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Effect of Dietary Fats on the Fatty Acid Compositions of Serum and Immune Tissues in Chickens1
Effect of Dietary Fats on the Fatty Acid Compositions of Serum and Immune Tissues in Chickens 1 KEVIN L. FRTTSCHE,2 NANCY A. CASSITY, and SHU-CAI HUANG Department of Animal Sciences, University of Missouri-Columbia, 110 Animal Sciences Research Center, Columbia, Missouri 65211 (Received for publication August 31, 1990)
1991 Poultry Science 70:1213-1222 INTRODUCTION
Apart from storing and transporting forms of metabolic fuel, the fatty acid portion of dietary fats serves several other important functions in the body. These include providing for the essential fatty acid requirements, acting as structural components of cell membranes, and serving as precursors for eicosanoid production. Eicosanoids, such as the prostaglandins (PG) and leukotrienes, are recognized as important modulators of cell-mediated immunity and humoral immunity (Goldyne and Stobo, 1981; Stenson and Parker, 1982; RolaPleszczynski, 1985). Considerable evidence has accumulated that suggests that dietary fats may influence the immune response through their ability to modulate eicosanoid production (Johnston, 1985, Kinsella et ai, 1990). It is widely recognized that the fatty acid moieties of phospholipids determine many membrane functions. The possible influence of lipids on
receptor binding, signal transmission, and lymphocyte proliferation makes knowing the fatty acid composition of immune cells of particular interest (Traill and Wick, 1984). Little is known about the influence that dietary fat source has on the fatty acid composition of chicken immune cells. The objective of the present study was to determine the effect of feeding various fat sources on the fatty acid composition of immune cells in chickens. The fats tested came from both animal sources [lard (LA) and menhaden fish oil (FO)] and vegetable sources [corn (CO), canola (CA), and linseed (LO)]. A wide range of fatty acid compositions are represented, including fats rich in saturated (LA), monounsaturated (CA), and polyunsaturated (CO, LO, FO) fatty acids (PUFA). The PUFA could be further separated into n-6 fatty acid rich (CO) or n-3 fatty acid rich (LO and FO) oils. MATERIALS AND METHODS
Contribution from the Missouri Agriculture Experiment Station; Journal Series Number 11,280. ^To whom correspondence should be addressed. ^ e n Roy Hatchery, Berger, MO 63014.
Animals and Diets One hundred female Leghorn-type chickens (Babcock B-300)3 were used for these studies.
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ABSTRACT The purpose of the present study was to measure the effect of dietary fat source on the fatty acid composition of immune cells in chickens. One-day-old female chicks were fed corn and soybean meal-based diets containing 7% of either lard, corn oil, canola oil, linseed oil (LO), or menhaden fish oil (FO). After being fed experimental diets for 3 to 4 wk, samples of serum, thymus glands, bursa of Fabricius glands, and splenocytes were collected. All samples were frozen and stored at -80 C until lipid analysis. Results indicate that the fatty acid composition of the sera and immune tissues of chickens reflected the fat in the diet The relative content of long-chain polyunsaturated fatty acids varied considerably among immune tissues, with, from greatest to least, spleen, bursa, and thymus. The young chick demonstrated a substantial capacity to elongate and desaturate linoleic (Cjg^-fi) and a-linolenic acids (Cig:3n.3). Feeding chicks fats rich in n-3 fatty acids (e.g., LO or FO) decreased significantly (P<.05) the level of arachidonic acid (C2o:4n-6) present in the serum and immune tissues by 50 to 75%. The levels of eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C20:6n-3) w e r e substantially increased (P<05) by FO and LO feeding. However, LO, which is rich in Ci8:3n_3, was generally only one-half to one-quarter as effective as FO in elevating EPA and DHA levels in immune tissues. The implications for these changes in serum and immune tissue fatty acid profiles are discussed briefly. (Key words: fatty acid composition, spleen, thymus, bursa of Fabricius, dietary fat)
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FRTTSCHE ET AL.
