: Nutrient or energy retained/Intake of digestible nutrient or digestible energy.
Protein and energy retention efficiencies tended to increase with decreasing dietary carbohydrate/lipid ratio (Table 3 ). Whole-body lipid gain in relation to digestible lipid intake (calculated as for protein and energy retention efficiencies) was elevated in fish fed diets with the highest digestible carbohydrate levels ( 136.62 13.9% with diet HC and 109.3 + 7.0% with diet MC) relative to those fed diet LC (70.9? 5.4%). Compositions of whole bodies were not influenced by dietary treatment (Table 4). By contrast, liver size in relation to body weight (hepatosomatic index) and hepatic lipid content were significantly elevated (P< 0.05) in fish fed diets HC and MC. Water content was inversely related to lipid level in the liver and dorsal and ventral muscle. Irrespective of dietary treatment, ventral muscle had more lipid than dorsal muscle. Muscle lipid concentration was increased significantly in fish fed diet MC. After a 48-h fasting period, plasma glucose was higher in trout previously fed diets HC and MC than in fish fed diet LC (Fig. 1). Within each group, an increase in plasma glucose was observed after feed intake. As expected, fish fed diet LC had low values for plasma glucose and those fed diet HC always had the highest plasma glucose. Within each dietary treatment, mean values for plasma glucose concentration at each sampling time were compared. According to the results of the statistical
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Table 4 Whole-body and tissue levels of proximate constituents (on wet weight basis) and hepatosomatic indices (HSI) of trout fed the experimental diets for 9 weeks
Whole body Water (%) Protein (%) Lipid (%) Ash (%) Energy (W/100 g)
Diet HC
Diet MC
71.lf 0.5 17.0+ 0.2 8.7& 0.3 3.2+ 0.1 747 k16
70.4i 17.0’1 9.5f. 3.1+ 764 +ll
Diet LC
0.4 0.4 0.2 0.1
71.4’r 0.4 17.35 0.1 8.4+ 0.4 3.0f 0.1 729 +17
Liver HSI (%) Water (O/o) Lipid (mg/g)
2.8 ‘I 0.2” 72.1 &0.9b 52.620.4”
2.5kO.l” 72.4k0.1b 49.8 + 2.7a
1.3fO.Ob 74.9 f 0.2” 44.5 & 0.9b
Dorsal muscle Water (%) Lipid (mg/g)
75.5 f0.2” 49.2+ 3.2
74.8+0.1b 51.6kO.3
75.320.2” 45.1 f 1.4
Ventral muscle Water (%) Lipid (mg/g)
73.9fO.l” 68.Of 1.9b
73.0k0.2b 76.8+ 1.5”
74.3kO.3” -63.8 + 2.6b
Values (mean + s.e.m., n = 3 for whole body and n = 6 for tissues) with different superscript letters in the same row are significantly different (PcO.05).
300
3250 2 $200 8 d B ml50 E ta -ii 100
50 0
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 time (h)
Fig. 1. Postprandial pattern of glycaemia in trout fed the experimental diets. For each sample time, values (mean + s.e.m., n = 5 ) with the same superscript letter are not significantly different (P> 0.05 ) between the dietary treatments. -IX- diet HC; -O-diet MC, -V-diet LC.
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test (not shown on the figure), the higher the dietary level of digestible carbohydrate, the later the attainment of maximum plasma glucose level. Plasma glucose concentration returned to the fasting level 30 h after food ingestion in fish fed diet MC. However, the plasma glucose level in fish fed diet HC at this time was about two-fold higher than in those fed diet MC. For fish fed diet LC, plasma glucose returned to the fasting level 12 h after the meal and subsequently increased again 16 and 30 h after the meal.
4. Discussion Digestibility of starch is related to the physical state of the starch (i.e. crude or gelatinized). The improvement in complex carbohydrate digestibility by gelatinization has been established in freshwater species (Takeuchi et al., 1990; Medale et al., 199 1; Bergot, 1993) and in marine species (Jeong et al., 199 1). The mixed starch diet had a higher ADC value for starch and energy than expected. This result may have been due to partial gelatinization of crude starch during the processing of this diet. The digestibility of protein and lipid was not affected by the type of dietary carbohydrate or by the carbohydrate/lipid ratio. Protein and lipid ADC values were low compared to those generally found in rainbow trout reared in freshwater (Cho et al., 1982). MacLeod ( 1977) reported that salinity decreased the absorption efficiency of protein and energy in rainbow trout. Such a diminution in protein digestibility due to salinity has also been shown in the same species by La11 and Bishop ( 1976). On the other hand, the ADC value of gelatinized starch in this study was higher than the values found for trout grown in freshwater at the same temperature (Brauge, unpublished data). According to these data, the effect of salinity on specific digestive enzymes needs further investigations. The growth rate of trout was not affected by the dietary levels and sources of non-protein energy used in the present study. Edwards et al. ( 1977) observed that, up to 25% of “metabolizable energy” (calculated value) provided by carbohydrate, dietary carbohydrate/lipid ratio had no influence on growth of trout, either in diluted seawater or in freshwater. Our results agree with these data, showing that rainbow trout reared in seawater are able to efficiently utilize high amounts of digestible carbohydrate, unlike chinook salmon. Mazur et al. ( 1992) found that gelatinized starch providing 22.5% of the “metabolizable energy” had an adverse effect on growth of chinook salmon. Moreover, the feed intake of chinook salmon was higher when they were fed a high-lipid diet. In the present study, the voluntary feed intake of trout tended to be depressed for the diet containing the highest level of lipid. Feed efficiency of trout was improved slightly when they ingested the diet containing the highest lipid level, but there was no significant effect of dietary treatment. .However, the production of 1 kg of rainbow trout required 475 2 7 g of digestible protein with diet HC and only 419 + 21 g with diet LC. Further, 18.8 2 0.3 MJ of digestible energy per kilogram weight gain were needed for fish
