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Department of Avian Sciences, University of California, Davis, CA 95616
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
FOOTNOTES
LITERATURE CITED
ABSTRACT 
Two experiments were designed to determine the effects of dietary (n-3) fatty acids and grain source on the growth-suppressive effects of the inflammatory response and indices of specific immunity. In Experiment 1, chicks were fed diets containing 0.5, 1, or 2 g/100 g of either corn oil or fish oil. In Experiment 2, chicks were fed diets containing up to 2 g/100 g of either fish oil, linseed oil or corn oil as the source of dietary fat, in either cereal grain- or corn-based diets. In each experiment, subsets of chicks within each dietary treatment were either vaccinated with infectious bronchitis virus (IBV) vaccine, injected with Salmonella typhimurium lipopolysaccharide (LPS), heat-killed Staphylococcus aureus, or remained noninjected. Increasing dietary fish oil, but not corn oil increased body weight and lessened the growth-suppressing effect of heat-killed S. aureus or S. typhimurium LPS. Increasing the concentration of dietary fish oil decreased febrile response, circulating hemopexin and metallothionein concentrations. Dietary fish oil resulted in decreased release relative to dietary corn oil of interleukin-1 by peritoneal macrophages. Although IBV titers were not significantly affected by dietary oil treatment, phytohemagglutination-induced wattle swelling was greater among chicks fed fish oil. In Experiment 2, the modulating effects of fish oil on the immune system were dependent on the type of grain used in the diet, with fish oil/cereal diets resulting in greater cell-mediated immunity and lower indices of inflammation than fish oil/corn diets. Inclusion of increasing amounts of fish oil in the diet improved performance, decreased indices of the inflammatory response and either improved or did not change indices of the specific immune response of growing chicks.
KEY WORDS: chicks · fish oil · inflammatory response · (n-3) polyunsaturated fatty acids · immune systemINTRODUCTION
An inflammatory response can decrease feed consumption and muscle protein accretion, and increase metabolic rate, synthesis of acute phase proteins and organ mass relative to body mass (Klasing and Korver 1997
, Roura et al. 1992). This change in the partitioning of nutrients away from growth and toward processes associated with the acute phase response is evident as a decrease in the efficiency of food use for growth. Strategies that minimize the diversion of nutrients away from growth and muscle deposition are important in animal production.
In mammals, the fatty acid composition of phospholipid membranes in immune cells can affect the degree of inflammatory response to a challenge with an immunogen, either in vitro (Billiar et al. 1988, Prescott 1984) or in vivo (German et al. 1987). Immune cells with membranes enriched in (n-3) polyunsaturated fatty acids (PUFA5) at the expense of (n-6) PUFA release lower amounts as well as less potent mediators of inflammation (Billiar et al. 1988, Prescott 1984). These mediators, the eicosanoids, are involved in the release and function of pro-inflammatory cytokines such as tumor necrosis factor 
(TNF ) (Scales et al. 1989), interleukin-1 (IL-1; Knudsen et al. 1986, Kunkel et al. 1987), and IL-6 (Navarra et al. 1992). Two eicosanoids important in the inflammatory response are prostaglandins of the E series (PGE) and leukotrienes of the B series (LTB).
Inclusion of fish oil in the diet has been shown to increase the proportion of (n-3) PUFA relative to (n-6) PUFA in the tissues of humans (Schmidt et al. 1991), rats (Billiar et al. 1988), mice (German et al. 1987, Whelan et al. 1991), and poultry (Chanmugam et al. 1992, Friedman and Sklan 1995, Fritsche et al. 1991b). Although most research of dietary oils has utilized high levels (> 5 g/100 g) of inclusion in the diet, in mice the ratio of (n-3):(n-6) PUFA appears to be more important in modulating eicosanoid biosynthesis than the absolute concentration of (n-3) PUFA in the diet (Boudreau et al. 1991, Broughton et al. 1991). German et al. (1988) demonstrated that at high concentrations of dietary linoleic acid, fish oil supplementation had a minimal effect on leukotriene production relative to the same concentration of fish oil with lower concentrations of linoleic acid. In chickens fed corn-wheat-soy diets, increasing the (n-3):(n-6) PUFA ratio from 0.07 (the lowest possible in this type of diet) to 0.33 resulted in much greater liver (n-3) PUFA and much lower (n-6) PUFA (Korver 1997). Increases in the (n-3):(n-6) PUFA ratio to 0.66 and to 1.00 resulted in further, although more subtle, increases in hepatic (n-3):(n-6) PUFA ratio.
