Essential fatty acid nutrition and defence mechanisms in rainbow trout Oncorhynchus mykiss

Camp. Biodwn.

Pergamon 0300-%29(95)00042-9

Physiol. Vol. I I IA, No. 3. pp. 361-367. 1995 Copyright 0 1995 Elsevier ScienceLtd Printed in Great Britain. All rights reserved 0300-9629/95 $9.50 + 0.00

Essential fatty acid nutrition and defence mechanisms in rainbow trout Oncorhynchus wtykks Viswanath Kiron, Hideo Fukuda, Takeshi Watanabe

Toshio

Takeuchi

and

Department of Aquatic Biosciences, Tokyo University of Fisheries, Konan 4, Minato, Tokyo 108, Japan The influence of saturated and polyunsaturated fatty acids on immune responses of rainbow trout was investigated. In vitro killing of bacteria by macrophages and antibody production were compromised by dietary essential fatty acid deficiency in fish. Polyunsaturated fatty acids fed fish were stronger in resisting pathogens, but excess levels of n-3 highly unsaturated fatty acids may not be effective. Essential fatty acids enhance immunocompetence. Key words: Antibody

Macrophages; Comp. Biochem.

production; Essential fatty acids; Health; Immune responses; Infection; Nutrition; Rainbow trout. Physiol.

IllA,

361-367,

1995.

Introduction The firm links established between nutrition and immune function in higher vertebrates have caused a growing realization that such dependence might as well be prevalent in fish. The complexity of nutritional influence on the functional capacities of the immune system in particular and fish health in general is an area of recent research interest. However, the focus of most of the studies (Blazer and Wolke, 1984a,b; Durve and Lovell, 1982; Hardie et al., 1990, 1991; La11et al., 1985, 1990; Li and Lovell, 1985; Navarre and Halver, 1989; Ndoye et al., 1990; Obach and Baudin Laurencin, 1992; Patterson et al., 1985; Scarpa et al., 1992) has been vitamin C, E or minerals. The importance of dietary lipid on disease resistance factors in fish has recently been investigated by Blazer and her team (Blazer et al., 1989; Blazer, 1991; Sheldon and Blazer, 1991), Erdal et al. (1991) and Waagber et al. (1993a).

The relationship of lipids to the immune functions in man and animals is multifactorial (Gurr, 1983); involving not only the dietary lipid-immune system interaction, but also the biochemical initiations at the cellular level, considering that many essential and non-essential fatty acids are contained in the lymphocytes. Various events involved in lymphocyte activation and function are related to arachidonic vertebrates metabolism in higher acid (Gershwin et al., 1985). Lipids can exert important regulatory activity on the fixed cells of the reticula-endothelial system, thereby influencing the immunologic response to antigen (Gross and Newberne, 1980). The different effects produced by polyunsaturated fatty acids (PUFA) on immunologic function have been summarized by Meade and Mertin (1976) from their own work. Gross and Newberne (1980) have suggested in their review that PUFA exert a part of their effects, through spleen immunoregulatory mechanisms, or directly on lymphocyte plasma membranes, or indirectly by acting as precursors for the synthesis of prostaglandins. The in vitro and in vivo effects of fatty acids have been reviewed by Gershwin et al.

Correspondence IO: V. Kiron, Dept of Aquatic Biosciences, Tokyo University of Fisheries, Konan 4, Minato. Tokyo 108. Japan. Tel. 3 5463 0555; Fax 3 5463 0553. Received 27 June 1994; revised 19 December 1994: accepted 20 December 1994. 361

362

V. Kiron

(1985). The importance of lipid in nutritional immunology has been evaluated lately by Maki and Newberne (1992). The n-3 fatty acids are important for both fresh water and marine fish. Watanabe (1982) has shown that the n-3 HUFA has a higher essential fatty acid (EFA) value for rainbow trout, and the satisfying requirement level was 0.5% in the diet. Our experiments were intended to examine the impact of essential fatty acid nutritional status on different mechanisms involved in operating the immune system of Oncorhynchus

mykiss.

Materials and methods Eyed eggs of rainbow trout obtained from two different hatcheries were transported to the fish-care unit of our laboratory. The eggs originating either from the Okutama or the Fuji population were hatched out and weaned on commercial diets. The experimental fish were chosen from the Okutama population (unless otherwise stated) and subjected to the different dietary treatments as per the design. The formulations used in this experiment did not have any bearing of the rest&s from the accompanying paper (Kiron et al., 1995). The diets were made from purified ingredients, varying the lipid composition for the different experiments, and pelleted to suit the different size groups of fish. The casein based diets contained about 50% protein and 5% lipid as frequently employed in nutritional experiments with rainbow trout (Takeuchi et al., 1978). The fish were fed to satiation at 09.00 and 17.00 hr.

et al.

acid; LA), IF, (linolenic acid; LNA), and IF, (n-3 highly unsaturated fatty acids; n-3 HUFA), the latter three the polyunsaturated fatty acid (PUFA) groups. Group IF, was the only essential fatty acid (EFA) deficient group, whereas the other three (IF,, IF1 and IF,) were the EFA receiving fish. The diets were offered to the groups twice a day to satiation. The mean fish weights in the groups IF,, IF,, IF, and IF, prior to macrophage isolation were 177 g, 257 g, 225 g and 245 g, respectively. By the end of 13 weeks, the fish were collected to remove the pronephros for macrophage isolation. The fish were anaesthetised in 300 ppm phenoxyethanol, and the body of the fish was doused with 70% ethyl alcohol before moving it to the sterile facilities for dissection of the head kidney. The macrophage isolation and killing activity determination in vitro were performed according to the description of Secombes (1990) with some modifications. Extra care was taken to conduct the operation using sterile procedures. The head kidney was gently pushed with Teflon tabs through a fine meshed steel sieve into L-15 medium (Gibco Laboratories) containing 2% fetal calf serum (FCS, Gibco Laboratories), 100 U/ml penicillin/streptomycin (P/S, Banyu Pharma, Meiji Seikha Kaisha) and 10 U/ml heparin (Sigma Chemical Company). This cell suspension was carefully layered onto 34%/51% (v/v) Percoll gradient (Sigma

Table I. Composition of the preparatory diet, basal diet and the diets in the experiment to assess the in vitro killing activity of rainbow trout macrophages Quantity

Defence

mechanisms

Different feeding experiments were conducted with rainbow trout to understand the influence of saturated and polyunsaturated fatty acids on macrophage function and antibody production. In vitro macrophuge activity. Fish from the stock grown on commercial rations were selected at a weight of about 80 g. They were then gradually trained on the purified casein-based lipid deleted diet (Table 1) which was continued thereafter for 19 weeks, twice a day. At the end of this preparatory period the fish size range was 159-191 g. Ten fish were each then kept in 60 1 flow-through tanks as four dietary treatments. The rearing temperature during the 13-week experimental period was 15 f 2°C. The experimental diets were formulated by replacing 5 g dextrin in the preparatory diet with a corresponding amount of lipid, constituted by the respective fatty acids (Table 1). The four diet groups were designated as IF, (palmitic acid; PA), the saturated fatty acid group; IF, (linoleic

Ingredient Casein Amino acid mixture* cc-Starch Dextrin Lipid? Vitamin mixturei Mineral mixture* Cellulose

Preparatory 52.0 1.3 15.0 20.0 0.0 1.6 5.0 5.1

diet

Basal diet 52.0 1.3

IS.0 15.0 5.0 1.6 5.0 5.1

*As described in Kiron et a/. (1995). tMethyl esters of the following fatty acids were added to the respective diets: Group IF,, 5g Palmitic acid (purity 96%. Tokyo Chemical Industry); Group IF?, 1 g linoleic acid (purity 95%, Tokyo Chemical Industry): Group IF,, 1g linolenic acid (purity 99%, Sigma Chemical Company); Group IF,, 1 g n-3 HUFA (purity 85%. Nippon Chemical Feed Company). Remaining portion of lipid in Groups IF,. IF, & IF, constituted by palmitic acid. $Vitamin mixture: 1.0 g vitamin blend; 0.1 g DL-C( tocopherol (50% purity); 0.5 g choline chloride. The vitamin blend included the respective amount of the following vitamins per 100 g of diet:-B, 6 mg; II, IO mg; B, 4 mg; Blz 0.01 mg; C 500mg; niacin 40 mg; calcium pantothenate 10 mg; inositol 200 mg; biotin 0.6 mg; folic acid I .5 mg; PABA 5 mg; K, 5 mg; A 4000 IU; D, 4000 IU.

EFA nutrition

and immune

Chemical Company) in tubes. This was centrifuged (400g) for 25 min at 4°C and the cell band at the 34-5 1% region was harvested for macrophages. After the cell count with Trypan Blue, the suspension was adjusted with L-15 (containing 0.1% FCS and 100 U/ml P/S) to provide 2 x IO’ viable cells/ml. The cell suspension was delivered (100 ~1) to each of the 96 wells in a microtitre plate. After incubating the plate for 2 hr at 15°C the wells were washed repeatedly three times with L-l 5 medium, primarily to remove unattached cells. The layer of cells that remained attached in the well was provided with 100 ~1 of another L-l 5 preparation (containing 5% FCS) and then left again at 15°C. The plates were kept for about 60 hr before using them for the bactericidal assay. After confirming the attached macrophage cell number from duplicate sample wells per plate by nuclei release count to fall within the same range, the plates were washed twice with plain L-l 5; and the cells were supplemented with L-15 medium (containing 5% FCS). Aeromonas salmonicida cultured in trypto soy broth (TSB) were collected and bacterial dilution from 10’ cells/ml (6.25 x lo4 cells) was added to each well. The plates were shaken and soon centrifuged at 15Og for 5 min. The cells were incubated at 15°C for 0 and 5 hr, allowing the bactericidal action to take effect. At the two time limits, the supernatants were removed from the wells and the macrophages were lysed by adding 50 ~1 of 0.2% Tween 20 to stop the killing. One hundred microlitres of TSB were added to the wells to help the surviving bacteria to grow for 20 hr at 15°C. The bacterial number was estimated by adding 10 ~1 of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT, Dojin Kagaku Kenkyujo; 0.5% w/v in distilled water) to every well. The plate was mechanically shaken for exactly 15 min and immediately read at 620 nm in a plate reader (Tosoh MPR A4). The survival percentage was calculated based on the 0 and 5 hr values. Mean values were calculated from eight fish per treatment. Antibody production. The Okutama fish population weaned on a commercial trout ration was gradually offered a fat-free casein based preparatory diet (Table 1) for about 24 weeks. Thereafter, at a weight of about 70 g, they were separated into six diet groups, 10 fish per tank in duplicates. The experimental feeding period of 9 weeks was initiated at this juncture. Group IIF, was continued on the fat-free diet mentioned above. Group IIF, received palmitic acid, IIFz linoleic acid, IIF linolenic acid and IIF, n-3 highly unsaturated fatty acid 0.5% (Table 2). IIF, and IIF, were the EFA deficient

responsesin

363

trout

Table 2. Lipid composition of the diet* fed to the different groups in the experiment to determine antibody response Diet groups Fatty (g)

acid?

Palmitic acid Linoleic acid Linolenic acid n-7 HUFA

IIF,

-

IIF,

IIF,

IIF,

IIF,

5.0

4.0 1.0 -

4.0 I.0

4.5 0.5

-

*The basal diet composition is same as shown tMethyl esters of fatty acids; see also footnote

in Table of Table

I.

I.

groups, whereas the rest received an adequate amount of dietary EFA. The mean size of the fish ranged from 96 g in Group IIF, to 173 g for Group IIF, at the end of 9 weeks. One lot of fish from each dietary group was immunized with formalin-killed Aeromonas salmonicida. The other group which served as the control fish was inoculated with an identical volume of Freund’s complete adjuvant. The antibody production in both immunized and non-immunized fish was measured 6 weeks later by enzyme-linked immunosorbent assay (ELISA). The methodological details were presented in the former publication (Kiron et al., 1995). The values were expressed as OD at 492 nm relative to the titres of the non-immunized serum. Disease resistance capacity

Trout hatchlings were weaned on preparatory microdiets (Table 1) until they were separated for the experiment. Two experiments were run with rainbow trout: Trial l-Fuji population, initial weight 0.39 g and Trial 2-Okutama population, initial weight 0.38 g, to evaluate the resistivity of fish against the IHN virus in the early stages provided by dietary fatty acids. In the first infectivity trial, four diet groups were set up with 15 fish in duplicate tanks. The variation in the dietary fatty acid composition is shown in Table 3. In the first trial, Group IIIF, was EFA deficient, whereas IIIF2, IIIF, and IIIF4 were groups receiving the nutritionally important fatty acids. The experimental diets were provided for 4 weeks before subjecting them to pathogenic challenge. In the second infectivity trial, the EFA-deprived group was IVF, , whereas the others (IVF,, IVF, and IVF4) had the essential fatty acids included in the diets that were provided for 6 weeks, after which they were exposed to the IHN virus. The procedure adopted for disease challenge was similar to our previous description (Kiron et al., 1995). The progressive loss of fish due to the infection was monitored every day, and the mean mortality from duplicate tanks as a percentage represented the results of the challenge experiment.

364

V. Kiron Table 3. Lipid composition

et al.

of diets* fed to the different groups trout

in the pathogenic

challenge

tests on rainbow

Diet groups Trial Fatty

1

Trial 2

acid?

(g) Palmitic acid Linoleic acid Linolenic acid n-3 HUFA

IIIF,

IIIF,

IIIF,

IIIF,

IVF,

IVF,

IVF,

IVF,

5.0 -

4.0 1.0 -

4.0

4.5

5.0

4.0 1.0 -

4.0

4.0

1.0 -

1.0

1.0 0.5

*The basal diet composition is same as shown in Table I. tMethy1 esters of fatty acid; see also footnote of Table 1.

Statistics

The effect of diet on the various aspects was checked by analysis of variance, and significant differences were separated by Duncans’ multiple range test.

Results

IIF, (mean values were 0.225 and 0.242, respectively; Fig. 2) when compared with fish receiving LNA (IIF,) and n-3 HUFA (IIF,). The value in Group IIF, was 0.404 + 0.042 and in Group IIF,, it was 0.375 + 0.033. Though not statistically significant, the lower titre compared with IIF, in the n-3 HUFA fed group is worth a mention.

Defence mechanisms

Disease resistance capacity

In vitro macrophage activity. The killing capacity of the macrophages isolated from fish receiving various fatty acids in their diet was different from each other (Fig. 1). The percentages of surviving bacteria in the EFA deficient group (IF,) were the maxima (84.25 + 13.31%). The killing activity was not dissimilar among n-3 polyunsaturated fatty acid fed fish (IF, = 56.75 f 9.28%, IF, = 52.13 + 15.60%). The number of surviving bacteria (73.12 f 13.56%) was greater in the LA fed fish (IF,) compared with the other EFA groups (IF,, P < 0.05; IF4, P -C0.01) indicating a reduced bactericidal activity. Antibody production. The antibody response as OD values at 492 nm was distinctly lower (P < 0.01) in the EFA deficient groups IIF, and

There was a wide difference in mortality rates between the two trials. In the first trial employing the Fuji trout population (Fig. 3), the mortality rates in fish receiving n-3 polyunsaturated fatty acids were much lower (10% for IIIF, and 15% for IIIF,) compared with the saturated fatty acid fed fish (IIIF, = 25%) and n-6 unsaturated fatty acid fed fish (IIIF, = 30%). In the subsequent challenge experiment, using the Okutama population, all fish provided 1.O% n-3 HUFA (group IVF,), died on the eighth day after infection (Fig. 4). Mortality rates approached 100% in the EFA deficient group (IVF, ) by the 21st day, post-infection. The figures were around 80% after 21 days for groups IVF, and IVF, which had received 1% of LA and LNA, respectively. 0‘J -

.rn -L-

0.4

i$

80

1.

.m

.

E

8 d

60

.2 .e

46

5 Co

. .

El

0 .3

% . 8 02

20 0 0 ., -

IF1

IF2

IF3

IF4

Diet groups Fig. 1. In vitro killing of bacteria by macrophages isolated from pronephros of rainbow trout fed on different fatty acids. IF,: palmitic acid, IF,: linoleic acid, IF,: linolenic acid, IF,: n-3 HUFA. Values in parentheses are standard deviations.

IIF,

IIF,

IIF,

IIF,

IIF,

Diet groups Fig. 2. Antibody production in rainbow trout fed different fatty acid diets. Titres are expressed as the optical density at 492 nm, IIF,: lipid deprived, IIF,: palmitic acid, IIF,: linoleic acid, IIF,: linolenic acid, IIF,: n-3 HUFA. The horizontal line across the bar indicates the mean.

EFA nutrition and immune responses

g -

W--

lllF2

+-

lllF3

25

OL_7

8

9

10

11 12 13

Days,

14 15

16

17 18

19 20

21

post-infection

Fig. 3. Mortality rates after IHNV pathogenic challenge on rainbow trout groups fed different fatty acid diets in Trial 1. IIIF,: palmitic acid. IIIF:: iinoleic acid, IIIF,: linolenic acid, IIF,: 0.5% n-3 HUFA. Mean of duplicate tanks per treatment.

Discussion Fletcher (1986) reviewed the modulation of defence mechanisms in fish by season, nutrition. hormones and pollutants. Therein, she suggested that provision of various fatty acids in fish diet would probably reveal dietary links that could be manipulated for better protection. The current study was undertaken to investigate if such a relationship existed in the defence system of rainbow trout. The leucocytic cells, particularly neutrophils and macrophages, are capable of killing and digesting foreign matter (Cohn, 1978). This process of cellular ingestion and digestion of particulate matter is the most common defence reaction occurring in animals (MacArthur and Fletcher, 1985). Macrophages are also important in the initiation of the immune response (Varesio et al., 1980). In fish, the macrophages are involved in immune responses as accessoryand antigen-presenting cells and also as immune-activated effector cells (Clem et al., 1985;

“3

4

5

6

7

8

-

IVFl

t

IVF2

+

IVF3

9 10 11 12 13 14 16 16 17 18 19 20 21

Days,

post-infection

Fig. 4. Mortality rates after IHNV pathogenic challenge on rainbow trout groups fed different fatty acid diets in Trial 2. IVF,: palmitic acid, IVF?: linoleic acid, IVF,: linolenic acid, IVF,: 1% n-3 HUFA. Mean of duplicate tanks per treatment.

in trout

365

Graham and Secombes, 1990; Miller ef al., 1985). The phagocytic activity of the macrophage is a membrane-dependent process as the lipid phase of the membrane plays a prominent role in the cellular endocytic mechanism (Schroit and Gallily, 1979). The bactericidal activity of macrophage cells in vitro was reduced when the diet was EFA deficient. A maximum number of Aeromonas salmonicida survived in this group of fish. Macrophages of fish receiving LNA or n-3 HUFA were more effective in killing the bacteria in t&o. The effectiveness of n-3 series fatty acids in intracellular killing has been demonstrated in a study by Sheldon and Blazer (199 I ) on channel catfish. Using purified diets containing either menhaden oil, soybean oil or beef tallow at 7%, they correlated enhanced bactericidal activity to increasing levels of HUFAs and this was independent of the rearing temperature. Recently, WaagbPr er al. (1993a) observed in Atlantic salmon that the bacterial killing activity of macrophages at 12°C was reduced in fish fed on sardine oil which contained more n-3 PUFA compared with fish receiving capelin oil which was lower in n-3 PUFA content. However, there was no difference in the activity of the macrophages at a higher temperature of 18’~C. In the present study on rainbow trout, the macrophage activity in vitro was superior in all the groups receiving EFA (LA, LNA and n-3 HUFA) compared with the deficient group receiving palmitic acid. Studies by DiLuzio and Blickens (1966) have shown that methyl palmitate markedly depresses the phagocytic activity of the human reticuloendothelial system. Further work on isolated human neutrophils (Hawley and Gordon, 1976) demonstrated that unsaturated fatty acid (oleic acid) caused no change in bactericidal activity, whereas saturated fatty acid (PA) moderately depressed the bactericidal action. Schroit and Gallily ( 1979) have commented that the enrichment of membrane phosphatides with unsaturated fatty acids enhanced macrophage phagocytic activity in mice. They further explained that a ‘fluid’ membrane which affords rapid membrane component movement and diffusability would be crucial for the ingestion process of macrophages. To put it simply, immunomodulation by dietary lipids is effected by changes in the plasma membrane lipid structure of lymphocyte subpopulations. This is the reason why the macrophages of rainbow trout in our study receiving the PUFAs responded better against the bacteria. However, caution has to be exercised in such dietary manipulations as it has been shown that high fat concentration in the diet, particularly PUFA, can suppress lymphocyte functions when EFA requirements are met.

366

V. Kiron

This has been demonstrated by Erickson et al. (1983) in mice. The antibody production was reduced in rainbow trout which received EFA-deficient diets in the present experiment. The values obtained in the LNA and n-3 HUFA groups were significantly higher. In an earlier study on disease resistance in channel catfish, Blazer et al. (1989) had indicated that dietary lipids may influence the levels of circulating antibody in fish. They compared a laboratory diet and two commercial formulations and found variations in the antibody titres, and assumed that it could be due to the difference in lipid levels and ratio of n-3, n-6 fatty acids. In subsequent research, Erdal et al. (1991) illustrated a relationship between n-3 fatty acids and the immune response in Atlantic salmon. The fish were fed diets with various types of oils containing increasing amounts of n-3 fatty acids, and they noted an immunosuppressive effect. The amount of n-3 fatty acids in the diets of Atlantic salmon ranged between 16 and 24% (as a percentage of total dietary fatty acids), whereas the rainbow trout in our experiment received only 10% dietary n-3 HUFA and this may be why we did not observe any adverse effect. Increasing the amounts of n-3 PUFA (1 l-35%) in the diets of Atlantic salmon was again cited as the reason for a reduction in specific antibody production against Vibrio salmonicida (Waagbs et al., 1993b). It should be noted that our diets just satisfied the EFA requirement of rainbow trout (Watanabe, 1982) and probably, if an excess of n-3 HUFA were offered to this fish, it might be immunosuppressive. Though it may be so, one has also to consider the finding of Henderson et al. (1992) that antibody production was not affected by dietary n-3 PUFA in rainbow trout vaccinated with Yersinia ruckeri. In an investigation by DeWille et al. (1979) on mice, it was shown that diets deficient in EFA significantly reduced the humoral response against T-cell-dependent and T-cellindependent antigens. Simultaneously, they found that various levels of polyunsaturated fatty acids did not reduce antibody production. These observations were further consolidated (DeWille et al., 1981) when they found that in vivo T-cell function (delayed type hypersensitivity) was reduced during EFA deficiency. However, in another examination, Friend et al. (1980) highlighted that a 20% fat diet, rich in unsaturated fatty acids, reduced the ability of guinea-pigs to form antibodies, apart from reducing the delayed hypersensitivity responses. Even as the essentiality of lipids in immunomodulation is undisputed, the conflicting reports regarding n-3 PUFA may be due to their interaction with other nutrients like vitamin E.

et al.

The two pathogenic challenge tests on small rainbow trouts produced different mortality patterns. The difference could be traced to the fish population, the Okutama group (Trial 2) being less resistant to the virus as noted in the earlier experiment (Kiron et al., 1995) and thereby resulting in a greater mortality than the Fuji trouts. The results of Trial 1 using the Fuji population exhibited a link to the observations on macrophage activity and antibody production which were lowered in EFA deficient fish. The fish receiving dietary LNA and n-3 HUFA had lower mortality rates probably due to the efficiency of the defence mechanisms aided by the dietary nutrients. The mortality in PA fed fish was high in both trials. However, in Trial 2, fish fed 1% n-3 HUFA experienced total mortality in relatively less time. We can only speculate that EFA above the required level might have impaired the defence mechanisms. Erdal et al. (1991) also reported an increased mortality on infection with Yersinia ruckeri in Atlantic salmon fed a greater amount of n-3 HUFA. Further experiments are needed to support our hypothesis. From the several observations made in this study, it appears that dietary EFA are generally important in maintaining uncompromised immune responses. Research on homeotherms has clarified that fatty acid composition of the cell membrane is crucial since several of the disease resistance mechanisms are membranerelated. In addition to the membrane changes discussed earlier, it has been established that fatty acids are precursors for the production of eicosanoids-prostaglandins, thromboxanes and leukotrienes (Johnston, 1985). Macrophages are actively involved in the production of these substances in man. Since nutritional immunology in fish is a relatively new area of investigation, future research might touch upon several of these aspects. In conclusion, there appears to be a clear relationship between EFA in diet and improved immune responses in rainbow trout Oncorhynchus mykiss.

References Blazer V. S. (1991) Piscine macrophage function and nutritional influences: A review. J. Aquat. Anim. Hlrh 3, 77-86. Blazer V. S., Ankley G. T. and Finco-Kent D. (1989) Dietary influences on disease resistance factors in channel catfish. Derll. camp. Immunol. 13, 43-48. Blazer V. S. and Wolke R. E. (1984a) Effect of diet on the immune response of rainbow trout (S&no gairdneri). Can. J. Fish Aquat. Sci. 41, 1244-1247. Blazer V. S. and Wolke R. E. (1984b) The effect of tocopherol on the immune response and non-specific resistance factors of rainbow trout (Salmo gairdneri). Aquaculture. 37, 1-9.

EFA nutrition

and imm ume responses

Clem L. W.. Sizemore R. C.. Ellsaesser C. F. and Miller N. W. ( 1985) Monocytes as accessory cells in fish immune responses. De&. camp. Inzmuno/. 9, 8033809. Cohn 2. (1978) The activation of mononuclear phagocytes: fact, fancy and future. .I. Immunol. 121, 813-816. DeWille J. W.. Fraker P. J. and Romsos D. R. (1979) Effects of essential fatty acid deficiency. and various levels of dietary polyunsaturated fatty acids, on humoral immunity in mice. I. Nutr. 109, 1018-1027. DeWille J. W.. Fraker P. J. and Romsos D. R. (1981) Effects of dietary fatty acids on delayed-type hypersensitivity in mice. I. Nutr. 111, 203992043. DiLuzio N R. and Blickens D. A. (1966) Influence of intravenously administered lipids on reticuloendothelial function. .I. Reticuloendotheliul Sot. 3, 250-270. Durve V. S. and Love11 R. T. (1982) Vitamin C and disease resistance in channel catfish (Ictctlurus pimctutus). Can I. Ayuur. SCi. 39, 948 951. Erdal J. 1.. Evenson 0.. Kaurstad 0. K., Lillehaug A., Solbakken R. and Thorud K. (1991) Relationship between diet and immune response in Atlantic salmon (Solmo .su/o~ L.) after feeding various levels of ascorbic acid and omega-3 fatty acids. Aquoculrure 98, 363 379. Erickson K. L.. Adams D. A. and McNeil1 C. J. (1983) Dietary lipid modulation of immune responsiveness. Lipid., 18, 468m 474. Fletcher T C. (1986) Modulation of non-specific host defenses m fish. Vet. Immunol. Immunopatho~ 12, 59961. Friend J. V . Lock S. 0.. Gurr M. I. and Parish W. E. (1980) Effect of different dietary lipids on the immune responses of Hartley strain guinea pigs. Inl. Arch. Allergy uppl. Immunol 62, 29230 I. Gershwin M. E.. Beach R. S. and Hurley L. S. (1985) Nutririon und Immunitv. pp. 259 283. Academic Press. New York. Graham S. and Secombes C. J. (1990) Cellular requirements for lymphokine secretion by rainbow trout Salnzo gctirdnrvt leucocytes. Derl. camp. Immunol. 14, 59968. Gross R. L. and Newberne P. M. (1980) Role of nutrition in immunologic function. Pilysiol. Rev. 60, 188-302. Gurr M. I. ( 1983) The role of lipids in the regulation of the immune system. Progr. Lipid‘ Rc~s. 22, 257-287. Hardie L. J.. Fletcher T. C. and Secombes C. J. (1990) The etfect of vitamin E on the immune response of the Atlantic salmon (Sulmo .wdar L.). Ayuaculrure 87, l-13. Hardie L. J.. Fletcher T. C. and Secombes C. J. (1991) The effect of vitamin C on the immune response of the Atlantic salmon (Solmo .ra/ar L.). Aquaculrurr 95, 201 214

Hawley H. P. and Gordon G. B. (1976) The effects of long chant free fatty acids on human neutrophil function and structure. Luh. Inwsr. 34. 216-222. Henderson R. J..Tatner M. F.. and Lin W. (1992) Antibody production in relation to lipid composition in rainbow trout fed diets of different (n-3) polyunsaturated fatty acid content. Aqucrculrurr 100, 232. Johnston P. V. (1985) Dietary fat, eicosanoids. and immunity. .Idc. Lipid Rex. 21, 103-141. Kiron V., Watanabe T.. Fukuda H.. Okamoto N. and Takeuchi T. (1995) Protein nutrition and defence mechanisms in rainbow trout Oncorh~nchus mykiss. Camp. Biochcvw P/~wio/. 11 IA. 35 I- 359. bll S. P.. Oliver G.. Weerakoon D. E. M. and Hines J. A. (1990) The effect of vitamin C deficiency and excess on the immune response in Atlantic salmon (Salmo .sa/ur L.). In T/w Cwrm/ S/ufus r!/ Fish Nutrition in Aquuculturr (Edited by Takeda M. and Watanabe T.), pp. 4X--441. Japan Translation Centre, Tokyo. Lall S. P.. Paterson W. D.. Hines J. A. and Adams N. J.

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