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fed diet HC whereas those fed diet LC required 16.0 ? 0.8 MJ DE/kg weight gain. The foregoing results suggest that energy from lipid spares protein more effectively than energy from carbohydrate when trout are held in seawater. Whole-body composition was not influenced by the dietary carbohydrate/lipid ratios, indicating increased lipid synthesis in fish fed diets high in digestible carbohydrate content. This conclusion is supported by the high “lipid retention efficiencies”, i.e. whole-body lipid gain in relation to digestible lipid intake (above 100 o/o),noted in fish fed the diets high in digestible carbohydrate. In fact, “retention efficiency” is not a meaningful term for lipid since deposited fatty acids can originate directly in dietary lipid or be synthesized from dietary carbohydrate. The amount of fat deposited seems, therefore, to be connected to the amount of non-protein digestible energy in the diet rather than to the sources of the nonprotein digestible energy. In contrast to the situation for the whole body, liver and muscle composition varied with the dietary treatments. Hepatosomatic index and liver lipid content were higher for fish fed the diets high in digestible carbohydrate. It is well known that liver size is directly related to hepatic glycogen level in salmonids (Bergot, 1979a; Pieper and Pfeffer, 1980; Dixon and Hilton, 1985; Kim and Kaushik, 1992). Absorbed carbohydrate which is not utilized to provide energy can be deposited in the liver both as glycogen and as lipid after conversion. According to Henderson and Sargent ( 198 1)) the liver is the main site of fatty acid synthesis in salmonids. In the present study, the liver composition data suggest that a high dietary carbohydrate/lipid ratio enhances lipid synthesis and deposition in the liver. The diet containing the highest level of lipid led to the lowest level of liver lipid, suggesting that lipid deposition from dietary lipid does not occur in liver but probably in adipose tissue. We did not measure the lipid content of the whole digestive tract, but many studies have shown an increase in perivisceral adipose tissue after salmonids have been fed high-fat diets (see Watanabe, 1982). The lipid content of ventral muscle was surprisingly high in fish fed the diet with the intermediate carbohydrate/lipid ratio. This may have stemmed from a combination of deposition of dietary lipid and lipogenesis from carbohydrate since the diet contained both gelatinized starch and 10% lipid. Indeed, in vitro data obtained by Henderson and Sargent ( 198 1) showed that glucose exerted no inhibitory effect on lipogenesis when dietary lipid level was 10% or less. Plasma glucose levels were related to the digestible carbohydrate content of the diets and remained high 24 h after feeding the high-carbohydrate diets. This prolonged hyperglycaemia, previously observed in trout reared in freshwater (Palmer and Ryman, 1972; Bergot, 1979b; Himick et al., 1991; Higgs et al., 1992), has been described as an “ill-effect” of digestible carbohydrate on glucose metabolism. In the present study, the term “ill-effect” does not seem to be appropriate since no pathological signs were noted and the trout grew well. The high plasma glucose concentrations induced by high dietary levels of digestible carbohydrate were initially thought to be due to a lack of insulin secretion (Palmer and Ryman, 1972 ). Recent studies have clearly demonstrated that salmonids do have the ability to secrete insulin and to increase insulin secretion in response to high levels of
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dietary carbohydrate (Hilton et al., 1987 ) . Impaired binding of insulin to its receptors in skeletal muscle was suspected initially as the problem (Plisetskaya, 1989). However, it now appears that the problem is impairment of one or more post-receptor events (Gutierrez et al., 199 1) . The hyperglycaemia could also result from a suppression of insulin secretion by elevated somatostatin secretion (Harmon et al., 1991). The observed increases in plasma glucose concentration in trout fed the diet low in digestible carbohydrate, 16 and 30 h after feed intake, suggest that glucose can be provided by gluconeogenesis when it is not supplied directly by dietary carbohydrate, as has been described in freshwater fish by Walton and Cowey (1982) andMalinovskaya (1989). Little is known about the effect of salinity on glycaemia. Sweeting et al. ( 1985) did not observe any change in plasma glucose of coho salmon smolts after a 24-h acclimation period in seawater. In the present study, the peak of plasma glucose was reached earlier than in trout fed the same experimental diets and reared at the same temperature in freshwater (Brauge, unpublished data). Therefore, carbohydrate absorption may be stimulated in trout held in seawater. According to Garin ( 1984), increased water salinity led to an elevation in utilization rate of glucose in seabass. Such an effect of salinity, however, did not seem to exist in rainbow trout since plasma glucose remained high 30 h after feeding the diet with high levels of digestible starch. Acknowledgements
