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Woodall, A. N., Ashley, L. M., Halver, J. E., Olcott, H. S. & Van der Veen, J. (1964). Nutrition of salmonid fishes. XIII. The α-tocopherol requirement of chinook.
Fish & Shellfish Immunology (2000) 10, 293–307 doi:10.1006/fsim.1999.0238 Available online at http://www.idealibrary.com on

High dietary intake of -tocopherol acetate enhances the non-specific immune response of gilthead seabream (Sparus aurata L.) J. ORTUÑO, M. A. ESTEBAN

AND

J. MESEGUER*

Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain (Received 10 May 1999, accepted 19 July 1999 ) To determine the e#ects of three high levels of dietary intake of -tocopherol acetate (vitamin E) on the non-specific immune response of gilthead seabream (Sparus aurata L.), specimens were fed a commercial diet (100 mg -tocopherol kg 1) as control, or vitamin E supplemented diets (600, 1200 or 1800 mg -tocopherol acetate kg 1) for 15, 30 or 45 days. Growth, serum -tocopherol levels, natural haemolytic complement activity and head–kidney leucocyte migratory, respiratory burst and phagocytic activities were studied at each of the assay times. A positive correlation between -tocopherol acetate intake and serum -tocopherol levels was observed, the increase being linked to both the dosage and length of treatment. Specimens fed the diet supplemented with 600 mg vitamin E kg 1 showed no enhancement in any of their immune parameters, while those fed the diet supplemented with 1200 mg vitamin E kg 1 presented a slightly higher (but not statistically significant) specific growth rate than fish fed the other diets. In addition, serum haemolytic activity and the phagocytosis of head–kidney leucocytes were enhanced by the dietary intake of 1200 mg vitamin E kg 1 after 30 and 45 days of treatment, although leucocyte migration and respiratory burst activity remained una#ected. The highest vitamin E dietary dose used, 1800 mg kg 1, unexpectedly provoked no immunostimulation. These results indicate that a moderate level of vitamin E in the diet (1200 mg kg 1) stimulates the seabream’s non-specific immune system after 30 days of administration. Lower or higher vitamin E concentrations may not be so e#ective, because of an imbalance in the vitamin E ratio with other antioxidants. The proposed dietary levels of vitamin together with the indicated administration time could be useful for reducing the susceptibility of farmed fish to infectious diseases.  2000 Academic Press Key words:

vitamin E, non-specific immune response, complement, leucocytes, gilthead seabream (Sparus aurata L.)

I. Introduction Infectious diseases are the main cause of economic loss in aquaculture (Blazer & Wolke, 1984) and, as a consequence, many studies have looked into the modulation of the fish immune system in order to prevent the outbreak of disease. Evidence from homeotherms and, more recently, from fish, reveal that *Corresponding author. E-mail:[email protected] 1050–4648/00/040293+15 $35.00/0

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diet a#ects the immune system (Chandra, 1988; Landolt, 1989; Chew, 1995; Ortun˜ o et al., 1999). Furthermore, because it is well known that the deficiency of some micronutrients produces pathological signs and immunodepression, vitamins and minerals are now included in farmed fish food to promote optimal growth and health. At present, the e#ects of these micronutrients on the fish immune system are not well established since they are not always apparent or predictable. Although the minimum requirements for these micronutrients have been established in some fish species (Cowey et al., 1981; Wilson et al., 1984), very few studies have focused on the relationship between the immune system and dietary supplements above the minimum levels established for such micronutrients (Montero et al., 1999). Vitamin E is essential for most vertebrate species studied (Turner & Finch, 1990; Le Grusse & Watier, 1993). It is a structural component of the membranes, and also has antioxidant properties which prevent unsaturated fatty acid peroxidation (Le Grusse & Watier, 1993). Since fish have high levels of unsaturated fatty acids to maintain cell membrane fluidity especially at low temperatures (Blazer, 1992), it is assumed that vitamin E plays an important role. Indeed, pathological signs, such as growth reduction, haematological alterations, anaemia, epithelial pigmentation loss, ascites, exophtalmia and apathy appear in fish fed vitamin E deficient diets (Wilson et al., 1984; Hamre et al., 1997). In addition, a marked immunodepression has been observed when vitamin E levels are depleted (Blazer & Wolke, 1984; Hardie et al., 1990), a finding which confirms its interaction with the immune system. It is well established that a high dietary vitamin E supplementation enhances both humoral and cellular defences in mammals (Tengerdy et al., 1981; Panush & Delafuente, 1985; Tengerdy, 1989; Bendich, 1990; Moriguchi et al., 1990). Taking into account that fish possess a well developed immune system as well as hydrolytic and transport mechanisms for vitamin E similar to those found in other vertebrates (Gallo-Torres, 1980), the fish immune system might be expected to respond in the same way as in homeotherms. Contrary to the above hypothesis, Hardie et al. (1990) observed no immunostimulation in rainbow trout fed a high dose of vitamin E. However, few studies have paid attention to the e#ects of a high dietary vitamin E supplementation on the immune system of farmed fish (Montero et al., 1999). In an attempt to shed more light on this question, the aim of this study was to provide more information related to the impact of vitamin E on the nonspecific immune system of teleosts. For this, di#erent dietary vitamin E doses, above the minimum requirement established for other species, were tested over various times in order to study their e#ect on both the humoral and cellular non-specific immune responses of the sparid gilthead seabream (Sparus aurata L.). II. Materials and Methods ANIMALS

