Time course deposition of conjugated linoleic acid in market size rainbow trout (Oncorhynchus mykiss) muscle

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Aquaculture 274 (2008) 366 – 374 www.elsevier.com/locate/aqua-online

Time course deposition of conjugated linoleic acid in market size rainbow trout (Oncorhynchus mykiss) muscle A. Ramos a,b , N.M. Bandarra c , P. Rema a,d , P. Vaz-Pires a,e , M.L. Nunes c , A.M. Andrade c , A.R. Cordeiro c , L.M.P. Valente a,e,⁎ CIIMAR — Centro Interdisciplinar de Investigação Marinha e Ambiental, Rua dos Bragas, 177, 4050-123 Porto, Portugal b ESAC — Escola Superior Agrária de Coimbra, Bencanta, 3040-316 Coimbra, Portugal INIAP — Instituto Nacional de Investigação Agrária e das Pescas, Departamento de Inovação Tecnológica e Valorização dos Produtos da Pesca, Av. Brasília, 1449-006 Lisboa, Portugal d UTAD — Universidade de Trás-os-Montes e Alto-Douro, Departamento de Zootecnia, Apartado 1013, 5000-911 Vila Real, Portugal e ICBAS — Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal a

c

Received 19 October 2007; received in revised form 26 November 2007; accepted 27 November 2007

Abstract Previous studies clearly suggested that conjugated linoleic acid (CLA) could be successfully incorporated up to 1% in rainbow trout diets contributing to the production of a functional food. The determination of the time course deposition of CLA has never been evaluated in fish. Hence, homogenous groups of 43 rainbow trout (Oncorhynchus mykiss) with an average initial body weight of 216.5 ± 19.6 g were randomly distributed among 6 square fibre glass tanks (250 l), in an open flow-through system. Triplicate groups of fish were fed commercial extruded diets containing 0% CLA (control group) or 1% CLA, by hand to apparent satiety, for 12 weeks. Every 2 weeks, 18 trout from each treatment were sampled to evaluate whole body composition, muscle fatty acid profile and sensory properties. At the end of the experiment all groups of fish weighted more than 400 g and no significant differences were detected in growth performance, feed conversion, nutrient or energy utilization and body composition among treatments. In general terms, the muscle total saturated, monounsaturated and polyunsaturated fatty acids did not vary among dietary treatments, despite the increasing concentration levels of CLA. The muscle incorporation of the two biological active isomers increased gradually during the 12 weeks of feeding CLA reaching the maximum level (2.7% of total fatty acids) after 12 weeks. Nevertheless, after 8 weeks of feeding, the observed value (2.2% of total fatty acids) was not significantly different from the final. At every sampling point, sensory data indicated no significant differences between animals fed control and CLA diets. The present results suggest that feeding market size rainbow trout with 1% CLA, during an 8-week period, is enough to attain muscle CLA levels similar to those observed at 12 weeks. Moreover, a fast accumulation of CLA isomers in the muscle was registered, reaching 1.3% of total fatty acids just after 2 weeks of supplementation, which reinforces the potential of aquaculture fish to supply those bioactive fatty acids, becoming functional foods. © 2007 Elsevier B.V. All rights reserved. Keywords: Rainbow trout; Conjugated linoleic acid (CLA); Sensory analysis; Muscle fatty acid profile; Functional food

1. Introduction Food quality is presently receiving more and more attention and aquaculture products are no exception. The consumer begins ⁎ 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 © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.11.040

to realize the influence of diet on his health and the benefits of some food components, searching for new and healthier products. Dietary formulations in aquaculture tend to increase the lipid content in diets to spare protein and to decrease the amount of waste produced by fish (Cho, 1992; Cho et al., 1994; Kaushik, 1998; Medale et al., 1998; Company et al., 1999). However, these diets alter body composition and slaughter quality, particularly through an increase of lipid deposition (Cowey, 1993; Hillestad and Johnsen, 1994; Vergara et al., 1999). Additionally, the fish oil

A. Ramos et al. / Aquaculture 274 (2008) 366–374

substitution by vegetable sources in fish diets, an actual tendency that will continue to grow, is accompanied by a reduction of valuable n−3 highly unsaturated fatty acids (HUFA) in edible products (Arzel et al., 1994; Bell et al., 2003; Izquierdo et al., 2003, 2005; Regost et al., 2004) that can induce detectable sensory alterations, since lipids, and fatty acids in particular are the major responsible for fish characteristic flavour and texture (Arzel et al., 1994; Sérot et al., 2001, 2002; Regost et al., 2004). The deleterious aspects of feeding either high-energy diets or vegetable oil diets, which are indispensable for the sustainability of the activity, can be compensated by the inclusion of functional dietary components that present beneficial effects for human health. Among the components that can receive such designation are conjugated linoleic acids (CLA), a generic designation for geometric and positional isomers of linoleic acid with conjugated double bonds. These conjugated fatty acids are receiving great attention by the scientific community, especially cis-9, trans-11 and trans-10, cis-12 CLA isomers. Several studies relate their involvement in health-promoting actions (reviewed by Belury, 2002; Rainer and Heiss, 2004), specially the anti-carcinogenic activity (Ha et al., 1990; Ochoa et al., 2004; Kuniyasu et al., 2006) and the modulating of body fat, reducing lipogenesis and fat accumulation (recently reviewed by Park and Pariza, 2007) in laboratory (Park et al., 1997; Sisk et al., 2001) and farm animals (Szymczyk et al., 2001; Ostrowska et al., 2003). In humans the results concerning the body fat decrease are limited and contradictory (Blankson et al., 2000; Gaullier et al., 2004; Terpstra, 2004). CLA, mainly cis-9, trans-11, are found naturally in ruminant lipids, being intermediary products of ruminal biohydrogenation of linoleic acid to stearic (Pariza et al., 2001; Fukuda et al., 2005). In dairy products, CLA cis-9, trans-11 level range from 0.4 to 0.8% of fat (Lin et al., 1995) and in ruminant meats, from 0.1 to 1.9% of total fatty acids, but is usually lower than 1% (Schmid et al., 2006). The interest of CLA enhancing or supplementation in food products is clear and an actual challenge in several animal production sectors (Bessa et al., 2000; Ostrowska et al., 2003; Cherian and Goeger, 2004; Raes et al., 2004) including aquaculture. In pork meat, Lauridsen et al. (2005) reported CLA levels of 0.44% of fatty acids (80% cis-9, trans-11 and 20% trans-10, cis-12) after 0.5% of dietary CLA supplementation. In broilers fed 0.5 to 1.5% CLA for 42 days, Szymczyk et al. (2001) observed CLA levels in meat from 3.1 to 9.8% of fatty acids (approximately 35% consisted of the two functional isomers, like in the diet). Raes et al. (2002) reported a mean level of cis9, trans-11 and trans-10, cis-12 CLA of 3.4% of yolk fatty acids, after feeding laying hens with 1% CLA for 28 days. In fish, several experiments tested increasing dietary CLA levels: rainbow trout (0.5 to 2% of CLA, Bandarra et al., 2006; Valente et al., 2007b), Atlantic salmon (0.5 to 4% of CLA, Berge et al., 2004; Kennedy et al., 2005; Leaver et al., 2006), hybrid striped bass (0.5 to 1% of CLA, Twibell et al., 2000), Atlantic cod (0.5 and 1% of CLA, Kennedy et al., 2007a), channel catfish (0.5 and 1% of CLA, Manning et al., 2006), yellow perch (0.5 and 1% of CLA, Twibell et al., 2001), European sea bass (0.5 to 2% of CLA, Valente et al., 2007a)