This study was supported by a research grant from IFREMER (Institut Francais de Recherches pour l’Exploitation de la Mer) and the Conseil Regional d’Aquitaine (France). The experiments were performed at the fish nutrition laboratory of IFREMER (Plouzane, France). We thank Dr. R. Metailler and his team for providing the rearing facilities and for their advice. Dr. S. Kaushik is also acknowledged with gratitude for his critical reading of the manuscript. References Bergot, F., 1979a. 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-l 67. Bergot, F., 1979b. Effects of dietary carbohydrates and of their mode of distribution on glycaemia in rainbow trout Salmo gairdneri (Richardson). Comp. Biochem. Physiol., 64A: 543-547. Bergot, F., 1993. Digestibility of native starches of various botanical origins by rainbow trout (Oncorhynch mykiss). In: S.J. Kaushik and P. Luquet (Editors), Fish Nutrition in Practice. INRA, Paris, pp. 857-865. Bolin, D.W., Ring, R.P. and Klosterman, W.W., 1952. A simplified method for the determination of chromic oxide ( CrzO,) when used as an inert substance. Science, 16: 634-635. Cho, C.Y., Slinger, S.J and Bayley, H.S., 1982. Bioenergetics of salmonid fishes: energy intake, expenditure and productivity. Comp. Biochem. Physiol., 73B: 25-41. Dixon, D.G. and Hilton, J.W., 1985. Effects of available carbohydrate and water temperature on the
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chronic toxicity of waterborne copper to rainbow trout (Salmo gairdneri). Can. J. Fish. Aquat. Sci., 42: 1007-1013. Edwards, D.J., Austreng, E., Risa, S. and Gjedrem, T., 1977. Carbohydrate in rainbow trout diets. I. Growth of fish of different families fed diets containing different proportions of carbohydrate. Aquaculture, 11: 31-38. Folch, J., Lees, M. and 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. Garin, D., 1984. Influence de la salinite sur l’utilisation oxydative du glucose et du glutamate chez le loup (Dicentrarchus labrax). Ichtyophysiol. Acta, 8: 58-74. Gutierrez, J., Asgard, T., Fabbri, E. and Plisetskaya, E.M., 1991. Insulin-receptor binding in skeletal muscle of trout. Fish Physiol. Biochem., 9: 351-360. Harmon, J.S., Eilertson, C.D., Sheridan, M.A. and Plisetskaya, E.M., 1991. Insulin suppression is associated with hypersomatostatinemia and hyperglucagonemia in glucose-injected rainbow trout. Am. J. Physiol. 261: R609-R613. Henderson, R.J. and Sargent, J.R., 1981. Lipid biosynthesis in rainbow trout, Salmo gairdnerii, fed diets of differing lipid content. Comp. Biochem. Physiol., 69C: 3 l-37. Higgs, D.A., Dosanjh, B.S., Uin, L.M., Himick, B.A. and Eales, J.G., 1992. Effects ofdietary lipid and carbohydrate levels and chronic 3,5,3’-triiodo-r_-thyronine treatment on growth, appetite, food and protein utilization and body composition of immature rainbow trout, Oncorhynchus mykiss, at low temperature. Aquaculture, 105: 175-190. Hilton, J.W. and Atkinson, J.L., 1982. Response of rainbow trout (Salmo gairdneri) to increased levels of available carbohydrate in practical trout diet. Br. J. Nutr., 47: 597-607. Hilton, J.W., Plisetskaya, E.M. and Leatherland, J.F., 1987. Does oral 3,5,3’-triiodo-L-thyronine affect dietary glucose utilization and plasma insulin levels in rainbow trout (Sabrzo gairdneri)? Fish Physiol. Biochem., 4: 113-l 20. Himick, B.A., Higgs, D.A. and Eales, J.G., 1991. The acute effects of alteration in the dietary concentrations of carbohydrate, protein and lipid on plasma Tq, TJ, and glucose levels in rainbow trout, Oncorhynchus mykiss. Gen. Comp. Endocrinol., 82: 45 l-458. Jeong, K.-S., Takeuchi, T. and Watanabe, T., 1991. Improvement of nutritional quality of carbohydrate ingredients by extrusion process in diets of red sea bream. Nippon Suisan Gakkaishi, 57: 1543-1549. Kaushik, S.J. and Oliva-Teles, A., 1985. Effect of digestible energy on nitrogen and energy balance in rainbow trout. Aquaculture, 50: 89-101. Kaushik, S.J., Medale, F., Fauconneau, B., and Blanc D., 1989. Effect of digestible carbohydrates on protein/energy utilization and on glucose metabolism in rainbow trout (Salmo gairdneri R.). Aquaculture, 79: 63-74. Kim, J.D. and Kaushik, S.J., 1992. Contribution of digestible energy from carbohydrates and estimation of protein/energy requirements for growth of rainbow trout (Oncorhynchus mykiss). Aquaculture, 106: 161-169. Lall, S.P. and Bishop, F.J., 1976. Studies on the nutrient requirements of rainbow trout (Salmo gairdneri) grown in seawater and freshwater. In: T.V.R. Pillay and Wm. A. Dill, (Editors). Advances in Aquaculture. Fishing New Books Ltd., Famham, Surrey, UK pp. 580-584. MacLeod, M.G., 1977. Effects of salinity on food intake, absorption and conversion in the rainbow trout Salmogairdneri. Mar. Biol., 43: 93-102. Malinovskaya, M.V., 1989. Pathways of carbohydrate metabolism in fishes in relation to temperature adaptation (a review). Hydrobiol. J., 597: 30-40. Maxime, V., Soulier, P., Quentel, C., Aldrin, J.F. and Peyraud, C., 1986. Etude comparative de la consommation d’oxygene et de quelques parametres hemochimiques et hematologiques chez la truite arc-en-ciel et la truite fario, en eau deuce et en eau de mer, au cours de l’augmentation estivale de temperature. Ichtyophysiol. Acta, 10: 185-200. Maynard, L.A. and Loosli, J.K., 1969. Animal Nutrition, 6th edn., MacGraw-Hill, New York, pp. 613. Mazur, C.N., Higgs, D.A., Plisetskaya, E. and March, B.E., 1992. Utilization of dietary starch and