Sixty specimens (15018 g mean weight) of the hermaphroditic protandrous seawater teleost gilthead seabream (Sparus aurata L.) obtained from

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Table 1. Composition and ingredients of the basic diet Composition Crude protein Crude fat Total ash Brute cellulose Additives

Ingredients 47% 21% 11% 2% 19%

Fish meal Oily meal Cereals Terrestrial animal subproducts Fish oil Vitamins: A, D, E (-tocopherol 100 mg kg 1) Minerals synthetic antioxidants (100 ppm)

(Data from ProAqua Nutrition S.A., Palencia, Spain.)

Culmarex S.A. (Murcia, Spain), were kept in running (flow rate 1500 l h 1) seawater (salinity 22%) aquaria, at 201 C and with a natural photoperiod. Fish were allowed to acclimate 30 days before the beginning of the experiment. FEEDING

Four experimental diets were prepared in the laboratory from a commercial pellet diet (-tocopherol content 100 mg kg 1, ProAqua Nutritición S.A., Palencia, Spain) (Table 1). For this, three vitamin E solutions of 24, 48 and 72 mg (+)--tocopherol acetate (Sigma) ml 1 fish oil were made. Supplemented diets were prepared daily by spraying the vitamin solutions uniformly on the feed at a ratio of 25 ml kg 1 dry weight to obtain final concentrations of 600, 1200 and 1800 mg vitamin E kg 1 diet. The non-supplemented diet was prepared by spraying the feed with fish oil only. The -tocopherol level of the fish oil used to dilute the vitamin E was less than 1 g ml 1 as determined by HPLC analysis, which was carried out as described below for serum vitamin E determination. The specimens were divided randomly into four groups and each group was fed one of the four di#erent diets. Fish were fed at a rate of 10 g dry diet kg 1 biomass each day for 15, 30 or 45 days. The biomass of fish in each aquarium was measured before the experiment and after each sampling, with the daily ration being adjusted accordingly. SAMPLES COLLECTION

Five fish of each group were randomly sampled after 15, 30 and 45 days of treatment, being kept 24 h without feeding before sampling. The fish were anaesthetised with benzocaine (4% in acetone) (Sigma), weighed and measured. Blood and head-kidney samples were obtained from each specimen and the immunological competence determined as described below. Blood samples were collected from the caudal vein with a 27 gauge needle and a 1 ml syringe. Blood samples were allowed to clot at room temperature for 4 h. Following centrifugation, the serum was removed and frozen at 80 C until use.

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Head-kidney leucocytes were isolated as described previously (Esteban et al., 1998). Briefly, the head-kidney was dissected out by a ventral incision, cut into small fragments and transferred to 8 ml sRPMI-1640 medium: RPMI1640 medium supplemented with 10 I.U. ml 1 heparin (Sigma), 100 I.U. ml 1 penicillin (Biochrom), 100 g ml 1 streptomycin (Biochrom), 2% foetal calf serum (FCS)(Gibco) and 0·35% sodium chloride (Sigma) to adjust medium osmolarity to gilthead seabream plasma osmolarity (353·33 mOs). Cell suspensions were obtained by forcing fragments of the organ through a nylon mesh (mesh size 100 m). Head-kidney cell suspensions were layered over a 34–51% Percoll density gradient (Pharmacia) and centrifuged at 400  g for 30 min at 4 C. After centrifugation, the bands of leucocytes above the 34–51% interfaces were collected with a Pasteur pipette, washed twice, counted and adjusted to 107 cells ml 1 in sRPMI. Cell viability was greater than 95%, as determined by the trypan blue exclusion test. GROWTH

After 45 days, specific growth rates (SGR, % body weight day 1) for each group were determined using the equation: SRG=100 (logn Wf – logn Wo) t 1, where Wo and Wf were the initial and final weights of each experimental group, respectively, after t days (Ricker, 1979). SERUM

-TOCOPHEROL LEVELS

Serum -tocopherol levels of all the experimental specimens were determined by HPLC analysis using a modification of the methods described by Cowey et al. (1981) and Indyk (1988). NATURAL HAEMOLYTIC COMPLEMENT ACTIVITY

The activity of the alternative complement pathway was assayed using sheep red blood cells (SRBC, Biomedics) as targets (Ortun˜ o et al., 1998). SRBC were washed in phenol red-free Hank’s bu#er (HBSS) containing Mg +2 and EGTA and resuspended at 3% in HBSS. Aliquots of 500 l of test serum as complement source, diluted in HBSS, was added to 500 l of SRBC to give final concentrations of 10, 5, 2·5, 1·25, 0·625, 0·313, 0·1565 and 0·078%. After incubation for 1 h at 22 C the samples were centrifuged at 800g for 5 min at 4 C to avoid unlysed erythrocytes. The relative haemoglobin content of the supernatants was assessed by measuring their optical density at 540 nm in a spectrophotometer. The values of maximum (100%) and minimum (spontaneous) haemolysis were obtained by adding 500 l of distilled water or HBSS to 500 l samples of SRBC, respectively. The degree of haemolysis (Y) was estimated and the lysis curve for each specimen was obtained by plotting Y/(1-Y) against the volume of serum added (ml) on a log-log scaled graph. The volume of serum producing 50% haemolysis (ACH50) was determined and the number of ACH50 units ml 1 was obtained for each experimental group. MIGRATION ASSAY

The migratory activity of seabream head-kidney leucocytes was studied in a 48 well microchemotaxis chamber (Neuroprobe)(Ortun˜ o et al., 1998). To the

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lower wells of the chamber 32 l of 10% diluted normal gilthead seabream serum was added as attractant. The wells were then covered with a 5 m pore size polyvinylpyrrolidone-free polycarbonate filter (Millipore) and 43 l of the leucocyte suspensions were placed in triplicate in the upper chamber. The cells were allowed to migrate for 90 min at 22 C in a 85% humidity and 5% CO2 atmosphere before the apparatus was dismantled. Aliquots of 32 l of RPMI-1640 culture medium were placed in the lower wells of the control wells. After incubation, the cells on the upper surface of the filter were removed by scraping with a rubber blade, and the filter fixed in methanol and stained with Giemsa (Merck). The cells visible on the lower surface of the filter covering each well were counted in three random fields of view (400) with a Nikon microscope. Data were expressed as mean number of cells per field. RESPIRATORY BURST

The respiratory burst activity of gilthead seabream head-kidney leucocytes was studied by flow cytometry. Each assay was carried out according to Banati et al. (1994). Dihydrorhodamine-123 (DHR)(Molecular Probes) was dissolved in N,N-dimethylformamide (Sigma) at a concentration of 1 mM (stock solution), and stored in 25 l aliquots at 80 C. Samples of 30 l head-kidney leucocyte suspensions (previously adjusted to 107 cell ml 1 in RPMI-1640 culture medium) were placed in 5 ml tubes (Falcon, Beckton Dickinson) and 5 l of the working DHR solution (10 M in HBSS) were added to each tube, before being incubated for 5 min at 22 C. After incubation, 300 l of phorbol 12-myristate 13-acetate (PMA, Sigma) solution (1 ng ml 1 in HBSS) were added to each sample (except control tubes, which received HBSS) and the tubes were incubated for an additional 30 min. Following incubation, samples were immediately analysed in a FACScan (Becton Dickinson) flow cytometer with an argon-ion laser adjusted to 488 nm. Instrumental settings were adjusted to obtain optimal discrimination of the di#erent cell populations present in seabream head-kidney leucocyte suspensions. DNA staining with propidium iodide (PI) (Sigma) was performed to exclude cell debris (Orpegen Pharma, 1995). Cell suspensions (106 cells 200 l 1 of PBS) were fixed in ethanol for 30 min at 4 C, washed by centrifugation and resuspended in 800 l of fresh PBS. Then 100 l of RNase (Sigma) (1 mg ml 1) and 100 l of PI (400 mg ml 1) were added to each sample, before the samples were incubated (37 C, 30 min), acquired and analysed. Data were collected in the form of two-parameter side scatter (granularity) (SSC) and forward scatter (size) (FSC), or green fluorescence (rhodamine 123) (FL1) and red fluorescence (PI) (FL2) dot plots and fluorescence histograms on a computerised system. Each of the analyses was performed on 10 000 cells, which were acquired at the rate of 300 cells s 1. Fluorescence histograms represented the relative fluorescence on a logarithmic scale. Standard samples of leucocytes not incubated with DHR were included in each assay. Samples incubated at 4 C were used as negative controls and samples containing DHR-labelled leucocytes and 30 l 1 mM hydrogen peroxide (Merck) as positive control. Activated leucocytes showed high respiratory burst activity, which allowed the oxidation of nonfluorescent DHR to the green fluorescent rhodamine 123. The percentage