Sample Collection
TABLE 1. Composition of chick starter diet1 Ingredients
g/kg
Corn Soybean meal (48% CP) DL-methionine Fat Dicalcium phosphate Limestone (ground) Salt (NaCl) Mineral mix 2 Vitamin mix3 Se premix (200 mg Se/kg) Choline chloride (70% aqueous solution)
575.3 319.9 .3 70.0 17.4 9.1 4.0 1.0 .5 1.0 13
'Diet provided approximately 3,200 kcal MEJkg and 20.4% crude protein. 2 Mineral premix from Calcium Carbonate Co., Quincy, IL 62301. Provides (in milligrams per kilogram of diet): Mn, 120 (manganous oxide); Zn, 80 (zinc oxide); Fe, 60 (iron sulfate); Cu, 10 (copper sulfate); I, 1 (calcium iodate); Ca, 160 (calcium carbonate). 3 Provides in milligrams per kilogram of diet (except as noted): all-frani-retinylpalmitate, 8,818 IU; cholecalciferoL 3,307 ICU; d-alpha-tocopheryl acetate, 9 5 IU; vitamin Bi2, 8.8 |ig; riboflavin, 5.5; niacin, 55; calcium d-pantothenate, 16.8; folic acid, 1.1; pyridoxine HCL 1.3; thiamine mononitrate, .6; menadione, .66 (menadione sodium bisulfate, 2.0); ethoxyquin, 55.
bursa glands were immediately placed in glass vials and snap frozen in a dry-ice acetone bath. Spleens were placed in 10 mL of sterile-filtered ice-cold Hanks balanced salt solution without Ca 2+ and Mg 2+ . Single-cell suspensions of splenocytes were made by forcing each spleen through a tissue sieve* equipped with an 80-mesh stainless-steel screen. Using a 10-mL syringe without a needle, cell clumps were dispersed by several gentle washings through the sieve. Red blood cells and dead cells were removed by centrifugation of the spleen cell suspension over lymphocyte separation medium, density 1.077 to 1.080 at 20 C as described by the manufacturer.5 Cells at the interface (predominantly lymphocytes and monocytes) were collected, washed twice, and resuspended in 1 mL of 10-mM EDTA. All samples were stored at -80 C prior to lipid extraction.
Four chicks were selected randomly from each dietary group and were anesthetized by CO2 inhalation. This process was repeated on five separate days. Blood (5 to 10 mL), collected by cardiac puncture, was allowed to clot at room temperature. Serum was collected following centrifugation (450 X g, 10 min) of the clotted blood. Immediately following blood collection, spleens, thymus glands, and bursa of Fabricius glands were removed and weighed. Thymus and Fatty Acid Analysis Samples of serum (1 mL) and splenocytes (-2-5 x 107 cells) were diluted 1:1 with buffer 4 (50 vaM Trizma-HCl; 1 vaM EDTA; .32 M Sigma, St. Louis, MO 63178-9916. 5 Organon Teknika Corp., Durham, NC 27704-0969. sucrose; pH 7.4). Thymus and bursa glands were
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Upon receipt, 1-day-old chicks were weighed and randomly assigned to one of five treatment groups (20 chicks per group). Chicks were fed diets that contained 1%, (wt/wt) LA, CO, CA, LO, or FO. Chicks were housed in a standard battery. Electric heaters were set at 90 to 95 C. Following an acclimation period of 3 to 5 days, chicks were housed individually in stainlesssteel wire hanging cages; uiey continued to receive the experimental diet originally assigned on Day 1. Throughout the study, the room was maintained at 80 to 85 C, with a relative humidity of 45 to 50%. A diurnal light cycle of 12 h was maintained. Chicks had free access to food and water until the time of sample collection. The diets were formulated to meet or exceed all the nutritional requirements of the growing chick (National Research Council, 1984). Composition of the diets is shown in Table 1. Fatty acid composition of individual fats was analyzed, as was the etfier extractable portion of the dry diet ingredients prior to the addition of fat. All diets contained 10% total lipids (3% in the dry ingredients to which 7% was added). The fatty acid composition of the various diets was then calculated based on the relative contribution of each of these sources (i.e., endogenous and exogenous; Table 2). Fresh feed was provided every other day; any feed remaining being discarded. Autoxidation of the oils in the diet was prevented by following previously described guidelines (Fritsche and Johnston, 1988). Peroxide content, as measured by peroxide value, of diets stored at 20 C or kept at room temperature for 48 h did not significantly increase. Chicks were weighed weekly.
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DIETARY FATS AND CHICKEN IMMUNE TISSUES TABLE 2. Fatty acid composition of chick
1
Sets
Treatment2 LA
C
.9 20.6 2.0 10.1 37.8 25.1 .6 .6 .2
14:0 16:0 Cl6:l Cl8:0 C 18:l C 18:2n-6 C 18:3n-3 C 20:l C20:4n-6 C 20:5n-3 022:511-3 c
CO
.
.