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glucose tolerance in juvenile chinook salmon (Oncorhynchus tshawytscha) of different strains in seawater. Fish Physiol. Biochem., 10: 303-3 13. Medale, F., Aguirre, P. and Kaushik, S.J., 199 1. Utilization of dietary carbohydrates by rainbow trout at two water temperatures. In: C. Wenk and M. Boessinger (Editors), Energy Metabolism of Farm Animals. European Association for Animal Production, Publ. 58: 392-395. Palmer, T.N. and Ryman, B.E., 1972. Studies on oral glucose intolerance in fish. J. Fish Biol., 4: 31 I319. Pieper, A. and Pfeffer, E., 1980. Studies on the comparative efficiency of utilization of gross energy from some carbohydrates, proteins and fats by rainbow trout (Salmo gairdneri, R. ) . Aquaculture, 20: 323-332. Plisetskaya, E.M., 1989. Physiology of fish endocrine pancreas. Fish Physiol. Biochem., 7: 39-48. SAS, 1987. SAS/Stat Guide for Personal Computers. SAS Inst. Inc., North Carolina, USA, 1028 pp. Sweeting, R.M., Wagner, C.F. and MacKeown, B.A., 1985. Changes in plasma glucose, amino acid, nitrogen and growth hormone during smoltification and seawater adaptation in coho salmon, Oncorhynchus kisutch. Aquaculture, 45: 185-197. Takeuchi, T., Jeong, K.-S. and Watanabe, T., 1990. Availability of extruded carbohydrate to rainbow trout (Oncorhynchus mykiss) and carp (Cyprinus carpio). Nippon Suisan Gakkaishi, 56: 18391845. Thivend, P., Mercier, C. and Guilbot, A., 1972. Determination of starch with glucoamylase. In: R.L. Whistler and J.N. Bemiller (Editors), Methods in Carbohydrate Chemistry, Vol. VI. Academic Press, New York-London, pp. 100-105. Walton, M.J. and Cowey, C.B., 1982. Aspects of intermediary metabolism in salmonid fish. Comp. Biochem. Physiol., 73B: 59-79. Watanabe T., 1982. Lipid nutrition in fish. Comp. Biochem. Physiol., 73B: 3-l 5.
Aquaculture 123 (1994) 109-120
Effect of dietary carbohydrate levels on growth, body composition and glycaemia in rainbow trout, Oncorhynchus mykiss, reared in seawater C. Brauge, F. Medale*, G. Corraze INRA, Fish Nutrition Laboratory, Hydrobiology Station, BP3, 64310 Saint Pie-sur-Nivelle, France (Accepted 13 January 1994)
Abstract Three experimental diets were fed to near-satiation twice a day for 9 weeks to triplicate groups of 40 rainbow trout (mean initial weight 230 g) reared in seawater (36.5 ppt) at 9°C. The diets were formulated to have the same levels of nitrogen (crude protein 43% DM) and energy (digestible energy: around 15 kJ/g DM) but they contained different amounts of digestible carbohydrate (8-24.4%) and fat (5.7-l 1.1%). Digestibility was measured using chromic oxide as a marker. Plasma glucose concentrations were measured in 48-h fasted trout and at selected intervals after a single meal. Digestibility of starch and energy was lowest in trout fed the diet containing the highest level of crude starch. Apparent digestibility values of protein (86.0-87.4%) and fat (76.8-8 1.5%) were similar for the three diets. Fish growth and body composition were not affected significantly (P> 0.05) by the non-protein energy sources of the diets. However, the amounts of digestible protein and digestible energy required per kilogram production were higher for fish fed the highcarbohydrate diet (475 ? 7 g DP and 18.8 ?- 0.3 MJ DE) than for fish fed the high-fat diet (4 19 f 2 1 g DP and 16.0 5 0.8 MJ DE). Liver size and liver lipid content increased in direct relationship to the dietary level of digestible carbohydrate. The highest percentage of muscle lipid was found in fish fed the diet containing 25% carbohydrate and 10% lipid as non-protein energy sources. Glycaemia was increased by elevated digestible carbohydrate intake. In conclusion, 230 g rainbow trout were able to utilize carbohydrate efficiently for growth in seawater (up to 25% of digestible carbohydrate in diet) under the conditions of this study. However, dietary lipid had a slightly better protein-sparing effect than digestible carbohydrate at a similar level of digestible energy intake. High dietary levels of digestible carbohydrate induced lipogenesis and enhanced lipid deposition in the liver. *Corresponding author. 0044-8486/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SsDIOO44-8486(94)00018-J
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1. Introduction Many previous studies have been conducted with the aim of decreasing the level of protein in fish diets by adding energy from non-protein sources. Energy from dietary lipid has been shown to effectively spare protein energy in several fish species (Watanabe, 1982). The effect of dietary carbohydrate on fish growth seems to depend upon the source, dietary concentration and digestibility, the level of dietary intake, rearing conditions and fish species. Hilton and Atkinson ( 1982) reported that high dietary levels ( > 14%) of cerelose depressed growth of rainbow trout. However, it has been demonstrated that digestible carbohydrate in the form of gelatinized starch improves protein and energy utilization in the same species (Kaushik and Oliva Teles, 1985; Kaushik et al., 1989; Kim and Kaushik, 1992). Also, according to Pieper and Pfeffer ( 1980), gelatinized starch is just as effective as lipid as an energy source for rainbow trout. These studies were carried out with small juvenile trout ( 15-40 g) in freshwater, and at high temperature ( 15- 18 “C) . Sea-ranching of large rainbow trout has been extended for the past 10 years in France. This type of ranching needs lower water temperature than in freshwater since euryhalinity of rainbow trout is reduced at high temperature (Maxime et al., 1986). Little is known about the effect of dietary carbohydrate level on growth of salmonids reared in seawater. Mazur et al. ( 1992)) however, found that lipid was a better non-protein energy source than digestible carbohydrate for chinook salmon held in seawater. Further data about nutrient utilization of large rainbow trout held in seawater are needed to improve production efficiency of trout sea-ranching. The aim of this study was to assess the effect of increasing dietary levels of digestible carbohydrate on growth, feed efficiency and body composition of rainbow trout reared in seawater at low temperature. Since high dietary levels of digestible carbohydrate have led to prolonged hyperglycaemia in salmon (Mazur et al., 1992), the effect of feeding the experimental diets to trout on plasma glucose level was also examined.