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of activated leucocytes was established as the percentage of green fluorescent cells within the total cell population. The degree of activation was assessed from the mean fluorescence intensity of the cells. BACTERIA

Vibrio anguillarum (now named Listonella anguillarum) strain R82 (serotype 01) (Toranzo & Barja, 1990) was grown in trypticase soy broth (TSB) (Gibco) on agar plates from 1 ml aliquots of stock cultures that had been frozen at 80 C. To label bacteria with fluorescein isothiocyanate (FITC) (Sigma), an isolated colony from each culture was expanded in TSB with FITC (50 or 100 g ml 1) and grown overnight at 20 C in a light-protected microenvironment. The optical density of V. anguillarum cell suspensions was measured at 600 nm, the number of cfu ml 1 being adjusted to 109 with standard curves of growth in phosphate bu#ered saline solution (PBS). After labelling, free FITC was removed by washing three times in PBS, and the bacteria were heat-killed at 60 C for 15 min. After inactivation, the bacteria were washed again. The staining was uniformity examined and then the bacterial cell suspensions were aliquoted and stored at 80 C. PHAGOCYTIC ACTIVITY

The phagocytic activity of gilthead seabream head-kidney leucocytes was studied by flow cytometry according to Esteban et al. (1998). Samples consisting of 100 l head-kidney leucocyte suspensions, previously adjusted to 107 cells ml 1 in sRPMI-1640, were placed in 5 ml Falcon tubes. To each tube 10 l of FITC-labelled bacteria was added and the samples were centrifuged (400g, 5 min, 22 C). Afterwards, the samples were resuspended and incubated at 22 C for 1 h. At the end of the incubation time the samples were placed on ice to stop phagocytosis and 500 l ice-cold PBS were added to each sample. The fluorescence of the extracellular bacteria (i.e. free bacteria and bacteria adhered to phagocytes but not ingested) was quenched by adding 8·5 l ice-cold trypan blue (0·4% in PBS) per sample. Immediately, the samples were mixed gently and analysed in the flow cytometer. Standard samples of FITC-labelled V. anguillarum cells or seabream leucocytes were included in each phagocytosis assay. Samples incubated at 4 C were used as negative controls. Phagocytic ability was defined as the percentage of cells with one or more ingested bacteria (green-FITC fluorescent cells) within the total cell population (10 000 cells). The number of ingested bacteria per cell (phagocytic capacity) was assessed from the mean fluorescence intensity of the cells. STATISTICAL ANALYSIS

All assays were performed in triplicate and the mean S.E. for each dietary group (five fish per group) was calculated. The data from the flow cytometric assays were studied by using the statistical option of the Lysis Software Package (Becton Dickinson). Data were analysed statistically by one-way analysis of variance (ANOVA) to observe any di#erence due to time or

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Table 2. Absolute values of serum -tocopherol levels in gilthead seabream specimens fed commerical diet (control) or -tocopherol acetate supplemented diets Vitamin E (mg kg 1) supplemental doses 0 (Control) 600 1200 1800

15 days

30 days

45 days

102·9a 183·2a,b 210·9a,b 335·2b

123·5a 376·3a,b 5215a,b 673b

142·8a 283·5a 6111a,b 8813b

Data are shown as mean S.E. g -tocopherol ml 1 serum.a,b,c represent statistical results (di#erent letters represent statistical di#erences due to treatment).

treatment. Bonferroni’s test was used to determine di#erences between groups. Di#erences were considered statistically significant when P