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and common carp (2.5 and 5% of CLA, Schwarz et al., 2002). Those trials (8 to 14 weeks, but mainly 12) resulted in no decrease of muscle lipid level, but in a successful incorporation of CLA in muscle, contributing to the production of a functional food. The sum of cis-9, trans-11 and trans-10, cis-12 CLA levels in flesh ranged from 1.8% of fatty acids in European sea bass fed 0.5% (Valente et al., 2007a) to 8.4% of fatty acids in Atlantic salmon fed 4% CLA (Leaver et al., 2006), being superior to values observed in natural sources of CLA, and similar or higher than those reported in pork or chicken meat where CLA content was enhanced (Szymczyk et al., 2001; Lauridsen et al., 2005). These results indicate that the ability of fish to incorporate CLA varies among species, but the developmental stage of the fish and the dietary lipid content also seems to affect the pattern of lipid metabolism. Previous studies with market size rainbow trout clearly showed that CLA could be incorporated up to 1% in high-energy diets, contributing to the production of a functional food with 2.1% of CLA (sum of cis-9, trans-11 and trans-10, cis-12) in fillet fatty acids, after 12 weeks supplementation (Valente et al., 2007b). The objective of the present experiment was to determine the minimal administration period of dietary CLA to obtain the desirable deposition levels of CLA in the muscle. The time course effects of supplementation market size rainbow trout with 1% CLA on whole body composition, CLA deposition levels, and sensory properties of fillets were evaluated over 12 weeks. 2. Materials and methods 2.1. Experimental diets A commercial extruded diet for rainbow trout was supplied by Sorgal S. A. (Ovar, Portugal). The CLA mixture (containing 70% CLA) was offered by Bioriginal Food and Science Corp., Saskatoon SK, Canada. Before oil coating, the pellets (5 mm of diameter) were analyzed for fat composition and then coated with 21.9% oil containing the different CLA levels (0, control or 1%). The CLA supplement was added to the diet at the expense of fish oil to maintain a constant lipid (27% DM) and energy (25–26 kJ/g DM) level among dietary treatments. Ingredients and proximate composition of the experimental diets are presented in Table 1 and the fatty acid profiles of the diets in Table 2.

2.2. Growth trial The trial was conducted in the University of Trás-os-Montes and AltoDouro (UTAD, Vila Real, Portugal) rearing facilities, with market size rainbow trout (Oncorhynchus mykiss) acquired from a trout farm. Fish were acclimated to the experimental conditions for a period of 2 weeks and fed the control diet until the beginning of the experiment. Six homogenous groups of 43 fish with an average initial body weight of 216.5 ± 19.6 g (mean ± S.D.) were randomly distributed among 6 square fibre glass tanks (250 l), in an open flow-through system. Triplicate groups of fish for each treatment were hand-fed to apparent satiety, two times a day (08:30 and 17:00 h) for 12 weeks. The pH, ammonia, nitrites, nitrates and phosphates were monitored during the entire trial and maintained at levels compatible with the species. The daily water temperature was 12 ± 1 °C and fish were exposed to natural photoperiod. A pooled sample of 6 fish from the initial stock at the beginning of the experiment was taken and stored at − 20 °C for subsequent whole body

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Table 1 Ingredients and proximate composition of the experimental diets

Ingredients (%) Fish meal Corn gluten meal Extruded pea meal a Brewer's yeast Soybean molasses 48 Fish oil CLA mixture b Vitamin c and mineral mix d Proximate composition Dry matter (DM, %) Crude protein (% DM) Crude fat (% DM) Ash (% DM) Gross energy (kJ g− 1 DM)

Control

1% CLA

47.0 9.7 6.9 1.0 9.6 21.9 0.0 3.9

47.0 9.7 6.9 1.0 9.6 20.4 1.4 3.9

91.8 46.4 27.2 8.5 25.2

91.8 46.7 27.3 8.4 25.9

a

Aquatex (20.5 Crude protein); Sotexpro, Bermericourt, France. CLA mixture: total CLA, 70%; 18:2 cis-9, trans-11, 31.7%; 18:2 trans-10, cis-12, 30.0%. c Vitamins (mg or IU/kg diet): retinyl acetate, 8000 IU; DL-cholecalciferol, 2000 IU; DL-alpha tocopheryl acetate, 100 mg; menadione sodium bisulfitete, 10 mg; cyanocobalamin, 0.02 mg; thiamine hydrochloride, 15 mg; riboflavin, 25 mg; pyridoxine hydrochloride, 15 mg; folic acid, 10 mg; biotin, 1 mg; ascorbic acid (Lutavit C monophosphate 35), 100 mg; betaine, 500 mg; inositol, 300 mg; nicotinic acid, 100 mg; pantothenic acid, 50 mg; choline chloride, 1000 mg. d Minerals (g or mg/kg diet): manganese sulphate, 20 mg; potassium iodide, 0.6 mg; copper sulphate, 5 mg; cobalt sulphate, 0.4 mg; magnesium sulphate, 500 mg; zinc (Bioplex, Alltech), 30 mg; selenium (Sel-Plex 2000, Alltech), 0.3 mg; iron sulphate, 40 mg; calcium carbonate, 2.15 g; dibasic calcium phosphate, 5 g; potassium chloride, 1 g; sodium chloride, 0.4 g. b

composition analyses. Every 2 weeks, after a fasting period of 24 h, fish were bulk weighted, and 18 trout from each treatment were sampled: 6 fish to evaluate whole body composition (immediately stored at − 20 °C), 6 fish for total lipids and fatty acid profile in muscle and hepatossomatic index determinations (immediately frozen in liquid nitrogen and stored at − 80 °C), and 6 fish to perform a sensory test (stored for 24 h at 4 °C, before sensory analysis). The sacrificed animals were previously anaesthetized by immersion in an ethylene glycol monophenyl ether (1:2500) bath, except those for sensory analysis that were killed by a sharp blow on the head. 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 Whole fish from each tank were ground, pooled and moisture content was determined (105 °C for 24 h). The homogenate was subsequently freeze-dried before further analysis. Ground diets and whole body samples were then analyzed for dry matter (105 °C for 24 h), ash by combustion in a muffle furnace (550 °C for 12 h), crude protein (Micro-Kjeldahl; N × 6.25) after acid digestion, lipid content by petroleum ether extraction (at Soxhlet 40–60 °C) and gross energy in an adiabatic bomb calorimeter (IKA, werke C2000). Total lipid determinations of diets and individual muscle samples were carried out following the Blight and Dyer (1959) method with small modifications. Fatty acid methyl esters (FAME) of diets were prepared by sulphuric acid modified transesterification during 2 h at 80 °C. For FAME analysis of trout muscle, base-catalysed transesterification was followed, 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

isomerization of CLA. FAME were analyzed in a Varian CP 3800 (Walnut Creek, CA, USA) gas chromatograph, equipped with an auto sampler and fitted with an injector and a flame ionisation detector both at 250 °C. The separation was achieved using a capillary column HP-INNOWAX (30 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 oven temperature was raised at 4 °C/min to 220 °C, and maintained at 220 °C for 25 min. The split ratio was 100:1 and the quantification was done using the area of the internal standard 21:0. All analytical determinations were done in triplicate.