2. Materials and methods
Experimental diets Three experimental diets were formulated to contain constant levels of crude protein (Table 1) and digestible energy (Tables 1 and 2 ). Increasing amounts of fish oil were included in the diets to obtain graded levels of lipid. The amount of digestible energy supplied by carbohydrate was adjusted by using either gelatinized wheat starch (diet HC), crude wheat starch (diet LC) or a nearly equal mixture of both kinds of starch (diet MC). The diets HC and MC were coldpelleted using a laboratory pellet mill ( Simon-Heesen, Oslo). Because of its high fat level, diet LC was drawn into spaghetti-like strands using a meat grinder after
C. Brauge et al. /Aquaculture, 123 (1994) 109-120 Table 1 Ingredients and proximate compositions Ingredients
(%)
Fish meal’ Fish oil* Gelatinized wheat starch Crude wheat starch Vitamin mix3 Mineral mix4 Sodium alginate
of the experimental
111
diets
Diet HC
Diet MC
Diet LC
60.1 0.0 30.1 6.8 1.0 1.0 1.0
60.1 4.9 16.6 15.4 1.0 1.0 1.0
60.1 9.8 0.6 26.5 1.0 1.0 1.0
87.9 44.1 7.0 30.1 12.1 19.1
91.8 42.9 10.4 25.8 12.2 20.1
92.4 42.8 14.4 22.8 12.1 21.1
Proximate composition Dry matter (% WW) Crude protein (%DM) Crude lipid (% DM) Starch (% DM ) Ash (O/oDM) Gross energy (W/g DM)
‘Norwegian herring fish meal (CP = 69.8% DM), Descal, Donzacq, France. ?Soprop&che, Boulogne-sur-mer, France. Acidity index= 6%, stabilized with 200 ppm ethoxyquin, peroxide index < 10, iodine index between 140 and 160. 3Composition of vitamin mix (mg/g): vitamin A acetate, 1 (500 UI); nL-cholecakiferol, 2 (200 UI); DL-cf-tocopheryl acetate, 5 (2.5 UI); menadione, 0.30; thiamine hydrochloride, 0.75; riboflavin, 1; pyridoxine hydrochloride, 0.75; cyanocobalamin, 0.0025; nicotinic acid, 10; ascorbic acid, 25; folic acid, 0.25; calcium pantothenate, 2.5; choline chloride, 100; inositol, 50; biotin, 0.12. All ingredients were diluted with cellulose to 1 g. 4Composition of mineral mix (mg/g): calcium biphosphate*2H,O, 500; calcium carbonate, 2 15; sodium chloride, 40; potassium chloride, 90; magnesium hydroxide, 124; zinc sulfatee7H,O, 4; manganese sulfate.H,O, 3; cuprous sulfate*5H,O, 3; cobaltous sulfate, 0.02; potassium iodide, 0.04; ferric citrate- 7Hz0, 20; sodium fluoride, 1.
the addition of 20% water. Diet LC was then dried in an oven at 35°C for 48 h and subsequently cut into pellets. Chromic oxide was added to a portion of each diet at a level of 1% as an inert marker for digestibility measurement. The three experimental diets, kept in a cold room (4’ C) until use, were analysed for proximate composition according to the following procedures: dry matter by drying at 110 oC for 24 h, ash by combustion at 550 “C in a muffle furnace for 24 h, crude protein by the Kjeldahl method after acid digestion ( %Nx 6.25)) lipid according to Folch et al. ( 1957), starch using amylase and glucose oxidase (Thivend et al., 1972)) and gross energy using an IRA adiabatic bomb calorimeter. Feeding trial
Rainbow trout (Oncorhynchus mykiss) were transferred from freshwater to diluted seawater ( 17 ppt). After a 7-day acclimation period, 369 rainbow trout were alloted randomly and equally to 9 tanks (water volume: 450 litres) which were each supplied by running seawater (water flow rate: 750 l/h, salinity 36.5 ppt ). The photoperiod was 10 h light/ 14 h darkness. Initial weighing was carried
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out after another 7-day acclimation period to tanks and salinity. Nine fish ( l/ tank) were sampled for determination of initial whole body composition. Triplicate groups of 40 trout (initial mean body weight 230 g) were fed one of three experimental diets to near-satiation twice a day for 9 weeks. Fish were groupweighed and counted every 3 weeks, and feed intake was recorded daily. Water temperature varied between 7.9 and 10.7 ‘C during the experiment. At the end of the feeding trial, 10 fish per tank were withdrawn randomly and killed after anaesthesia (ethylene glycol monophenyl ether 1:2500). Composition of whole bodies was determined on 3 groups of 8 fish per diet, using the same analytical procedures as for proximate composition of diets. Whole bodies were freeze-dried, ground, and pooled for each tank before analyses. Liver, dorsal and ventral muscles were removed from 6 fish per diet, frozen in liquid nitrogen and stored at - 20” C. Liver and muscle samples were then analysed individually for lipid content by the Folch method.