9.4 .1 1.7 27.2 58.6 1.0
CA
LO
FO
5.7 .1 2.0 51.8 31.0 5.5 1.1
6.3 .1 3.0 22.7 28.7 37.8
5.3 15.3 8.1 2.8 16.3 18.5 .9
9.3 22.8 66.5
.7 10.7 1.8 62 23.4 24.5 38.8
•
C22:fa-3
ZSAT4 XMONO5 ZPUFA6
31.6 40.4 25.9
11.1 27.3 59.6
7.7 53.0 36.5
'Fatty acid composition of individual fats was analyzed, as was the ether attractable portion of the dry diet ingredients prior to the addition of fat. All diets contained 10% total lipids (3% in the dry ingredients to which 7% was added). The fatty acid composition of the various diets was then calculated based on the relative contribution of each of these sources (i.e., endogenous and exogenous). Because all diets contained the same amount of fat (10%), the amount of individual fatty acids in grams per kilogram of diet can be determined by multiplying the weight percentage by a factor of .1. treatment groups are based on the source of added fat in the diet with LA = lard, CO = corn oil; CA = canola oil; LO = linseed oil; and FO = menhaden fish oil. 3 Fatty acids are denoted by the number of carbons: number of double bonds, followed by the position of the first double bond relative to the methyl end (n-). 4 XSAT = Sum total area percentage of CM-Q, Ci&o and Cig-g. 5 2MONO = Sum total area percentage of Cis : i, Ci8 : i, C20:i^ U F A = Sum total area percentage of C 1 8 : 2 n ^, C 18 . 3n _ 3 , C20:4n-6. Q20:5n-3. C22:5n-3> C22:6Q-3-
quickly thawed and homogenized in 10 volumes of the Tris, EDTA, and sucrose buffer with a mechanical homogenizer.6 Lipids in the diluted serum, splenocytes, or tissue homogenate (2 mL) were extracted with four volumes of chloroform and methanol (2:1, vol/vol). The organic phase containing the lipid extract was removed and the aqueous phase re-extracted with four volumes of chloroform, methanol, and 12N HC1 (2:1:.013, vol/vol/vol). The organic layers were pooled, filtered, and reduced in volume under N2. Total lipids were transmethylated with 4% sulfuric acid in methanol for 1 h in a 60 C water bath. The mixture was saponified according to the procedure of Kates (1986) by the addition of 33% potassium hydroxide and subsequent heating at 60 C for 1
^issumizer, Tekmar, Cincinnati, OH 45222. 'Hewlett-Packard, Inc., Avondale, PA 19311. 8 Supelco, Inc., Bellefonte, PA 16823-0048.
h. Heptane was used to remove nonsaponifiable material and the solution was acidified by addition of fW hydrochloric acid and the free fatty acids extracted with heptane. The free fatty acids were again transmethylated. Fatty acid methyl esters (FAME) were analyzed using a Hewlett-Packard gas-liquid chromatograph, Model 58907 equipped with a 30-m x .25-mm inside diameter fused silica capillary column (SUPELCOWAX 10).8 Helium was the carrier gas with the flow rate set at 1 mL/min. Oven temperature was 190 C (10 min) then raised 3 C/min to 230 C and held for 14 min. The FAME were identified by comparing relative retention times of commercially available standards (PUFA-1 and PUFA-2).8 Results, expressed as a percentage of total fatty acids, were determined using a Hewlett-Packard 3380A integrator.7 A flame ionization detector response of 1.000 was assumed for all FAME. Therefore, the relative amount of a given fatty acid is directly proportional to the area under the
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Fatty Acid J
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FRITSCHE EI AL. TABLE 3. Immune organ weights (as a percentage of body weight) of chickens fed different sources of far
Fat source
Spleen
Thymus
Bursa of Fabricius
Number of samples per group Lard Com oil Canola oil Linseed oil Fish oil SEM (pooled) Probability value
20 .150 .155 .148 .152 .170 .008 .09
13 .496 .493 .500 .408 .515 .025 .96
13 .665 .532 .563 .621 .611 .045 .24
TABLE 4. Distribution of total serum fatty acids of 3- to 4-wk-old chickens fed various
fin
sources^~
Fat source Fatty acids
LA
CO
CA
C14.0 C16.0 Clfrl C18.0 Cig.j Ci8-2n-6 Ci8:3n^ C 18 . 3n _ 3 C20:2n-6 C20:3n-6 C2o-4n^ C20:5n-3 C 22:4o -6 C22.5n_6 C22.Sn.3 C22:fe.3 XSAT4 EMONO5 HPUFA6 Zn-67 Zn-38
.2 b 17.5* 1.4b 13.7* 18.1* 23.4C -3 .4C -3 b l-4 a 14.5* -3° -8 a 1.1* .4 d 2.2C 31.4 19.5* 45.2° 41.8 b 3£|
.2 b 17.4* .3 d 12.4*b 12.9b 36.8* -2 .5C .4* 1.2* 12.8b -1° -8a 1.1* .3 d 1.0* 29.9 13.3° 55.4* 53.3* L9^
(% of total weightr .lb .2 b 14.7b 16.9* .9° .5 d 13.2* 13.3* 18.3* 14.1 b 27.5b 27.2 b .5 .2 1.5b 11.2" .3 b .2= 1.4* 1.0b b 12.0 3.9° -6" 4.3 b -5* .ld .4 b 0° .8° 1.5b 3.5b 3.3 b 28.0 29.1 19.3* 15.5b b 49.0 53.0* 42.6b 32.6C 64= 20.3 b
LO
FO
SEM3
.6* 17.8* 1.8* 10.3b 9.4C 16.9d .6 .5° .2C .5° 3.5C 14.8* .3" ,2 b 3.0 15.4* 28.8 ll.ld 55.9* 22.2 d 33/7*^
.1 .4 .1 .8 .5 1.0 .2 1.0 .1 .1 .5 .2 .1 .1 .1 .4 .9 .5 1.2 1.1 .6
Means within rows with no common superscripts are significantly different (P<.05). Chicks were fed diets containing 1% by weight of lard (LA), com oil (CO), canola oil (CA), linseed oil (LO), or a menhaden fish oil (FO) for 3 to 4 wk prior to sample collection and analysis. •'Values are expressed as means (n = 4 to 5). 3 Pooled SEM. 4 SSAT = Sum total area percentage of C^a, Ci&o, and C ^ . 5 ZMONO = Sum total area percentage of Cjg.i, Cig : i, and C2o:i. 1
6 2PUFA = Sum total C20:5n-3, C22:4n_6, C 2 2 : 5 n ^, 7 2n-6 = Sum total area 8 2n-3 = Sum total area
area percentage of Cig:2n_6, C ^ j ^ , Cig:3n_3, C20:2n-6. c20:3n-6. C2o:4n.6. C22:5n_3, C22:&i-3. percentage of Cig :2n ^, C 1 8 : 3 n ^, C ^ Q . ^ , Caoan* C^n-fr C a ^ , C 22: 5 n ^. percentage of C i g ^ j , C 20: 5 n . 3 , C22:5n-3. C22:6o-3-
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'Chicks were fed corn and soybean meal diets containing 1% by weight of the various fat sources as indicated for 3 to 4 wk before tissue collection. 2 Values are expressed as means; fat source did not significantly (P<05) affect organ weight
DIETARY FATS AND CHICKEN IMMUNE TISSUES
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TABLE 5. Distribution of total splenocyte fatty acids of 3- to 4-wk-old chickens fed various fat
sourcesr^
Fat source Fatty acids
LA
CO
CA
C
1.0* 25.3 2.2 a 10.7 22.2" 10.3C .1 0b .lb .5° 1.1 16.51* 3.8a 2° .9° 1.5d 37.0 24.5a 37.0b 32.3° 4.6C
.2° 19.9 .6 b 11.6 13.4b 17.7a .1 0b .lb 1.3" 1.9 21.7" 5.6a 0° 1.5*
.ic 17.8 .9 b 10.6 20.6 ab 13.8b .2 .4" .5 a 1.0b 1.9 21.l a 4.9 s .3C 1.9b S.l^ 28.4 22.0 ab 48.7a 42.9 ab 5.7°
LO
FO
SEM3
.4* 20.1 .9" 8.6 14.8b 18.1a .1 5.0" 0b .6° 1.9 12.7bc 1.3b 5.4b 5.4 a 3.7b 29.1 15.8b 54.1 a 34.7bc 19.5b
yab 23.3
.2 2.0 .4 1.1 2.4 .7 .1 .8 .1 .1 2 2.3 .7 .4 .5 .4 22 2.7 2.8 3.0 .9
n of
ZSAT 4 ZMONO5 SPUFA 6 Sn-6 7 £a-3 8
21cd
31.8 14.1b 51.9a 48.3 a 3.6C
19ab
7.8 13.9b 11.6° 0 .lb 0b .4C 1.2 7.0° .9 b 12.5" 6.1 a 9.3 a 31.8 15.7b 49.0 s 21.1 d 28.0 s
Means within rows with no common superscripts are significantly different (P<05). Chicks were fed diets containing 7% by weight lard (LA), corn oil (CO), canola oil (CA), linseed oil (LO), or a fish (menhaden) oil (FO) for 3 to 4 wk prior to sample collection and analysis. 2 Values are expressed as means (n = 4 to 5). 3 Pooled SEM. 4 ZSAT = Sum total area percentage of Ci4^>, Cig-o, and Ci 8 .o. 5 ZMONO = Sum total area percentage of C\^.\, C ^ i , and C20:i. 1
6 c
ZPUFA = Sum total area percentage of Ci g:2 n-6, C 1 8 : 3 n . 6 , C 1 8 : 3 n .3, C20:2n-6. C 2 o :3 n-6. C 20: 4 n .6,
20:5n-3.
C22:4n-6> C22:5n-6. c22:5n-3> c 22:6n-3-
7
Zn-6 = Sum total area percentage Of C18:2n_<;, C 1 8 : 3 l l ^ , C20:2n-6. C20 :3n -6. C20:4n-6. C22:4n-6.
8
2n-3 = Sum total area percentage of C 1 8 : 3 n . 3 , C 2 0 : 5 n . 3 , C22:5i,.3, C22:6n-3-
gas-chromatographic peak relative to the sum of all the FAME peaks. Statistical Analysis Fatty acid data were subjected to one-way ANOVA to test for an effect of dietary fat source (Steel and Tonie, 1980). When significant differences occurred (P<.05), treatment mean differences were identified by Fisher's least significant difference and Scheffe's F test. All analyses were conducted on a Macintosh n® computer using version 1.03 of StatView H 9
C22:5n-6-
RESULTS A N D DISCUSSION
Immune Organ Weight Feeding chickens various sources of fat did not significantly affect the weight of the spleen, thymus, and bursa (Table 3). Data are presented as a percentage of body weight to account for differences in body weight. Fat source did significantly affect body weight (Fritsche et al., 1991). The FO-fed chicks tended to be heavier and LO-fed chicks lighter compared with chicks fed LA, CO, and CA. The differences in body weights were also reflected by differences in feed intake. Serum Fatty Acid Profile
9
Abacus Concepts, Inc., Berkeley, CA 94701.
The effect of feeding various sources of fat on serum fatty acids of chicks is shown in Table 4.
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14:0 C 16:0 Cl6:l Cl8:0 C 18:l C 18:2n-6 c 18:3n-« C 18:3n-3 ^20:1 C20:2n-6 C20:3n-6 C 20:4n-6 C 22:4n-6 C 20:5n-3 C 22:5n-3 C 22:6n-3
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FRTTSCHE ET AL.
TABLE 6. Distribution of total bursa fatty acids of 3- to 4-wk-old chickens fed various fat sources1^ Fat source Fatty acids
LA
CO
CA
C
1.0 21.4 6.6 6.5 29.5" 14.3b .2" .3"
.8 21.4 4.6 7.9 22.2 b 20.7a
>
i!i a 1.8a 11.6a .4C 3.3 a 2 1.0° 1.2° 30.1 27.2° 41.9* 39.2a 2.9b
1.2 20.4 5.7 7.4 31.6a 15.7b .5 a l.l" .8 a .7" 1.0b 5.9b 1.3C 1.6b .6 1.4° 2.0 bc 29.0 38.1 a 31.9 b 26.0 b 5.8b
LO
FO
SEM3
.8 20.4 5.0 7.5 22.0 b 14.2b 2h 8.3 a 0" Jte 1.2b 4.7 b 6.0b
1.5 19.6 6.8 12 23.4 b 10.1° .3 b .8" .3 b
.4 1.3 .9 1.0 1.3 1.0 .1 .5 .2 .1 .1 .7 .6 .3 .2 2 .3 1.4 .8 1.7 1.2 1.0
f"n C
14:0
4 ab
l.l" 9.8 a
.6°
2.4 b .3 1.5C 2.0*° 29.0 36.5a 33.2b 28.7b 4.4 b
4 ab
b :s .4
ab
.6° .3 4.4 a 2.4 b 28.7 27.0° 42.7 a 21.7° 21.1"
.3°
.5C 22c 10.9 s Jf> .6 4.8 a 7.5 a 28.4 30.5 b 38.5 a 14.5d 24.0*
Means within rows with no common superscripts are significantly different (P<05). 'Chicks were fed diets containing 7% by weight lard (LA), corn oil (CO), canola oil (CA), linseed oil (LO), or a menhaden fish oil (FO) for 3 to 4 wk prior to sample collection and analysis. Values are expressed as means (n = 4 to 5). 3 Pooled SEM. 4 ZSAT = