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Influence of lipid class and fatty acid deficiency on survival, growth, and fatty acid composition in rainbow trout juveniles
Aquaculture 264 (2007) 363 – 371 www.elsevier.com/locate/aqua-online
Influence of lipid class and fatty acid deficiency on survival, growth, and fatty acid composition in rainbow trout juveniles J. Rinchard 1 , S. Czesny 2 , K. Dabrowski ⁎ School of Environment and Natural Resources, The Ohio State University, 2021 Coffee Road, Columbus, OH 43210, USA Received 27 July 2006; received in revised form 26 November 2006; accepted 26 November 2006
Abstract Triplicate groups of rainbow trout (182 ± 51 mg) were fed four experimental casein–gelatin based diets containing different fatty acid levels for 8 weeks. Diet 1, containing only the oleic acid methyl ester (18:1n − 9), was used as an essential fatty acids deficient diet. Diet 2, supplemented with olive and linseed oils was designed to provide moderate amount of linoleic (18:2n − 6) and linolenic (18:3n − 3) acids in the form of triglycerides. Diet 3 contained cod liver oil and provided highly unsaturated fatty acids (HUFA) as well as their precursors. Diet 4, supplemented with soy-refined lecithin, contained high levels of 18:2n − 6 in the form of phospholipids. At the end of the experiment, the growth of fish fed the lecithin supplemented diet was significantly higher (17 fold increase) than that of the other groups (6–14 fold increase). Survival was significantly lower in fish fed the diet 1 in comparison to fish fed the other experimental diets (57.8% vs. 84.4–86.7%). Fish fed diets 1 and 4 contained significantly less total and neutral lipids in the whole body than fish fed diets 2 and 3. Whole body fatty acid profiles from both neutral and phospholipid fractions closely reflected dietary fatty acid composition. Fish fed linoleate containing phospholipids preferentially deposited this fatty acid in neutral lipids (33.0 ± 1.6%) in comparison to body phospholipids deposition (21.2 ± 1.0%). An increase of dietary linoleate also resulted in a significantly higher percentage of arachidonate (20:4n − 6) in fish body phospholipids. In conclusion, selective accumulation of 18, 20, and 22 polyunsaturated fatty acids in body of juvenile rainbow trout is the function of fatty acid origin, e.g., lipid class, which has been very little studied in this species. © 2006 Elsevier B.V. All rights reserved. Keywords: Rainbow trout (Oncorhynchus mykiss); Fatty acid composition; Lecithin; Fish oil; Vegetable oil; Growth
1. Introduction The importance of highly unsaturated fatty acids (HUFA, n − 6 and n − 3 fatty acids, C20 and C22 with ⁎ Corresponding author. Tel.: +1 614 2924555; fax: +1 614 2927432. E-mail address: [email protected] (K. Dabrowski). 1 Present address: School of Natural Resources and Environment, University of Michigan and USGS Great Lakes Science Center, Ann Arbor, MI 48105, USA. 2 Present address: Illinois Natural History Survey, Lake Michigan Biological Station, Zion, IL 60099, USA. 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.11.024
more than 2 double bonds) in fish nutrition is well established (Henderson and Tocher, 1987; Sargent et al., 1993), and these fatty acids are required by fish to ensure optimal growth, development, and reproduction (Sargent et al., 1995, 1999). Vegetable oils, rich in linoleic (18:2n − 6) and/or linolenic (18:3n − 3) acids, are considered to be excellent substitutes for fish oil in freshwater fish feeds, because freshwater fish are able to convert dietary 18:2n − 6 and 18:3n − 3 to HUFA, such as arachidonic acid (ARA, 20:4n − 6), eicosapentaenoic acid (EPA, 20:5n − 3), and docosahexaenoic acid (DHA, 22:6n − 3) (Tocher, 2003).
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Several studies conducted on freshwater fish indicated that vegetable oils can successfully replace fish oil in fish feeds without affecting survival and growth (Wonnacott et al., 2004; Subhadra et al., 2006). Caballero et al. (2002) reported that in rainbow trout Oncorhynchus mykiss up to 80–90% of vegetable oils (e.g., soybean, rapeseed, olive, and palm oils) can be used without compromising their growth. The replacement of fish oil by vegetable oils has a profound impact on the fatty acid composition of fish tissues with an increase in 18:2n − 6 and 18:3n − 3 and a decrease in DHA and EPA (Greene and Selivonchick, 1990; Caballero et al., 2002). Caballero et al. (2003) also observed an accumulation of lipid droplets in the enterocytes related to a decrease in dietary n − 3 HUFA content, indicating that the lipid transport and/ or metabolism in fish appeared to be affected. Similarly, Ruyter et al. (2006) found that rainbow trout fed soybean oil accumulated 3 times more linoleate in triglycerides than in phospholipids in the intestine. Dietary lecithin supplementation is used to enhance growth and survival of fish (Poston, 1990; Kanazawa, 1997; Salhi et al., 1999; Cahu et al., 2003). Lecithin also prevents lipid oxidation and facilitates fat absorption. Salhi et al. (1999) suggested that the increase in dietary polar lipids may improve lipid transport from the enterocytes to the blood by enhancing chylomicron synthesis. There are inconsistent reports of the effect of lecithin on growth, due to differences in fish size among studies. Thus, Watanabe and Takeuchi (1976) did not observe enhancement of growth in juvenile rainbow trout averaging 1.5 g when fed diets supplemented with lecithin, whereas Poston (1991) noted a better growth when a lecithin supplemented diet (4 and 8%) was provided at an early stage (0.1 g). The aim of this study was to compare the effects of different sources of dietary lipids (phospholipids or triglycerides) on growth, survival, and fatty acid composition in rainbow trout juveniles. Fish were fed 4 semi-purified casein–gelatin based diets in which lipids were supplied either by oleic acid methyl ester, a mixture of two vegetable oils (linseed and olive oils) rich in 18:2n − 6 and 18:3n − 3, cod liver oil rich in HUFA (EPA and DHA), or soy-refined lecithin rich in 18:2n − 6 containing phospholipids. 2. Materials and methods 2.1. Experimental diets Four experimental casein–gelatin based diets were formulated to be isonitrogenous (65%) and isolipidic
(14%), but containing different sources of lipids (Table 1). The first diet contained only the oleic acid methyl ester (18:1n − 9), and was used as an essential fatty acids deficient diet (OA). The second diet supplemented with olive and linseed oil (O + L) was designed to provide moderate amount of linoleic (18:2n − 6) and linolenic (18:3n − 3) acids, but no HUFA. The third diet contained cod liver oil (CLO) and provided HUFA as well as their precursors. The fourth diet supplemented with soyTable 1 Composition (%) of the four experimental diets fed to rainbow trout Experimental diets
Ingredients Casein (vitamin-free) Gelatin Dextrin (80% water soluble) Wheat meal Fish protein concentrate a Esters of oleic acid Olive oil Linseed oil Cod liver oil Soy-refined lecithin Vitamin mixture b Mineral mixture c Ascorbic acid d Carboxymethylcellulose sodium salt L-arginine L-methionine L-lysine Choline chloride (99%)
OA
O+L
CLO
LE
40.00 8.00 6.25 15.00 5.00 14.00 0.00 0.00 0.00 0.00 4.00 3.00 0.05 2.00 0.50 0.40 0.80 1.00
40.00 8.00 6.25 15.00 5.00 0.00 8.75 5.25 0.00 0.00 4.00 3.00 0.05 2.00 0.50 0.40 0.80 1.00
40.00 8.00 6.25 15.00 5.00 0.00 0.00 0.00 14.00 0.00 4.00 3.00 0.05 2.00 0.50 0.40 0.80 1.00
40.00 8.00 6.25 15.00 5.00 0.00 0.00 0.00 0.00 14.00 4.00 3.00 0.05 2.00 0.50 0.40 0.80 1.00
64.6 12.6 5.1 7.6
64.4 14.4 5.3 7.1
65.2 11.2 7.5 9.1
Proximate composition (dry matter %) Crude protein 66.8 Crude lipid 12.1 Ash 5.2 Moisture 9.6
a Concentrate of fish soluble protein (CPSP 90: crude protein, 82– 84% WW; crude lipid, 9–13% WW), Sopropêche S.A., Boulogne-surmer, France. b Roche Performance Premix (Hoffman-La Roche, Inc., Nutley, New Jersey), composition per g of the vitamin mixture: vitamin A, 2645.50IU; vitamin D3, 220.46IU; vitamin E, 44.09IU; Vitamin B12, 13 μg; riboflavin, 13.23 mg; niacin, 61.73 mg; D-pantothenic acid, 22.05 mg; menadione, 1.32 mg; folic acid, 1.76 mg; pyridoxine, 4.42 mg; thiamine, 7.95 mg; D-biotin, 0.31 mg. c Bernhart Tomarelli salt mixture (ICN Pharmaceuticals, Costa Mesa, CA), composition (g/100 g): calcium carbonate, 2.1; calcium phosphate dibasic, 73.5; citric acid, 0.227; cupric citrate, 0.046; ferric citrate (16 to 17% Fe), 0.558; magnesium oxide, 2.5; manganese citrate, 0.835; potassium iodide, 0.001; potassium phosphate dibasic, 8.1; potassium oxide, 6.8; sodium chloride, 3.06; sodium phosphate, 2.14; and zinc citrate, 0.133. Five milligrams of Se in the form of sodium selenite was added per kilogram of the salt mixture. d Phosphitan C (Mg-L-ascorbyl-2-phosphate), Showa Denko K.K., Tokyo, Japan.
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refined lecithin (LE) containing high levels of 18:2n − 6 in the form of phospholipids. The methyl ester of oleic acid, olive oil, linseed oil, cod liver oil, and soyrefined lecithin were purchased from ICN (Aurora, OH, USA). 2.2. Fish and experimental conditions The experiment was carried out in a semi-closed recirculating-water system, composed of twelve 30-L glass aquaria, an Aquanetics Deluxe System Paks (model 166; Aquanetic Systems, San Diego, CA, USA), and a sedimentation tank. Each aquarium was equipped with continuous aeration, and water was supplied at a rate of 1 L min− 1. Throughout the experiment, water temperature ranged from 15 to 19 °C, whereas the photoperiod was maintained constant (12 h L/12 h D). Rainbow trout (182 ± 51 mg, n = 30), fed a commercial diet (Starter, BioOregon, OR, USA) for three days prior to the beginning of the present study, were randomly distributed among 12 aquaria (n = 50) and assigned to one of the four experimental diets with three aquaria per dietary treatment. Fish were fed by hand 3–4 times a day at a restricted ration of up to 90% satiation for 8 weeks. Aquaria were cleaned and feces discarded daily. 2.3. Sampling procedures, lipids and fatty acids analysis Every two weeks, fish from each aquarium were weighed, counted and feeding rate was readjusted. At week 4, fish density was reduced to 20 fish per aquarium. At week 6, 5 fish per aquarium were sacrificed by overdose of MS-222 and immediately frozen at − 80 °C for subsequent proximate and fatty acid analysis. At the end of the experiment (8 weeks), survival, weight gain (WG = (final weight − initial weight) × 100 / initial weight), specific growth rate (SGR = [loge final weight − loge initial weight] × 100 / duration of the experiment in days), and feed conversion rate (FCR = dry feed intake / weight gain) were calculated. Analysis of crude protein, moisture, and ash in diets and the whole body of fish was performed according to standard procedures (AOAC, 1995). Dietary and whole body lipids were extracted according to the procedure of Folch et al. (1957) with chloroform/ methanol (2:1 v/v) containing 0.01% of butylated hydroxytoluene (BHT) as antioxidant. The organic solvent was evaporated under a stream of nitrogen and the amount of lipid was determined gravimetrically.
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Whole body crude lipids were then separated into polar (phospholipids) and neutral (mostly triglycerides) lipids using Sep-Pak silica cartridges (Waters, Milford, MA, USA). Chloroform and methanol were used as the mobile phases for neutral and phospholipids, respectively (Juaneda and Rocquelin, 1985). Fatty acid methyl esters (FAME) were prepared following the methods of Metcalfe and Schmitz (1961). Prior to transmethylation, a known amount of nonadecanoate acid (19:0), proportional to the amount of total, neutral or phospholipids detected (8 mg per 50 mg of lipids), was added to the samples as the internal standard for the quantification of fatty acids. FAME were quantified using a Varian 3900 gas chromatograph (Varian, Inc., Walnut Creek, CA) equipped with a flame ionization detector, a capillary column (Varian Chrompack capillary column (WCOT fused silica 100 m × 0.25 mm coating CPSIL 88 for FAME, df = 0.2), and an auto-injector (CP-8410 AutoInjector, Varian, Inc.). Helium was used as a carrier gas at a flow of 30 ml/min. The injector and detector temperatures were 270 and 300 °C, respectively. Initial temperature of the oven was 175 °C for 26 min which increased to 205 °C by increments of 2 °C/min, then held at 205 °C for 24 min. The individual fatty acids were identified by comparing their retention times to that of a standard mix of fatty acids (Nu-Check-Prep, Inc., Elysian, MI, USA) and quantified by comparing their peak area with that of the internal standard. Fatty acids are expressed as percent of total identified FAME. 2.4. Statistical analysis Results are expressed as means ± SD (n = 3). Homogeneity of variance was verified for all data using Bartlett's test (Dagnelie, 1975). Percentage data were arc sin transformed prior to statistical analysis. Data were subjected to analysis of variance (ANOVA) and subsequent comparison of means by Fisher least significant differences test (P < 0.05). Linear regression analyses were performed between dietary and whole body fatty acid concentrations. Differences were accepted as statistically significant when P < 0.05. 3. Results 3.1. Proximate and fatty acid compositions of the experimental diets Although the experimental diets were designed to be isolipidic (14%), the LE diet contained the lowest lipid content and the CLO diet the highest level (Table 1). The
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fatty acid content of the diets clearly reflected the major fatty acids present in the lipid sources (Table 2). High levels of monounsaturated fatty acid (96.2%), mainly oleic acid (18:1n − 9), and low percentages of n − 3 and n − 6 fatty acids (1.0 and 1.5%, respectively) were found in the OA diet. Diet O + L provided moderate amounts of 18:2n − 6 and 18:3n − 3, both precursors of HUFA (14.2 and 18.5%, respectively). High percentages of EPA and DHA were present in the CLO diet (10.2 and 7.6%, respectively), while 18:2n − 6 and 18:3n − 3 only represented 6.5 and 2.4%, respectively. The highest level (64.8%) of polyunsaturated fatty acids (PUFA, fatty acids with 2 or more double bonds) was measured in the LE diet, mainly represented by 18:2n − 6 (53.7%). The concentrations of ARA, EPA and DHA were similar and low in the OA, O + L, and LE diets in comparison to the CLO diet.
Fig. 1. Changes of rainbow trout weight fed the four experimental diets for 8 weeks. At each sampling date means with different superscript letter are significantly different (P < 0.05).
3.2. Growth and survival of rainbow trout Table 2 Fatty acid composition (% of total fatty acids detected) in the four experimental diets Fatty acids
Experimental diets OA
O+L
CLO
LE
Saturated 14:0 16:0 17:0 18:0 ∑ Saturated
0.2 1.1 nd nd 1.3
0.3 11.6 0.1 2.0 14.0
6.5 18.3 0.2 2.9 27.9
0.9 19.3 0.2 4.0 24.8
Monounsaturated 16:1n − 9 and n − 7 18:1n − 9 and n − 7 20:1n − 9 22:1n − 11 ∑ Monounsaturated
0.2 96.0 nd nd 96.2
0.9 51.1 0.1 nd 52.1
7.7 26.9 1.5 1.9 38.0
0.5 13.5 0.3 0.1 14.4
Polyunsaturated 18:2n − 6 20:2n − 6 20:3n − 6 20:4n − 6 22:4n − 6 22:5n − 6 ∑ n−6 18:3n − 3 18:4n − 3 20:3n − 3 20:4n − 3 20:5n − 3 22:5n − 3 22:6n − 3 ∑ n−3 ∑ Polyunsaturated ∑ n − 3/∑ n − 6
1.2 nd nd 0.3 nd nd 1.5 0.3 nd nd nd 0.2 nd 0.5 1.0 2.5 0.7
14.2 nd nd 0.3 nd nd 14.5 18.5 nd nd nd 0.3 nd 0.5 19.3 33.8 1.3
6.5 0.1 0.1 1.0 nd 0.3 8.0 2.4 2.2 0.2 0.8 10.2 2.5 7.6 25.9 33.9 3.2
53.7 nd nd 0.4 nd nd 54.1 5.8 nd nd nd 0.4 nd 0.9 10.7 64.8 0.2
nd: not detected.
The first significant effect of dietary fatty acids on rainbow trout growth was observed after 4 weeks of feeding (Fig. 1). At that time, fish weight was significantly (P < 0.01) lower in fish fed the OA diet. At 6 weeks, the growth of fish fed the O + L and CLO diets were similar, whereas the weight of fish fed the LE diet was significantly (P < 0.01) higher than the other groups. Similar patterns were observed at the end of the experiment, however the growth of fish fed the CLO diet was significantly higher than the fish fed the O + L diet (Fig. 1 and Table 3). Specific growth rate and feed conversion ratio were significantly (P < 0.01) affected by dietary treatments (Table 3). Fish fed the LE diet have the highest SGR and the lowest FCR, whereas the opposite trend was found in fish fed the OA diet. Intermediate SGR and FCR were found in fish fed the Table 3 Growth of rainbow trout fed the four experimental diets for 8 weeks Experimental diets OA
O+L
CLO
LE
Final weight (g) 1.2 ± 0.1d 2.3 ± 0.2c 2.7 ± 0.1b 3.3 ± 0.3a d c b Weight 572 ± 78 1165 ± 133 1388 ± 31 1684 ± 137a gain (%)1 SGR (%)2 3.3 ± 0.2d 4.5 ± 0.2c 4.7 ± 0.1b 5.1 ± 0.1a FCR3 2.3 ± 0.3a 1.5 ± 0.2b 1.3 ± 0.0bc 1.1 ± 0.1c b a a Survival (%) 57.8 ± 3.8 84.4 ± 3.8 86.7 ± 7.7 84.4 ± 10.2a Weight gain = ((final weight − initial weight) × 100) / initial weight. Specific growth rate (SGR) = ((loge final weight − loge initial weight) × 100) / duration of the experiment in days. 3 Feed conversion ratio (FCR) = (dry feed intake) / wet weight gain. Means with different superscript letter in a row are significantly different (P < 0.05). 1
2
J. Rinchard et al. / Aquaculture 264 (2007) 363–371 Table 4 Moisture (%) and proximate composition (% of dry matter) of whole body rainbow trout fed the experimental diets for 6 weeks Parameters
Experimental diets OA
Crude protein Crude lipid Phospholipids (% of total lipids) Ash Moisture
O+L a
CLO c
77.0 ± 4.0 66.0 ± 2.4 21.1 ± 3.4b 27.9 ± 3.0a 27.5 ± 3.8a 19.4 ± 0.5b
LE c
65.2 ± 0.7 71.2 ± 1.3b 29.8 ± 2.9a 21.6 ± 1.7b 22.1 ± 3.1b 27.3 ± 1.9a
12.6 ± 0.9b 14.8 ± 0.7ab 16.3 ± 1.5a 16.1 ± 1.4a 94.2 ± 4.0a 85.3 ± 1.4b 85.9 ± 0.3b 86.3 ± 0.4b
Means with different superscript letter in a row are significantly different (P < 0.05).