Digestibility trial Apparent digestibility of nutrients and energy was determined using chromic oxide as an indigestible marker after a progressive acclimation period to salinity (36.5 ppt) of 14 days. One hundred and eighty trout (initial mean weight 230 g) were randomly and equally alloted to 6 tanks (water volume 260 litres) which were each supplied by running seawater (water flow rate 420 l/h, salinity 36.5 ppt ). Trout, in duplicate tanks per diet, were adapted to the chromic-oxide diets for 7 days. Faeces were then continously collected for 9 days by the faecal settling column method (Guelph system) according to Cho et al. ( 1982). Trout were fed twice a day a daily ration of 1.4% of body weight. Temperature ranged from 7.9 to 8.7”C during the trial. Faeces were freeze-dried, ground, and pooled for each tank before analyses. Chromic oxide was measured after perchloric acid digestion (Bolin et al., 1952). Apparent digestibility coefficients (ADC) were calculated according to Maynard and Loosli ( 1969).
Plasma glucose levels After the digestibility trial, trout were maintained on their respective experimental diets for another week. They were then fasted for 48 h. Blood samples were collected first on 5 fasted trout from each group (O-sampling time). These trout were then discarded while the others were fed one meal of the experimental diets at a ration level of 1.7% BW. Blood was sampled at each selected time interval (2,4,6, 8, 12, 16,24 and 30 h after feeding) from 5 fish per diet. These fish were removed randomly from the duplicate tanks. Fish were discarded after blood sampling in order to sample each trout once only. Blood samples were taken by caudal vessel puncture on anaesthetized fish (mean body weight 290 g). A mixture of potassium oxalate 4% and sodium fluoride 4% was used to prevent coagulation and glycolysis. Blood was immediately centrifuged and plasma glucose was analysed using a glucose analyser (Beckman II, USA). Water temperature was 8.2 oC during this study.
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Statistical analyses One-way analysis of variance was performed to assess the effect of the dietary treatment on the different parameters. The effect of sampling time on plasma glucose levels was also tested by one-way analysis of variance since each sample was taken from a different fish. When a significant effect was found, means were compared using the Neuman-Keuls multiple range test. Significance was tested at a 0.05 Type I error level. Statistical analyses were performed using the SAS package (1987).
3. Results The apparent digestibility coefficients for protein and lipid were not influenced by dietary treatment (Table 2). As expected, ADC of starch decreased significantly (P< 0.05 ) with increasing dietary levels of crude starch. Consequently, the ADC value of energy was the lowest for diet LC. The digestible energy provided by carbohydrate was 28,21 and 10% of total digestible energy for diets HC, MC and LC, respectively, and ratios of digestible carbohydrate to digestible lipid were 4.3,2.2 and 0.7, respectively (Table 2). Voluntary feed intake of fish fed diet LC, i.e. the diet containing the highest level of crude starch, was slightly but not significantly (P> 0.05) lower than found for fish fed the other diets (Table 3 ). Digestible protein intake was not changed, but digestible energy intake was significantly depressed (PC 0.05 ) in fish fed diet LC. Final body weights, specific growth rates and feed efficiencies tended to be lower for trout fed diet HC, but these differences were not significant (P> 0.05 ) (Table 3). Table 2 Apparent digestibility coefficients of the dietary components diets as digestible nutrients or energy Diet HC
and composition
of the experimental
Diet MC
Diet LC
Apparent digestibility coefficients (%) Protein Lipid Starch Energy
86.7 80.8 81.1” 78.3”
86.0 81.5 73.1b 76.9a
87.4 76.8 35.4” 67.2b
36.9 8.Sb 18.9b 1546”
37.4 11.1a 8.1’ 1418b
Digestible nutrients (%) or energy (W/100 g DM) in the diets Digestible Digestible Digestible Digestible
protein lipid carbohydrate energy
38.2 5.7” 24.4a 1496ab
Values (means of 2 tanks) with different (PiO.05).
superscripts
in the same row are significantly
different
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Table 3 Feed intake, growth performance diets for 9 weeks
and nutrient retention
efficiencies
in trout fed the experimental
Diet HC
Diet MC
Diet LC
12.1 5.4” 4.7 231.7 181.0”
12.3 5.3” 4.5 247.7 190.3a
11.5 4.9b 4.3 242.0 162.7b
230.5 f2.4 429.0 +5.1 1.04t0.02 0.79kO.02
227.6 i5.4 451.0 57.9 1.14f0.04 0.84kO.01
227.3 k9.8 444.1 k3.5 1.12+0.09 0.88 I!I0.05
33.9 f2.5 43.5 f2.5
36.6 k2.5 46.6 k2.5
Intake Feed (g DM/kg BW/d) Crude protein (g/kg BW/d) Digestible protein (g/kg BW/d) Gross energy (W/kg BW/d) Digestible energy (kJ/kg BW/d)
Growth performance Initial body weight (g) Final body weight (g ) Specific growth rate’ (o/o/d) Feed efficiency2
Retention ef$ciency as % of digestible intake Protein Energy
31.5 AI1.7 41.0 +2.6
Values (mean I!Istandard error of mean, n = 3) with different superscript letter in the same row are significantly different (PC 0.05 ) . ‘Specific growth rate: [ (In BWt end - In BWt start) /62 ] x 100. ‘Feed efficiency: wet weight gain/dry food intake. 3Retention efficiency coeficient: Nutrient or energy retained/Intake of digestible nutrient or digestible energy.
