Effect of dietary conjugated linoleic acid on muscle, liver and visceral lipid deposition in rainbow trout juveniles (Oncorhynchus mykiss)

Aquaculture 254 (2006) 496 – 505 www.elsevier.com/locate/aqua-online

Effect of dietary conjugated linoleic acid on muscle, liver and visceral lipid deposition in rainbow trout juveniles (Oncorhynchus mykiss) N.M. Bandarra a , M.L. Nunes a , A.M. Andrade a , J.A.M. Prates b , S. Pereira a , M. Monteiro a , P. Rema c,e , L.M.P. Valente d,e,⁎ a

b

INIAP-Investigação Agrária e das Pescas, Departamento de Inovação Tecnológica e Valorização dos Produtos da Pesca, Av. Brasília, 1449-006 Lisboa, Portugal CIISA-Faculdade de Medicina Veterinária, Rua Prof. Cid dos Santos, Pólo Universitário do Alto da Ajuda, 1300-477 Lisboa, Portugal c Departamento de Zootecnia, Universidade de Trás-os-Montes e Alto-Douro, Apartado 1013, 5001 Vila Real, Portugal d ICBAS-Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, 4000 Porto, Portugal e CIIMAR-Centro Interdisciplinar de Investigação Marinha e Ambiental, Rua dos Bragas, 177, 4050-123 Porto, Portugal Received 8 June 2005; received in revised form 20 October 2005; accepted 20 October 2005

Abstract Some of the conjugated linoleic acids (CLA), cis-9, trans-11 and trans-10, cis-12 isomers, may present a wide range of health benefits. Taking into account these beneficial effects and the relative low amounts of CLA in fish products, commercial diets for juvenile rainbow trout were supplemented with graded levels (0, 0.5, 0.75, 1 and 2%) of CLA. Duplicated groups of 5.3 g fish were fed to satiation, twice a day, over a period of 12 weeks with a CLA mixture, containing mainly the bioactive cis-9, trans-11 and trans-10, cis-12 isomers. The increase of dietary CLA levels did not affect growth performance, feed conversion ratio (FCR), hepatosomatic index (HSI) or viscerosomatic index (VSI) of the rainbow trout juveniles. Total lipids in muscle and liver were similar among treatments. In viscera, CLA supplementation reduced total lipids, but only the 0.5% CLA treatment differed significantly from the control. The fatty acid profile was affected by the dietary inclusion of CLA, resulting in a significant increase of the saturated and polyunsaturated fractions and a general decrease of the monounsaturated one, in both muscle and viscera. Nevertheless, in liver only fish fed 1% CLA had a significantly higher saturated fraction. The general decreases of monounsaturated fatty acid level in tissues with the saturated increase suggest a reduction of Δ-9 desaturase activity. CLA was deposited in all tissues and reflected the dietary CLA level. The highest concentration was attained when the 2% diet was used (6.9 g/100 g lipids in muscle). In conclusion, rainbow trout are capable of incorporating CLA in muscle, liver and viscera better than any other vertebrate, showing that this species is a potential route to increase human CLA ingestion. © 2005 Elsevier B.V. All rights reserved. Keywords: Conjugated linoleic acid (CLA); Fatty acids; Liver; Lipids; Muscle; Rainbow trout; Viscera

1. Introduction ⁎ Corresponding author. CIIMAR-Centro Interdisciplinar de Investigação Marinha e Ambiental, Rua dos Bragas, 177, 4050-123 Porto, Portugal. Tel.: +351 22 340 18 25; fax: +351 22 340 18 38. E-mail address: [email protected] (L.M.P. Valente). 0044-8486/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.10.034

Fish farming has expanded and intensified rapidly, in particular during the last two decades (Hannesson, 2003). In order to support the rise of this activity, in

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years to come, particular attention has to be paid to the development of more suitable diets for each species. On the other hand, consumers are becoming more exigent in terms of meat quality of farmed products and, as a rule, expect to get the health benefits generally associated with wild fish (Paterson et al., 1997; Hunter and Roberts, 2000). High-energy diets are widely used in farmed fish, since lipids represent the most concentrated source of energy (approximately 9 kcal/g, about double that supplied by both carbohydrate and protein). Nevertheless, such diets induce higher fat deposition either in muscle or belly of those fish. Taking into account that the composition of fish reflects the formulation of their diets, it is a challenge to include dietary health-promoting nutrients that can lead to tailor-made products with beneficial health effects. It is well known that conjugated linoleic acids (CLA) belong to this group of compounds and are responsible for anticarcinogenic, antiatherosclerotic, antioxidative, immunomodulative and antibacterial properties in humans (Jahreis et al., 2000; Pariza et al., 2001; Roche et al., 2001). CLA refers to a group of fatty acids, which exists as positional, and stereoisomer mixtures of octadecadienoic acids characterized by conjugated double bonds. Among the various CLA isomers the cis-9, trans-11 is the principal dietary form, but lower levels of trans-10, cis-12 are also present in some food products (milk, cheese, animal fat) (Ip et al., 1999; Jahreis et al., 2000; Roche et al., 2001; Loor et al., 2003). Both isomers are known to possess biological activity (Pariza et al., 2001). The effects of dietary CLA have mainly been studied in mammalian species, in which their conjugated structures are known to have an important role in fat deposition as they interfere with the activity of key enzymes involved in lipid mobilisation (Evans et al., 2002; Raes et al., 2004). In fish, studies with CLA are very scarce and with variable results. Choi et al. (1999) reported an improvement of the growth rate of carp fed diets containing 1% CLA, whereas Twibell et al. (2000) observed an inverse result during the feeding of hybrid striped bass with a diet containing the same CLA level. In what concerns the effect of CLA on the muscle lipid content and fatty acid composition the results are rather concordant, showing that feeding CLA elevated tissue concentration of these fatty acid isomers in several fish species: Atlantic salmon (Berge et al., 2004), hybrid striped bass (Twibell et al., 2000) and tilapia (Yasmin et al., 2004). However, each species seems to present specific metabolism towards CLA. Since fish are an important source of protein and n-3 PUFA (polyunsaturated fatty acids), a further increase