The enrichment of cell membrane (n-3) PUFA is associated with decreases in the inflammatory response, improvements in growth rate, and either increased or no change in specific immunity. The inclusion of fish oil in the diet of mammals appears to improve humoral immunity and ameliorate the suppression of the cellular immune response caused by PGE2 (Fritsche et al. 1992, Schmidt et al. 1991).
The endogenous mediators of inflammation can themselves be involved in the pathogenesis of several diseases, including rheumatoid arthritis (Ridderstad et al. 1991), systemic lupus erythematosus (Das 1994) and atherosclerosis (Makheja 1992). Mice suffering from murine lupus nephritis and fed diets containing fish oil have decreased expression of renal IL-1, IL-6 and TNF mRNAs compared to those fed diets containing corn oil (Chandresekar and Fernandes 1994). The consumption of fish oil not only affects the release of regulatory mediators from various immune tissues, but also modulates the response of target tissues to those mediators. Rats fed diets containing fish oil have decreased pyrogenic responses to exogenous IL-1 (Cooper and Rothwell 1993) and anorexic responses to exogenous TNF (Mulrooney and Grimble 1993).
Fish oil concentrations greater than 2 or 3 g/100 g of diet may result in fish flavors in poultry meat and are impractical because of decreased consumer acceptance (Dean et al. 1969, Miller et al. 1967). However, lower dietary concentrations enrich poultry in (n-3) fatty acids for human consumption and might also be beneficial to the productivity of the chicken. The first experiment presented here was conducted to determine if the inclusion of low amounts of fish oil in the diets of fast-growing broiler chickens could lessen the negative impact of an experimental inflammatory response on their growth rate, feed consumption, and body weight gain per unit feed consumption. The experiment was designed to relate any changes in these parameters to indices of inflammatory and specific immune responses. The second experiment was designed to examine the relative efficacy of fish oil in diets with different carbohydrate sources on modulation of the inflammatory and immune responses.
MATERIALS AND METHODS
) prior to the two experiments. When chicks were 3 d of age, experimental chicks were selected for uniform body weight from a twofold larger population and randomly assigned to dietary treatments. All experiments were approved by the University of California, Davis animal use committee.
Diets.
The corn-soy experimental diets used in Experiment 1 were based on the NRC (1984) standard research reference diet for chicks to which either corn oil (Best Foods, CPC International, Englewood Cliffs, NJ) or menhaden oil (Zapata-Haynie, Reedville, VA) was added at 0.5, 1, or 2 g/100 g diet. Table 1 shows composition and calculated (n-3):(n-6) PUFA ratio of each diet. Nine diets were used in Experiment 2 (Table 2), with menhaden, linseed (United States Biochemical Corp., Cleveland, OH), or corn oil as the source of dietary fat in either corn- or mixed cereal-based diets. Diets were kept isocaloric and isonitrogenous by adding appropriate amounts of corn starch and cellulose. Each of the six (Experiment 1) or nine (Experiment 2) experimental diets was fed to four pens of five chicks per pen.
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Table 1. Composition and calculated (n-3):(n:6) polyunsaturated fatty acid ratio of diets fed in Experiment 11,2 |
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Table 2. Composition and calculated (n-3):(n-6) polyunsaturated fatty acid ratio of diets fed in Experiment 21,2 |
. Briefly, chicks were injected on d 28 with 100 µL of PHA (500 µg/mL in phosphate-buffered saline) into the right wattle; only phosphate-buffered saline was injected into the left wattle. Twenty-four hours later, the thickness of each wattle was measured using a micrometer. The ratio of the thickness of the PHA-injected wattle to the thickness of the PBS-injected wattle is the wattle index. On d 29, Sephadex-elicited peritoneal macrophages were purified as described by Klasing and Peng (1987) and stimulated in vitro with heat-killed S. aureus to determine the capacity of these cells to produce interleukin-1 (IL-1). Briefly, macrophages were recruited by injecting into the peritoneal cavity of chicks 10 mL of a 50 g/L solution of Sephadex G-75 superfine (Pharmacia, Piscataway, NJ) in saline (9 g/L). Twenty-four hours later, cells were harvested from the peritoneum, washed, resuspended at 5 × 109 cells/L and cultured in RPMI 1640 medium containing per L 50 mL fetal bovine serum, 25 µmol 2-mercaptoethanol, 1 × 105 U