O + L and LE diets. Survival was significantly (P < 0.01) lower in fish fed the OA diet in comparison to fish fed all other experimental diets (Table 3). Mortality occurred after 2 weeks. 3.3. Proximate and fatty acid composition of rainbow trout
respectively) were detected in fish fed the OA diet. The highest percentages of 18:2n − 6 and 18:3n − 3 were found in fish fed the LE and O + L diets, respectively, whereas the highest concentrations of EPA and DHA were measured in fish fed the CLO diet. The highest concentration of ARA was found in fish fed LE diet. The percentage of saturated, monounsaturated and PUFA differed significantly (P < 0.01) among fish fed the four experimental diets. Neutral lipids of fish fed the CLO diet had the highest n − 3/n − 6 ratio, whereas neutral lipids of fish fed the LE diet had the lowest n − 3/n − 6 ratio. With respect to the phospholipid fraction, 16:0 and 18:1 (both n − 7 and n − 9) were the major saturated and monoene fatty acids regardless of the dietary lipid source. Concentrations of DHA and EPA did not differ significantly (P > 0.05) between fish fed OA and LE
Table 5 Fatty acid composition of neutral lipids (% of total lipids detected) of whole body rainbow trout fed the experimental diets for 6 weeks Fatty acids
The proximate composition of fish fed the O + L and CLO diets did not differ significantly (P > 0.05) (Table 4). Fish fed the OA and LE diets contained significantly (P < 0.01) higher protein levels and less lipids than fish fed the O + L and CLO diets. The percentages of polar lipids in fish fed the O + L and CLO diets were significantly (P < 0.01) lower in comparison to those measured in fish fed the OA and LE diets. Moisture level was the highest and ash content was the lowest in fish fed the OA diet and, these values were significantly (P < 0.01) different for all other experiment groups. Moisture and ash levels did not differ significantly (P > 0.05) among fish fed the three other experimental diets. Fatty acid composition of the neutral and phospholipid fractions of the whole body of rainbow trout, expressed as percentage of total fatty acids detected, reflected dietary fatty acid compositions (Tables 5 and 6). The fatty acid composition of rainbow trout phospholipids was less affected than neutral lipids by dietary fatty acid concentrations. The concentrations of ARA, EPA, and DHA were significantly higher in the phospholipids than in neutral lipids. With respect to the neutral lipid fraction, 18:1, both n − 7 and n − 9, were the predominant fatty acids measured in fish fed the OA, O + L and CLO diets, whereas 18:2n − 6 was the dominant fatty acid in fish fed the LE diet. High level of monounsaturated fatty acids (77.7%), mainly 18:1 (both n − 7 and n − 9), and low percentages of n − 3 and n − 6 fatty acids (3.0 and 1.6%,
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Experimental diets OA
O+L
Saturated 14:0 16:0 17:0 18:0 ∑ Saturated
CLO
LE
1.1 ± 0.6b 6.9 ± 0.5d 0.5 ± 0.1a 2.3 ± 0.3c 10.7 ± 1.0c
1.2 ± 0.2b 5.3 ± 0.2a 14.0 ± 0.8c 17.6 ± 0.7b 0.1 ± 0.0c 0.3 ± 0.0b 4.2 ± 0.2b 4.1 ± 0.1b b 19.5 ± 1.2 27.3 ± 0.8a
1.3 ± 0.1b 20.4 ± 1.7a 0.2 ± 0.0c 6.4 ± 0.2a 27.2 ± 2.3a
Monounsaturated 16:1n − 9 and n − 7 18:1n − 9 and n − 7 20:1n − 9 22:1n − 11 ∑ Monounsaturated
5.0 ± 0.3b 77.7 ± 1.0a 1.2 ± 0.1a 0.3 ± 0.1b 84.2 ± 0.8a
3.5 ± 0.3c 8.3 ± 0.4a b 51.4 ± 1.1 32.5 ± 0.9c 1.4 ± 0.2a 1.8 ± 0.0a b 0.3 ± 0.1 0.9 ± 0.3a 56.5 ± 0.9b 43.5 ± 1.1c
2.7 ± 0.4d 24.2 ± 4.1d 0.5 ± 0.1c 0.3 ± 0.1b 27.8 ± 3.7d
Polyunsaturated 18:2n − 6 20:2n − 6 20:3n − 6 20:4n − 6 22:4n − 6 22:5n − 6 ∑ n−6 18:3n − 3 18:4n − 3 20:3n − 3 20:4n − 3 20:5n − 3 22:5n − 3 22:6n − 3 ∑ n−3 ∑ Polyunsaturated ∑ n − 3/∑ n − 6
0.8 ± 0.1d 0.4 ± 0.3a nd 0.1 ± 0.0d 0.2 ± 0.2b 0.5 ± 0.2b 1.6 ± 0.2d 1.4 ± 0.2b 0.1 ± 0.0b 0.2 ± 0.2b 0.3 ± 0.1a 0.3 ± 0.0b 0.2 ± 0.1b 0.6 ± 0.1d 3.0 ± 0.3d 5.1 ± 0.3d 1.9 ± 0.4b
9.6 ± 1.5b 5.6 ± 0.3c 0.3 ± 0.1a 0.6 ± 0.0a b 0.3 ± 0.1 0.2 ± 0.0b 0.4 ± 0.1c 0.9 ± 0.1b nd 0.5 ± 0.1a 0.5 ± 0.0b 0.5 ± 0.0b b 10.8 ± 1.6 7.8 ± 0.2c 8.5 ± 0.8a 1.7 ± 0.1b b 0.6 ± 0.0 1.9 ± 0.1a 0.4 ± 0.1b 0.2 ± 0.0b a 0.5 ± 0.3 0.5 ± 0.0a 0.5 ± 0.1b 5.8 ± 0.3a b 0.2 ± 0.0 1.6 ± 0.0a 1.9 ± 0.0b 9.2 ± 0.2a b 12.7 ± 1.3 20.8 ± 0.5a 24.0 ± 0.3c 29.2 ± 0.6b 1.2 ± 0.3c 2.7 ± 0.1a
33.0 ± 1.6a nd 2.1 ± 0.2a 1.2 ± 0.2a 0.2 ± 0.0b 0.9 ± 0.1a 36.5 ± 1.4a 1.8 ± 0.9b 2.0 ± 0.8a 1.3 ± 0.1a 0.2 ± 0.1a 0.5 ± 0.2b 0.2 ± 0.1b 1.5 ± 0.3c 7.5 ± 0.9c 45.0 ± 1.9a 0.2 ± 0.0d
Means with different superscript letter in a row are significantly different (P < 0.05). nd: not detected.
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Table 6 Fatty acid composition of phospholipids (% of total lipids detected) of whole body rainbow trout fed the experimental diets for 6 weeks Fatty acids
Experimental diets OA
O+L
CLO
LE
Saturated 14:0 16:0 17:0 18:0 Σ Saturated
1.3 ± 0.5a 14.7 ± 0.5b 0.1 ± 0.0a 3.9 ± 0.4c 20.0 ± 1.1b
1.3 ± 0.4a 21.2 ± 0.6a 0.1 ± 0.0a 5.9 ± 0.5b 28.3 ± 0.3a
1.3 ± 0.1a 21.6 ± 0.6a 0.2 ± 0.2a 5.4 ± 0.8b 28.5 ± 1.4a
1.4 ± 0.1a 20.9 ± 0.4a 0.2 ± 0.0a 6.4 ± 0.2a 28.9 ± 0.8a
Monounsaturated 16:1n − 9 and n − 7 18:1n − 9 and n − 7 20:1n − 9 22:1n − 11 ∑ Monounsaturated
5.8 ± 0.2a 44.6 ± 0.4a 1.0 ± 0.8a 0.1 ± 0.0b 51.6 ± 0.5a
3.3 ± 0.1c 23.3 ± 0.4b 1.2 ± 0.2a 0.5 ± 0.3a 28.3 ± 0.3b
4.0 ± 1.0b 17.0 ± 0.7c 1.2 ± 0.3a 0.1 ± 0.0b 22.3 ± 1.2c
1.9 ± 0.1d 13.2 ± 1.0d 0.3 ± 0.0b 0.1 ± 0.0b 15.6 ± 1.1d
Polyunsaturated 18:2n − 6 20:2n − 6 20:3n − 6 20:4n − 6 22:4n − 6 22:5n − 6 ∑ n−6 18:3n − 3 18:4n − 3 20:3n − 3 20:4n − 3 20:5n − 3 22:5n − 3 22:6n − 3 ∑ n−3 ∑ Polyunsaturated ∑ n − 3/∑ n − 6
1.2 ± 0.1c 1.1 ± 0.6a 3.6 ± 0.2a 2.1 ± 0.2b 1.2 ± 0.1a 1.4 ± 0.5b 10.5 ± 0.6b 1.2 ± 0.0b 0.3 ± 0.1b 0.6 ± 0.1c 0.7 ± 1.0a 1.6 ± 0.2c 0.9 ± 0.3a 12.6 ± 0.7c 17.9 ± 1.4d 28.4 ± 0.8d 1.7 ± 0.2bc
6.3 ± 0.4b 0.4 ± 0.1a 0.1 ± 0.1c 2.0 ± 0.1b 0.1 ± 0.0b 0.9 ± 0.1c 9.7 ± 0.3b 4.1 ± 0.2a 0.5 ± 0.1ab 1.1 ± 0.1b 0.8 ± 0.1a 3.2 ± 0.3b 0.7 ± 0.1a 23.2 ± 0.4b 33.6 ± 0.3b 43.3 ± 0.1c 3.5 ± 0.1b
2.1 ± 0.2c 0.9 ± 0.6c 0.2 ± 0.2b 1.5 ± 0.5b 0.2 ± 0.3c 0.7 ± 0.2a 5.7 ± 1.6c 0.7 ± 0.3c 0.5 ± 0.1ab 0.4 ± 0.1c 0.6 ± 0.3a 8.4 ± 0.2a 1.0 ± 0.4a 31.9 ± 1.1a 43.5 ± 1.5a 49.2 ± 0.1b 8.2 ± 2.8a
21.2 ± 1.0a 0.2 ± 0.0b 2.4 ± 0.1b 4.5 ± 0.4a 0.1 ± 0.0b 5.9 ± 0.4a 34.3 ± 0.5a 1.3 ± 0.1b 0.7 ± 0.3a 3.8 ± 0.1a 0.2 ± 0.0a 1.2 ± 0.3c 0.3 ± 0.1a 13.5 ± 1.3c 21.2 ± 1.4c 55.5 ± 1.8a 0.6 ± 0.0c
Means with different superscript letter in a row are significantly different (P < 0.05).