Protein and energy retention efficiencies tended to increase with decreasing dietary carbohydrate/lipid ratio (Table 3 ). Whole-body lipid gain in relation to digestible lipid intake (calculated as for protein and energy retention efficiencies) was elevated in fish fed diets with the highest digestible carbohydrate levels ( 136.62 13.9% with diet HC and 109.3 + 7.0% with diet MC) relative to those fed diet LC (70.9? 5.4%). Compositions of whole bodies were not influenced by dietary treatment (Table 4). By contrast, liver size in relation to body weight (hepatosomatic index) and hepatic lipid content were significantly elevated (P< 0.05) in fish fed diets HC and MC. Water content was inversely related to lipid level in the liver and dorsal and ventral muscle. Irrespective of dietary treatment, ventral muscle had more lipid than dorsal muscle. Muscle lipid concentration was increased significantly in fish fed diet MC. After a 48-h fasting period, plasma glucose was higher in trout previously fed diets HC and MC than in fish fed diet LC (Fig. 1). Within each group, an increase in plasma glucose was observed after feed intake. As expected, fish fed diet LC had low values for plasma glucose and those fed diet HC always had the highest plasma glucose. Within each dietary treatment, mean values for plasma glucose concentration at each sampling time were compared. According to the results of the statistical
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Table 4 Whole-body and tissue levels of proximate constituents (on wet weight basis) and hepatosomatic indices (HSI) of trout fed the experimental diets for 9 weeks
Whole body Water (%) Protein (%) Lipid (%) Ash (%) Energy (W/100 g)
Diet HC
Diet MC
71.lf 0.5 17.0+ 0.2 8.7& 0.3 3.2+ 0.1 747 k16
70.4i 17.0’1 9.5f. 3.1+ 764 +ll
Diet LC
0.4 0.4 0.2 0.1
71.4’r 0.4 17.35 0.1 8.4+ 0.4 3.0f 0.1 729 +17
Liver HSI (%) Water (O/o) Lipid (mg/g)
2.8 ‘I 0.2” 72.1 &0.9b 52.620.4”
2.5kO.l” 72.4k0.1b 49.8 + 2.7a
1.3fO.Ob 74.9 f 0.2” 44.5 & 0.9b
Dorsal muscle Water (%) Lipid (mg/g)
75.5 f0.2” 49.2+ 3.2
74.8+0.1b 51.6kO.3
75.320.2” 45.1 f 1.4
Ventral muscle Water (%) Lipid (mg/g)
73.9fO.l” 68.Of 1.9b
73.0k0.2b 76.8+ 1.5”
74.3kO.3” -63.8 + 2.6b
Values (mean + s.e.m., n = 3 for whole body and n = 6 for tissues) with different superscript letters in the same row are significantly different (PcO.05).
300
3250 2 $200 8 d B ml50 E ta -ii 100
50 0
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 time (h)
Fig. 1. Postprandial pattern of glycaemia in trout fed the experimental diets. For each sample time, values (mean + s.e.m., n = 5 ) with the same superscript letter are not significantly different (P> 0.05 ) between the dietary treatments. -IX- diet HC; -O-diet MC, -V-diet LC.
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test (not shown on the figure), the higher the dietary level of digestible carbohydrate, the later the attainment of maximum plasma glucose level. Plasma glucose concentration returned to the fasting level 30 h after food ingestion in fish fed diet MC. However, the plasma glucose level in fish fed diet HC at this time was about two-fold higher than in those fed diet MC. For fish fed diet LC, plasma glucose returned to the fasting level 12 h after the meal and subsequently increased again 16 and 30 h after the meal.
4. Discussion Digestibility of starch is related to the physical state of the starch (i.e. crude or gelatinized). The improvement in complex carbohydrate digestibility by gelatinization has been established in freshwater species (Takeuchi et al., 1990; Medale et al., 199 1; Bergot, 1993) and in marine species (Jeong et al., 199 1). The mixed starch diet had a higher ADC value for starch and energy than expected. This result may have been due to partial gelatinization of crude starch during the processing of this diet. The digestibility of protein and lipid was not affected by the type of dietary carbohydrate or by the carbohydrate/lipid ratio. Protein and lipid ADC values were low compared to those generally found in rainbow trout reared in freshwater (Cho et al., 1982). MacLeod ( 1977) reported that salinity decreased the absorption efficiency of protein and energy in rainbow trout. Such a diminution in protein digestibility due to salinity has also been shown in the same species by La11 and Bishop ( 1976). On the other hand, the ADC value of gelatinized starch in this study was higher than the values found for trout grown in freshwater at the same temperature (Brauge, unpublished data). According to these data, the effect of salinity on specific digestive enzymes needs further investigations. The growth rate of trout was not affected by the dietary levels and sources of non-protein energy used in the present study. Edwards et al. ( 1977) observed that, up to 25% of “metabolizable energy” (calculated value) provided by carbohydrate, dietary carbohydrate/lipid ratio had no influence on growth of trout, either in diluted seawater or in freshwater. Our results agree with these data, showing that rainbow trout reared in seawater are able to efficiently utilize high amounts of digestible carbohydrate, unlike chinook salmon. Mazur et al. ( 1992) found that gelatinized starch providing 22.5% of the “metabolizable energy” had an adverse effect on growth of chinook salmon. Moreover, the feed intake of chinook salmon was higher when they were fed a high-lipid diet. In the present study, the voluntary feed intake of trout tended to be depressed for the diet containing the highest level of lipid. Feed efficiency of trout was improved slightly when they ingested the diet containing the highest lipid level, but there was no significant effect of dietary treatment. .However, the production of 1 kg of rainbow trout required 475 2 7 g of digestible protein with diet HC and only 419 + 21 g with diet LC. Further, 18.8 2 0.3 MJ of digestible energy per kilogram weight gain were needed for fish