penicillin, and 100 mg streptomycin. Cells were incubated at 42°C and 5% CO2 ; after 6 h, nonadherent cells were washed off. Cells were stimulated to produce IL-1 by addition of 100 heat-killed S. aureus/macrophage for 4 h. Monolayers were washed twice with media and incubated an additional 16 h. S. aureus were grown in nutrient broth and washed three times before being killed at 85°C for 10 min. The bacteria were rewashed and suspended in medium. Culture supernatants were dialyzed (MW cut-off 2000) against phosphate-buffered saline, and biologically active IL-1 in the samples was measured by PHA-induced comitogenesis of thymocytes (Klasing and Peng 1987). Briefly, several thymic lobes were removed from chicks, placed in medium and teased apart using forceps. Debris was removed by sedimentation for 10 min, after which a single-cell suspension was collected and washed three times by centrifugation at 600 × g for 10 min. Red blood cells were concentrated at the bottom of the centrifuge tube and discarded after each wash. Cells (50 µL of cell suspension) were incubated in 96-well plates at 2 × 106 cells per well. Fifty µL of cell culture supernatants and 50 µL of PHA (4 mg/L final concentration), and 50 µL treatments or medium were added resulting in a final volume of 200 µL. Cells were incubated at 42°C and 5% CO2 for a total of 72 h; the final 18 h in the presence of 27 Bq [3H]thymidine (20 µL, New England Nuclear, Boston, MA). Cells were harvested onto glass fiber filter mats using a Skatron cell harvester (Skatron Co., Sterling, VA). IL-1 activity is reported as the stimulation index, which is the ratio of [3H]thymidine incorporated into DNA of thymocytes incubated with the IL-1 source plus PHA to [3H]thymidine in thymocytes incubated in medium plus PHA.
Inflammatory response.
A second group of chicks was fed the same six (Experiment 1) or nine (Experiment 2) diets in 12 pens of five chicks per diet, starting when chicks were 3 d of age. When chicks were 10 d of age, four pens per diet were injected intraperitoneally with 3 mL of a 100 mg/L solution of S. typhimurium LPS; four pens were injected with 3 × 109 heat-killed Staphylococcus aureus/kg body weight; four pens were not injected and served as controls. Injections were repeated when chicks were 12 and 14 d of age to stimulate an authentic infectious challenge. S. typhimurium LPS was reconstituted in saline (9 g/L) to 100 mg/L and sterilized by passing through a 0.45 µm filter. S. aureus were grown in nutrient broth, washed in saline, heat-killed at 85°C for 10 min and suspended in saline (9 g/L) at 1012 cells/L. When chicks were 15 d of age, they were bled, killed by cervical dislocation. Livers were removed, freeze-clamped in liquid nitrogen, and stored frozen until analysis.
1) of this second group of chickens were determined between days 10 and 15 of the experiment. Concentrations of the acute phase protein, hemopexin, in the plasma taken on the final day of the experiment were determined to give an index of the acute phase response. Hemopexin concentrations were determined by rocket gel electrophoresis using a rabbit anti-chicken hemopexin antibody. The concentration of the acute phase protein metallothionein in the liver was assessed by the 109Cd affinity assay described by Eaton and Toal (1982). Cloacal temperature was determined 6 h following the first immunogen injection to provide an index of the responsiveness of the hypothalamus to cytokines released during the inflammatory stress.
) using SAS. All dependent variables were also analyzed by a one-way ANOVA with 18 unrelated treatments (Experiment 1) or 27 unrelated treatments (Experiment 2). Single degree of freedom orthogonal contrasts (Steel and Torrie 1980) were made for indicated comparisons using the GLM procedure of SAS.
RESULTS
Table 3.
Effect of dietary oil source on indices of specific immunity and inflammatory responses of broiler chicks (Experiment 1)
DISCUSSION ACKNOWLEDGMENTS 
Fig. 2.
Effect of dietary oil source, grain type and immunogen challenge on growth rate, feed consumption and feed conversion efficiency of broiler chicks in Experiment 2. Panel A: Body weight gain (g/chick·day). Main effect and interaction P values: diet, P = 0.09; immunogen, P < 0.01; diet by immunogen, P = 0.03. Panel B: Feed consumption (g/chick·day). Main effect and interaction P values: diet, P = 0.36; immunogen, P < 0.04; diet by immunogen, P = 0.09. Panel C: Feed conversion efficiency (g body weight gain/g feed consumed). Main effect and interaction P values: diet, P = 0.17; immunogen, P < 0.01; diet by immunogen, P = 0.05. In each panel, individual bars represent the mean + SEM of four replicates.
[View Larger Version of this Image (53K GIF file)]
Fig. 3.
Effect of dietary oil source and immunogen challenge on indices of the inflammatory response of broiler chicks in Experiment 1. Panel A: Body temperature 6 h postinjection. Significant main effect and interaction P values: immunogen, P < 0.01; source by level, P = 0.04; source by level by immunogen, P = 0.04. Panel B: Plasma hemopexin (nmol/L plasma) 24 h after the third injection. Significant main effect and interaction P values: source, P = 0.05; immunogen, P < 0.01. Panel C: Hepatic metallothionein (nmol/g liver) 24 h after the third injection. Significant main effect and interaction P values: source, P = 0.04; immunogen, P < 0.01; source by level, P = 0.01; source by immunogen, P = 0.01; source by level by immunogen, P = 0.01. In each panel, individual bars represent the mean + SEM of four replicates. Abbreviations: Hpx = hemopexin; MT = metallothionein.
[View Larger Version of this Image (44K GIF file)]
Fig. 4.
Effect of dietary oil source, grain type and immunogen challenge on indices of the inflammatory response of broiler chicks in Experiment 2. Panel A: Body temperature 6 h postinjection. Main effect and interaction P values: diet, P = 0.11; immunogen, P < 0.01; diet by immunogen, P = 0.11. Panel B: Plasma hemopexin (nmol/L plasma) 24 h after the third injection. Main effect and interaction P values: diet, P = 0.27; immunogen, P < 0.01; diet by immunogen, P = 0.04. Panel C: Hepatic metallothionein (nmol/g liver) 24 h after the third injection. Main effect and interaction P values: diet, P = 0.16; immunogen, P < 0.01; diet by immunogen, P = 0.06. In each panel, individual bars represent the mean + SEM of four replicates. Abbreviations: Hpx = hemopexin; MT = metallothionein.
[View Larger Version of this Image (55K GIF file)]
Fig. 5.
Effect of dietary oil source, grain type and immunogen challenge on indices of the immune response of broiler chicks in Experiment 2. Panel A: Circulating levels of antibodies to infectious bronchitis virus (IBV). Chicks were vaccinated at 14 d of age with IBV, and antibody titers at d 28 were determined by ELISA. Main effect P value: diet, P = 0.19. Panel B: Wattle index, where index is the ratio of phytohemagglutinin (PHA)-induced swelling at 24 h postinjection of the injected wattle relative to the vehicle-injected contralateral wattle. Main effect P value: diet, P < 0.01. Panel C: In vitro release of interleukin-1-like (IL-1) activity from sephadex-elicited peritoneal macrophages, expressed as stimulation index (SI). Cells were stimulated with S. aureus for 18 h, and the supernatants were assayed via thymocyte comitogenesis bioassay for IL-1 activity. SI is the ratio of [3H]thymidine incorporated into the DNA of thymocytes in the presence of the IL-1 source and PHA to the [3H]thymidine incorporated into the DNA of thymocytes in the presence of PHA. Main effect P value: diet, P = 0.03. In each panel, individual bars represent the mean + SEM of four replicates. Different letters indicate means differ significantly, P < 0.05.
[View Larger Version of this Image (37K GIF file)]
, Klasing et al. 1987, Roura et al. 1992, Takahashi et al. 1995), pigs (McCracken et al. 1995), and rats (Peisen et al. 1995). When chicks were challenged with either LPS or S. aureus, the inclusion of fish oil in the diet partially mitigated the decrease in body weight gain and feed conversion efficiency. In Experiment 1, a bacterial challenge simulated by injecting LPS resulted in about a 15% decrease in the rate of gain of chicks consuming 2 g corn oil/100 g diet. This effect was lessened by feeding 2 g fish oil/100 g diet, resulting in only 10% lower rate of weight gain. In the second experiment, the efficacy of fish oil was examined with two different dietary backgrounds, either cereal or corn. Menhaden oil was effective at ameliorating LPS-induced growth depression, and this interaction was more pronounced with the cereal diets than the corn diets. Possibly this was due to the lower level of (n-6) fatty acids, including linoleic acid, found in the cereal diets relative to the corn diets, however the slightly slower growth rates in the absence of a challenge in cereal fed chicks could also contribute to this observation.