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in CLA content could be of great interest to enhance the nutritional value of fish meat for human consumption. The effects of dietary CLA in performance or tissue composition of most species of interest for European aquaculture have not yet been reported. An experiment to evaluate the effects of graded dietary levels of CLA (0, 0.5%, 0.75%, 1% or 2%) on rainbow trout juvenile growth, body composition, utilization of nutrients and energy and lipogenic enzymes activities has been described by Figueiredo-Silva et al. (2005). In that experiment, fish were sampled to study tissue lipid deposition and fatty acid profile in muscle, liver and viscera. The results of these analyses are presented here. 2. Material and methods 2.1. Experimental diets A commercial extruded diet without fish oil added was supplied from Sorgal (Ovar, Portugal). The pellets (2 mm of diameter) were then coated with 10% oil containing the different CLA levels (0, 0.5%, 0.75%, 1% or 2%) to obtain five isonitrogenous (50% crude protein) and isolipidic (16% total lipid) diets. The CLA supplement was added to the diets at the expense of fish oil to maintain a constant energy level among dietary treatments. The CLA mixture (Table 1) was a gift from Bioriginal Food and Science Corp., Saskatoon SK, Canada. Ingredients and proximate composition of diets are present in Table 2 and the fatty acid profiles in Table 3. 2.2. Growth trial The trial was undertaken with rainbow trout (Oncorhynchus mykiss) juveniles produced in the University of Trás-os-Montes e Alto-Douro (UTAD) rearing facilities. Fish were held under optimal conditions and fed the control diet (0% CLA) for a period of 2 weeks before the beginning of the experiment. Homogenous groups of 50 juveniles with an average initial body weight of 5.30 ± 0.02 g (mean ± S.D.) were then randomly distributed among 10 square fibreglass tanks (200 l), in an open circulation water system. Duplicated groups of fish for each treatment were fed by hand to apparent satiety, two times a day (09.30 and 18.00 h) for 12 weeks and feed intake was recorded. The pH, ammonia, nitrites, nitrates and phosphates in the water were monitored during the entire trial and maintained at levels compatible with this species. The daily water temperature was 1 ± 1 °C and trout were exposed to natural photoperiod. At the end of the experiment, fish were

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Table 1 Distribution of different CLA isomers (% total CLA) present in the CLA mixture used for diet supplementation. Values are mean ± S.D. (n = 3) CLA isomers

Distribution (%)

trans-12, trans-14 trans-11, trans-13 trans-10, trans-12 trans-9, trans-1 trans-8, trans-10 trans-7, trans-9 trans-6, trans-8 Total trans, trans cis-12, trans-14 trans-11, cis-13 cis-11, trans-13 trans-10, cis-12 cis-9, trans-11 trans-8, cis-10 trans-7, cis-9 Total cis, trans cis-11, cis-13 cis-10, cis-12 cis-9, cis-11 cis-8, cis-10 Total cis, cis Total CLA

0.01 ± 0.01 0.07±0.06 0.48 ± 0.02 3.12 ± 0.01 2.96 ± 0.06 0.48 ± 0.06 0.09 ± 0.01 7.21 ± 0.12 0.17 ± 0.14 0.12 ± 0.03 1.65 ± 0.27 42.24 ± 1.13 44.65 ± 1.26 n.d. 1.60 ± 0.12 90.43 ± 0.13 n.d. 1.09 ± 0.01 1.28 ± 0.00 n.d. 2.36 ± 0.01 100.00

of CLA fatty acids in supplement). After leaving the mixture overnight at room temperature, 5 ml of a solution of NaCl (5%) solution was added and the required esters extracted with hexane (2 × 5 ml). The organic phase was collected, filtered through activated charcoal and dried over anhydrous sodium sulphate. Solvent was removed under nitrogen and the methyl esters dissolved in 2 ml of n-heptane. For FAME and CLA analysis of trout muscle, liver and viscera, extracted total lipids were used for methyl esters preparation by base-catalysed transesterification, with sodium methoxide 0.5 M solution in anhydrous methanol (2 h at 30 °C), as proposed by Park et al. (2001) and Kramer et al. (2002), in order to avoid isomerisation of CLA. FAME analyses were performed in a Varian Star 3400 Cx (Walnut Creek, CA, USA) gas chromatograph equipped with an auto sampler and fitted with a flame ionisation detector at 250 °C. The separation was achieved using a capillary column HP-INNOWAX (30 Table 2 Ingredient and proximate composition of diets with different CLA incorporation levels (control, 0.5%, 0.75%, 1.0% or 2.0%)

n.d.—not detected.

individually weighed for growth determination. Prior to sampling, fish were fasted for 24 h and then anaesthetized by immersion in an ethylene glycol monophenyl ether (1:2500) bath. Ten randomly sampled fish were collected from each dietary group and muscle, liver and viscera were withdrawn and weighed. Samples were immediately frozen in liquid nitrogen and stored at − 80 °C prior to analysis. Experiments were conducted according to the European Council Directive 86/609/ EEC regarding the protection of animals used for experimental and other scientific purposes. 2.3. Analytical methods Total lipid and protein determinations of diets and fish samples were carried out following the Bligh and Dyer method (1959), with small modifications, and the methodology referred in AOAC (1998), respectively. Muscle and viscera of 10 fish were analysed individually. In the case of liver analysis, two pools with five livers per dietary treatment were prepared and analysed individually. Fatty acid methyl esters (FAME) of diets were prepared by the addition of 4 ml of sulphuric acid (0.04 M) in dry methanol to 100 mg of sample, in order to obtain methyl esters from free fatty acids (chemical structure

Dietary treatments Control 0.5

0.75

1.0

2.0

Ingredients (%) Fish meal LT (68–70)SP Fish meal 60 Soybean meal 48 Wheat meal Soluble fish protein concentrate Fish oil CLA Vitamin a and mineral Mix b

31.9 21.3 21.3 10.6 2.7 10.0 0.0 2.2

31.9 21.3 21.3 10.6 2.7 9.5 0.5 2.2

31.9 21.3 21.3 10.6 2.7 9.25 0.75 2.2

31.9 21.3 21.3 10.6 2.7 9.0 1.0 2.2

31.9 21.3 21.3 10.6 2.7 8.0 2.0 2.2

Proximate composition Dry matter (%) Crude protein (% DM) Crude fat (% DM) Ash (% DM) Gross energy (kJ g− 1 DM)

89.1 50.1 16.1 12.0 23.0

89.2 50.8 16.9 11.6 22.8

88.8 50.1 16.8 11.5 22.8

88.8 50.7 16.5 11.7 23.0

89.3 50.9 16.3 11.7 22.6

Vitamin mixture (IU or mg kg− 1 diet): DL-alpha tocopherol acetate, 60 IU; sodium menadione bisulphate, 5 mg; retinyl acetate, 15000 IU; DL-cholecalciferol, 3000 IU; thiamin, 15 mg; riboflavin, 30 mg; pyridoxine, 15 mg; cyanocobalamin, 0.05 mg; nicotinic acid, 175 mg; folic acid, 5 mg; ascorbic acid, 500 mg; inositol, 1000 mg; biotin, 2.5 mg; calcium panthotenate, 50 mg; choline chloride, 2000 mg. b Mineral mixture (g or mg kg− 1 diet): calcium carbonate (40% Ca), 2.15 g; magnesium oxide (60% Mg), 1.24 g; ferric citrate, 0.2 g; potassium iodide (75% I), 0.4 mg; zinc sulphate (36% Zn), 0.4 g; copper sulphate (25% Cu), 0.3 g; manganese sulphate (33% Min), 0.3 g; dibasic calcium phosphate (20% Ca, 18% P), 5 g; cobalt sulphate, 2 mg; sodium selenite (30% Se), 3 mg; potassium chloride, 0.9 g; sodium chloride, 0.4 g. a

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Table 3 Fatty acid profile (% total fatty acids) and CLA content (% total lipids) of diets with different CLA incorporation levels (control, 0.5%, 0.75%, 1.0% or 2.0%) Dietary treatments

Fatty acids (%) 16:0 18:0 ΣSaturated 1 16:1n-7 18:1n-9 ΣMonounsaturated 2 18:2n-6 18:3n-3 20:4n-3 20:4n-6 20:5n-3 22:5n-3 22:5n-6 22:6n-3 ΣPolyunsaturated 3 Total CLA (% total lipids) 4

Control

0.5

0.75

1.0

2.0

14.73 ± 0.44a 3.92 ± 0.03a 22.65 ± 0.72 4.40 ± 0.67 15.17 ± 0.19 24.34 ± 1.08 13.04 ± 0.13a 1.71 ± 0.01a 0.41 ± 0.02 7.45 ± 0.93a 4.28 ± 0.24 0.44 ± 0.11 0.84 ± 0.14 8.59 ± 1.01a 40.21 ± 2.06a 0.62 ± 0.03a

12.79 ± 0.30a,b 3.15 ± 0.04a,b 20.75 ± 0.49 3.28 ± 0.03 15.54 ± 0.50 22.83 ± 0.14 10.34 ± 0.72b 1.22 ± 0.14b 0.34 ± 0.01 5.88 ± 0.44a,b 3.45 ± 0.12 0.76 ± 0.09 0.32 ± 0.04 7.10 ± 0.58a,b 48.68 ± 0.73b 6.39 ± 0.09b

11.00 ± 0.27a,b 2.77 ± 0.07b 18.41 ± 1.28 1.65 ± 1.52 13.88 ± 0.03 19.79 ± 2.10 8.30 ± 1.05b 0.96 ± 0.19b 0.54 ± 0.32 5.09 ± 0.41a,b 3.25 ± 0.04 1.01 ± 0.45 0.43 ± 0.17 5.97 ± 0.30b 49.37 ± 0.48b 8.96 ± 0.01c

9.98 ± 0.88b 2.62 ± 0.37b 16.79 ± 1.72 2.96 ± 0.62 14.38 ± 0.40 21.27 ± 1.68 8.54 ± 0.25b 1.02 ± 0.01b 0.48 ± 0.21 4.99 ± 0.62b 3.43 ± 0.75 0.54 ± 0.03 0.40 ± 0.08 5.52 ± 0.06b 55.19 ± 2.09c 10.12 ± 0.36d

12.43 ± 1.91a,b 3.27 ± 0.44a,b 20.24 ± 2.31 3.27 ± 0.62 15.01 ± 1.23 22.70 ± 1.69 10.35 ± 0.50b 1.36 ± 0.02a 1.47 ± 0.52 5.26 ± 0.36a,b 3.48 ± 0.07 0.64 ± 0.15 0.28 ± 0.03 6.35 ± 0.77a,b 57.70 ± 0.16d 23.22 ± 0.49e

The values are mean ± S.D. (n = 3). Different superscript letters within rows represent significant differences between treatments (P b 0.05). 1 Saturated: 12:0, 14:0, 15:0, 16:0, 17:0, 18:0, 19:0, 20:0, 22:0. 2 Monounsaturated: 16:1n-7, 17:1n-8, 18:1n-9, 18:1n-7, 20:1n-9, 20:1n-7, 22:1n-11, 22:1n-9. 3 Polyunsaturated: 16:4n-3, 18:2n-6, 18:3n-6, 18:3n-3, 18:4n-3, 20:2n-6, 20:4n-6, 20:3n-3, 20:4n-3, 20:5n-3, 22:4n-6, 22:5n-6, 22:5n-3, 22:6n-3 and total CLA. 4 Sum of isomers trans-10, cis-12 and cis-9, trans-11.

m length, 0.25 mm internal diameter and 0.25 μm film thickness) from Agilent (Albertville, MN, USA). After holding at 180 °C for 5 min, the temperature was raised at 4 °C/min to 220 °C, and maintained at 220 °C for 25 min with the injector at 250 °C. The split ratio was 100:1 and the quantitation was done using the area of the internal standard 21:0. All analytical determinations were done in triplicate. The methyl esters of CLA isomers were individually separated by triple column silver-ion in series (ChromSpher 5 Lipids, 250 mm × 4.6 mm i.d., 5 μm particle size, Chrompack, Bridgewater, NJ, USA), using an HPLC system (Agilent 1100 Series, Agilent Technologies Inc., Palo Alto, CA, USA) equipped with autosampler and diode array detector (DAD) adjusted at 233 nm. The mobile phase was 0.1% acetonitrile in n-hexane maintained at a flow rate of 1 ml/min and injection volumes of 20 μl were used. The identification of the individual CLA isomers was achieved by comparison of their retention times with commercial standards (18:2 cis-9, trans-11, 18:2 trans-10, cis-12, 18:2 cis-9, cis-11 and 18:2 trans-9, trans-11 CLA isomers, obtained from Matreya Inc., Pleasant Gap, PA, USA) and with values published in the literature, as well as by spectral analysis. Total and individual CLA isomer contents in samples were quantified based on the external standard

technique and on the method of area normalization (AOAC 963.22, 2000). 2.4. Statistical analysis Data were analysed using an ANOVA with the Statistics 6.0 for Windows package. Previously, normality and homogeneity of variances were verified by Kolmogorov-Smirnov and Bartlett tests, respectively. When data did not meet the assumptions of ANOVA, the nonparametric ANOVA equivalent (Kruskal-Wallis test) was performed. When these tests showed significance (P b 0.05), individual means were compared using Tukey or Dunn Test, respectively (Zar, 1996). 3. Results The isomer distribution of the CLA mixture used for diet supplementation is presented in Table 1. The cis, trans group is the major one, representing 90% of total, followed by trans, trans with 7%. The CLA biologically active isomers trans-10, cis-12 and cis-9, trans-11 were the most important, contributing with 86.9% for total, but several other CLA isomers could be detected in the mixture. Besides the two biological active isomers (trans-10, cis-12 and cis-9, trans-11) it was

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Table 4 Effect of dietary CLA incorporation levels (control, 0.5%, 0.75%, 1.0% or 2.0%) on body weight, feed convertion ratio (FCR), hepatosomatic index (HSI) and viscerosomatic index (VSI) of rainbow trout fed during 12 weeks Dietary treatments

Initial body weight (g) Final body weight (g) FCR HSI VSI

Control

0.5

0.75

1.0

2.0

5.34 ± 0.01 70.21 ± 1.10 0.74 ± 0.01 1.10 ± 0.15 7.20 ± 1.10

5.32 ± 0.02 65.24 ± 1.69 0.73 ± 0.01 1.21 ± 0.07 6.78 ± 0.99

5.33 ± 0.01 62.54 ± 2.08 0.74 ± 0.01 1.14 ± 0.02 7.32 ± 0.58

5.33 ± 0.02 64.49 ± 1.92 0.72 ± 0.002 1.21 ± 0.07 7.27 ± 0.97

5.34 ± 0.02 63.44 ± 2.16 0.73 ± 0.01 1.10 ± 0.05 7.32 ± 0.58

The values are mean ± S.D. (n = 2 except for HSI and VSI where n = 10). Absence of superscript letters within rows indicates no significant differences between treatments (P N 0.1). FCR = dry feed intake/weight gain. HSI = 100 × (liver weight / body weight). VSI = 100 × (viscera weight/body weight).

possible to identify the trans-10, trans-12; trans-9, trans-11; trans-8, trans-10; cis-10, cis-12; cis-9, cis-11 and cis-11, trans-13 and trans-7, cis-9 isomers. The fatty acid profile of diets (Table 3) indicated a majority of polyunsaturated fatty acids (PUFA) followed by monounsaturated and saturated fatty acids. Linoleic acid (18:2n-6; LA) showed the highest level

followed by docosahexaenoic acid (22:6n-3; DHA), arachidonic acid (20:4n-6; AA) and eicosapentaenoic acid (20:5n-3; EPA).Total CLA level registered reflected CLA supplementation. The increase of dietary CLA levels (control, 0.5%, 0.75%, 1% and 2%) did not affect (P N 0.05) growth performance, feed conversion ratio (FCR),

Table 5 Main fatty acids (% total fatty acids), total CLA (% total lipids), total lipids (% wet weight) and protein content (% wet weight) in muscle of rainbow trout fed graded dietary levels of CLA (control, 0.5%, 0.75%, 1.0% or 2.0%) during 12 weeks Dietary treatments

Fatty acids (%) 16:0 18:0 ΣSaturated 1 16:1n-7 18:1n-9 ΣMonounsaturated 2 18:2n-6 18:3n-3 18:4n-3 20:4n-3 20:4n-6 20:5n-3 22:5n-3 22:5n-6 22:6n-3 ΣPolyunsaturated 3 Total CLA 4 Total lipids (%) Proteins (%)

Control

0.5

0.75

1.0

2.0

18.41 ± 0.92 3.96 ± 0.06a 27.28 ± 1.33a 5.33 ± 0.15a 20.31 ± 0.28a 31.25 ± 0.35a 5.69 ± 0.11 1.02 ± 0.07a 0.64 ± 0.03 0.67 ± 0.03a 0.92 ± 0.06 3.43 ± 0.13 1.38 ± 0.30a 0.67 ± 0.03a 20.67 ± 0.62a 38.08 ± 0.82a 0.42 ± 0.02a 4.57 ± 1.34 21.80 ± 0.64a,b

18.93 ± 0.44 5.81 ± 0.09b 29.88 ± 0.39b 4.42 ± 0.14b 18.17 ± 0.23b 27.84 ± 0.27b 5.71 ± 0.10 0.96 ± 0.01a 0.63 ± 0.01 0.62 ± 0.01a 1.08 ± 0.25 3.35 ± 0.11 1.13 ± 0.01a,b 0.63 ± 0.02a,b,c 20.28 ± 0.18a,b 39.13 ± 0.32a,b 1.85 ± 0.10b 4.12 ± 1.06 22.67 ± 0.38a

18.38 ± 0.46 5.64 ± 0.37b 28.67 ± 0.77a,b 4.24 ± 0.16b 17.68 ± 0.25c 27.12 ± 0.38b 5.76 ± 0.16 0.97 ± 0.04a 0.62 ± 0.06 0.67 ± 0.01a 1.00 ± 0.15 3.44 ± 0.16 1.15 ± 0.03a,b 0.64 ± 0.02a,c 20.64 ± 0.77a 40.68 ± 0.80b,c 4.65 ± 0.02c 4.22 ± 0.97 21.32 ± 0.88b

18.39 ± 0.72 5.93 ± 0.18b 29.25 ± 1.11b 4.24 ± 0.20b 17.65 ± 0.24c 27.25 ± 0.33b 5.84 ± 0.25 0.96 ± 0.04a 0.63 ± 0.02 0.66 ± 0.05a 0.87 ± 0.08 2.75 ± 1.55 1.12 ± 0.02a,b 0.59 ± 0.01b 19.16 ± 0.48b,c 40.14 ± 1.06a,b,c 5.06 ± 0.39c 4.83 ± 1.92 21.75 ± 0.58a,b

18.78 ± 0.59 6.08 ± 0.37b 29.82 ± 0.83b 3.85 ± 0.29c 16.25 ± 0.15d 24.45 ± 0.74c 5.66 ± 0.11 0.85 ± 0.01b n.d. 0.05 ± 0.01b 0.88 ± 0.08 3.13 ± 0.23 1.00 ± 0.08b 0.56 ± 0.02b 18.67 ± 0.82c 41.72 ± 0.99c 6.88 ± 0.55d 4.16 ± 1.29 22.33 ± 0.85a,b

The values are mean ± S.D. (n = 10). Different superscript letters within rows represent significant differences between treatments (P b 0.05). n.d.— not detected. 1 Saturated: 12:0, 14:0, 15:0, 16:0, 17:0, 18:0, 19:0, 20:0, 22:0. 2 Monounsaturated: 16:1n-7, 17:1n-8, 18:1n-9, 18:1n-7, 20:1n-9, 20:1n-7, 22:1n-11, 22:1n-9. 3 Polyunsaturated: 16:4n-3, 18:2n-6, 18:3n-6, 18:3n-3, 18:4n-3, 20:2n-6, 20:4n-6, 20:3n-3, 20:4n-3, 20:5n-3, 22:4n-6, 22:5n-6, 22:5n-3, 22:6n-3 and total CLA. 4 Sum of isomers trans-10, cis-12 and cis-9, trans-11.

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Table 6 Distribution of CLA isomers (% total CLA) in muscle of rainbow trout fed graded dietary levels of CLA (control, 0.5%, 0.75%, 1.0%, or 2.0 %) during 12 weeks CLA Isomers (%)

Dietary treatments Control

trans-12, trans-14 trans-11, trans-13 trans-10, trnas-12 trans-9, trans-11 trans-8, trans-10 trans-7, trans-9 trans-6, trans-8 Total trans, trans cis-12, trans-14 trans-11, cis-13 cis-11, trans-13 trans-10, cis-12 cis-9, trans-11 trans-8, cis-10 trans-7, cis-9 Total cis, trans cis-11, cis-13 cis-10, cis-12 cis-9, cis-11 cis-8, cis-10 Total cis, cis

0.5 a

1.97 ± 0.03 6.70 ± 0.11a 1.29 ± 0.44a 2.12 ± 0.30a 13.02 ± 0.65a 38.69 ± 2.17a n.d. 63.79 ± 2.22a 0.21 ± 0.29 1.10 ± 0.87 1.97 ± 0.46 11.00 ± 3.14a 19.18 ± 1.78a n.d. 0.89 ± 1.26 34.34 ± 2.05a n.d. 0.00 ± 0.00a 1.87 ± 0.17a n.d. 1.87 ± 0.17a

0.75 b

0.42 ± 0.04 1.42 ± 0.07b 2.80 ± 0.01b 2.73 ± 0.01b 2.39 ± 0.10b 5.36 ± 0.23b n.d. 15.12 ± 0.01b 0.53 ± 0.75 1.03 ± 1.03 2.14 ± 1.39 34.57 ± 0.35b 42.74 ± 0.37b n.d. 1.80 ± 1.11 82.80 ± 0.00b n.d. 0.85 ± 0.02b 1.22 ± 0.03b n.d. 2.07 ± 0.01a,b

1.0 b

0.32 ± 0.02 1.11 ± 0.01c 2.82 ± 0.00b 2.73 ± 0.00b 1.92 ± 0.05b,c 4.14 ± 0.41b n.d. 13.04 ± 0.48b,c 0.79 ± 0.42 1.18 ± 0.64 1.40 ± 0.28 36.68 ± 0.67b 43.36 ± 0.02b n.d. 1.35 ± 0.59 84.76 ± 0.45b n.d. 0.93 ± 0.03b,c 1.27 ± 0.00b n.d. 2.20 ± 0.03a,b

2.0 b,c

0.25 ± 0.00 0.95 ± 0.04c,d 2.75 ± 0.02b 2.73 ± 0.01b 1.57 ± 0.02b,c 3.16 ± 0.22b n.d. 11.42 ± 0.29b,c 0.72 ± 0.38 1.25 ± 0.75 1.43 ± 0.37 37.39 ± 0.90b 44.21 ± 0.12b n.d. 1.43 ± 0.21 86.44 ± 0.23b,c n.d. 0.94 ± 0.01b,c 1.20 ± 0.04b n.d. 2.14 ± 0.06a,b

0.19 ± 0.01c 0.75 ± 0.04d 2.69 ± 0.01b 2.74 ± 0.03b 1.12 ± 0.07c 1.51 ± 0.11b n.d. 9.00 ± 0.20c 0.74 ± 0.41 1.30 ± 0.75 1.36 ± 0.44 38.55 ± 0.32b 45.41 ± 0.12b n.d. 1.36 0± .42 88.71 ± 0.14c n.d. 1.00 ± 0.04c 1.29 ± 0.02b n.d. 2.29 ± 0.07b

The values are mean ± S.D. (n = 10). Different superscript letters within rows represent significant differences between treatments (P b 0.05). n.d.— not detected. Table 7 Main fatty acids (% total fatty acids), total CLA (% total lipids) and total lipids (% wet weight) in liver of rainbow trout fed graded dietary levels of CLA (control, 0.5%, 0.75%, 1.0% or 2.0%) during 12 weeks Dietary treatments

Fatty acids (%) 16:0 18:0 ΣSaturated 1 16:1n-7 18:1n-9 ΣMonounsaturated 2 18:3n-3 18:4n-3 20:4n-3 20:4n-6 20:5n-3 22:5n-3 22:5n-6 22:6n-3 ΣPolyunsaturated 3 Total CLA 4 Total lipids (%)

Control

0.5

0.75

1.0

2.0

14.49 ± 0.09a,b 5.18 ± 0.17 22.23 ± 0.39a 1.75 ± 0.14 11.64 ± 1.42 16.92 ± 1.24 0.43 ± 0.02 0.14 ± 0.02 0.40 ± 0.03a 3.54 ± 0.18 3.15 ± 0.10 1.05 ± 0.03 1.37 ± 0.04a,b 38.58 ± 0.01 53.99 ± 0.16 0.17 ± 0.01a 3.85 ± 0.49

12.94 ± 0.67a 9.27 ± 0.29 24.37 ± 1.04a,b 1.50 ± 0.28 10.52 ± 0.28 14.87 ± 0.04 0.37 ± 0.11 n.d. 0.39 ± 0.01a 3.43 ± 0.04 3.40 ± 0.09 0.92 ± 0.08 1.50 ± 0.00a 39.13 ± 0.45 54.83 ± 0.57 0.69 ± 0.07b 2.82 ± 0.25

14.35 ± 0.21a,b 8.80 ± 0.94 25.51 ± 0.47a,b 1.47 ± 0.35 10.48 ± 1.07 14.54 ± 1.60 0.39 ± 0.04 n.d. 0.49 ± 0.01b 4.82 ± 1.18 3.57 ± 0.32 1.00 ± 0.05 1.26 ± 0.09b 37.23 ± 1.11 55.27 ± 0.29 1.48 ± 0.16c 4.76 ± 0.61

15.47 ± 0.67b 9.92 ± 1.98 28.34 ± 1.90b 1.08 ± 0.10 10.39 ± 1.13 13.99 ± 1.29 0.31 ± 0.01 n.d. 0.44 ± 0.02a,b 2.57 ± 0.53 3.53 ± 0.24 0.87 ± 0.08 1.31 ± 0.03a,b 37.99 ± 1.48 53.21 ± 1.62 1.98 ± 0.03c 4.05 ± 0.71

13.93 ± 0.08a,b 9.79 ± 1.78 25.93 ± 1.93a,b 1.04 ± 0.11 9.13 ± 0.43 12.47 ± 0.47 0.29 ± 0.01 n.d. 0.56 ± 0.03c 3.81 ± 0.25 3.44 ± 0.07 0.91 ± 0.01 1.32 ± 0.03a,b 37.99 ± 0.97 55.93 ± 0.78 3.11 ± 0.01d 3.92 ± 0.18

The values are mean ± S.D. (n = 2). Different superscript letters within rows represent significant differences between treatments (P b 0.05). n.d.—not detected. 1 Saturated: 12:0, 14:0, 15:0, 16:0, 17:0, 18:0, 19:0, 20:0, 22:0. 2 Monounsaturated: 16:1n-7, 17:1n-8, 18:1n-9, 18:1n-7, 20:1n-9, 20:1n-7, 22:1n-11, 22:1n-9. 3 Polyunsaturated: 16:4n-3, 18:2n-6, 18:3n-6, 18:3n-3, 18:4n-3, 20:2n-6, 20:4n-6, 20:3n-3, 20:4n-3, 20:5n-3, 22:4n-6, 22:5n-6, 22:5n-3, 22:6n-3 and total CLA. 4 Sum of isomers trans-10, cis-12 and cis-9, trans-11.

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hepatosomatic index (HSI) and viscerosomatic index (VSI) of rainbow trout juveniles (Table 4). There were no significant differences in total lipids in muscle and liver of fish fed the five diets (Tables 5 and 7). The dietary inclusion of CLA reduced viscera total lipids, although results were only significant (P b 0.05) for fish fed the 0.5% level (Table 8). The fatty acid profile of muscle is summarized in Table 5 and reflects, in general terms, diet composition. The differences were due to the high level of DHA in this tissue, which attained three times the level in each diet (Table 3). The dietary inclusion of CLA modified (P b 0.05) total percentages of the main groups of fatty acids. Both saturated and polyunsaturated fractions increased significantly with CLA incorporation, whereas monounsaturated fatty acids showed a significant reduction. No differences were detected in the PUFA fraction between the control and 0.5% CLA or among the 0.75%, 1% and 2% of CLA fed fish. However, fish fed the control diet exhibited a lower proportion (P b 0.05) of PUFA compared with those fed 0.75% and 2% CLA (Table 5). Although no differences (P N 0.05) were found in muscle deposition of AA and EPA among treatments, fish fed the 1% and 2% CLA diet exhibited a lower DHA level (P b 0.05) than the

controls. Total CLA was affected by dietary treatment attaining the highest levels when the 2% diet was used. The main CLA isomers detected in muscle are presented in Table 6. Dietary CLA supplementation mainly resulted in a significant increase in the biologically active trans-10, cis-12 and cis-9, trans-11 CLA isomers. The fatty acid distribution in liver is presented in Table 7. The main fraction is composed by PUFA, attaining more that 50% of the total, higher than that observed in muscle, followed by saturated and monounsaturated fatty acids. The only n-3 fatty acid that showed a significant increase with CLA supplementation was 20:4 n-3, when the 0.75% and 2% diets were fed. The other PUFAs, in particular AA, EPA and DHA, did not vary significantly with dietary CLA inclusion. The same trend was observed for the monounsaturated fraction. Nevertheless, there was a CLA dietary influence in the saturated fraction, which increased with CLA supplementation. Livers of fish fed 1% CLA had a significantly higher saturated fraction than the control. Total CLA in liver was affected by dietary treatment attaining the highest levels when the 2% diet was fed. The fatty acid profile of viscera is presented in Table 8. In general, the distribution of fatty acids was similar

Table 8 Main fatty acids (% total fatty acids), total CLA (% total lipids) and total lipids (% wet wt) in viscera of rainbow trout fed graded dietary levels of CLA (control, 0.5%, 0.75%, 1.0% or 2.0%) during 12 weeks Dietary treatments

Fatty acids (%) 16:0 18:0 ΣSaturated 1 16:1n-7 18:1n-9 ΣMonounsaturated 2 18:2n-6 18:3n-3 20:4n-3 20:4n-6 20:5n-3 22:5n-3 22:5n-6 22:6n-3 ΣPolyunsaturated 3 Total CLA 4 Total lipids (%)

Control

0.5

0.75

1.0

2.0

16.82 ± 0.46a,b 3.81 ± 0.09a 25.58 ± 0.62a 6.53 ± 0.12a 23.73 ± 0.36a 37.48 ± 0.45a 7.13 ± 0.11 1.30 ± 0.05a 0.72 ± 0.01 0.94 ± 0.08 3.31 ± 0.04a 1.27 ± 0.03a 0.50 ± 0.02a 15.56 ± 0.23a 35.01 ± 0.37a 0.53 ± 0.16a 43.20 ± 7.75a

17.20 ± 0.30b 5.65 ± 0.17b 27.88 ± 0.61b 5.39 ± 0.11b 21.16 ± 0.18b 33.20 ± 0.25b 6.99 ± 0.18 1.23 ± 0.06a 0.68 ± 0.03 0.92 ± 0.07 3.27 ± 0.20a 1.14 ± 0.04b 0.51 ± 0.03a 15.61 ± 0.20a 36.82 ± 0.68b 1.95 ± 0.45b 32.09 ± 5.99b

17.04 ± 0.20b 5.63 ± 0.41b 27.76 ± 0.38b,c 5.33 ± 0.15b,c 20.52 ± 0.15c 32.50 ± 0.17c 7.20 ± 0.23 1.26 ± 0.06a 0.72 ± 0.01 1.05 ± 0.08 3.30 ± 0.04a 1.12 ± 0.02b,c 0.48 ± 0.04a 14.84 ± 0.33b 37.63 ± 0.24b,c 4.03 ± 0.83c 34.99 ± 7.53a,b

17.05 ± 0.66b 5.81 ± 0.04b 28.02 ± 0.98b 5.13 ± 0.16c 19.87 ± 0.29d 31.46 ± 0.61d 7.01 ± 0.20 1.20 ± 0.05b 0.70 ± 0.01 0.95 ± 0.10 3.19 ± 0.13a 1.08 ± 0.04c 0.47 ± 0.02a,b 14.63 ± 0.27b 37.85 ± 0.69c 4.60 ± 0.45c 40.19 ± 3.03a,b

16.26 ± 0.20a 5.92 ± 0.21b 26.76 ± 0.25c 4.67 ± 0.11d 19.21 ± 0.20e 29.66 ± 0.15e 7.25 ± 0.20 1.19 ± 0.04b 0.71 ± 0.03 0.95 ± 0.31 2.92 ± 0.11b 0.97 ± 0.02d 0.43 ± 0.03b 13.30 ± 0.13c 40.65 ± 0.47d 9.50 ± 1.18d 37.40 ± 6.63a,b

The values are mean ± S.D. (n = 10). Different superscript letters within rows represent significant differences between treatments (P b 0.05). 1 Saturated: 12:0, 14:0, 15:0, 16:0, 17:0, 18:0, 19:0, 20:0, 22:0. 2 Monounsaturated: 16:1n-7, 17:1n-8, 18:1n-9, 18:1n-7, 20:1n-9, 20:1n-7, 22:1n-11, 22:1n-9. 3 Polyunsaturated: 16:4n-3, 18:2n-6, 18:3n-6, 18:3n-3, 18:4n-3, 20:2n-6, 20:4n-6, 20:3n-3, 20:4n-3, 20:5n-3, 22:4n-6, 22:5n-6, 22:5n-3, 22:6n-3 and total CLA. 4 Sum of isomers trans-10, cis-12 and cis-9, trans-11.

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to that observed in muscle. Total percentage of monounsaturated fatty acids decreased (P b 0.05) with the dietary presence of CLA. An opposite trend was observed in saturated and polyunsaturated fractions (P b 0.05). Total CLA viscera level was strongly affected by the dietary treatment (P b 0.05), attaining the highest values when the 2% diet was fed. 4. Discussion The dietary supplementation with increasing levels of CLA did not affect growth performance or feed utilization of rainbow trout (Table 4), as has been discussed in our previous work (Figueiredo-Silva et al., 2005). Similar results have been attained in various fish species fed 0.5–5% of CLA (Twibell et al., 2001; Twibell and Wilson, 2003; Berge et al., 2004; Yasmin et al., 2004). However, other previous studies indicated that growth of fish could be affected by the presence of CLA (Choi et al., 1999; Twibell et al., 2000). With regard to the influence of dietary treatments on tissue composition, no differences were observed in total lipid level in muscle and liver of fish fed the five diets. Fat content of muscle ranged between 4.12% and 4.83%, whereas in liver the minimum and maximum levels were 2.82% and 4.76%, respectively. In fact, the HSI value was also similar among all fish, reflecting the liver lipid content. This is in accordance with earlier reports by Jones et al. (1999) and Yasmin et al. (2004), who observed that CLA supplementation did not affect liver weight of rats and tilapia juveniles, respectively. In contrast, Twibell et al. (2000) reported a 2.5 fold reduction in liver lipid concentration of hybrid striped bass fed with 1% CLA compared to fish fed the control diet. In the present study, lipid content in viscera showed a slight decrease with CLA inclusion in fish fed the control compared to those fed the 0.5% diet. However, these results did not affect the VSI, which was not modified by CLA supplementation. Different results were reported by Twibell et al. (2000), who observed a significant reduction in intraperitoneal fat ratio in hybrid striped bass fed 1% CLA compared to fish fed no CLA. Various animal model and human studies also confirm that dietary CLA decreases adiposity (Evans et al., 2002). Protein content in muscle varied between 21.4% and 22.7% and fish fed the 0.75% CLA diet had a significant lower level than those fed 0.5% CLA. No dietary influence of CLA was noticed, since there were no significant differences between the control and any other treatment. This observation is in agreement with previous studies in Atlantic salmon (Berge et al., 2004)

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and channel catfish juveniles (Twibell and Wilson, 2003), where protein level in muscle was not significantly affected by dietary CLA. The present results correspond well with earlier observations in rainbow trout that reported an absence of differences in whole body composition among fish fed increasing levels of CLA (Figueiredo-Silva et al., 2005). The total percentages of the main groups of fatty acids in the different tissues were affected by dietary inclusion of CLA. In the present study, the CLA supplement was added to the diets at the expense of fish oil. Substitution of fish oil with various amounts of CLA resulted in dose-dependent changes in several essential fatty acids (Table 3). Thus, some differences in the fatty acid profiles of the various tissues are expected due to differences in the fatty acid profile of the diets. Therefore, care should be taken in interpretating the effects of CLA supplementation on changes in the profiles of other fatty acids as was recommended by Ostrowska et al. (2003). In muscle and viscera the saturated fraction of the control group was significantly lower than that found in fish fed the supplemented diet. Nevertheless, in liver only fish fed 1% CLA had a significantly higher saturated fraction. This was a clear effect of dietary CLA, since the control diet contained higher amounts of saturated fatty acids. In muscle, the main fatty acid responsible for these differences was 18:0, since 16:0 was rather constant among treatments. In viscera, both 16:0 and the 18:0 were responsible for increasing the saturated fraction in fish fed CLA. Concomitantly to the increase in the saturated fraction, a general decrease of the monounsaturated fatty acids (16:1n-7 and 18:1n-9) with CLA supplementation was observed. Similar results have been reported for hybrid striped bass (Twibell et al., 2000), yellow perch (Twibell et al., 2001), juvenile Atlantic salmon (Berge et al., 2004), barrows (Lauridsen et al., 2005), pigs (Bee, 2001) and rats (Bretillon et al., 1999), and were related to the putative inhibition of Δ-9 desaturase activity. The dietary incorporation of CLA led to a general increase of total PUFA level in muscle and viscera, mainly due to the higher deposition of these conjugated fatty acids in that lipid fraction. However, with CLA supplementation a decrease of DHA was observed in fish fed the two highest CLA levels (1% and 2% CLA) compared with the control group. Similar results were reported by Twibell et al. (2000) in muscle of hybrid striped bass fed 1% CLA. This was probably a result of the reduction of this fatty acid in the diets containing CLA. However, according to Berge et al. (2004) and taking into consideration the dietary fatty acids level, it

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is possible to verify that the inclusion of CLA in diets affected lipid metabolism contributing to a more efficient use of essential fatty acids with a prominent biosynthesis and deposition of DHA. This elevated DHA conversion could be a result of higher Δ-5 and Δ-6 fatty acyl desaturase activity in this species as pointed out by Pereira et al. (2003). In liver, most of the PUFA levels were not affected by dietary CLA supplementation, with 20:4n-3 being the only n-3 fatty acid that showed an increase with CLA inclusion. Twibell et al. (2000) has previously reported that n-3 fatty acids appeared to be sequestered in the liver of hybrid striped bass resulting in decreasing concentrations in muscle. The present results indicated that dietary CLA was primarily incorporated into both muscle and visceral fat and to a lesser extent in the liver. Similar results were reported for hybrid striped bass (Twibell et al., 2000). Muscle CLA concentrations reflected dietary CLA level and attained 5.06 and 6.88 g/100 g lipids in fish fed 1% and 2% CLA, respectively. Muscle CLA concentrations were 2.92 g/100 g fatty acids in perch (Twibell et al., 2001) and 7.97 g/100 g fatty acids in hybrid striped bass (Twibell et al., 2000). Berge et al. (2004) reported 3.8 and 7.3 g/100 g fatty acids in whole body of Atlantic salmon fed 1% and 2% CLA, respectively. These results indicate that the ability of fish to incorporate CLA varies among species, being a result of the different energetic metabolism or diet composition, but in any case is superior to that observed in other vertebrates. Ostrowska et al. (2003) reported 2.88 mg/g fat in muscle of pigs fed 1% CLA while in adipose tissue of pigs fed diet containing 1.5% CLA the level was 4.2 g/100 g total lipids (Smith et al., 2002). Several recent studies have reported that the natural intramuscular CLA content of beef and lamb varied between 0.2 and 1 g/100 g of total fatty acids (Raes et al., 2004) whereas the natural CLA content of cheese and milk is 4.1–7.0 mg/g fat (Chin et al., 1992). The incorporation of individual and total isomers of CLA in muscle varied with dietary treatment, showing some selectivity in the uptake or incorporation of certain isomers, especially the cis, trans. The cis-9, trans-11 isomer seemed to be more efficiently incorporated than trans-10, cis-12, as also reported in mammalian species (Ostrowska et al., 2003; Lauridsen et al., 2005). This is of particular value with respect to potential health benefits related to its anti-cancer properties (Pariza et al., 2001). Different results were observed in hybrid striped bass, which showed a preferential muscle deposition of the trans-10, cis-12 isomer. In muscle, the main CLA isomers detected in

control samples belonged to the trans, trans group with 63.79%, followed by cis, transwith 34.34%. Cis, cis CLA isomers presented a very low percentage, around 2%. The trans-7, trans-9 represented the main isomer identified in the control diet (39%), but with CLA supplementation its relative importance decreased to values as low as 2–5%. This isomer probably has an endogenous origin, as its level in the analysed CLA mixture was very low. With the dietary CLA supplementation a decrease was recorded in total trans, trans isomers. Twibell et al. (2001) reported the absence of CLA trans-9, trans-11 and trans-10, trans12 in juvenile perch muscle and justified it with the preferential oxidation of these trans, trans fatty acids, because cis double bounds of fatty acids must be converted to the transform to complete β-oxidation reaction. However, in rainbow trout muscle these two isomers were the only trans, trans forms displaying an increase with dietary CLA supplementation compared to the control. In conclusion, rainbow trout are capable of absorbing and deposit CLA in muscle, liver and viscera better than other vertebrate, showing that this species is a potential source to increase human CLA ingestion. Future studies must be undertaken to ascertain whether commercial diets of a higher fat content could result in desirable CLA deposition levels in market size fish. Acknowledgements Special thanks for Sorgal, S.A., Ovar, Portugal, for supplying the extruded diet before coating, and to Bioriginal Food and Science Corp., Saskatoon SK, Canada, for providing the CLA mixture. We also thank António Júlio Pinto for his technical assistance during the growth trial and samplings and Claúdia FigueiredoSilva for her critical reading of the manuscript. This work was supported by project POCTI/CVT/39237/ 2001 (FCT, Portugal, with the support of the European fund FEDER). References AOAC, 1998. Official Methods of Analysis, 16th, 4th Revision, Vol I and II. Association of Official Analytical Chemistry. AOAC International, Washington, DC. AOAC, 2000. Official Methods of Analysis, 17th. Association of Official Analytical Chemistry. AOAC International, Gaithersburg, MD. Bee, G., 2001. Dietary conjugated linoleic acid affects tissue lipid composition but not the novo lipogenesis in finishing pigs. Anim. Res. 50, 383–399. Berge, G.M., Ruyter, B., Torbjø´ rn, Å., 2004. Conjugated linoleic acid in diets for juvenile Atlantic salmon (Salmo salar); effects on fish

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