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fed diet HC whereas those fed diet LC required 16.0 ? 0.8 MJ DE/kg weight gain. The foregoing results suggest that energy from lipid spares protein more effectively than energy from carbohydrate when trout are held in seawater. Whole-body composition was not influenced by the dietary carbohydrate/lipid ratios, indicating increased lipid synthesis in fish fed diets high in digestible carbohydrate content. This conclusion is supported by the high “lipid retention efficiencies”, i.e. whole-body lipid gain in relation to digestible lipid intake (above 100 o/o),noted in fish fed the diets high in digestible carbohydrate. In fact, “retention efficiency” is not a meaningful term for lipid since deposited fatty acids can originate directly in dietary lipid or be synthesized from dietary carbohydrate. The amount of fat deposited seems, therefore, to be connected to the amount of non-protein digestible energy in the diet rather than to the sources of the nonprotein digestible energy. In contrast to the situation for the whole body, liver and muscle composition varied with the dietary treatments. Hepatosomatic index and liver lipid content were higher for fish fed the diets high in digestible carbohydrate. It is well known that liver size is directly related to hepatic glycogen level in salmonids (Bergot, 1979a; Pieper and Pfeffer, 1980; Dixon and Hilton, 1985; Kim and Kaushik, 1992). Absorbed carbohydrate which is not utilized to provide energy can be deposited in the liver both as glycogen and as lipid after conversion. According to Henderson and Sargent ( 198 1)) the liver is the main site of fatty acid synthesis in salmonids. In the present study, the liver composition data suggest that a high dietary carbohydrate/lipid ratio enhances lipid synthesis and deposition in the liver. The diet containing the highest level of lipid led to the lowest level of liver lipid, suggesting that lipid deposition from dietary lipid does not occur in liver but probably in adipose tissue. We did not measure the lipid content of the whole digestive tract, but many studies have shown an increase in perivisceral adipose tissue after salmonids have been fed high-fat diets (see Watanabe, 1982). The lipid content of ventral muscle was surprisingly high in fish fed the diet with the intermediate carbohydrate/lipid ratio. This may have stemmed from a combination of deposition of dietary lipid and lipogenesis from carbohydrate since the diet contained both gelatinized starch and 10% lipid. Indeed, in vitro data obtained by Henderson and Sargent ( 198 1) showed that glucose exerted no inhibitory effect on lipogenesis when dietary lipid level was 10% or less. Plasma glucose levels were related to the digestible carbohydrate content of the diets and remained high 24 h after feeding the high-carbohydrate diets. This prolonged hyperglycaemia, previously observed in trout reared in freshwater (Palmer and Ryman, 1972; Bergot, 1979b; Himick et al., 1991; Higgs et al., 1992), has been described as an “ill-effect” of digestible carbohydrate on glucose metabolism. In the present study, the term “ill-effect” does not seem to be appropriate since no pathological signs were noted and the trout grew well. The high plasma glucose concentrations induced by high dietary levels of digestible carbohydrate were initially thought to be due to a lack of insulin secretion (Palmer and Ryman, 1972 ). Recent studies have clearly demonstrated that salmonids do have the ability to secrete insulin and to increase insulin secretion in response to high levels of
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dietary carbohydrate (Hilton et al., 1987 ) . Impaired binding of insulin to its receptors in skeletal muscle was suspected initially as the problem (Plisetskaya, 1989). However, it now appears that the problem is impairment of one or more post-receptor events (Gutierrez et al., 199 1) . The hyperglycaemia could also result from a suppression of insulin secretion by elevated somatostatin secretion (Harmon et al., 1991). The observed increases in plasma glucose concentration in trout fed the diet low in digestible carbohydrate, 16 and 30 h after feed intake, suggest that glucose can be provided by gluconeogenesis when it is not supplied directly by dietary carbohydrate, as has been described in freshwater fish by Walton and Cowey (1982) andMalinovskaya (1989). Little is known about the effect of salinity on glycaemia. Sweeting et al. ( 1985) did not observe any change in plasma glucose of coho salmon smolts after a 24-h acclimation period in seawater. In the present study, the peak of plasma glucose was reached earlier than in trout fed the same experimental diets and reared at the same temperature in freshwater (Brauge, unpublished data). Therefore, carbohydrate absorption may be stimulated in trout held in seawater. According to Garin ( 1984), increased water salinity led to an elevation in utilization rate of glucose in seabass. Such an effect of salinity, however, did not seem to exist in rainbow trout since plasma glucose remained high 30 h after feeding the diet with high levels of digestible starch. Acknowledgements
This study was supported by a research grant from IFREMER (Institut Francais de Recherches pour l’Exploitation de la Mer) and the Conseil Regional d’Aquitaine (France). The experiments were performed at the fish nutrition laboratory of IFREMER (Plouzane, France). We thank Dr. R. Metailler and his team for providing the rearing facilities and for their advice. Dr. S. Kaushik is also acknowledged with gratitude for his critical reading of the manuscript. References Bergot, F., 1979a. 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-l 67. Bergot, F., 1979b. Effects of dietary carbohydrates and of their mode of distribution on glycaemia in rainbow trout Salmo gairdneri (Richardson). Comp. Biochem. Physiol., 64A: 543-547. Bergot, F., 1993. Digestibility of native starches of various botanical origins by rainbow trout (Oncorhynch mykiss). In: S.J. Kaushik and P. Luquet (Editors), Fish Nutrition in Practice. INRA, Paris, pp. 857-865. Bolin, D.W., Ring, R.P. and Klosterman, W.W., 1952. A simplified method for the determination of chromic oxide ( CrzO,) when used as an inert substance. Science, 16: 634-635. Cho, C.Y., Slinger, S.J and Bayley, H.S., 1982. Bioenergetics of salmonid fishes: energy intake, expenditure and productivity. Comp. Biochem. Physiol., 73B: 25-41. Dixon, D.G. and Hilton, J.W., 1985. Effects of available carbohydrate and water temperature on the
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