2.4. Sensory analysis The same protocol of slaughter and filleting was strictly applied to all fish. The fillets were skinned, washed with tap water and hand-cut into portions of 20 g. They were then put into small plastic cups previously coded with threedigit numbers and microwaved until homogeneous cooking (15–20 s at maximum power of 750 W). Samples were randomized and served hot in a consumer type test to 30 regular fish eating panelists who were asked to compare 4 unknown samples with an identified control (0% CLA), in a room designed for sensory analysis. The codified samples to be classified consisted in a hidden control, from the same fish; a sample from another trout fed 0% CLA and 2 other samples from 2 different animals fed 1% CLA. An explanation sheet with detailed instructions and questions to be answered was given to each panelist. The samples were evaluated using a scale from 0 (similar to control) to 6 (extremely different from control). If sensory differences were detected between samples, panelists were asked to describe them, to obtain a general idea of the

Table 2 Fatty acid profile (% total fatty acids) and total CLA content (% total lipids) of diets with 0% (control) or 1% CLA

Fatty acids (%) 14:0 16:0 18:0 Σ saturated a 16:1 18:1n − 9 20:1 Σ monounsaturated b 16:2n − 4 16:3n − 3 18:2n − 6 18:2 cis-9, trans-11(%) 18.2 trans-10, cis-12(%) 18:3n − 3 18:4n − 3 20:4n − 6 20:4n − 3 20:5n − 3 22:5n − 6 22:5n − 3 22:6n − 3 Σ polyunsaturated c Σn − 3 Σn − 6 n − 3/n − 6

Control

1% CLA

6.0 17.4 3.4 28.6 7.8 14.6 4.8 32.5 0.3 0.3 3.1 0.0 0.0 1.1 2.6 1.0 0.9 12.3 0.3 1.6 10.2 35.8 30.2 5.3 5.7

5.2 16.4 3.6 27.0 6.7 14.2 4.7 30.6 0.2 0.3 3.6 2.4 2.3 1.0 2.8 0.9 0.9 11.3 0.3 1.5 9.9 39.4 28.9 5.6 5.1

a Saturated: 12:0, 14:0, 14:0 isobranched, 15:0, 16:0, 16:0 isobranched, 17:0, 18:0, 19:0, 20:0, 22:0 and 24:0. b Monounsaturated: 16:1n − 7, 17:1n − 8, 18:1n − 9, 18:1n − 7, 20:1n − 9, 20:1n − 7, 22:1n − 11 and 22:1n − 9. c Polyunsaturated: 16:2n − 4, 16:3−n n − 3, 16:4n − 3, 18:2n − 6, 18:2CLA, 18:3n − 6, 18:3n − 3, 18:4n − 3, 20:2n − 6, 20:3n − 3, 20:4n − 6, 20:4n − 3, 20:5n − 3, 22:2n − 6, 22:4n − 6, 22:5n − n − 6, 22:5n − 3 and 22:6n − 3.

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2-way ANOVA with time and diet as main effects. When these tests showed significance (P b 0.05), individual means were compared using the Tukey test. The results of sensory analysis were subjected to two-way ANOVA to test the effects of experimental diets and assessors followed by a Dunnett test. Significant differences were considered when P b 0.05.

Table 3 Effects of dietary CLA incorporation level (0 or 1%) on growth, efficiency, feed intake and retention in rainbow trout growth over 12 weeks

Growth IBW a FBW b HSI c Final length (cm) Final condition factor d FCR e DGI f PER g

Control

1% CLA

ANOVA P-value

216.5 ± 0.6 412.2 ± 25.0 1.0 ± 0.1 31.4 ± 0.8 1.3 ± 0.0 1.4 ± 0.1 1.7 ± 0.2 1.6 ± 0.2

216.3 ± 0.5 413.6 ± 42.5 1.0 ± 0.2 31.4 ± 0.9 1.3 ± 0.1 1.3 ± 0.2 1.7 ± 0.3 1.7 ± 0.2

0.643 0.962 0.980 0.992 0.918 0.599 0.970 0.596

9.4 ± 0.5 4.4 ± 0.2 2.6 ± 0.1 243.5 ± 13.1

0.124 0.158 0.142 0.349

25.4 ± 3.7 57.5 ± 4.6 14.7 ± 1.5

0.955 0.092 0.806

Intake (g/kg or kJ/kg ABW h/day) Dry matter 10.0 ± 0.2 Protein 4.6 ± 0.1 Lipid 2.7 ± 0.0 Energy 251.9 ± 4.0 Retention (% intake) Protein Lipid Energy

25.3 ± 2.8 47.8 ± 6.0 14.3 ± 2.3

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3. Results 3.1. Growth performance Rainbow trout growth was not affected by the inclusion of 1% CLA displaying similar daily growth index (1.7%), feed conversion ratio (1.3–1.4), protein efficiency ratio (1.6–1.7), feed intake (9.4–9.9 g kg− 1 day− 1) or nutrient retention to the control (Table 3). Also, hepatossomatic index (0.96) was not affected by the dietary inclusion of CLA. 3.2. Whole body composition The evolution of whole body composition of rainbow trout fed the different diets, during 12 weeks, showed similar trends and was not affected by the dietary inclusion of CLA (Table 4). By the end of the feeding trial, protein and lipid content ranged from 16.6 to 16.2% and from 13.0 to 14.2%, respectively. Protein content varied through time, but no clear trend could be established. Body lipid (from 8.7 to 14%) and energy content (from 7 to 10 kJ g− 1) increased, contrasting to the decrease observed in moisture (from 69.3 to 65–66%).

The values are mean ± standard deviation (n = 3, except for HSI where n = 6). In each line, absence of superscripts letters indicates no significant differences between treatments (P N 0.05). a IBW, Initial mean body weight. b FBW, Final mean body weight. c HSI, Hepatossomatic index = 100 × (liver weight / body weight), %. d Condition factor = [weight (g) × 100] / lenght3 (cm). e FCR, Feed conversion ratio = dry feed intake / weight gain. f DGI, Daily growth index = 100 × ((Final body weight)1/3− (Initial body weight)1/3) / days, %/day. g PER, Protein efficiency ratio = weight gain / crude protein intake. h ABW, Average body weight = (IBW + FBW) / 2.

3.3. Muscle lipid deposition Total lipid and fatty acid profile of rainbow trout muscle are presented in Table 5. Muscle total lipids, saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acid fractions were not significantly affected by the dietary incorporation of 1% CLA. Nevertheless, several individual fatty acids were altered. The most evident change was the significant increase of the two biologically active CLA isomers cis-9, trans-11 and trans-10, cis12, in the muscle of trout fed CLA. Two weeks of CLA supplementation led to a significant increase of these two isomers in the muscle (1.3% of total fatty acids), being the maximum levels attained after 12 weeks of feeding (2.7% of total fatty acids). Nevertheless, 8 weeks of feeding were enough to obtain a CLA deposition level (2.2%; 1 mg g− 1 of muscle) that did not differ significantly from the maximum level registered at 12 weeks (2.7%; 0.9 mg g− 1 of muscle). This increasing muscle deposition of CLA was followed by a general increase of palmitic (16:0) and stearic

attributes that contributed to that difference. In the last sampling, fillets of 3 fish per treatment were frozen at −20 °C for 7 months, when a last sensory evaluation was done, applying the same protocol.