), and along with IL-6 and TNF, the synthesis of acute phase proteins such as hemopexin (Baumann and Gauldie 1994) and metallothionein (Bremner and Beattie 1990, Klasing 1984). In Experiment 1, the in vitro release of IL-1 from macrophages isolated from chicks fed fish oil was less than that from corn oil-fed chicks. Thus it is likely that the blunted acute phase response observed in vivo in fish oil-fed chicks was at least partly due to lower levels of inflammatory cytokines such as IL-1. Dietary (n-3) fatty acids have been shown to decrease interleukin-1 and tumor necrosis factor production by cultured human mononuclear cells (Endres et al. 1989). The mechanism by which (n-3) fatty acids specifically decrease the inflammatory response was not investigated in these experiments. Increasing the (n-3):(n-6) concentration increases the (n-3) content of membrane phospholipids of both target and effector cells. This in turn may result in decreased pro-inflammatory signals released by effector cells, and may also decrease responsiveness of target cells to pro-inflammatory signals.
, Klasing 1994). In our control birds, which were raised in environments that were extremely clean with minimal exposure to infectious challenges, there was no benefit or detriment in terms of growth to enriching the diet with (n-3) fatty acids. When the birds were challenged, however, the fish oil diets resulted in greater growth rates than did the corn oil diets. Thus, in practical poultry husbandry, fish oil may benefit growth when the birds are challenged by pathogens. Additionally, when birds are reared in commercial-type environments with the build-up of dust, dander, and feces, the inflammatory response is constantly stimulated. These nonpathogen environmental immunogens increase the level of the catabolic cytokine, IL-1, altering the birds' metabolism and redirecting nutrients away from growth and toward an inflammatory response (Roura et al. 1992). Under practical poultry production conditions, fish oil might be fed to minimize the catabolic effect of both pathogens and environmental immunogens by decreasing production of pro-inflammatory cytokines and acute phase proteins, permitting a higher growth rate.
reported that chicks fed a diet containing 7 g menhaden oil/100 g diet have higher antibody responses to sheep red blood cells than did chicks fed the same concentration of either lard, corn oil or canola oil. Cellular immune response as measured by antibody-dependent cell cytotoxicity of splenocytes is decreased in broilers fed 7 g fish oil versus those fed 7 g corn oil/100 g diet, although cytotoxicity of peripheral blood leukocytes is not affected by dietary treatment (Fritsche and Cassity 1992). Thus, high levels of dietary fish oil apparently have different immunomodulatory effects than lower levels. In mammals, diets containing fish oil either improve, decrease, or do not affect indices of specific immunity depending on the index of immune function, the amount of fish oil inclusion in the diet, and the concentration of dietary fat. Anti-sheep red blood cell antibody responses of rats fed 17 g fish oil + 3 g corn oil/100 g diet and either 30 or 90 mg vitamin E/100 g diet are significantly higher than corn oil-fed rats fed the same amounts of vitamin E (Fritsche et al. 1992). In noninfected mice, feeding a high (n-3) PUFA diet (20 g fish oil/100 g diet) results in the greatest percentage and number of T cells, but in Listeria-infected mice, this diet results in the lowest percentage of T cells in the peritoneum when compared to mice fed the same concentration of sunflower oil and coconut oils. B cell populations are not affected by dietary fat in noninfected mice, but the fish oil diet results in the highest percentage of B cells in infected mice (Huang et al. 1992). Splenocyte natural killer cell activity of mice fed 10 g fish oil/100 g diet is decreased 25% compared to that of mice fed a the same concentration of corn oil, although cell-mediated cytotoxicity of cytotoxic T lymphocytes and peritoneal cells is not affected (Fritsche and Johnston 1990). Level of inclusion appears to play a role in the effect of fish oil, since this oil is immunosuppressive in the host vs. graft model in mice only at high concentrations (10 g/100 g of diet) (Hinds and Sanders 1993). In humans, inclusion of fish oil at 0.54% of total energy in a low fat diet decreases T cell proliferation in response to Concanavalin A and PHA, while inclusion of only 0.13% of calories as fish oil in a similar diet results in an increase in the same indices. Delayed-type hypersensitivity is decreased versus baseline at the higher level of fish oil, but there was no change at the low level of fish oil (Meydani et al. 1993).
) and S. enteritidis (Kogut et al. 1995, Tellez et al. 1994). The net benefit of immunomodulatory nutrients such as fish oil in natural disease challenges remains to be characterized. Under the conditions of these experiments, lessening the inflammatory response improved performance characteristics of broiler chickens.
FOOTNOTES
.
Manuscript received 2 December 1996. Initial reviews completed 23 January 1997. Revision accepted 12 June 1997.
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