Vegetable lipid sources for gilthead seabream (Sparus aurata): effects on fish health

Aquaculture 225 (2003) 353 – 370 www.elsevier.com/locate/aqua-online

Vegetable lipid sources for gilthead seabream (Sparus aurata): effects on fish health D. Montero a,*, T. Kalinowski a, A. Obach b, L. Robaina a, L. Tort c, M.J. Caballero a, M.S. Izquierdo a a

Grupo de Investigacio´n en Acuicultura, ULPGC and ICCM, P.O. Box 56, 35200, Telde, Las Palmas de Gran Canaria, Canary Islands, Spain b Nutreco Aquaculture Research Centre A/S, P.O. Box 48, N-4001 Stavanger, Norway c Departamento de Biologia Celular i Fisiologia, Facultad de Ciencias, Universitat Auto´noma de Barcelona, 08193, Cerdanyola, Barcelona, Spain

Abstract Commercial feeds for gilthead seabream are highly energetic, containing fish oil as the main lipid source. The steady production and raising prices of fish oil encourage the inclusion of vegetable oils in fish feeds. Fish oil could be at least partially substituted by vegetable oils in diets for marine species, being this substitution resulted in good feed utilization and maintenance of fish health, since imbalances in dietary fatty acids may alter the immunological status and stress resistance in fish. In order to evaluate the effect of vegetable oils on gilthead seabream health, fish were fed different isonitrogenous and isocaloric diets for 101 days (Experiment I) and 204 days (Experiment II). In Experiment I, diets were formulated to contain 60% of the fish oil used in the control diet (FO) as soybean oil (Diet 60SO), rapeseed oil (60RO), linseed oil (60LO) or a blend of those oils (Mix). In Experiment II, the same diets plus two which contained 80% of the fish oil as soybean oil (80SO) and linseed oil (80LO), respectively, were assayed. At the end of both experiments, basal levels of different immunological parameters were determined, including both humoral immunity (alternative complement pathway activity and serum lysozyme activity) and cellular immunity (circulating neutrophil activity and phagocytic index of head kidney macrophages). In addition, response to a confinement stress was assayed in terms of variations in plasma cortisol. The effect of dietary vegetable oils on fatty acid composition of head kidney macrophages and circulating red blood cells (RBC) was also studied. No effects of dietary vegetable oils were found in fish fed the experimental diets for a medium period. Feeding dietary vegetable oils for a long period did not affect lysozyme or neutrophil activity. However, in Experiment II, the inclusion of soybean oil reduced both serum alternative complement pathway activity (from 249 IU/ml (FO2) down to 153.8 IU/ml (60SO2)) and head kidney phagocytic

* Corresponding author. Tel.: +34-928-132-900; fax: +34-928-132-908. E-mail address: [email protected] (D. Montero). 0044-8486/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0044-8486(03)00301-6

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activity (from 25.75% (FO2) down to 14.58% (80SO2). Inclusion of rapeseed oil reduced phagocytic activity. Fish fed vegetable oil-containing diets showed different patterns of stress response, especially those fish fed the linseed oil diets that showed a significant increase in plasma cortisol level after stress. The fatty acid composition of head kidney macrophages reflected the fatty acids content of the respective diets, but a selective incorporation of essential fatty acids into these cells was observed. The same trend was found in circulating red blood cells, indicating the important role of essential fatty acids on these cells. Sixty percent of fish oil can be replaced by a blend of different vegetable oils without affecting gilthead seabream health. However, if single vegetable oil is used to replace 60% of fish oil, fish health can be affected in terms of immunosuppression or stress resistance. Rapeseed oil affected head kidney macrophages activity, soybean oil affected serum alternative complement pathway activity and linseed oil altered stress response of fish. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Vegetable oils; Sparus aurata; Immunology; Phagocytic index; Stress; Arachidonic acid; DHA; EPA

1. Introduction Traditionally, fish oil has constituted the main lipid source added to compound aquafeeds for marine fish, due to its high digestibility and n-3 HUFA content, essential fatty acids for marine fish species, and to the acceptable market price of fish oil in comparison to other animal-consumed fats. The relative lipid content in marine fish diets has being raised during the last years due to a general trend to produce more energetic diets. But the increase in the global demand of fish oil for human and animal consumption, together with the rather stable production of fish oil, has produced a steady increase in the market price of this ingredient since early 1995. As a consequence, there is increased interest in the inclusion of vegetable oils in marine fish diets to partially replace and reduce the dependency on fish oil. Variations in the dietary fatty acid profiles caused by the inclusion of vegetable oil sources may alter the fish metabolism, which may affect fish health and stress resistance. The inclusion of vegetable oils can produce inadequate ratios of n-3 and n-6 fatty acids which could affect fish health by altering the synthesis of eicosanoids (Fracalossi et al., 1994). Arachidonic acid (20:4n-6) (AA) and eicosapentaenoic acid (20:5n-3) (EPA) are precursors of two groups of eicosanoids, prostaglandins and leukotrienes, which among other functions stimulate macrophages and other leucocytes to destroy bacteria. When the intake of n-6 fatty acid increases, a higher level of AA-derived eicosanoids may be observed in Atlantic salmon (Bell et al., 1993). In addition, a decrease of in vitro production of AA-derived prostaglandins has been described in astroglial cells of turbot cultured in EPA enriched medium (Bell et al., 1994). However, the role of n-3 and n-6 fatty acids in fish immune response is unclear, reports are not conclusive and are very often contradictory. Some authors have reported negative effects of high dietary levels of n-3 PUFAs in channel catfish (Erdal et al., 1991; Fracalossi and Lovell, 1994; Li et al., 1994), whereas other reports show positive effects of n-3 fatty acids on the immune response of fish. For instance, high levels of dietary n-3 fatty acids increased activity of head kidney macrophages of channel catfish (Sheldon and Blazer, 1991) and rainbow trout

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(Ashton et al., 1994). In addition, inadequate levels of dietary n-3 PUFAs reduced antibody production and in vitro killing of bacteria by macrophages in rainbow trout (Kiron et al., 1995) and depleted alternative complement pathway activity in gilthead seabream (Montero et al., 1998). Thus, replacement of marine fish oils with alternate lipids of plant origin in feed of farmed fish should be studied not only to supply lipids at the correct level with the proper balance of EFA for optimum growth but also to maintain proper immune function in fish. The objective of the present study was to examine the effect of replacing dietary fish oil with vegetable oils on gilthead seabream health.

2. Materials and methods 2.1. Experiment I 2.1.1. Experimental diets Five isoenergetic and isonitrogenous experimental diets were formulated with a constant lipid content of about 25% (Table 1). Peruvian anchovy oil was the lipid source in the control diet (Diet FO1). In the other experimental diets fish oil was included at a level high enough (40% of Diet FO1) to keep the n-3 HUFA levels well over 3% in order to meet the essential fatty acid requirement of gilthead seabream (Montero et al., 1996). Thus, whereas Diet FO1 contained 6.71% n-3 HUFA on dry weight basis, the other experimental diets contained from 3.5% to 4% (Table 1). Soybean, rapeseed, linseed and a combination of the three oils were used in Diets 60SO1, 60RO1, 60LO1 and Mix1, respectively, as lipid sources to replace the remaining 60% of fish oil used in Diet FO1. Mainly due to the different linoleic acid content of the vegetable oils, the n-3/n-6 differed among the experimental diets, ranging from 0.8 in Diet 60SO1 to 3.3 in Diet FO1 (Table 2). The diets were produced at the Nutreco Technology Centre (Stavanger, Norway) as extruded, sinking pellets. All the experimental diets were made from the same basal feed. After extrusion, the feed kernels were coated with different oils. Diets were kept refrigerated at Instituto Canario de Ciencias Marinas (ICCM) (Canary Islands, Spain) during the experimental period. 2.1.2. Feeding trial Gilthead seabream (Sparus aurata) juveniles (10 g initial body weight) were distributed in 15 tanks of 100 l (20 fish/tank, each diet assayed by triplicates) supplied with continuous seawater (1.65 l/min), with temperature ranged from 19.5 to 23.8 jC and aeration (oxygen ranged from 6.9 to 8.3 mg/l during the experimental period). Fish were fed the experimental diets to apparent satiation (three times a day, 6 days a week) during 101 days. 2.2. Experiment II 2.2.1. Experimental diets Seven isoenergetic and isonitrogenous experimental diets with a lipid content of about 22% were formulated (Table 3). Peruvian anchovy oil was lipid source in Diet FO2. All

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Table 1 Composition (g/kg) of experimental diets containing different lipid sources used in Experiment I Ingredient

Fish meal (LT) Corn gluten Wheat Lysine (99%) Premixb Anchovy oil Soybean oilc Rapeseed oilc Linseed oilc

Diet FO1a

60SO1

60RO1

60LO1

Mix1

361.3 259.8 165.0 7.2 10.0 196.7

361.3 259.8 165.0 7.2 10.0 78.7 118.0

361.3 259.8 165.0 7.2 10.0 78.7

361.3 259.8 165.0 7.2 10.0 78.7

361.3 259.8 165.0 7.2 10.0 78.7 11.8 35.4 70.8

118.0 118.0

a

FO1 = 100% fish oil; SO1 = 60% soybean oil; RO1 = 60% rapeseed oil; 60LO1 = 60% linseed oil; Mix1 = 60% of blend of different vegetable oils. b Premix of vitamins and minerals according to NCR (1993) recommendation for fish. c Crude vegetable oils.

other diets contained vegetable oils to substitute for 60% of the anchovy oil used in Diet FO2 (60SO2, 60RO2, 60LO2 and 60Mix2) and 80% (diets 80SO and 80LO). Fish oil was included in diets 60SO2, 60RO2, 60LO2 and 60Mix2 at a level high enough to meet the EFA requirements of this species (Table 4). The diets were produced at the Nutreco Technology Centre as extruded, sinking pellets. All the experimental diets were made from the same basal feed. After extrusion, the feed kernels were coated with different oils (Table 3). The diets were kept refrigerated at ICCM (Canary Islands, Spain) during the experimental period.

Table 2 Composition of selected fatty acids (g FA/100 g total FA) of diets containing fish oil or fish oil in combination with different vegetable lipid sources and fed to gilthead seabream in Experiment I Diet

16:0 18:1n-9 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3 Saturated Monounsaturated Total n-3 Total n-6 Total n-9 n-3 HUFA

FO1a

60SO1

60RO1

60LO1

Mix1

15.5 11.4 7.3 3.8 0.6 13.9 8.9 25.0 25.9 31.2 9.4 14.2 25.2

13.4 14.4 26.2 4.7 0.4 8.1 5.6 20.9 24.8 21.3 27.5 16.7 15.2

11.3 27.8 16.0 5.2 0.3 7.5 5.4 17.8 39.5 20.7 17.2 30.9 14.2

11 15.1 13.0 23.0 0.3 7.3 5.3 17.6 25.1 38.2 14.1 17.7 13.9

11.2 17.8 14.6 18.2 0.3 7.3 5.4 17.8 28.2 33.4 15.7 20.5 14.0

a FO1 = 100% fish oil; SO1 = 60% Soybean oil; RO1 = 60% rapeseed oil; 60LO1 = 60% linseed oil; Mix1 = 60% of blend of different vegetable oils.

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Table 3 Composition (g/kg) of experimental diets containing different lipid sources used in Experiment II Ingredient

Diet FO2

60SO2

60RO2

60LO2

Mix2

80SO

80LO

Fish meal (LT) Corn gluten Wheat Lysine (99%) Premixa Anchovy oil Soybean oilb Rapeseed oilb Linseed oilb

381 260 150.6 7.23 25 176

381 260 150.6 7.23 25 70.4 105.6

381 260 150.6 7.23 25 70.4

381 260 150.6 7.23 25 70.4

381 260 150.6 7.23 25 70.4 10.56 31.68 63.36

381 260 150.6 7.23 25 35.2 140.8

381 260 150.6 7.23 25 35.2

105.6 105.6

140.8

a

Premix of vitamins and minerals according to NCR (1993) recommendations for fish. b Crude vegetable oils.

2.2.2. Feeding trial Gilthead seabream juveniles (85 g initial body weight) were distributed in 24 tanks of 500 l (65 fish/tank, each diet fed to three tanks) supplied with continuous seawater (8.3 l/ min), with temperature ranged from 19.5 to 23.8 jC and aeration (Oxygen ranged from 6.5 to 7.9 mg/l during experimental period). Fish were fed the experimental diets until apparent satiation (three times a day, 6 days a week), until commercial size (204 days). 2.2.3. Blood collection and sample preparation Fish were individually sampled from each tank. No anaesthetic was used in order to avoid the effect of anaesthesia on blood parameters. In Experiment I, three fish were sampled from each tank. Fish handling time was less than 1 min per fish. Thus, total capture time was less than 8 min per tank to minimize capture stress effects on analyzed Table 4 Main fatty acid composition (g FA/100 g total FA) of diets fed to gilthead seabream in Experiment II Diet

16:0 18:1n-9 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3 Saturated Monounsaturated Total n-3 Total n-6 Total n-9 n-3 HUFA

FO2a

60SO2

60RO2

60LO2

Mix2

80SO

80LO

17.51 13.64 4.70 0.64 0.74 16.16 7.09 28.31 29.47 31.56 7.98 16.48 26.17

14.26 17.07 30.42 3.42 0.42 7.85 4.24 21.14 26.46 18.54 32.65 19.05 13.09

11.24 37.10 14.69 5.03 0.36 7.10 4.31 18.99 43.66 19.89 16.34 39.66 12.98

11.48 16.32 11.48 28.09 0.32 6.94 3.98 18.46 25.05 42.23 13.15 18.32 12.24

11.86 22.29 14.53 19.97 0.30 6.92 3.84 18.51 31.25 33.59 15.55 24.44 11.72

13.45 18.24 38.52 4.39 0.30 5.02 2.43 19.13 26.42 13.83 39.79 19.87 8.16

9.58 15.99 13.24 36.69 0.25 4.30 3.31 15.26 23.54 46.07 14.71 17.84 8.23

a FO2 = 100% fish oil; 60SO2 = 60% soybean oil; 60RO2 = 60% rapeseed oil; 60LO2 = 60% linseed oil; Mix2 = 60% of blend of different vegetable oils; 80SO = 80% soybean oil; 80LO = 80 % linseed oil.

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parameters (Sumpter, 1997). Blood was obtained by caudal sinus puncture with 1 ml plastic syringe and three different aliquots of blood were used for different analyses. The first aliquot was transferred to an Eppendorf tube coated with lithium heparin as anticoagulant and was used for hematological determination. A second aliquot was transferred to another Eppendorf tube without anticoagulant and was used for serum parameters. In Experiment II, the same procedure described above was used for blood collection, except that the first aliquot of blood was used for nitro blue tetrazolium (NBT) determination, which measures the activity of circulating neutrophils. 2.2.4. Haematology and NBT index Haematocrit (Ht) was measured by microcentrifugation (3000 rpm, 10 min). Circulating red blood cell (RBC) number and total haemoglobin (Hb) were determined by a haematological counter System 800. The reduction of NBT to formazan caused by oxygen radicals from circulating neutrophils was measured spectrophotometrically as described by Siwicki et al. (1993). 2.2.5. Serum determinations The second aliquot of blood was allowed to clot at 4 jC for 2 h. Serum was then separated by centrifugation at 3000 rpm for 10 min, and stored at 80 jC until analysis. Alternative complement pathway activity using rabbit RBC was determined as described by Sunyer and Tort (1995) for gilthead seabream. The reciprocal of the serum dilution causing 50% lysis of RBC is designated as the ACH50 and results are presented as ACH50 units/ml. Lysozyme activity was assayed by a turbidimetric assay as described by Anderson and Siwicki (1994), by measuring the lytic activity of the gilthead seabream serum against Microccocus lysodeikticus, using hen egg white lysozyme as standard. 2.2.6. Macrophage phagocytic activity In Experiment II, head kidney of six fish per diet was removed and macrophages were isolated using a percoll gradient. Macrophage solution was incubated with Vibrio anguillarum as described by Esteban and Mesenger (1997). Phagocytic activity was measured as described by Blazer (1991). One hundred macrophages per fish were counted and the phagocytic capacity was determined as the percentage of macrophages with phagocytic ability. 2.2.7. Fatty acid analysis In Experiment II, samples of head kidney macrophages from six fish per tank were collected, pooled and stored at 80 jC until analyses. Lipids from the experimental diets and head kidney macrophages from both species were extracted with a chloroform:methanol (2:1 v/v) mixture as described by Folch et al. (1957). Fatty acid methyl esters were obtained by transmethylation as described by Christie (1982) and identified using gas chromatography under the conditions previously described (Izquierdo et al., 1990). 2.2.8. Stress test At the end of each feeding period, fish were subjected to an acute stress (5 min of net chasing in Experiment I and overcrowding for 2 h in Experiment II). Blood samples were

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obtained to determine plasma cortisol concentration after being subjected to the stress (Experiment I: 1, 2 and 3 h; Experiment II: 2.5, 4 and 8 h). Fish were not reused and blood sampling procedures were similar to those described above. Blood was placed in an Eppendorf tube coated with lithium heparin and was immediately centrifuged at 3000 rpm during 10 min. The plasma obtained was stored at 80 jC until analysis. Plasma cortisol concentration was determined by RIA as described by Molinero and Gonzalez (1995), using trysin – antitrypsin method. 2.2.9. Statistical analysis All the data was statistically treated using ANOVA and Tukey’s multiple range test (Sokal and Rolf, 1995).

3. Results Replacing 60% of fish oil by vegetable oils for 101 days did not affect fish growth (Izquierdo et al., in press), whereas 80% of fish oil replacement by either soybean or linseed oil for 204 days significantly ( P < 0.05) reduced gilthead seabream growth (Izquierdo, personal communication). 3.1. Haematology No significant differences were found in the haematocrit value or the haemoglobin content among blood samples from fish fed the different experimental diets in Experiment I. However, the number of circulating erythrocytes (RBC) was higher in fish fed Diet FO1 when compared to fish fed Diets 60SO1 and 60LO1 (4.50  106 against 3.49  106 and 3.67  106 cells/mm3) (Table 5). 3.2. Humoral immunity Regarding serum immunology, no significant differences were found in the activity of the serum alternative complement pathway in fish from Experiment I (Table 5). However, in Experiment II, fish fed soybean oil-containing diets (60SO2 and 80SO) showed the

Table 5 Haematological parameters and serum alternative complement pathway activity of fish fed the experimental diets (Experiment I) (mean F S.D.)

Haematocrit (%) Haemoglobin (g/dl) RBC ( 106/mm3) ACH50 (units/ml)

FO11

60SO1

60RO1

60LO1

MIX1

44.56 F 3.25 8.53 F 1.21 4.50 F 0.70a 119.7 F 34.9

43.13 F 2.45 7.16 F 0.21 3.49 F 0.12b 107.5 F 32.5

44.00 F 8.10 7.48 F 1.14 4.02 F 0.15ab 108.0 F 32.5

44.67 F 6.14 7.17 F 1.23 3.67 F 0.79b 111.3 F 30.5

44.44 F 2.51 7.77 F 1.03 3.89 F 0.85ab 96.1 F 44.7

Different letter within a line denotes significant differences ( P < 0.05). 1 FO1 = 100% fish oil; SO1 = 60% Soybean oil; RO1 = 60% rapeseed oil; 60LO1 = 60% linseed oil; Mix1 = 60% of blend of different vegetable oils.

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Table 6 NBT activity, serum parameters, phagocytic index and plasma cortisol values after stress of fish fed the experimental diets

NBT (abs nm) ACH50 (units/ml) Lysozyme (KU/ml) Phagocytic capacity (%) Plasma cortisol (ng/ml)2

60SO2

60RO2

60LO2

Mix2

80SO 0.94 F 0.01

80LO

0.93 F 0.03

0.88 F 0.02

0.87 F 0.20

0.97 F 0.02

0.91 F 0.02

0.96 F 0.01

249.9 F 46.9a

153.8 F 80.7b

244.25 F 58.3ab

236.5 F 59.9ab

235.3 F 27.5ab

217.0 F 60.13ab

241.5 F 41.5a

82.31 F 55.34

71.94 F 39.94

69.76 F 51.08

73.72 F 45.31

72.76 F 46.41

69.86 F 47.05

70.59 F 45.18

25.75 F 7.23a

18.52 F 9.64ab

15.98 F 5.80b

20.00 F 4.97ab

19.00 F 2.94ab

14.58 F 3.37b

23.25 F 1.25a

3.54 F 0.24

3.98 F 0.64

4.79 F 0.87

4.48 F 1.54

6.17 F 1.24

5.39 F 2.01

4.26 F 0.53

Experiment II (mean F S.D.). Different letter within a line denotes significant differences ( P < 0.05). 1 FO2 = 100% fish oil; 60SO2 = 60% soybean oil; 60RO2 = 60% rapeseed oil; 60LO2 = 60% linseed oil; Mix2 = 60% of blend of different vegetable oils; 80SO = 80% soybean oil; 80LO = 80 % linseed oil. 2 Values obtained just before stress induction (mean F S.E.).

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Table 7 Fatty acid composition of head kidney macrophages from seabream fed different vegetable lipid sources (g FA/ 100 g total FA) Fatty acid

Diet FO2

14:0 15:0 16:0 16:1n-7 16:2 17:0 17:1 16:4n-3 18:0 18:1n-9 18:1n-7 18:2n-9 18:2n-6 18:3n-6 18:4n-6 18:3n-3 18:4n-3 18:4n-1 20:0 20:1n-9 20:1n-7 20:2n-9 20:2n-6 20:3n-6 20:4n-6 20:3n-3 20:4n-3 20:5n-3 22:1n-11 22:1n-7 22:3n-6 22:4n-6 22:5n-6 22:5n-3 22:6n-3 Saturated Monoenoics S n-3 S n-6 S n-9 n-3 HUFA DHA/EPA AA/EPA AA/DHA

60SO2 a

3.10 0.29 21.13 5.12 0.57 0.35 0.52 1.69 8.08 14.33a 3.33 0.15 3.87a 0.22 0.21 0.35a 0.53 0.26 0.16 1.31 0.18 0.19 0.17 0.25 3.16a 0.05 0.51 10.30a 0.79 0.32 0.26 0.25 0.31 2.89 12.94a 33.11 26.04 29.33a 8.70a 16.19a 23.25a 1.26 0.31 0.24

c

1.92 0.17 18.69 3.32 0.30 0.24 0.33 1.25 7.75 16.67a 3.12 0.13 18.62d 0.27 0.12 1.29b 0.37 0.17 0.20 1.16 0.16 0.20 0.61 0.42 1.59b 0.14 0.34 6.67b 0.73 0.37 0.16 0.16 0.15 2.56 7.88b 28.96 25.97 20.64b 22.08c 18.27a 14.55b 1.18 0.24 0.20

60RO2 bc

2.12 0.21 19.74 0.15 0.33 0.26 0.28 2.14 7.75 25.08b 3.41 0.20 10.21bc 0.19 0.15 1.60b 0.29 0.17 0.19 1.41 0.15 0.18 0.37 0.31 2.06b 0.17 0.34 6.53b 0.68 0.41 0.13 0.19 0.19 2.27 8.20b 30.29 31.73 21.54b 13.80b 26.99b 14.73b 1.26 0.32 0.25

60LO2 c

1.86 0.20 21.25 3.37 0.29 0.30 0.25 2.04 10.48 17.72a 2.93 0.13 8.58b 0.16 0.00 7.51c 0.27 0.14 0.17 1.10 0.14 0.15 0.29 0.33 1.66b 0.68 0.37 5.49b 0.78 0.38 0.24 0.34 0.23 1.84 5.22b 34.26 26.78 23.48b 11.83b 19.32a 10.71b 0.95 0.30 0.32

Different letters in the same line denote significant differences ( P < 0.05).

Mix2

80SO ab

2.60 0.20 18.75 3.81 0.29 0.20 0.35 1.31 7.14 24.11b 2.14 0.11 11.30c 0.16 0.11 8.44c 0.37 0.15 0.16 1.27 0.12 0.16 0.33 0.29 1.51b 0.50 0.37 5.71b 0.74 0.35 0.18 0.22 0.20 2.22 4.69b 29.05 30.81 23.69b 14.30b 25.83ab 10.41b 0.82 0.26 0.32

c

1.19 0.13 18.96 1.68 0.19 0.20 0.17 1.66 8.92 14.70a 3.24 0.10 22.27e 0.25 0.11 1.48b 0.20 0.14 0.17 0.87 0.00 0.13 0.91 0.67 1.75b 0.20 0.29 5.74b 0.49 0.24 0.10 0.17 0.19 2.23 7.87b 29.57 21.39 19.66b 26.43d 15.89a 13.61b 1.37 0.30 0.22

80LO 1.37c 0.19 19.66 2.04 0.26 0.28 0.18 2.42 10.71 14.49a 2.72 0.13 8.61b 0.19 0.09 7.99c 0.27 0.10 0.16 0.98 0.13 0.11 0.33 0.46 1.84b 0.91 0.47 7.24b 0.58 0.28 0.12 0.19 0.21 2.12 8.28b 32.37 21.46 29.71a 12.02b 15.99a 15.52b 1.14 0.25 0.22

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lowest values of ACH50. Fish fed 60SO2 diet had significantly lower ( P < 0.05) ACH50 value than fish fed diet FO2 (153.8 and 249.9 units/ml, respectively) (Table 6). The inclusion of linseed or rapeseed oil in seabream diets did affect on the activation of the serum alternative complement pathway (Table 6). No effect of dietary vegetable oils was observed on seabream serum lysozyme activity (Table 6). Fish fed 100% fish oil-containing diet showed the highest (but not significant) value of lysozyme activity in serum. 3.3. Cellular immunity There was no effect of vegetable oils on the macrophage respiratory burst activity of fish fed the experimental diets as measured by NBT reduction (Table 6). However, the cellular immunity, measured as phagocytic activity of head kidney macrophages, was affected in fish fed either rapeseed or soybean oil-containing diets (Diets 60RO2 and 80SO, respectively), fish fed these diets showed significantly ( P < 0.05) lower values of phagocytic activity against V. anguillarum than those showed by fish fed FO2 diet (Table 6). 3.4. Fatty acid composition of total lipids from head kidney macrophages Inclusion of vegetable oils increased linoleic and linolenic acids in macrophages of fish fed diets with soybean (diets 60SO2 and 80SO) or linseed (diets 60LO2 and 80LO) oil, respectively. Oleic acid (18:1n-9) was increased in macrophages of fish fed rapeseed oil (60RO2), whereas fish fed FO1 diet showed the highest n-3 HUFA proportion in macrophages (Table 7). There were no significant differences in the DHA content of head kidney macrophages of fish fed the vegetable oil-containing diets of Experiment II (Table 7). Macrophages from fish fed FO2 diets had significantly ( P < 0.05) higher DHA content. Fish fed FO2 diets had significantly ( P < 0.05) more AA in the head kidney macrophages than the fish fed vegetable oil-containing diets (Table 7). Macrophages from fish fed vegetable oilcontaining diets showed lower quantities of EPA when compared with those from fish fed FO2 diet (Table 7).

Table 8 Head kidney macrophage fatty acid/dietary fatty acid ratios for the main fatty acids of fish fed the experimental diets (Experiment II) Macrophage FA/dietary FA

DIETS FO2a

60SO2

60RO2

60LO2

Mix2

80SO

80LO

18:1n-9 18:2n-6 18:3n-3 AA (20:4n-6) EPA (20:5n-3) DHA (22:6n-3)

1.09 0.85 0.56 4.38 0.66 1.91

1.02 0.50 0.31 3.10 0.69 1.51

0.70 0.71 0.32 5.89 0.94 1.94

1.1 0.76 0.27 5.19 0.81 1.34

1.09 0.78 0.42 5.03 0.83 1.23

0.84 0.60 0.35 6.03 1.19 3.36

0.93 0.67 0.22 7.67 1.73 2.58

a FO2 = 100% fish oil; 60SO2 = 60% soybean oil; 60RO2 = 60% rapeseed oil; 60LO2 = 60% linseed oil; Mix2 = 60% of blend of different vegetable oils; 80SO = 80% soybean oil; 80LO = 80 % linseed oil.

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The ratio AA/EPA tended to be constant among fish fed the experimental diets. Values obtained in the different experimental groups were close to 0.3. The same tendency could be observed for the AA/DHA ratio, which ranged from 0.2 to 0.32 (Table 7).

Table 9 Fatty acid composition of erythrocytes from seabream fed different vegetable lipid sources (g FA/100 g total FA) Fatty acid

14:0 15:0 16:0 16:1n-7 16:2 17:0 17:1 16:4n-3 18:0 18:1n-9 18:1n-7 18:2n-9 18:2n-6 18:3n-6 18:4n-6 18:3n-3 18:4n-3 18:4n-1 20:0 20:1n-9 20:1n-7 20:2n-9 20:2n-6 20:3n-6 20:4n-6 20:3n-3 20:4n-3 20:5n-3 22:1n-11 22:1n-7 22:3n-6 22:4n-6 22:5n-6 22:5n-3 22:6n-3 Saturated Monoenes S n-3 S n-6 S n-9 n-3 HUFA

Diet FO2

60SO2

60RO2

60LO2

Mix2

80SO

80LO

1.35a 0.15 22.35 1.71 0.25a 0.38 0.22 0.32 11.99 9.68a 2.04ab 0.21 2.00a 0.21 0.12 0.16a 0.19 0.17 0.24ab 0.82ab 0.13 0.20 0.13 0.21 1.72 0.15 0.66 15.38a 0.43 0.27 0.21 0.84 0.26 2.86 21.23 36.99 15.38a 40.90 5.70a 11.05a 40.23a

0.91bc 0.10 21.48 0.92 0.15ab 0.27 0.13 0.23 11.43 12.01ab 1.90ab 0.15 11.78d 0.22 0.23 0.47a 0.13 0.09 0.24ab 0.95ab 0.10 0.18 0.52 0.41 1.62 0.09 0.36 9.46b 0.46 0.28 0.18 0.61 0.30 2.50 18.53 34.81 16.83a 31.89 15.87d 13.36ab 31.05b

1.02ab 0.11 24.61 1.10 0.17ab 0.27 0.11 0.29 12.92 17.41c 2.34b 0.23 6.33bc 0.20 0.08 0.79a 0.09 0.07 0.34b 1.37c 0.09 0.20 0.33 0.28 1.51 0.15 0.31 8.32b 0.66 0.29 0.08 0.36 0.20 1.88 16.99 39.63 23.38b 26.83 9.39bc 19.27c 25.66b

0.96bc 0.12 25.21 1.05 0.14ab 0.32 0.09 0.24 14.35 11.59ab 0.08a 0.14 4.93b 0.16 0.07 4.74bc 0.16 0.06 0.21ab 0.79a 0.07 0.13 0.23 0.20 1.32 0.61 0.54 8.89b 0.43 0.26 0.12 0.44 0.23 2.13 16.83 41.63 15.95a 34.15 7.70ab 12.72ab 29.02b

0.83bc 0.09 19.43 0.48 0.13ab 0.26 0.12 0.23 11.18 13.40ab 1.84ab 0.16 6.14bc 0.16 0.07 3.55b 0.16 0.07 0.21ab 0.99ab 0.08 0.16 0.30 0.25 1.63 0.51 0.48 10.69b 0.59 0.24 0.10 0.51 0.24 2.63 21.53 32.43 17.79a 39.78 9.41bc 14.79ab 35.84a

0.66c 0.30 20.18 0.41 0.09b 0.26 0.09 0.18 11.77 11.64ab 0.81a 0.14 15.53e 0.27 0.05 0.79a 0.10 0.05 0.23ab 0.84ab 0.07 0.14 0.67 0.55 1.48 0.16 0.37 7.80b 0.54 0.24 0.18 0.64 0.30 2.14 18.74 33.86 15.51a 30.34 19.64e 12.77ab 29.26b

0.72bc 0.08 19.48 0.66 0.09b 0.21 0.15 0.22 12.39 14.15b 1.54a 0.14 7.48c 0.16 0.05 6.78c 0.14 0.05 0.19a 1.06b n.d. 0.14 0.35 0.28 1.51 0.75 0.76 8.66b 0.62 0.15 0.21 0.28 0.21 1.81 18.19 33.51 18.17a 37.35 10.51c 15.53b 30.21b

Different letters in the same line denote significant differences ( P < 0.05).

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The fatty acid in macrophages/fatty acid in diet ratio was lower than 1 for the nonessential fatty acids (oleic, linolenic and linoleic acids) (Table 8). However, for the essential fatty acids, this ratio was higher than 1: macrophage DHA/dietary DHA ratio is around 2, AA macrophage/dietary AA ratios ranged from 3 to 7 (being the highest ratios from those fish fed the diets with lower AA content), The EPA macrophage/dietary EPA ratio was higher than 1 in those fish fed the lowest EPA contents in diet (Diets 80S and 80L) (Table 8). 3.5. Fatty acid composition of erythrocytes As described above for the macrophages, dietary fatty acids from vegetable oil increased both linoleic and linolenic acids in erythrocytes of fish fed diets with soybean (diets 60SO2 and 80SO) or linseed oil (diets 60LO2 and 80LO), respectively (Table 9). Monoenoic fatty acids were increased in erythrocytes of fish fed rapeseed oil (60RO2), mainly due to an increase of oleic acid. Fish fed FO2 diet showed the highest n-3 HUFA proportion in erythrocytes (Table 9), mainly due to an increase of EPA, since there were no significant differences among groups in DHA content (Table 9).

Fig. 1. Time course of plasma cortisol concentrations in gilthead seabream fed different vegetable lipid sources during a 101-day experimental period (Experiment I). Asterisk denotes significant differences ( P < 0.05) from control fish at the same hour.

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Fig. 2. Evolution of plasma cortisol concentrations in gilthead seabream fed different vegetable lipid sources during a 204-day period) (Experiment II). Asterisk denotes significant differences ( P < 0.05) from control fish at the same hour.

3.6. Stress test Different patterns in the evolution of plasma cortisol levels were observed after acute stress induction in fish fed the vegetable lipid sources in comparison to fish fed fish oil (Figs. 1 and 2). The highest values of plasma cortisol were found 1 h after the stress in fish fed fish oil, whereas in diets containing vegetable lipid sources these values were highest after 2 h of stress induction in Experiment I. At this sampling point, fish fed linseed oil showed significant ( P < 0.05) higher post stress plasma cortisol values when compared to fish fed 100% fish oil diets (Fig. 1). Maximum values of plasma cortisol were detected in Experiment II after 2.5 h of stress induction and were significantly higher ( P < 0.05) in fish fed both diets containing linseed oil when compared to fish fed FO2 diet (Fig. 2).

4. Discussion Some evidence of an effect of dietary fatty acid imbalances on different mechanisms of the immune system of cultured fish has been reported (Erdal et al., 1991; Sheldon and Blazer, 1991; Kiron et al., 1995; Montero et al., 1999), including the activation of the alternative complement pathway activity (Montero et al., 1998). However, little is known

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about the specific role of dietary fatty acids in the different processes involved in the immune system of fish. Feeding diets with a 60% fish oil replacement by soybean, linseed or rapeseed oils for 101 days (Experiment I) did not significantly depressed humoral immune response in comparison to diets containing 100% fish oil. However, the inclusion of soybean oil or rapeseed oil in the diet during prolonged feeding periods (204 days) affected both humoral and cellular immunology in seabream (Experiment II). Extreme substitution levels may alter the production of compounds with relevant physiological activity, such as eicosanoids, resulting in imbalances of both AA-derived and EPA-derived eicosanoids (Ashton et al., 1994), which may act as inmunomodulators and affect fish resistance to diseases. As an example, the inclusion of 7% linseed oil in diets for catfish reduced fish survival to infection with Edwarsiella ictaluri at a temperature of 28 jC, as well as the ability of fish to produce antibodies (Fracalossi and Lovell, 1994). The lack of n-3 HUFA in rainbow trout diet decreased the resistance of fish to IHN virus (Kiron et al., 1995). In our study, although there was no effect of vegetable oil on serum lysozyme activity, the inclusion of soybean oil in the diet depleted the alternative complement pathway activity. Similar effects have been described for cod, showing the complement activity of this species as a negative correlation with monoenes present in serum (Waagboo et al., 1995). Cellular immunity has been described to be affected by the inclusion of vegetable oil in the diet. The bactericidal activity of macrophages from catfish decreased in fish fed soybean oil-containing diet instead of a menhaden oil-based diet (Sheldon and Blazer, 1991). In our experiment, the phagocytic activity of head kidney macrophages was affected in fish fed either rapeseed or soybean oil-containing diets. The high content of either linolenic or monoenes (mainly oleic acid) in the cell membrane and imbalances among fatty acids could influence membrane physical properties and, hence, phagocytic activity. These effects of dietary oils on macrophage-dependent both humoral and cellular immunity could be produced by an imbalance in the fatty acid composition of membrane pospholipids, affecting the physical properties of the membrane (i.e. permeability and fluidity), and the activity of membrane-associated receptors, which could be the reason for a decrease in complement activity. In our study, the nature of the dietary oils determined the fatty acid profile of macrophages as has been observed in other species (Waagboo et al., 1995; Farndale et al., 1999). However, a selective incorporation of certain fatty acids can be observed in head kidney macrophages. DHA is preferentially incorporated and retained into macrophages, since macrophage DHA/dietary DHA ratio is around 2, denoting the importance of this fatty acid in this type of cell. Similar results have been described for cod (Waagboo et al., 1995) and European seabass (Farndale et al., 1999). DHA is the main component of phosphoglycerides of fish biomembranes (Henderson and Tocher, 1987) and a preferential retention of this fatty acid has been observed under dietary essential fatty acid deficiency (Kanazawa, 1985; Izquierdo, 1996; Montero et al., 2001). Furthermore, AA incorporation into macrophages was even more marked, denoting the importance of this fatty acid as a precursor of eicosanoids and intracellular messenger and its role in fish immune regulation. In our study, macrophage AA/dietary AA ratios ranged from 3 to 7, whereas these values ranged from 4.8 to 6.8 for European seabass (Farndale et al., 1999)

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and 5 to 14 in head kidney of cod (Waagboo et al., 1995). AA is selectively incorporated in phosphatidylinositol of plaice neutrophils (Tocher and Sargent, 1986) indicating that these cells have a high affinity for this fatty acid (Thompson et al., 1995). AA has been found to regulate the production of O2-radicals and degranulation of polymorphonuclear neutrophils (Ely et al., 1995). Thus, the preferential incorporation of these fatty acids into macrophages may prevent excessive production of free radicals by these cells, explaining the lack of significant differences in the NBT of circulating neutrophils. Due to the high content of dietary EPA, most of the fish showed lower EPA in head kidney macrophages than that found in each diet except for those fish fed diets 80SO and 80LO, where the dietary EPA content was the lowest, with macrophage EPA/dietary EPA ratios higher than 1. This could be suggesting that EPA is also selectively incorporated into head kidney macrophages and, taking into account that the AA/EPA ratio in fish fed all diets tend to be equal to 0.3, this selective incorporation of EPA could be related to AA incorporation. This is in contrast with those data found for European seabass by Farndale et al. (1999), who found a direct relationship of dietary EPA in circulating leucocytes of European seabass, although the lipidic sources used by these authors were rich in EPA (i.e. krill). Selective incorporation of certain fatty acids could also be observed in red blood cells, where DHA content remained constant regardless of inclusion of vegetable oil indicating the importance of this fatty acid in the maintenance of red blood cell membrane properties, such as osmotic resistance (Hagave et al., 1991). Similar results were found for Atlantic cod fed diets containing soybean, capelin or sardine oils (Waagboo et al., 1995) and Atlantic salmon (Waagboo et al., 1993). Klinger et al. (1996) found less susceptibility to lysis in the erythrocytes of channel catfish fed a menhaden oil-based diet when compared with fish fed a soybean diet. Haematology (in terms of haematocrit and haemoglobin) was not affected by dietary lipid, as described by Greene and Selivonchick (1990) for rainbow trout, but the RBC number was higher in fish fed fish oil-based diet which could be related to a higher oxygen requirement due to a higher peroxisomal h-oxidation (Waagboo et al., 1995). In agreement with this hypothesis, Grisdale-Helland et al. (2002) found a lower oxygen consumption in Atlantic salmon fed 50% soybean oil-containing diet when compared with 100% fish oil-containing diet. Dietary lipid also seemed to affect the resistance of gilthead seabream to stressful conditions, possibly by altering the activation of the hypothalamic – pituitary – adrenal (HPA) axis as is well known in mammals (Tannenbaum et al., 1997; Kamara et al., 1998), and subsequently modifying the plasma cortisol concentration after stress (Montero et al., 1998). However, the role of specific fatty acids on the activation of the stress response remains unclear. Tannenbaum et al. (1997) found that high-fat diets (based mainly on an increase of corn oil from 4% to 20% of the diet) produced a pattern of effects on rats similar to that observed after chronic stress. These authors proposed that elevation in basal corticosterone, as well as the increased stress-induced hypersecretion of both corticosterone and ACTH can be affected by high fat-induced elevation of blood fatty acids, due to their electrophysiological effects on cells of the ventromedial hypothalamus, inhibiting the neuronal firing rate in this area and hence the inhibitory control of those cells on the activation of the HPA axis (Dallman, 1984).

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In both experiments, fish fed linseed oil-containing diets showed an increase on plasma cortisol concentration after stress. At present, there is no evidence of a specific role of linolenic acid in the stress response and further experiments are required to elucidate the role of each fatty acid on the activation of the hypothalamic – pituitary –interrenal axis of fish. In summary, fish oil may be partially replaced by vegetable oil in diets for gilthead seabream, but long periods of feeding soybean oil at a 60% replacement level may include immunosuppression in seabream. Rapeseed oil may reduce the phagocytic activity of head kidney macrophages of seabream, and linseed oil alters the stress response of seabream. Finally, fish fed a blend of the three vegetable oils used in the present study performed better in terms of health and stress resistance than those fed vegetable oil. Acknowledgements This study was partially supported by research grants from EU and the Spanish Government under FEDER program, project no. 1FD1997-1774 and from the European Union under ‘‘Quality of Life and Management of Living resources’’ through the project Q5RS-2000-30058 ‘‘RAFOA’’. References Anderson, D.P., Siwicki, A.K., 1994. Simplified assays for measuring non-specific defense mechanisms in fish. Am. Fish. Soc. Fish Health Section. Spec. Publ., 26 – 35 (Seattle, WA). Ashton, I., Clements, K., Barrow, S.E., Secombes, C.J., Rowley, A.F., 1994. Effects of dietary fatty acids on eicosanoid-generating capacity, fatty acid composition and chemotactic activity of rainbow trout (Oncorhynnchus mykiss) leucoytes. Biochim. Biophys. Acta 1214, 253 – 262. Bell, J.G., McVicar, A.H., Sargent, J.R., Thompson, K.D., 1993. Dietary sunflower, linseed and fish oils affect phospholipid fatty acid compostition, development of cardiac lessions, phospholipase activity and eicosanoid production in Atlantic salmon (Salmo salar). Prostaglandins, Leucotrienes Eicosanoid Fatty Acids 49, 665 – 673. Bell, J.G., Tocher, D.R., Sargent, J.R., 1994. Effect of supplementation with 20:3 (n-6), 20:4(n-6) and 20:5(n-3) on the production of prostaglandins E and F of the 1-, 2- and 3-series in turbot (Scophthalmus maximus) brain astroglial cells in primary culture. Biochem. Biophys. Acta 1211, 335 – 342. Blazer, V.S., 1991. Piscine macrophage function and nutritional influences: a review. J. Aquat. Anim. Health 3, 77 – 86. Christie, W.W., 1982. Lipid Analysis. Pergamon, Oxford. Dallman, M.F., 1984. Viewing the ventromedial hypothalamus from the adrenal gland. Am. J. Physiol. 246, 1 – 12. Ely, E.W., Seeds, M.C., Chilton, F.H., Bass, D.A., 1995. Neutrophil release of arachidonic acid, oxidants, and proteinases: casually related or independent. Biochim. Biophys. Acta 1258, 135 – 144. Erdal, J.I., Evensen, O., Kaurstad, O.K., Lillehaug, A., Solbakken, R., Throud, K., 1991. Relationship between diet and immune response in Atlantic salmon (Salmo salar, L.) after feeding various levels of ascorbic acid and omega-3 fatty acids. Aquaculture 98, 363 – 379. Esteban, M.A., Mesenger, J., 1997. Factors influencing phagocytic response of macrophages from the sea bass (Dicentrarchus labrax L.): an ultrastructural and quantitative study. Anat. Rec. 248, 533 – 541. Farndale, B.M., Bell, J.G., Bruce, M.P., Bromage, N.R., Oyen, F., Zanuy, S., Sargent, J.R., 1999. Dietary lipid composition affects blood leucocyte fatty acid compositions and plasma eicosanoid concentrations in European sea bass (Dicentrarchus labrax). Aquaculture 179, 335 – 350.

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