2.5. Statistical analysis Statistical analyses followed methods outlined by Zar (1999) and were performed with Statistica 7.0 package. All data were tested for normality and homogeneity of variances by Kolmogorov–Smirnov and Bartlett tests, respectively. The growth and feed intake parameters at the end of the 12 weeks trial were submitted to a one-way analysis of variance (ANOVA). Data on body composition, lipids and fatty acids in muscle were submitted to a

Table 4 Evolution of whole body composition of rainbow trout fed diets with 0% (control) or 1% CLA during a 12 weeks trial Whole body composition

Moisture (%) Crude protein (%) Crude lipid (%) Gross energy (kJ/g) Ash (%)

Control

1% CLA

Sampling time (weeks)

SEM

Sampling time (weeks)

2

4

6

8

10

12

2

4

6

8

10

12

68.9 16.5 8.3 7.7 2.4

67.1 17.2 11.5 8.8 2.5

67.1 17.1 11.7 9.1 2.1

66.2 16.8 13.9 11.0 2.2

64.9 17.2 14.4 11.3 2.1

65.7 16.6 13.0 10.1 2.4

67.9 16.5 9.4 7.6 2.5

68.0 16.8 9.8 8.4 2.1

65.8 17.1 13.0 9.6 2.4

65.0 16.8 14.5 11.2 2.4

64.7 16.6 14.2 10.6 2.3

64.9 16.2 14.2 9.8 2.4

0.3 0.1 0.4 0.2 0.5

ANOVA P-value Diet

Time

Diet × time

0.13 0.10 0.49 0.58 0.58

0.00 0.03 0.00 0.00 0.36

0.60 0.72 0.50 0.69 0.09

The values are mean (n = 3) and pooled standard error of mean (SEM). In each line, absence of superscript letters indicates absence of significant differences between sampling times, independently of the dietary treatments. Initial body composition was: moisture 69.3%, crude protein 17.0%, crude lipid 8.7%, gross energy 7.0 kJ/g, ash 2.5%.

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Table 5 Evolution of fatty acid profile (% total fatty acids) and total lipids (% wet weight) in muscle of rainbow trout fed the experimental diets with 0% (control) or 1% CLA, during 12 weeks Fatty acids (%)

14:0 16:0 18:0 ∑ SFA1 16:1 18:1 20:1 ∑ MUFA2 16:3n − 3 18:2n − 6 18:2 c9t11 18:2 t10c12 18:2 CLA3 18:3n − 3 18:4n − 3 20:4n − 6 20:4n − 3 20:5n − 3 22:5n − 6 22:5n − 3 22:6n − 3 ∑ PUFA4 Not ident. ∑ n−3 ∑ n−6 n − 3/n − 6 Total lipid

Control

1% CLA

Sampling time (weeks)

SEM

Sampling time (weeks)

2

4

6

8

10

12

2

4

6

8

10

12

3.6 15.0 3.2 25.8 5.0 17.3 3.4 26.5 0.5 10.1 0.3ef 0.1e 0.5f 1.5 1.0 0.7 1.1 6.6 0.3ab 2.2 17.4 43.7 4.0 31.2 12.0a 2.6 4.5

3.8 16.0 3.5 27.6 5.2 16.7 3.5 26.2 0.6 7.9 0.4ef 0.1e 0.5f 1.3 1.0 0.8 1.2 7.3 0.3ab 2.3 17.1 42.3 3.9 31.9 9.9ac 3.2 4.9

3.8 16.0 3.4 27.5 5.6 17.1 3.4 26.8 0.6 7.6 0.4ef 0.1e 0.5f 1.3 1.0 0.7 1.1 7.1 0.2b 2.5 17.9 42.7 3.0 32.5 9.7ac 3.5 5.4

3.8 15.9 3.4 27.4 5.3 15.7 3.4 25.3 0.5 6.3 0.4ef 0.1e 0.5f 1.2 1.0 0.7 1.4 8.3 0.3ab 2.6 18.0 42.7 4.6 34.3 8.0bc 4.4 4.0

3.9 15.9 3.4 27.2 5.8 16.9 3.6 27.0 0.6 6.7 0.5df 0.1e 0.6ef 1.3 1.0 0.7 1.2 7.6 0.2b 2.7 17.5 41.9 3.9 32.3 8.3ac 4.0 5.7

3.9 16.4 3.7 28.6 5.4 16.0 3.6 25.6 0.5 5.2 0.3 f 0.1e 0.4f 1.0 1.0 0.7 1.0 6.4 0.8a 2.5 17.0 40.5 5.2 30.2 9.8ac 3.3 4.1

3.7 16.3 4.0 27.8 4.8 17.5 3.5 27.2 0.4 9.6 0.7de 0.6d 1.3de 1.3 1.1 0.6 1.0 5.6 0.5ab 2.2 16.3 41.1 4.0 28.4 11.4a 2.5 3.8

3.7 16.5 4.1 27.9 4.8 16.7 3.4 26.2 0.4 9.6 0.8cd 0.8cd 1.6cd 1.4 1.1 0.6 1.0 6.1 0.2b 2.2 16.8 42.3 3.6 29.5 11.2ab 2.7 4.3

3.8 15.9 4.0 28.2 5.0 16.8 3.7 26.3 0.4 8.4 1.1bc 1.0bc 2.0bc 1.3 1.0 0.6 1.0 6.3 0.3b 2.2 16.2 41.7 3.8 29.3 10.4ab 2.9 5.1

3.9 16.4 4.5 27.8 5.4 17.0 3.8 28.4 0.4 7.1 1.2ab 1.0abc 2.2abc 1.2 1.2 0.7 1.0 6.4 0.2b 2.2 15.9 40.1 3.7 28.9 8.9ac 3.3 6.3

4.1 16.5 4.4 29.0 5.5 17.1 3.8 27.6 0.5 7.1 1.3ab 1.2ab 2.5ab 1.2 1.0 0.6 1.1 6.6 0.2b 2.4 15.2 40.1 3.3 28.8 8.8ac 3.4 6.5

4.2 16.9 4.7 29.6 5.4 15.9 3.7 26.3 0.4 5.7 1.4a 1.3a 2.7a 1.1 1.2 0.7 1.1 7.1 0.3b 2.4 15.3 39.4 4.7 29.1 7.5c 3.9 5.6

0.0 0.1 0.1 0.3 0.1 0.2 0.0 0.3 0.0 0.2 0.1 0.1 0.1 0.0 0.0 0.0 0.0 0.2 0.0 0.0 0.3 0.4 0.2 0.5 0.2 0.1 0.2

Anova P-value Diet

Time

Diet × time

0.12 0.04 0.00 0.06 0.03 0.55 0.01 0.20 0.00 0.04 0.00 0.00 0.00 0.98 0.05 0.02 0.00 0.01 0.15 0.03 0.03 0.12 0.41 0.00 0.86 0.07 0.26

0.05 0.26 0.14 0.17 0.00 0.12 0.44 0.75 0.84 0.00 0.00 0.00 0.00 0.00 0.65 0.96 0.39 0.26 0.02 0.09 0.95 0.53 0.04 0.88 0.00 0.01 0.29

0.82 0.77 0.81 0.92 0.41 0.83 0.48 0.61 0.93 0.52 0.00 0.00 0.00 0.46 0.77 0.33 0.08 0.16 0.03 0.58 0.96 0.97 0.72 0.88 0.03 0.19 0.37

Mean values (n = 6) and pooled standard error of mean (SEM). In each line, superscript letters indicates significant differences between treatments and time (P b 0.05). SFA = Saturated fatty acids: 12:0, 14:0, 14:0 isobranched, 15:0, 16:0, 16:0 isobranched, 17:0, 18:0, 19:0, 20:0, 22:0 and 24:0. 2 MUFA = monounsaturated fatty acids: 16:1n−7, 17:1n−8, 18:1n−9, 18:1n−7, 20:1n−9, 20:1n−7, 22:1n−11 and 22:1n−9. 318:2 c9t11+ 18:2 t10c12. 4 PUFA = polyunsaturated: 16:2n−4, 16:3n−3, 16:4n−3, 18:2n−6, 18:2CLA, 18:3n−6, 18:3n−3, 18:4n−3, 20:2n−6, 20:4n−6, 20:3n−3, 20:4n−3, 22:2n−6, 20:5n−3, 22:4n−6, 22:5n−6, 22:5n−3 and 22:6n−3. Not ident. = not identified. 1

acid (18:0) but a decrease of unsaturated palmitoleic (16:1), linoleic (18:2n − 6) acid, EPA (20:5n − 3) and DHA (22:6n − 3). These changes contributed to an overall reduction of total − 3 fatty acids accompanied by a decrease of total n − 6 fatty acids in the CLA group, and hence the n − 3/n − 6 remained unaffected.

3.4. Sensory analysis Results obtained in the 7 sensory tests realized are presented in Fig. 1. At every sampling point significant differences were detected between animals within treatments, but not among

Fig. 1. Sensory evaluation of rainbow trout fed 0% (control) and 1% CLA diets over 12 weeks. Values are means ± confidence interval (95%); n = 30. Scale reflects the magnitude of difference of each sample to a given control (fed 0% CLA), representing: 0 — no difference, 1 — small difference, 2 — small/moderate difference, 3 — moderate difference, 4 — moderate/large difference, 5 — large difference, 6 — very large difference. ⁎ samples significantly different from the control (P b 0.05).

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panelists or dietary treatments. All fillet samples were classified as slightly to moderately different from control, irrespectively of their dietary treatment. Sensory data indicate that the observed differences could not be attributed to the dietary treatment. Moreover, no influence of dietary CLA in the sensory attributes of frozen filets was registered.

4. Discussion The growth performance and nutrient utilization of rainbow trout was not affected by the incorporation of 1% CLA, throughout the 12-week period, confirming previous observations in both market size (Kennedy et al., 2007b) and smallersized fish (Figueiredo-Silva et al., 2005; Valente et al., 2007b) fed the same dietary level of CLA. Similar results were also described in various fish species fed 1% CLA (Choi et al., 1999; Twibell et al., 2001; Twibell and Wilson, 2003; Berge et al., 2004; Kennedy et al., 2005, 2007a; Manning et al., 2006; Valente et al., 2007a). In respect of whole body composition and muscle lipid level, no significant changes were induced by dietary CLA, in accordance with the majority of published works in several fish species (Twibell et al., 2000, 2001; Schwarz et al., 2002; Twibell and Wilson, 2003; Berge et al., 2004; Yasmin et al., 2004; Bandarra et al., 2006; Manning et al., 2006; Kennedy et al., 2007a; Valente et al., 2007a,b). Different results were reported by Kennedy et al. (2005) that registered an increase of muscle lipid levels, but no effect on whole body proximate composition of Atlantic salmon fed 1 and 2% CLA for 12 weeks. Nevertheless, another study with the same species described a decrease of whole body lipid content, accompanied by an increase of protein level, and similar muscle lipid content, after 12 weeks of CLA supplementation (Leaver et al., 2006). Previous studies showed that dietary CLA inclusion affects the muscle fatty acid profile in fish independently of any effect on body or muscle lipid level (Twibell et al., 2000, 2001; Berge et al., 2004; Bandarra et al., 2006; Valente et al., 2007a,b) and the very same was observed in the present work. Although whole body composition remained unaffected, muscle CLA deposition level (sum of cis-9, trans-11 and trans-10, cis-12 isomers) increased through time, reaching the maximum of 2.7% of muscle fatty acids (0.9 mg g− 1 of muscle) after 12 weeks. However, after 8 weeks of supplementation CLA deposition level already reached 2.2% of total fatty acids (1 mg g− 1of muscle) and no further significant increase was observed. Interestingly, after 2 weeks of CLA supplementation muscle CLA content was significantly superior to control, representing 1.3% of total fatty acids. That level is already higher than the observed in most of the natural CLA sources (Lin et al., 1995; Schmid et al., 2006). The CLA level detected in the control fish could be due to vegetable protein and/or vegetable oil incorporated in the commercial diets fed to those fish before starting the experiment. The final muscle CLA content registered in this experiment is well within values previously registered for smaller size rainbow trout (2.1% of total fatty acids) after 12 weeks of feeding CLA (Valente et al., 2007b). Moreover, Kennedy et al. (2007b) has recently reported a slightly lower deposition level of CLA (1.6% of total fatty

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acids), in the flesh of 800 g rainbow trout fed 1% CLA at similar dietary lipid level just for 8 weeks. However, when values are expressed in terms of fillet CLA content (1.05 mg of CLA per g of muscle) results become very similar to the ones presently observed in smaller size rainbow trout (360 g) as these fish exhibited a lower muscle fat content (5.6 vs 7.7% in 800 g trout). SFA, MUFA and PUFA levels were not affected by dietary CLA inclusion, although muscle CLA deposition level increased, like in previous studies (Berge et al., 2004; Kennedy et al., 2005; Valente et al., 2007b). Nevertheless there were significant variations in some fatty acids: in particular the increase of both palmitic (16:0) and stearic acids (18:0) and the decrease of palmitoleic acid (16:1), EPA (20:5n − 3) and DHA (22:6n − 3) at the end of 12 weeks of supplementation. The significant decrease of linoleic acid throughout time, in both groups, reflected the low dietary level of this fatty acid. Fish were previously fed on commercial diets that included vegetable oils, rich in linoleic acid, that can explain the initial values for this fatty acid. A significant increase of saturates and decrease of MUFA by dietary CLA has been often reported in mammals (Yamasaki et al., 2000) and fish (Twibell et al., 2001; Schwarz et al., 2002; Berge et al., 2004; Bandarra et al., 2006; Kennedy et al., 2007a; Valente et al., 2007a,b). CLA exert an inhibition of the Δ9-desaturase activity, that reduces MUFA level and hence increases saturates (Choi et al., 2002). The slight, but significant, decrease of the two most important n − 3 HUFA, EPA and DHA, in muscle, was also previously reported in rainbow trout juveniles (Bandarra et al., 2006), Atlantic salmon (Kennedy et al., 2005) and hybrid striped bass (Twibell et al., 2000). These changes are not probably a direct effect of CLA, but a result of the dietary CLA inclusion at the expense of fish oil, rich in EPA and DHA, causing a dietary reduction of both n − 3 HUFA. Nevertheless, CLA supplementation does not always affect the muscle levels of those two valuable HUFA (Twibell et al., 2001; Berge et al., 2004; Kennedy et al., 2007b; Valente et al., 2007a,b). Hence, the alteration of EPA and DHA seems to depend on the fish species, the developmental stage of the fish, the dietary lipid content and the extension of dietary lipid replacement. It is well known that dietary lipid sources influence the fatty acid composition of fish and hence the flesh sensory qualities (Bell et al., 2001, 2003; Regost et al., 2003, 2004; Turchini et al., 2003; Liu et al., 2004; Izquierdo et al., 2005), given the relationship between fish typical flavour and its volatile compounds resulting from n − 3 PUFA oxidation (Sérot et al., 2001, 2002). In the present study, the panelists considered all trout samples as slightly to moderately different from the control sample, irrespectively of their dietary treatment. That reflects a natural difference between individuals that is more pronounced than that attributed to the dietary treatment. Previous observations in rainbow trout (Valente et al., 2007b) revealed significant differences between animals fed increasing dietary CLA levels (0.5, 0.75 and 1%) and the identified control (0% CLA). Nevertheless, in that sensory test both the hidden control and the identified control came from the very same fish and, hence, did not allow noticing any possible natural inter-

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individual differences. Schwarz et al. (2002) did not observe any sensory differences in carp fed 0, 2.5 or 5% CLA. Similarly, Schabbel et al. (2004) did not find any differences in trout previously fed with diets containing 0, 1 and 3% CLA, nevertheless the flavour of deep frozen fillets with 3% CLA and 600 mg of vitamin E was considered positively distinct. In contrast, the present results report no influence of dietary CLA in the sensory attributes of frozen filets during 7 months, in market size rainbow trout. It could be thought of some influence of dietary CLA in the sensory attributes of frozen filets, given the anti-oxidant (Ha et al., 1990; Flintoff-Dye and Omaye, 2005) and/or pro-oxidant (Van den Berg et al., 1995) proprieties attributed to CLA and the connection between the muscle fatty acid oxidation and fillet aroma (Sérot et al., 2001, 2002), but the present results do not support such a conclusion. In summary, the results obtained in this experiment showed that the muscle incorporation of the two biological active CLA isomers increased gradually during the 12 weeks of feeding reaching the maximum level (2.7% of total fatty acids) after 12 weeks. Nevertheless, after 8 weeks of feeding the observed value (2.2% of total fatty acids; 1 mg g− 1of muscle) was not significantly different from the final. Those observations indicate that feeding rainbow trout with 1% CLA for a period between 6 to 8 weeks would be the appropriate for achieving a functional marketable trout. Optimal human dietary CLA intake remains to be established, but the average daily CLA intake estimated range between 95 and 440 mg, differing from country to country (reviewed by Schmid et al., 2006). It was hypothesised that 95 mg CLA per day is enough to show positive effects in the reduction of breast cancer, but several authors extrapolated from animal results that 3–3.5 g/day were needed to promote human health benefits (Williams, 2000; Terpstra, 2004). Using the results obtained after 8 weeks of feeding 1% CLA, this means that a single meal of a 250 g rainbow trout would supply around 150 mg of CLA, which is more than 50% of the total human daily intake in some Western populations. Moreover, CLA can be incorporated up to 1% in rainbow trout diets without any detectable major effects on whole body composition or sensory attributes, even in frozen fillets. In this trial, a fast accumulation of CLA isomers in the muscle was observed, reaching 1.3% of total fatty acids just after 2 weeks of supplementation. These results reinforce the potential of aquaculture fish to supply those bioactive fatty acids, becoming functional foods. CLA retaining time in muscle after stopping dietary supplementation, and the isomers wash-out period from fish tissues warrants further research. 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, (business@bioriginal. com) for offering the CLA mixture. We also thank António Júlio Pinto for his technical assistance during the growth trial and samplings, and Engs. Maria João Monteiro e Susana Teixeira, from Escola Superior de Biotecnologia, Universidade Católica

Portuguesa, for precious work and facilities for sensory tests. This work was supported by project POCTI/CVT/39237/2001 (FCT, Portugal, with the support of the European fund FEDER). References Arzel, J., Martinez Lopez, F.X., Metailler, R., Stephan, G., Viau, M., Gandemer, G., Guillaume, J., 1994. Effect of dietary lipid on growth performance and body composition of brown trout (Salmo trutta) reared in seawater. Aquaculture 123, 361–375. Bandarra, N.M., Nunes, M.L., Andrade, A.M., Prates, J.A.M., Pereira, S., Monteiro, M., Rema, P., Valente, L.M.P., 2006. Effect of dietary conjugated linoleic acid on muscle, liver and visceral lipid deposition in rainbow trout juveniles (Oncorhynchus mykiss). Aquaculture 254, 496–505. Bell, J.G., McEvoy, J., Tocher, D.R., McGhee, F., Campbell, P.J., Sargent, J.R., 2001. Replacement of fish oil with rapeseed oil in diets of Atlantic salmon (Salmo salar) affects tissue lipid compositions and hepatocyte fatty acid metabolism. J. Nutr. 131, 1535–1543. Bell, J.G., Tocher, D.R., Henderson, R.J., Dick, J.R., Crampton, V.O., 2003. Altered fatty acid compositions in Atlantic salmon (Salmo salar) fed diets containing linseed and rapeseed oils can be partially restored by a subsequent fish oil finishing diet. J. Nutr. 133, 2793–2801. Belury, M.A., 2002. Dietary conjugated linoleic acid in health: physiological effects and mechanisms of action. Annu. Rev. Nutr. 22, 505–531. Berge, G.M., Ruyter, B., Asgard, T., 2004. Conjugated linoleic acid in diets for juvenile Atlantic salmon (Salmo salar); effects on fish performance, proximate composition, fatty acid and mineral content. Aquaculture 237, 365–380. Bessa, R.J.B., Santos-Silva, J., Ribeiro, J.M.R., Portugal, A.V., 2000. Reticulorumen biohydrogenation and the enrichment of ruminant edible products with linoleic acid conjugated isomers. Livest. Prod. Sci. 63, 201–211. Blankson, H., Stakkestad, J.A., Fagertun, H., Thom, E., Wadstein, J., Gudmundsen, O., 2000. Conjugated linoleic acid reduces body fat mass in overweight and obese humans. J. Nutr. 130, 2943–2948. Blight, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Cherian, G., Goeger, M., 2004. Hepatic lipid characteristics and histopathology of laying hens fed CLA or n − 33 fatty acids. Lipids 39, 31–36. Cho, C.Y., 1992. Feeding systems for rainbow trout and other salmonids with reference to current estimates of energy and protein requirements. Aquaculture 100, 107–123. Cho, C.Y., Hynes, J.D., Wood, K.R., Yoshida, H.K., 1994. Development of high-nutrient-dense, low-pollution diets and prediction of aquaculture wastes using biological approaches. Aquaculture 124, 293–305. Choi, B.D., Kang, S.J., Ha, Y.L., Ackman, R.G., 1999. Accumulation of conjugated linoleic acid (CLA) in tissues of fish fed diets containing various levels of CLA. In: Xiong, Y.L., Ho, C.T., Shahidi, F. (Eds.), Quality Attributes of Muscle Foods. Kluwer Academic/Plenum Publishers, New York. Choi, Y., Park, Y., Storkson, J.M., Pariza, M.W., Ntambi, J.M., 2002. Inhibition of stearoyl-CoA desaturase activity by the cis-9, trans-11 isomer and the trans-10, cis-12 isomer of conjugated linoleic acid in MDA-MB-231 and MCF-7 human breast cancer cells. Biochem. Biophys. Res. Commun. 294, 785–790. Company, R., Calduch-Giner, J.A., Perez-Sanchez, J., Kaushik, S.J., 1999. Protein sparing effect of dietary lipids in common dentex (Dentex dentex): a comparative study with sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax). Aquat. Living Resour. 12, 23–30. Cowey, C.B., 1993. Some effects of nutrition on flesh quality of cultured fish. In: Kaushik, S.J., Luquet, P. (Eds.), Fish Nutrition in Practice. INRA, Paris, pp. 227–236. Figueiredo-Silva, A.C., Rema, P., Bandarra, N.M., Nunes, M.L., Valente, L.M.P., 2005. Effects of dietary conjugated linoleic acid on growth, nutrient utilization, body composition, and hepatic lipogenesis in rainbow trout juveniles (Oncorhynchus mykiss). Aquaculture 248, 163. Flintoff-Dye, N.L., Omaye, S.T., 2005. Antioxidant effects of conjugated linoleic acid isomers in isolated human low-density lipoproteins. Nutr. Res. 25, 1–12.

A. Ramos et al. / Aquaculture 274 (2008) 366–374 Fukuda, S., Furuya, H., Suzuki, Y., Asanuma, N., Hino, T., 2005. A new strain of Butyrivibrio fibrisolvens that has high ability to isomerize linoleic acid to conjugated linoleic acid. J. Gen. Appl. Microbiol. 51, 105–113. Gaullier, J.-M., Halse, J., Hoye, K., Kristiansen, K., Fagertun, H., Vik, H., Gudmundsen, O., 2004. Conjugated linoleic acid supplementation for 1 year reduces body fat mass in healthy overweight humans. Am. J. Clin. Nutr. 79, 1118–1125. Ha, Y.L., Storkson, J., Pariza, M.W., 1990. Inhibition of benzo(a)pyreneinduced mouse forestomach neoplasia by conjugated dienoic derivatives of linoleic-acid. Cancer Res. 50, 1097–1101. Hillestad, M., Johnsen, F., 1994. High-energy/low-protein diets for Atlantic salmon: effects on growth, nutrient retention and slaughter quality. Aquaculture 124, 109. 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. Aquacult. Nutr. 9, 397. Izquierdo, M.S., Montero, D., Robaina, L., Caballero, M.J., Rosenlund, G., Gines, R., 2005. Alterations in fillet fatty acid profile and flesh quality in gilthead seabream (Sparus aurata) fed vegetable oils for a long term period. Recovery of fatty acid profiles by fish oil feeding. Aquaculture 250, 431. Kaushik, S.J., 1998. Nutritional bioenergetics and estimation of waste production in non salmonids. Aquat. Living Resour. 11, 211–217. Kennedy, S.R., Campbell, P.J., Porter, A., Tocher, D.R., 2005. Influence of dietary conjugated linoleic acid (CLA) on lipid and fatty acid composition in liver and flesh of Atlantic salmon (Salmo salar). Comp. Biochem. Physiol. B-Biochem. Mol. Biol. 141, 168–178. Kennedy, S.R., Bickerdike, R., Berge, R.K., Porter, A.R., Tocher, D.R., 2007a. Influence of dietary conjugated linoleic acid (CLA) and tetradecylthioacetic acid (TTA) on growth, lipid composition and key enzymes of fatty acid oxidation in liver and muscle of Atlantic cod (Gadus morhua L.). Aquaculture 264, 372–382. Kennedy, S.R., Bickerdike, R., Berge, R.K., Dick, J.R., Tocher, D.R., 2007b. Influence of conjugated linoleic acid (CLA) or tetradecylthioacetic acid (TTA) on growth, lipid composition, fatty acid metabolism and lipid gene expression of rainbow trout (Oncorhynchus mykiss L.). Aquaculture 272, 489–501. Kramer, J., Blackadar, C., Zhou, J., 2002. Evaluation of two GC columns (60-m SUPELCOWAX 10 and 100-m CP sil 88) for analysis of milkfat with emphasis on CLA, 18:1, 18:2 and 18:3 isomers, and short-and long-chain FA. Lipids 37, 823–835. Kuniyasu, H., Yoshida, K., Sasaki, T., Sasahira, T., Fujii, K., Ohmori, H., 2006. Conjugated linoleic acid inhibits peritoneal metastasis in human gastrointestinal cancer cells. Int. J. Cancer 118, 571–576. Lauridsen, C., Mu, H., Henckel, P., 2005. Influence of dietary conjugated linoleic acid (CLA) and age at slaughtering on performance, slaughter-and meat quality, lipoproteins, and tissue deposition of CLA in barrows. Meat Sci. 69, 393–399. Leaver, M.J., Tocher, D.R., Obach, A., Jensen, L., Henderson, R.J., Porter, A.R., Krey, G., 2006. Effect of dietary conjugated linoleic acid (CLA) on lipid composition, metabolism and gene expression in Atlantic salmon (Salmo salar) tissues. Comp. Biochem. Physiol. A-Mol. Integr. Physiol. 145, 258–267. Lin, H., Boylston, D., Chang, M.J., Luedecke, L.O., Shultz, T.D., 1995. Survey of the conjugated linoleic acid contents of dairy products. J. Dairy Sci. 78, 2358–2365. Liu, K.K.M., Barrows, F.T., Hardy, R.W., Dong, F.M., 2004. Body composition, growth performance, and product quality of rainbow trout (Oncorhynchus mykiss) fed diets containing poultry fat, soybean/corn lecithin, or menhaden oil. Aquaculture 238, 309–328. Manning, B.B., Li, M.H., Robinson, E.H., Peterson, B.C., 2006. Enrichment of channel catfish (Ictalurus punctatus) fillets with conjugated linoleic acid and omega-3 fatty acids by dietary manipulation. Aquaculture 261, 337–342. Medale, F., Boujard, T., Vallee, F., Blanc, D., Mambrini, M., Roem, A., Kaushik, S.J., 1998. Voluntary feed intake, nitrogen and phosphorus losses in rainbow trout (Oncorhynchus mykiss) fed increasing dietary levels of soy protein concentrate. Aquat. Living Resour. 11, 239–246. Ochoa, J.J., Farquharson, A.J., Grant, I., Moffat, L.E., Heys, S.D., Wahle, K.W.J., 2004. Conjugated linoleic acids (CLAs) decrease prostate cancer

373

cell proliferation: different molecular mechanisms for cis-9, trans-11 and trans-10, cis-12 isomers. Carcinogenesis 25, 1185–1191. Ostrowska, E., Suster, D., Muralitharan, M., Cross, R.F., Leury, B.J., Bauman, D.E., Dunshea, F.R., 2003. Conjugated linoleic acid decreases fat accretion in pigs: evaluation by dual-energy X-ray absorptiometry. Br. J. Nutr. 89, 219–229. Pariza, M.W., Park, Y., Cook, M.E., 2001. The biologically active isomers of conjugated linoleic acid. Prog. Lipid Res. 40, 283–298. Park, Y., Pariza, M.W., 2007. Mechanisms of body fat modulation by conjugated linoleic acid (CLA). Food Res. Int. 40, 311–323. Park, Y., Albright, K., Liu, W., Storkson, J., Cook, M., Pariza, M., 1997. Effect of conjugated linoleic acid on body composition in mice. Lipids 32, 853–858. Park, Y., Albright, K.J., Cai, Z.Y., Pariza, M.W., 2001. Comparison of methylation procedures for conjugated linoleic acid and artifact formation by commercial (trimethylsilyl)diazomethane. J. Agric. Food Chem. 49, 1158–1164. Raes, K., Huyghebaert, G., De Smet, S., Nollet, L., Arnouts, S., Demeyer, D., 2002. The deposition of conjugated linoleic acids in eggs of laying hens fed diets varying in fat level and fatty acid profile. J. Nutr. 132, 182–189. Raes, K., De Smet, S., Demeyer, D., 2004. Effect of dietary fatty acids on incorporation of long chain polyunsaturated fatty acids and conjugated linoleic acid in lamb, beef and pork meat: a review. Anim. Feed Sci. Technol. 113, 199–221. Rainer, L., Heiss, C.J., 2004. Conjugated linoleic acid: health implications and effects on body composition. J. Am. Diet. Assoc. 104, 963–968. Regost, C., Arzel, J., Cardinal, M., Rosenlund, G., Kaushik, S.J., 2003. Total replacement of fish oil by soybean or linseed oil with a return to fish oil in turbot (Psetta maxima): 2. Flesh quality properties. Aquaculture 220, 737–747. Regost, C., Jakobsen, J.V., Rora, A.M.B., 2004. Flesh quality of raw and smoked fillets of Atlantic salmon as influenced by dietary oil sources and frozen storage. Food Res. Int. 37, 259–271. Schabbel, W., Maaß, D., Steinhart, H., Reiter, R., Schwarz, F.J., 2004. Performance carcass quality and fatty acid composition of trout (Salmo gairdneri) as affected by dietary conjugated linoleic acid and vitamin E supplementation. Proceedings of Society of Nutrition Physiology, p. 13. Schmid, A., Collomb, M., Sieber, R., Bee, G., 2006. Conjugated linoleic acid in meat and meat products: a review. Meat Sci. 73, 29–41. Schwarz, F.J., Maass, D., Schabbel, W., Steinhart, H., 2002. Dietary conjugated linoleic acid supplementation and the effects on performance, body composition, fatty acid pattern and sensory quality of carp (Cyprinus carpio). 10th International Symposium on Nutrition and Feeding in Fish, Rhodes, p. 79. Sérot, T., Regost, C., Prost, C., Robin, J., Arzel, J., 2001. Effect of dietary lipid sources on odour-active compounds in muscle of turbot (Psetta maxima). J. Sci. Food Agric. 81, 1339–1346. Sérot, T., Regost, C., Arzel, J., 2002. Identification of odour-active compounds in muscle of brown trout (Salmo trutta) as affected by dietary lipid sources. J. Sci. Food Agric. 82, 636–643. Sisk, M.B., Hausman, D.B., Martin, R.J., Azain, M.J., 2001. Dietary conjugated linoleic acid reduces adiposity in lean but not obese zucker rats. J. Nutr. 131, 1668–1674. Szymczyk, B., Pisulewski, P.M., Szczurek, W., Hanczakowski, P., 2001. Effects of conjugated linoleic acid on growth performance, feed conversion efficiency, and subsequent carcass quality in broiler chickens. Br. J. Nutr. 85, 465–473. Terpstra, A.H.M., 2004. Effect of conjugated linoleic acid on body composition and plasma lipids in humans: an overview of the literature. Am. J. Clin. Nutr. 79, 352–361. Turchini, G.M., Mentasti, T., Froyland, L., Orban, E., Caprino, F., Moretti, V.M., Valfre, F., 2003. Effects of alternative dietary lipid sources on performance, tissue chemical composition, mitochondrial fatty acid oxidation capabilities and sensory characteristics in brown trout (Salmo trutta L.). Aquaculture 225, 251–267. Twibell, R.G., Wilson, R.P., 2003. Effects of dietary conjugated linoleic acids and total dietary lipid concentrations on growth responses of juvenile channel catfish, Ictalurus punctatus. Aquaculture 221, 621–628.

374

A. Ramos et al. / Aquaculture 274 (2008) 366–374

Twibell, R.G., Watkins, B.A., Rogers, L., Brown, P.B., 2000. Effects of dietary conjugated linoleic acids on hepatic and muscle lipids in hybrid striped bass. Lipids 35, 155–161. Twibell, R.G., Watkins, B.A., Brown, P.B., 2001. Dietary conjugated linoleic acids and lipid source alter fatty acid composition of juvenile yellow perch, Perca flavescens. J. Nutr. 131, 2322–2328. Valente, L.M.P., Bandarra, N.M., Figueiredo-Silva, A.C., Cordeiro, A.R., Simoes, R.M., Nunes, M.L., 2007a. Influence of conjugated linoleic acid on growth, lipid composition and hepatic lipogenesis in juvenile European sea bass (Dicentrarchus labrax). Aquaculture 267, 225–235. Valente, L.M.P., Bandarra, N.M., Figueiredo-Silva, A.C., Rema, P., Vaz-Pires, P., Martins, S., Prates, J.A.M., Nunes, M.L., 2007b. Conjugated linoleic acid in diets for large-size rainbow trout (Oncorhynchus mykiss): effects on growth, chemical composition and sensory attributes Br. J. Nutr. 97, 289–297.

Van den Berg, J., Cook, N., Tribble, D., 1995. Reinvestigation of the antioxidant properties of conjugated linoleic acid. Lipids 30, 599–605. Vergara, J.M., Lopez-Calero, G., Robaina, L., Caballero, M.J., Montero, D., Izquierdo, M.S., Aksnes, A., 1999. Growth, feed utilization and body lipid content of gilthead seabream (Sparus aurata) fed increasing lipid levels and fish meals of different quality. Aquaculture 179, 35. Williams, C.M., 2000. Dietary fatty acids and human health. Ann. Zootech. 49, 165–180. Yamasaki, M., Mansho, K., Ogino, Y., Kasai, M., Tachibana, H., Yamada, K., 2000. Acute reduction of serum leptin level by dietary conjugated linoleic acid in Sprague–Dawley rats. J. Nutr. Biochem. 11, 467–471. Yasmin, A., Takeuchi, T., Hayashi, M., Hirota, T., Ishizuka, W., Ishida, S., 2004. Effect of conjugated linoleic and docosahexaenoic acids on growth of juvenile tilapia Oreochromis niloticus. Fisheries Sci. 70, 473–481. Zar, J.H., 1999. Biostatistical analysis. Prentice Hall, London. 663 pp.