diets, but were significantly (P < 0.01) higher in fish fed the O + L and CLO diets. Saturated fatty acid concentrations were significantly (P < 0.01) lower in fish fed OA diet in comparison to fish fed all other diets. The percentage of polyunsaturated fatty acids was significantly (P < 0.01) affected by dietary treatment, however, the main contributors to PUFA were different. Fatty acids from the n − 3 family dominated PUFA in fish fed CLO diet, while fatty acids of the n − 6 family were mostly in fish fed the LE diet. Finally, as reported for the neutral lipid fraction, the highest n − 3/n − 6 ratio was detected in fish fed the CLO diet and the lowest in fish fed the LE diet. Regression analysis was used to identify dose– response relationship between dietary and body fatty acids. As reported in Table 7, most of the individual
Table 7 Correlation between dietary fatty acid concentrations and fatty acid concentrations in whole body rainbow trout fed the experimental diets for 6 weeks Fatty acids 18:1n − 9 and n − 7 18:2n − 6 18:3n − 3 20:4n − 6 20:5n − 3 22:6n − 3 ∑ Saturated ∑ Monounsaturated ∑ Polyunsaturated ∑ n−3 ∑ n−6
Neutral lipids 2
Phospholipids
Slope
r
P
Slope
r2
P
0.64 0.59 0.41 0.33 0.53 1.06 0.62 0.66 0.66 0.66 0.64
1.00 1.00 0.94 0.11 1.00 0.98 0.99 1.00 0.99 0.96 1.00
<0.01 <0.01 0.03 0.66 <0.01 <0.01 <0.01 <0.01 <0.01 0.02 <0.01
0.37 0.38 0.18 − 1.36 0.63 2.02 0.31 0.43 0.45 1.01 0.51
0.97 1.00 0.91 0.26 0.92 0.67 0.80 0.99 0.94 0.97 0.90
0.01 <0.01 0.05 0.49 0.04 0.18 0.10 <0.01 0.03 0.01 0.05
J. Rinchard et al. / Aquaculture 264 (2007) 363–371
dietary fatty acids were linearly correlated to their concentrations in whole body of rainbow trout. 4. Discussion The four experimental diets used in this study differed only by their lipid sources, but the sources differed both in lipid classes and fatty acid composition. As reported in several species (Bell et al., 1991, 1994; Sargent et al., 1995), fatty acid concentrations of rainbow trout were influenced by the fatty acid composition of the dietary lipids. After 8 weeks of feeding, no sign of EFA deficiency, such as fin erosion and bacterial disease, was observed in rainbow trout fed the OA diet. However, these symptoms were only observed by Castell et al. (1972) in fish fed essential fatty acid (EFA) deficient diet over an 8-month period. Growth and survival was significantly lower in fish fed the OA diet. Similar growth depression has been observed in other studies where fish were fed n − 3 HUFA deficient diets (Watanabe et al., 1989; Lee et al., 2003). In this study, higher water and protein contents were found in fish fed the OA diet in comparison to fish fed the three other diets. Similar results were found by Castell et al. (1972) when rainbow trout were fed EFA diets. Both the O + L and CLO diets contained approximately the same amount of PUFA (32.7 and 33.9%, respectively). However, their fatty acid composition was different. PUFA of the O + L diet consisted primarily of 18:3n − 3 and 18:2n − 6, whereas PUFA of the CLO diet were mainly 20:5n − 3 and 22:6n − 3. Fish fed the O + L diet showed lower levels of n − 3 fatty acids and higher levels of C18 fatty acids in comparison to fish fed the CLO diet. The concentration of individual n − 3 HUFA in fish fed the O + L diet was higher than in their diet indicating that these fatty acids are mainly retained in the case of dietary shortages. It was also confirmed that rainbow trout are able to convert, through desaturation and elongation, 18:3n − 3 to 22:6n − 3 (via C24 intermediates; Buzzi et al., 1996, 1997). Bell et al. (2003) reported that fish size also affects DHA synthesis, where small rainbow trout (0.8 g) synthesized DHA ten times more efficiently than larger individuals (6 g). Although in the present study the requirements of both EFA were met (Castell et al., 1972; AOAC, 1995), growth of fish fed O + L diet was lower than that of fish fed the CLO diet. Increased levels of dietary n − 3 HUFA (e.g., EPA and DHA) have been reported to improve growth in several fish species (Bell and Sargent, 2003; Izquierdo et al., 2003; Lee et al., 2003). However, our results
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indicate that soy-refined lecithin (rich in 18:2n − 6) used as the only source of dietary lipid significantly improved growth performance and reduced body fat in early feeding of rainbow trout in comparison to diets rich in n − 3 PUFA (O + L and CLO diets). Similar results were observed in sea bass Dicentrarchus labrax larvae (Cahu et al., 2003) and common carp Cyprinus carpio juveniles (Geurden et al., 1997). Lecithin is known to be a source of bioavailable phospholipids, and improves intestinal absorption of cholesterol. Dietary phospholipids also provide specific lipid classes to produce lipoproteins. The lower efficiency of the O + L and CLO diets in promoting growth could be associated with their deficiency of phospholipids and cholesterol. Growth and survival of fish have been improved with dietary lecithin supplementation (Poston, 1990; Kanazawa, 1997; Salhi et al., 1999; Cahu et al., 2003). The major component of soy lecithin responsible for promoting growth is the phosphatidylcholine. Cahu et al. (2003) offered evidence that dietary phospholipids which contained negligible amounts of EPA and DHA resulted in better growth and survival than fish fed fish oil containing HUFA. Rainbow trout fed higher than optimal levels of n − 3 HUFA (2 to 6 times more than their requirement levels) showed poor growth and low feed conversion (Takeuchi and Watanabe, 1979). Similarly, Ibeas et al. (2000) attributed the growth reduction to an increase of n − 3 HUFA in phospholipids, which created an unbalance of the unsaturation level in the membrane phospholipids. Fish fed the LE diet presented very high levels of ARA in the phospholipid fraction indicating considerable conversion of 18:2n − 6 to ARA. This is not entirely new finding in salmonids as Bell et al. (1992) reported significantly increased levels of ARA in phosphoinositol and phophatidylcholine in heart of Atlantic salmon fed diets enriched in plant oils (containing 46% of linoleate). In contrast, in fish fed the CLO diet, we did not observe this conversion because of the higher dietary 18:3n − 3 provided, which is competing with the 18:2n − 6 for desaturation (Olsen and Ringo, 1992). ARA plays an important role in fish membranes, is the main precursor of eicosanoids, and is the main component of phosphatidylinositol (Sargent et al., 1999). Several studies also demonstrated the role of ARA in stress reduction (Koven et al., 2003; Van Anholt et al., 2004a,b). In the present study, fish fed the LE diet, which did not contain ARA, exhibited the highest levels of this fatty acid in both neutral and phospholipid fractions in comparison to the other groups (Table 6). Similarly, growth and survival of turbot Scophthalmus maximus was improved when ARA was fed as the only
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source of HUFA compared to fish fed DHA alone or ARA and DHA mixtures (Castell et al., 1994). However, the mechanism of ARA action on growth improvement is still unknown (Bell and Sargent, 2003). In conclusion, our results demonstrate for the first time that a diet supplemented with 14% lecithin fed during the early life stage of rainbow trout results in an excellent growth of fish, decreases body lipid accumulations and secures a high concentration of ARA in body phospholipids. Long-term studies with this or higher inclusion levels of lecithin in dietary formulations are required. Acknowledgements This work was supported by the North Central Regional Aquaculture Center, USDA, East Lansing, Michigan and the Great Lakes Fishery Trust, Lansing, Michigan. Salaries were provided in part by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, Wooster, Ohio. References AOAC (Association of Official Analytical Chemists), 1995. Official Methods of Analysis, 16th edition. AOAC, Arlington, Virginia. Bell, J.G., Sargent, J.R., 2003. Arachidonic acid in aquaculture feeds: current status and future opportunities. Aquaculture 218, 491–499. Bell, J.G., McVicar, A.H., Park, M.T., Sargent, J.R., 1991. High dietary linoleic acid affects the fatty acid compositions of individual phospholipids from tissues of Atlantic salmon (Salmo salar): association with stress susceptibility and cardiac lesion. J. Nutr. 121, 1163–1172. Bell, J.D., Dick, J.R., Sargent, J.R., McVicar, A.H., 1992. Dietary linoleic acid affects phospholipid fatty acid composition in heart and eicosanoid production in cardiomyocytes from Atlantic salmon (Salmo salar). Comp. Biochem. Physiol. 103A, 337–342. Bell, J.D., Tocher, D.R., McDonald, F.M., Sargent, J.R., 1994. Effects of diets rich in linoleic (18:2n − 6) and a-linoleic (18:3n − 3) acids on the growth, lipid class and fatty acid compositions and eicosanoid production in juvenile turbot (Scophthalmus maximus). Fish Physiol. Biochem. 13, 105–118. Bell, M.V., Dick, J.R., Porter, A.E.A., 2003. In vitro assays of docosahexaenoic acid biosynthesis in fish. In: Browman, H.I., Skiftesvik, A.B. (Eds.), The Big Fish Bang, Proceedings of the 26th Annual Larval Fish Conference, Bergen, Norway, July 22– 26, 2002, pp. 229–237. Buzzi, M., Henderson, R.J., Sargent, J.R., 1996. The desaturation and elongation of linoleic acid and eicosapentaenoic acid by hepatocytes and liver microsomes from rainbow trout (Oncorhynchus mykiss) fed diets containing fish oil or olive oil. Biochim. Biophys. Acta 1299, 235–244. Buzzi, M., Henderson, R.J., Sargent, J.R., 1997. Biosynthesis of docosahexaenoic acid in trout hepatocytes proceeds via 24-carbon intermediates. Comp. Biochem. Physiol. 116B, 263–267. Caballero, M.J., Obach, A., Rosenlund, G., Montero, D., Gisvold, M., Izquierdo, M.S., 2002. Impact of different dietary lipid sources on
growth, lipid digestibility, tissue fatty acid composition and histology of rainbow trout, Oncorhynchus mykiss. Aquaculture 214, 253–271. Caballero, M.J., Izquierdo, M.S., Kjorsvik, E., Montero, D., Socorro, J., Fernandez, A.J., Rosenlund, G., 2003. Morphological aspects of intestinal cells from gilthead seabream (Sparus aurata) fed diets containing different lipid sources. Aquaculture 225, 325–340. Cahu, C.L., Zambonino, J.L., Barbosa, V., 2003. Effect of dietary phospholipids level and phospholipids:neutral lipid value on the development of sea bass (Dicentrarchus labrax) larvae fed a compound diet. Br. J. Nutr. 90, 21–28. Castell, J.D., Lee, D.J., Sinnhuber, R.O., 1972. Essential fatty acids in the diet of rainbow trout: lipid metabolism and fatty acid composition. J. Nutr. 102, 93–99. Castell, J.D., Bell, J.G., Tocher, D.R., Sargent, J.R., 1994. Effects of purified diets containing different combinations of arachidonic and docosahexaenoic acid on survival, growth and fatty acid composition of juvenile turbot (Scophthalmus maximus). Aquaculture 128, 315–333. Dagnelie, P., 1975. Théorie et Méthodes Statistiques. Applications Agronomiques. Volume II: Les Méthodes de l'Inférence Statistique. Les Presses Agronomiques de Gembloux, Gembloux, Belgium. Folch, J., Lees, M., Stanley, G.H.S., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509. Geurden, I., Charlon, N., Marion, D., Bergot, P., 1997. Influence of purified soybean phospholipids on early development of common carp. Aquac. Int. 5, 137–149. Greene, D.H.S., Selivonchick, D.P., 1990. Effects of dietary vegetable, animal and marine lipids on muscle lipid and hematology of rainbow trout (Oncorhynchus mykiss). Aquaculture 89, 165–182. Henderson, R.J., Tocher, D.R., 1987. The lipid composition and biochemistry of freshwater fish. Prog. Lipid Res. 26, 284–347. Ibeas, C., Rodriguez, C., Badia, P., Cejas, J.R., Santamaria, F.J., Lorenzo, A., 2000. Efficacy of dietary methyl esters of n − 3 HUFA vs. triacylglycerols of n − 3 HUFA by gilthead seabream (Sparus aurata L.) juveniles. Aquaculture 190, 273–287. Izquierdo, M.S., Obach, A., Arantzamendi, L., Montero, D., Robaina, L., Rosenlund, G., 2003. Dietary lipid sources for seabream and seabass: growth performance, tissue composition and flesh quality. Aquac. Nutr. 9, 397–407. Juaneda, P., Rocquelin, G., 1985. Rapid and convenient separation of phospholipids and non phosphorus lipids from rat heart using silica cartridges. Lipids 30, 40–41. Kanazawa, A., 1997. Effects of docosahexaenoic acid and phospholipids on stress tolerance of fish. Aquaculture 155, 129–134. Koven, W., Van Anholt, R., Lutzky, S., Atia, I.B., Nixon, O., Ron, B., Tandler, A., 2003. The effect of dietary arachidonic acid on growth, survival, and cortisol levels in different-age gilthead seabream larvae (Sparus auratus) exposed to handling or daily salinity change. Aquaculture 228, 307–320. Lee, S.-M., Lee, J.H., Kim, K.-D., 2003. Effect of dietary essential fatty acids on growth, body composition and blood chemistry of juvenile starry flounder (Platichthys stellatus). Aquaculture 225, 269–281. Metcalfe, L.D., Schmitz, A.A., 1961. The rapid preparation of fatty acid esters for gas chromatographic analysis. Anal. Chem. 33, 363–364. Olsen, R.E., Ringo, E., 1992. Lipids of Arctic charr, Salvelinus alpinus (L.) II. Influence of dietary fatty acids on the elongation and
J. Rinchard et al. / Aquaculture 264 (2007) 363–371 desaturation of linoleic and linolenic. Fish Physiol. Biochem. 9, 393–399. Poston, H.A., 1990. Effect of body size, on growth, survival, and chemical composition of Atlantic salmon fed soy lecithin and choline. Prog. Fish-Cult. 52, 226–230. Poston, H.A., 1991. Response of rainbow trout to soy lecithin, choline, and autoclaved isolated soy protein. Prog. Fish-Cult. 53, 85–90. Ruyter, B., Moya-Falcon, C., Rosenlund, G., Vegusdal, A., 2006. Fat content and morphology of liver and intestine of Atlantic salmon (Salmo salar): effects of temperature and dietary soybean oil. Aquaculture 252, 441–452. Salhi, M., Hernandez-Cruz, C.M., Bessonart, M., Izquierdo, M.S., Fernandez-Palacios, H., 1999. Effect of different dietary polar lipid levels and different n − 3 HUFA content in polar lipids on gut and liver histological structure of gilthead seabream (Sparus aurata) larvae. Aquaculture 179, 253–263. Sargent, J.R., Bell, J.G., Bell, M.V., Henderson, R.J., Tocher, D.R., 1993. The metabolism of phospholipids and polyunsaturated fatty acids in fish. In: Lahlou, B., Vitiello, P. (Eds.), Aquaculture: Fundamental and Applied Research. . Coastal and Estuarine Studies, vol. 43. American Geophysical Union, Washington DC, pp. 103–124. Sargent, J.R., Bell, J.G., Bell, M.V., Henderson, R.J., Tocher, D.R., 1995. Requirement criteria for essential fatty acids. J. Appl. Ichthyol. 11, 183–198. Sargent, J.R., Bell, J.G., McEvoy, L., Tocher, D., Estevez, A., 1999. Recent developments in the essential fatty acid nutrition of fish. Aquaculture 177, 191–199.
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Subhadra, B., Lochmann, R., Rawles, S., Chen, R.G., 2006. Effect of dietary lipid source on the growth, tissue composition and hematological parameters of largemouth bass (Micropterus salmoides). Aquaculture 255, 210–222. Takeuchi, T., Watanabe, T., 1979. Effect of excess amounts of essential fatty acids on growth of rainbow trout. Bull. Jpn. Soc. Sci. Fish. 45, 977–982. Tocher, D.R., 2003. Metabolism and functions of lipids and fatty acids in teleost fish. Rev. Fish. Sci. 11, 107–184. Van Anholt, R.D., Spanings, F.A.T., Koven, W.M., Nixon, O., Wendelaar Bonga, S.E., 2004a. Arachidonic acid reduces the stress response of gilthead seabream Sparus aurata L. J. Exp. Biol. 207, 3419–3430. Van Anholt, R.D., Spanings, F.A.T., Koven, W.M., Wendelaar Bonga, S.E., 2004b. Dietary supplementation with arachidonic acid in tilapia (Oreochromis mossambicus) reveals physiological effects not mediated by prostaglandins. Gen. Comp. Endocrinol. 139, 215–226. Watanabe, T., Takeuchi, T., 1976. Evaluation of Pollack liver oil as a supplement to diets for rainbow trout. Bull. Jpn. Soc. Sci. Fish. 42, 893–906. Watanabe, T., Thongrod, S., Takeuchi, T., Satoh, S., Kubota, S.S., Fujimaki, Y., Cho, C.Y., 1989. Effect of dietary n − 6 and n − 3 fatty acids on growth, fatty acid composition and histological changes of white fish Coregonus lavaretus maraena. Nippon Suisan Gakkaishi 55, 1977–1982. Wonnacott, E.J., Lane, R.L., Kohler, C.C., 2004. Influence of dietary replacement of menhaden oil with canola oil on fatty acid composition of sunshine bass. N. Am. J. Aquac. 66, 243–250.
Influence of lipid class and fatty acid deficiency on survival, growth, and fatty acid composition in rainbow trout juveniles J. Rinchard 1 , S. Czesny 2 , K. Dabrowski ⁎ School of Environment and Natural Resources, The Ohio State University, 2021 Coffee Road, Columbus, OH 43210, USA Received 27 July 2006; received in revised form 26 November 2006; accepted 26 November 2006
Abstract Triplicate groups of rainbow trout (182 ± 51 mg) were fed four experimental casein–gelatin based diets containing different fatty acid levels for 8 weeks. Diet 1, containing only the oleic acid methyl ester (18:1n − 9), was used as an essential fatty acids deficient diet. Diet 2, supplemented with olive and linseed oils was designed to provide moderate amount of linoleic (18:2n − 6) and linolenic (18:3n − 3) acids in the form of triglycerides. Diet 3 contained cod liver oil and provided highly unsaturated fatty acids (HUFA) as well as their precursors. Diet 4, supplemented with soy-refined lecithin, contained high levels of 18:2n − 6 in the form of phospholipids. At the end of the experiment, the growth of fish fed the lecithin supplemented diet was significantly higher (17 fold increase) than that of the other groups (6–14 fold increase). Survival was significantly lower in fish fed the diet 1 in comparison to fish fed the other experimental diets (57.8% vs. 84.4–86.7%). Fish fed diets 1 and 4 contained significantly less total and neutral lipids in the whole body than fish fed diets 2 and 3. Whole body fatty acid profiles from both neutral and phospholipid fractions closely reflected dietary fatty acid composition. Fish fed linoleate containing phospholipids preferentially deposited this fatty acid in neutral lipids (33.0 ± 1.6%) in comparison to body phospholipids deposition (21.2 ± 1.0%). An increase of dietary linoleate also resulted in a significantly higher percentage of arachidonate (20:4n − 6) in fish body phospholipids. In conclusion, selective accumulation of 18, 20, and 22 polyunsaturated fatty acids in body of juvenile rainbow trout is the function of fatty acid origin, e.g., lipid class, which has been very little studied in this species. © 2006 Elsevier B.V. All rights reserved. Keywords: Rainbow trout (Oncorhynchus mykiss); Fatty acid composition; Lecithin; Fish oil; Vegetable oil; Growth
1. Introduction The importance of highly unsaturated fatty acids (HUFA, n − 6 and n − 3 fatty acids, C20 and C22 with ⁎ Corresponding author. Tel.: +1 614 2924555; fax: +1 614 2927432. E-mail address: [email protected] (K. Dabrowski). 1 Present address: School of Natural Resources and Environment, University of Michigan and USGS Great Lakes Science Center, Ann Arbor, MI 48105, USA. 2 Present address: Illinois Natural History Survey, Lake Michigan Biological Station, Zion, IL 60099, USA. 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.11.024
more than 2 double bonds) in fish nutrition is well established (Henderson and Tocher, 1987; Sargent et al., 1993), and these fatty acids are required by fish to ensure optimal growth, development, and reproduction (Sargent et al., 1995, 1999). Vegetable oils, rich in linoleic (18:2n − 6) and/or linolenic (18:3n − 3) acids, are considered to be excellent substitutes for fish oil in freshwater fish feeds, because freshwater fish are able to convert dietary 18:2n − 6 and 18:3n − 3 to HUFA, such as arachidonic acid (ARA, 20:4n − 6), eicosapentaenoic acid (EPA, 20:5n − 3), and docosahexaenoic acid (DHA, 22:6n − 3) (Tocher, 2003).
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Several studies conducted on freshwater fish indicated that vegetable oils can successfully replace fish oil in fish feeds without affecting survival and growth (Wonnacott et al., 2004; Subhadra et al., 2006). Caballero et al. (2002) reported that in rainbow trout Oncorhynchus mykiss up to 80–90% of vegetable oils (e.g., soybean, rapeseed, olive, and palm oils) can be used without compromising their growth. The replacement of fish oil by vegetable oils has a profound impact on the fatty acid composition of fish tissues with an increase in 18:2n − 6 and 18:3n − 3 and a decrease in DHA and EPA (Greene and Selivonchick, 1990; Caballero et al., 2002). Caballero et al. (2003) also observed an accumulation of lipid droplets in the enterocytes related to a decrease in dietary n − 3 HUFA content, indicating that the lipid transport and/ or metabolism in fish appeared to be affected. Similarly, Ruyter et al. (2006) found that rainbow trout fed soybean oil accumulated 3 times more linoleate in triglycerides than in phospholipids in the intestine. Dietary lecithin supplementation is used to enhance growth and survival of fish (Poston, 1990; Kanazawa, 1997; Salhi et al., 1999; Cahu et al., 2003). Lecithin also prevents lipid oxidation and facilitates fat absorption. Salhi et al. (1999) suggested that the increase in dietary polar lipids may improve lipid transport from the enterocytes to the blood by enhancing chylomicron synthesis. There are inconsistent reports of the effect of lecithin on growth, due to differences in fish size among studies. Thus, Watanabe and Takeuchi (1976) did not observe enhancement of growth in juvenile rainbow trout averaging 1.5 g when fed diets supplemented with lecithin, whereas Poston (1991) noted a better growth when a lecithin supplemented diet (4 and 8%) was provided at an early stage (0.1 g). The aim of this study was to compare the effects of different sources of dietary lipids (phospholipids or triglycerides) on growth, survival, and fatty acid composition in rainbow trout juveniles. Fish were fed 4 semi-purified casein–gelatin based diets in which lipids were supplied either by oleic acid methyl ester, a mixture of two vegetable oils (linseed and olive oils) rich in 18:2n − 6 and 18:3n − 3, cod liver oil rich in HUFA (EPA and DHA), or soy-refined lecithin rich in 18:2n − 6 containing phospholipids. 2. Materials and methods 2.1. Experimental diets Four experimental casein–gelatin based diets were formulated to be isonitrogenous (65%) and isolipidic
(14%), but containing different sources of lipids (Table 1). The first diet contained only the oleic acid methyl ester (18:1n − 9), and was used as an essential fatty acids deficient diet (OA). The second diet supplemented with olive and linseed oil (O + L) was designed to provide moderate amount of linoleic (18:2n − 6) and linolenic (18:3n − 3) acids, but no HUFA. The third diet contained cod liver oil (CLO) and provided HUFA as well as their precursors. The fourth diet supplemented with soyTable 1 Composition (%) of the four experimental diets fed to rainbow trout Experimental diets
Ingredients Casein (vitamin-free) Gelatin Dextrin (80% water soluble) Wheat meal Fish protein concentrate a Esters of oleic acid Olive oil Linseed oil Cod liver oil Soy-refined lecithin Vitamin mixture b Mineral mixture c Ascorbic acid d Carboxymethylcellulose sodium salt L-arginine L-methionine L-lysine Choline chloride (99%)
OA
O+L
CLO
LE
40.00 8.00 6.25 15.00 5.00 14.00 0.00 0.00 0.00 0.00 4.00 3.00 0.05 2.00 0.50 0.40 0.80 1.00
40.00 8.00 6.25 15.00 5.00 0.00 8.75 5.25 0.00 0.00 4.00 3.00 0.05 2.00 0.50 0.40 0.80 1.00
40.00 8.00 6.25 15.00 5.00 0.00 0.00 0.00 14.00 0.00 4.00 3.00 0.05 2.00 0.50 0.40 0.80 1.00
40.00 8.00 6.25 15.00 5.00 0.00 0.00 0.00 0.00 14.00 4.00 3.00 0.05 2.00 0.50 0.40 0.80 1.00
64.6 12.6 5.1 7.6
64.4 14.4 5.3 7.1
65.2 11.2 7.5 9.1
Proximate composition (dry matter %) Crude protein 66.8 Crude lipid 12.1 Ash 5.2 Moisture 9.6
a Concentrate of fish soluble protein (CPSP 90: crude protein, 82– 84% WW; crude lipid, 9–13% WW), Sopropêche S.A., Boulogne-surmer, France. b Roche Performance Premix (Hoffman-La Roche, Inc., Nutley, New Jersey), composition per g of the vitamin mixture: vitamin A, 2645.50IU; vitamin D3, 220.46IU; vitamin E, 44.09IU; Vitamin B12, 13 μg; riboflavin, 13.23 mg; niacin, 61.73 mg; D-pantothenic acid, 22.05 mg; menadione, 1.32 mg; folic acid, 1.76 mg; pyridoxine, 4.42 mg; thiamine, 7.95 mg; D-biotin, 0.31 mg. c Bernhart Tomarelli salt mixture (ICN Pharmaceuticals, Costa Mesa, CA), composition (g/100 g): calcium carbonate, 2.1; calcium phosphate dibasic, 73.5; citric acid, 0.227; cupric citrate, 0.046; ferric citrate (16 to 17% Fe), 0.558; magnesium oxide, 2.5; manganese citrate, 0.835; potassium iodide, 0.001; potassium phosphate dibasic, 8.1; potassium oxide, 6.8; sodium chloride, 3.06; sodium phosphate, 2.14; and zinc citrate, 0.133. Five milligrams of Se in the form of sodium selenite was added per kilogram of the salt mixture. d Phosphitan C (Mg-L-ascorbyl-2-phosphate), Showa Denko K.K., Tokyo, Japan.
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refined lecithin (LE) containing high levels of 18:2n − 6 in the form of phospholipids. The methyl ester of oleic acid, olive oil, linseed oil, cod liver oil, and soyrefined lecithin were purchased from ICN (Aurora, OH, USA). 2.2. Fish and experimental conditions The experiment was carried out in a semi-closed recirculating-water system, composed of twelve 30-L glass aquaria, an Aquanetics Deluxe System Paks (model 166; Aquanetic Systems, San Diego, CA, USA), and a sedimentation tank. Each aquarium was equipped with continuous aeration, and water was supplied at a rate of 1 L min− 1. Throughout the experiment, water temperature ranged from 15 to 19 °C, whereas the photoperiod was maintained constant (12 h L/12 h D). Rainbow trout (182 ± 51 mg, n = 30), fed a commercial diet (Starter, BioOregon, OR, USA) for three days prior to the beginning of the present study, were randomly distributed among 12 aquaria (n = 50) and assigned to one of the four experimental diets with three aquaria per dietary treatment. Fish were fed by hand 3–4 times a day at a restricted ration of up to 90% satiation for 8 weeks. Aquaria were cleaned and feces discarded daily. 2.3. Sampling procedures, lipids and fatty acids analysis Every two weeks, fish from each aquarium were weighed, counted and feeding rate was readjusted. At week 4, fish density was reduced to 20 fish per aquarium. At week 6, 5 fish per aquarium were sacrificed by overdose of MS-222 and immediately frozen at − 80 °C for subsequent proximate and fatty acid analysis. At the end of the experiment (8 weeks), survival, weight gain (WG = (final weight − initial weight) × 100 / initial weight), specific growth rate (SGR = [loge final weight − loge initial weight] × 100 / duration of the experiment in days), and feed conversion rate (FCR = dry feed intake / weight gain) were calculated. Analysis of crude protein, moisture, and ash in diets and the whole body of fish was performed according to standard procedures (AOAC, 1995). Dietary and whole body lipids were extracted according to the procedure of Folch et al. (1957) with chloroform/ methanol (2:1 v/v) containing 0.01% of butylated hydroxytoluene (BHT) as antioxidant. The organic solvent was evaporated under a stream of nitrogen and the amount of lipid was determined gravimetrically.
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Whole body crude lipids were then separated into polar (phospholipids) and neutral (mostly triglycerides) lipids using Sep-Pak silica cartridges (Waters, Milford, MA, USA). Chloroform and methanol were used as the mobile phases for neutral and phospholipids, respectively (Juaneda and Rocquelin, 1985). Fatty acid methyl esters (FAME) were prepared following the methods of Metcalfe and Schmitz (1961). Prior to transmethylation, a known amount of nonadecanoate acid (19:0), proportional to the amount of total, neutral or phospholipids detected (8 mg per 50 mg of lipids), was added to the samples as the internal standard for the quantification of fatty acids. FAME were quantified using a Varian 3900 gas chromatograph (Varian, Inc., Walnut Creek, CA) equipped with a flame ionization detector, a capillary column (Varian Chrompack capillary column (WCOT fused silica 100 m × 0.25 mm coating CPSIL 88 for FAME, df = 0.2), and an auto-injector (CP-8410 AutoInjector, Varian, Inc.). Helium was used as a carrier gas at a flow of 30 ml/min. The injector and detector temperatures were 270 and 300 °C, respectively. Initial temperature of the oven was 175 °C for 26 min which increased to 205 °C by increments of 2 °C/min, then held at 205 °C for 24 min. The individual fatty acids were identified by comparing their retention times to that of a standard mix of fatty acids (Nu-Check-Prep, Inc., Elysian, MI, USA) and quantified by comparing their peak area with that of the internal standard. Fatty acids are expressed as percent of total identified FAME. 2.4. Statistical analysis Results are expressed as means ± SD (n = 3). Homogeneity of variance was verified for all data using Bartlett's test (Dagnelie, 1975). Percentage data were arc sin transformed prior to statistical analysis. Data were subjected to analysis of variance (ANOVA) and subsequent comparison of means by Fisher least significant differences test (P < 0.05). Linear regression analyses were performed between dietary and whole body fatty acid concentrations. Differences were accepted as statistically significant when P < 0.05. 3. Results 3.1. Proximate and fatty acid compositions of the experimental diets Although the experimental diets were designed to be isolipidic (14%), the LE diet contained the lowest lipid content and the CLO diet the highest level (Table 1). The
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fatty acid content of the diets clearly reflected the major fatty acids present in the lipid sources (Table 2). High levels of monounsaturated fatty acid (96.2%), mainly oleic acid (18:1n − 9), and low percentages of n − 3 and n − 6 fatty acids (1.0 and 1.5%, respectively) were found in the OA diet. Diet O + L provided moderate amounts of 18:2n − 6 and 18:3n − 3, both precursors of HUFA (14.2 and 18.5%, respectively). High percentages of EPA and DHA were present in the CLO diet (10.2 and 7.6%, respectively), while 18:2n − 6 and 18:3n − 3 only represented 6.5 and 2.4%, respectively. The highest level (64.8%) of polyunsaturated fatty acids (PUFA, fatty acids with 2 or more double bonds) was measured in the LE diet, mainly represented by 18:2n − 6 (53.7%). The concentrations of ARA, EPA and DHA were similar and low in the OA, O + L, and LE diets in comparison to the CLO diet.
Fig. 1. Changes of rainbow trout weight fed the four experimental diets for 8 weeks. At each sampling date means with different superscript letter are significantly different (P < 0.05).
3.2. Growth and survival of rainbow trout Table 2 Fatty acid composition (% of total fatty acids detected) in the four experimental diets Fatty acids
Experimental diets OA
O+L
CLO
LE
Saturated 14:0 16:0 17:0 18:0 ∑ Saturated
0.2 1.1 nd nd 1.3
0.3 11.6 0.1 2.0 14.0
6.5 18.3 0.2 2.9 27.9
0.9 19.3 0.2 4.0 24.8
Monounsaturated 16:1n − 9 and n − 7 18:1n − 9 and n − 7 20:1n − 9 22:1n − 11 ∑ Monounsaturated
0.2 96.0 nd nd 96.2
0.9 51.1 0.1 nd 52.1
7.7 26.9 1.5 1.9 38.0
0.5 13.5 0.3 0.1 14.4
Polyunsaturated 18:2n − 6 20:2n − 6 20:3n − 6 20:4n − 6 22:4n − 6 22:5n − 6 ∑ n−6 18:3n − 3 18:4n − 3 20:3n − 3 20:4n − 3 20:5n − 3 22:5n − 3 22:6n − 3 ∑ n−3 ∑ Polyunsaturated ∑ n − 3/∑ n − 6
1.2 nd nd 0.3 nd nd 1.5 0.3 nd nd nd 0.2 nd 0.5 1.0 2.5 0.7
14.2 nd nd 0.3 nd nd 14.5 18.5 nd nd nd 0.3 nd 0.5 19.3 33.8 1.3
6.5 0.1 0.1 1.0 nd 0.3 8.0 2.4 2.2 0.2 0.8 10.2 2.5 7.6 25.9 33.9 3.2
53.7 nd nd 0.4 nd nd 54.1 5.8 nd nd nd 0.4 nd 0.9 10.7 64.8 0.2
nd: not detected.
The first significant effect of dietary fatty acids on rainbow trout growth was observed after 4 weeks of feeding (Fig. 1). At that time, fish weight was significantly (P < 0.01) lower in fish fed the OA diet. At 6 weeks, the growth of fish fed the O + L and CLO diets were similar, whereas the weight of fish fed the LE diet was significantly (P < 0.01) higher than the other groups. Similar patterns were observed at the end of the experiment, however the growth of fish fed the CLO diet was significantly higher than the fish fed the O + L diet (Fig. 1 and Table 3). Specific growth rate and feed conversion ratio were significantly (P < 0.01) affected by dietary treatments (Table 3). Fish fed the LE diet have the highest SGR and the lowest FCR, whereas the opposite trend was found in fish fed the OA diet. Intermediate SGR and FCR were found in fish fed the Table 3 Growth of rainbow trout fed the four experimental diets for 8 weeks Experimental diets OA
O+L
CLO
LE
Final weight (g) 1.2 ± 0.1d 2.3 ± 0.2c 2.7 ± 0.1b 3.3 ± 0.3a d c b Weight 572 ± 78 1165 ± 133 1388 ± 31 1684 ± 137a gain (%)1 SGR (%)2 3.3 ± 0.2d 4.5 ± 0.2c 4.7 ± 0.1b 5.1 ± 0.1a FCR3 2.3 ± 0.3a 1.5 ± 0.2b 1.3 ± 0.0bc 1.1 ± 0.1c b a a Survival (%) 57.8 ± 3.8 84.4 ± 3.8 86.7 ± 7.7 84.4 ± 10.2a Weight gain = ((final weight − initial weight) × 100) / initial weight. Specific growth rate (SGR) = ((loge final weight − loge initial weight) × 100) / duration of the experiment in days. 3 Feed conversion ratio (FCR) = (dry feed intake) / wet weight gain. Means with different superscript letter in a row are significantly different (P < 0.05). 1
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J. Rinchard et al. / Aquaculture 264 (2007) 363–371 Table 4 Moisture (%) and proximate composition (% of dry matter) of whole body rainbow trout fed the experimental diets for 6 weeks Parameters
Experimental diets OA
Crude protein Crude lipid Phospholipids (% of total lipids) Ash Moisture
O+L a
CLO c
77.0 ± 4.0 66.0 ± 2.4 21.1 ± 3.4b 27.9 ± 3.0a 27.5 ± 3.8a 19.4 ± 0.5b
LE c
65.2 ± 0.7 71.2 ± 1.3b 29.8 ± 2.9a 21.6 ± 1.7b 22.1 ± 3.1b 27.3 ± 1.9a
12.6 ± 0.9b 14.8 ± 0.7ab 16.3 ± 1.5a 16.1 ± 1.4a 94.2 ± 4.0a 85.3 ± 1.4b 85.9 ± 0.3b 86.3 ± 0.4b
Means with different superscript letter in a row are significantly different (P < 0.05).
O + L and LE diets. Survival was significantly (P < 0.01) lower in fish fed the OA diet in comparison to fish fed all other experimental diets (Table 3). Mortality occurred after 2 weeks. 3.3. Proximate and fatty acid composition of rainbow trout
respectively) were detected in fish fed the OA diet. The highest percentages of 18:2n − 6 and 18:3n − 3 were found in fish fed the LE and O + L diets, respectively, whereas the highest concentrations of EPA and DHA were measured in fish fed the CLO diet. The highest concentration of ARA was found in fish fed LE diet. The percentage of saturated, monounsaturated and PUFA differed significantly (P < 0.01) among fish fed the four experimental diets. Neutral lipids of fish fed the CLO diet had the highest n − 3/n − 6 ratio, whereas neutral lipids of fish fed the LE diet had the lowest n − 3/n − 6 ratio. With respect to the phospholipid fraction, 16:0 and 18:1 (both n − 7 and n − 9) were the major saturated and monoene fatty acids regardless of the dietary lipid source. Concentrations of DHA and EPA did not differ significantly (P > 0.05) between fish fed OA and LE
Table 5 Fatty acid composition of neutral lipids (% of total lipids detected) of whole body rainbow trout fed the experimental diets for 6 weeks Fatty acids
The proximate composition of fish fed the O + L and CLO diets did not differ significantly (P > 0.05) (Table 4). Fish fed the OA and LE diets contained significantly (P < 0.01) higher protein levels and less lipids than fish fed the O + L and CLO diets. The percentages of polar lipids in fish fed the O + L and CLO diets were significantly (P < 0.01) lower in comparison to those measured in fish fed the OA and LE diets. Moisture level was the highest and ash content was the lowest in fish fed the OA diet and, these values were significantly (P < 0.01) different for all other experiment groups. Moisture and ash levels did not differ significantly (P > 0.05) among fish fed the three other experimental diets. Fatty acid composition of the neutral and phospholipid fractions of the whole body of rainbow trout, expressed as percentage of total fatty acids detected, reflected dietary fatty acid compositions (Tables 5 and 6). The fatty acid composition of rainbow trout phospholipids was less affected than neutral lipids by dietary fatty acid concentrations. The concentrations of ARA, EPA, and DHA were significantly higher in the phospholipids than in neutral lipids. With respect to the neutral lipid fraction, 18:1, both n − 7 and n − 9, were the predominant fatty acids measured in fish fed the OA, O + L and CLO diets, whereas 18:2n − 6 was the dominant fatty acid in fish fed the LE diet. High level of monounsaturated fatty acids (77.7%), mainly 18:1 (both n − 7 and n − 9), and low percentages of n − 3 and n − 6 fatty acids (3.0 and 1.6%,
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Experimental diets OA
O+L
Saturated 14:0 16:0 17:0 18:0 ∑ Saturated
CLO
LE
1.1 ± 0.6b 6.9 ± 0.5d 0.5 ± 0.1a 2.3 ± 0.3c 10.7 ± 1.0c
1.2 ± 0.2b 5.3 ± 0.2a 14.0 ± 0.8c 17.6 ± 0.7b 0.1 ± 0.0c 0.3 ± 0.0b 4.2 ± 0.2b 4.1 ± 0.1b b 19.5 ± 1.2 27.3 ± 0.8a
1.3 ± 0.1b 20.4 ± 1.7a 0.2 ± 0.0c 6.4 ± 0.2a 27.2 ± 2.3a
Monounsaturated 16:1n − 9 and n − 7 18:1n − 9 and n − 7 20:1n − 9 22:1n − 11 ∑ Monounsaturated
5.0 ± 0.3b 77.7 ± 1.0a 1.2 ± 0.1a 0.3 ± 0.1b 84.2 ± 0.8a
3.5 ± 0.3c 8.3 ± 0.4a b 51.4 ± 1.1 32.5 ± 0.9c 1.4 ± 0.2a 1.8 ± 0.0a b 0.3 ± 0.1 0.9 ± 0.3a 56.5 ± 0.9b 43.5 ± 1.1c
2.7 ± 0.4d 24.2 ± 4.1d 0.5 ± 0.1c 0.3 ± 0.1b 27.8 ± 3.7d
Polyunsaturated 18:2n − 6 20:2n − 6 20:3n − 6 20:4n − 6 22:4n − 6 22:5n − 6 ∑ n−6 18:3n − 3 18:4n − 3 20:3n − 3 20:4n − 3 20:5n − 3 22:5n − 3 22:6n − 3 ∑ n−3 ∑ Polyunsaturated ∑ n − 3/∑ n − 6
0.8 ± 0.1d 0.4 ± 0.3a nd 0.1 ± 0.0d 0.2 ± 0.2b 0.5 ± 0.2b 1.6 ± 0.2d 1.4 ± 0.2b 0.1 ± 0.0b 0.2 ± 0.2b 0.3 ± 0.1a 0.3 ± 0.0b 0.2 ± 0.1b 0.6 ± 0.1d 3.0 ± 0.3d 5.1 ± 0.3d 1.9 ± 0.4b
9.6 ± 1.5b 5.6 ± 0.3c 0.3 ± 0.1a 0.6 ± 0.0a b 0.3 ± 0.1 0.2 ± 0.0b 0.4 ± 0.1c 0.9 ± 0.1b nd 0.5 ± 0.1a 0.5 ± 0.0b 0.5 ± 0.0b b 10.8 ± 1.6 7.8 ± 0.2c 8.5 ± 0.8a 1.7 ± 0.1b b 0.6 ± 0.0 1.9 ± 0.1a 0.4 ± 0.1b 0.2 ± 0.0b a 0.5 ± 0.3 0.5 ± 0.0a 0.5 ± 0.1b 5.8 ± 0.3a b 0.2 ± 0.0 1.6 ± 0.0a 1.9 ± 0.0b 9.2 ± 0.2a b 12.7 ± 1.3 20.8 ± 0.5a 24.0 ± 0.3c 29.2 ± 0.6b 1.2 ± 0.3c 2.7 ± 0.1a
33.0 ± 1.6a nd 2.1 ± 0.2a 1.2 ± 0.2a 0.2 ± 0.0b 0.9 ± 0.1a 36.5 ± 1.4a 1.8 ± 0.9b 2.0 ± 0.8a 1.3 ± 0.1a 0.2 ± 0.1a 0.5 ± 0.2b 0.2 ± 0.1b 1.5 ± 0.3c 7.5 ± 0.9c 45.0 ± 1.9a 0.2 ± 0.0d
Means with different superscript letter in a row are significantly different (P < 0.05). nd: not detected.
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Table 6 Fatty acid composition of phospholipids (% of total lipids detected) of whole body rainbow trout fed the experimental diets for 6 weeks Fatty acids
Experimental diets OA
O+L
CLO
LE
Saturated 14:0 16:0 17:0 18:0 Σ Saturated
1.3 ± 0.5a 14.7 ± 0.5b 0.1 ± 0.0a 3.9 ± 0.4c 20.0 ± 1.1b
1.3 ± 0.4a 21.2 ± 0.6a 0.1 ± 0.0a 5.9 ± 0.5b 28.3 ± 0.3a
1.3 ± 0.1a 21.6 ± 0.6a 0.2 ± 0.2a 5.4 ± 0.8b 28.5 ± 1.4a
1.4 ± 0.1a 20.9 ± 0.4a 0.2 ± 0.0a 6.4 ± 0.2a 28.9 ± 0.8a
Monounsaturated 16:1n − 9 and n − 7 18:1n − 9 and n − 7 20:1n − 9 22:1n − 11 ∑ Monounsaturated
5.8 ± 0.2a 44.6 ± 0.4a 1.0 ± 0.8a 0.1 ± 0.0b 51.6 ± 0.5a
3.3 ± 0.1c 23.3 ± 0.4b 1.2 ± 0.2a 0.5 ± 0.3a 28.3 ± 0.3b
4.0 ± 1.0b 17.0 ± 0.7c 1.2 ± 0.3a 0.1 ± 0.0b 22.3 ± 1.2c
1.9 ± 0.1d 13.2 ± 1.0d 0.3 ± 0.0b 0.1 ± 0.0b 15.6 ± 1.1d
Polyunsaturated 18:2n − 6 20:2n − 6 20:3n − 6 20:4n − 6 22:4n − 6 22:5n − 6 ∑ n−6 18:3n − 3 18:4n − 3 20:3n − 3 20:4n − 3 20:5n − 3 22:5n − 3 22:6n − 3 ∑ n−3 ∑ Polyunsaturated ∑ n − 3/∑ n − 6
1.2 ± 0.1c 1.1 ± 0.6a 3.6 ± 0.2a 2.1 ± 0.2b 1.2 ± 0.1a 1.4 ± 0.5b 10.5 ± 0.6b 1.2 ± 0.0b 0.3 ± 0.1b 0.6 ± 0.1c 0.7 ± 1.0a 1.6 ± 0.2c 0.9 ± 0.3a 12.6 ± 0.7c 17.9 ± 1.4d 28.4 ± 0.8d 1.7 ± 0.2bc
6.3 ± 0.4b 0.4 ± 0.1a 0.1 ± 0.1c 2.0 ± 0.1b 0.1 ± 0.0b 0.9 ± 0.1c 9.7 ± 0.3b 4.1 ± 0.2a 0.5 ± 0.1ab 1.1 ± 0.1b 0.8 ± 0.1a 3.2 ± 0.3b 0.7 ± 0.1a 23.2 ± 0.4b 33.6 ± 0.3b 43.3 ± 0.1c 3.5 ± 0.1b
2.1 ± 0.2c 0.9 ± 0.6c 0.2 ± 0.2b 1.5 ± 0.5b 0.2 ± 0.3c 0.7 ± 0.2a 5.7 ± 1.6c 0.7 ± 0.3c 0.5 ± 0.1ab 0.4 ± 0.1c 0.6 ± 0.3a 8.4 ± 0.2a 1.0 ± 0.4a 31.9 ± 1.1a 43.5 ± 1.5a 49.2 ± 0.1b 8.2 ± 2.8a
21.2 ± 1.0a 0.2 ± 0.0b 2.4 ± 0.1b 4.5 ± 0.4a 0.1 ± 0.0b 5.9 ± 0.4a 34.3 ± 0.5a 1.3 ± 0.1b 0.7 ± 0.3a 3.8 ± 0.1a 0.2 ± 0.0a 1.2 ± 0.3c 0.3 ± 0.1a 13.5 ± 1.3c 21.2 ± 1.4c 55.5 ± 1.8a 0.6 ± 0.0c
Means with different superscript letter in a row are significantly different (P < 0.05).
diets, but were significantly (P < 0.01) higher in fish fed the O + L and CLO diets. Saturated fatty acid concentrations were significantly (P < 0.01) lower in fish fed OA diet in comparison to fish fed all other diets. The percentage of polyunsaturated fatty acids was significantly (P < 0.01) affected by dietary treatment, however, the main contributors to PUFA were different. Fatty acids from the n − 3 family dominated PUFA in fish fed CLO diet, while fatty acids of the n − 6 family were mostly in fish fed the LE diet. Finally, as reported for the neutral lipid fraction, the highest n − 3/n − 6 ratio was detected in fish fed the CLO diet and the lowest in fish fed the LE diet. Regression analysis was used to identify dose– response relationship between dietary and body fatty acids. As reported in Table 7, most of the individual
Table 7 Correlation between dietary fatty acid concentrations and fatty acid concentrations in whole body rainbow trout fed the experimental diets for 6 weeks Fatty acids 18:1n − 9 and n − 7 18:2n − 6 18:3n − 3 20:4n − 6 20:5n − 3 22:6n − 3 ∑ Saturated ∑ Monounsaturated ∑ Polyunsaturated ∑ n−3 ∑ n−6
Neutral lipids 2
Phospholipids
Slope
r
P
Slope
r2
P
0.64 0.59 0.41 0.33 0.53 1.06 0.62 0.66 0.66 0.66 0.64
1.00 1.00 0.94 0.11 1.00 0.98 0.99 1.00 0.99 0.96 1.00
<0.01 <0.01 0.03 0.66 <0.01 <0.01 <0.01 <0.01 <0.01 0.02 <0.01
0.37 0.38 0.18 − 1.36 0.63 2.02 0.31 0.43 0.45 1.01 0.51
0.97 1.00 0.91 0.26 0.92 0.67 0.80 0.99 0.94 0.97 0.90
0.01 <0.01 0.05 0.49 0.04 0.18 0.10 <0.01 0.03 0.01 0.05
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dietary fatty acids were linearly correlated to their concentrations in whole body of rainbow trout. 4. Discussion The four experimental diets used in this study differed only by their lipid sources, but the sources differed both in lipid classes and fatty acid composition. As reported in several species (Bell et al., 1991, 1994; Sargent et al., 1995), fatty acid concentrations of rainbow trout were influenced by the fatty acid composition of the dietary lipids. After 8 weeks of feeding, no sign of EFA deficiency, such as fin erosion and bacterial disease, was observed in rainbow trout fed the OA diet. However, these symptoms were only observed by Castell et al. (1972) in fish fed essential fatty acid (EFA) deficient diet over an 8-month period. Growth and survival was significantly lower in fish fed the OA diet. Similar growth depression has been observed in other studies where fish were fed n − 3 HUFA deficient diets (Watanabe et al., 1989; Lee et al., 2003). In this study, higher water and protein contents were found in fish fed the OA diet in comparison to fish fed the three other diets. Similar results were found by Castell et al. (1972) when rainbow trout were fed EFA diets. Both the O + L and CLO diets contained approximately the same amount of PUFA (32.7 and 33.9%, respectively). However, their fatty acid composition was different. PUFA of the O + L diet consisted primarily of 18:3n − 3 and 18:2n − 6, whereas PUFA of the CLO diet were mainly 20:5n − 3 and 22:6n − 3. Fish fed the O + L diet showed lower levels of n − 3 fatty acids and higher levels of C18 fatty acids in comparison to fish fed the CLO diet. The concentration of individual n − 3 HUFA in fish fed the O + L diet was higher than in their diet indicating that these fatty acids are mainly retained in the case of dietary shortages. It was also confirmed that rainbow trout are able to convert, through desaturation and elongation, 18:3n − 3 to 22:6n − 3 (via C24 intermediates; Buzzi et al., 1996, 1997). Bell et al. (2003) reported that fish size also affects DHA synthesis, where small rainbow trout (0.8 g) synthesized DHA ten times more efficiently than larger individuals (6 g). Although in the present study the requirements of both EFA were met (Castell et al., 1972; AOAC, 1995), growth of fish fed O + L diet was lower than that of fish fed the CLO diet. Increased levels of dietary n − 3 HUFA (e.g., EPA and DHA) have been reported to improve growth in several fish species (Bell and Sargent, 2003; Izquierdo et al., 2003; Lee et al., 2003). However, our results
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indicate that soy-refined lecithin (rich in 18:2n − 6) used as the only source of dietary lipid significantly improved growth performance and reduced body fat in early feeding of rainbow trout in comparison to diets rich in n − 3 PUFA (O + L and CLO diets). Similar results were observed in sea bass Dicentrarchus labrax larvae (Cahu et al., 2003) and common carp Cyprinus carpio juveniles (Geurden et al., 1997). Lecithin is known to be a source of bioavailable phospholipids, and improves intestinal absorption of cholesterol. Dietary phospholipids also provide specific lipid classes to produce lipoproteins. The lower efficiency of the O + L and CLO diets in promoting growth could be associated with their deficiency of phospholipids and cholesterol. Growth and survival of fish have been improved with dietary lecithin supplementation (Poston, 1990; Kanazawa, 1997; Salhi et al., 1999; Cahu et al., 2003). The major component of soy lecithin responsible for promoting growth is the phosphatidylcholine. Cahu et al. (2003) offered evidence that dietary phospholipids which contained negligible amounts of EPA and DHA resulted in better growth and survival than fish fed fish oil containing HUFA. Rainbow trout fed higher than optimal levels of n − 3 HUFA (2 to 6 times more than their requirement levels) showed poor growth and low feed conversion (Takeuchi and Watanabe, 1979). Similarly, Ibeas et al. (2000) attributed the growth reduction to an increase of n − 3 HUFA in phospholipids, which created an unbalance of the unsaturation level in the membrane phospholipids. Fish fed the LE diet presented very high levels of ARA in the phospholipid fraction indicating considerable conversion of 18:2n − 6 to ARA. This is not entirely new finding in salmonids as Bell et al. (1992) reported significantly increased levels of ARA in phosphoinositol and phophatidylcholine in heart of Atlantic salmon fed diets enriched in plant oils (containing 46% of linoleate). In contrast, in fish fed the CLO diet, we did not observe this conversion because of the higher dietary 18:3n − 3 provided, which is competing with the 18:2n − 6 for desaturation (Olsen and Ringo, 1992). ARA plays an important role in fish membranes, is the main precursor of eicosanoids, and is the main component of phosphatidylinositol (Sargent et al., 1999). Several studies also demonstrated the role of ARA in stress reduction (Koven et al., 2003; Van Anholt et al., 2004a,b). In the present study, fish fed the LE diet, which did not contain ARA, exhibited the highest levels of this fatty acid in both neutral and phospholipid fractions in comparison to the other groups (Table 6). Similarly, growth and survival of turbot Scophthalmus maximus was improved when ARA was fed as the only
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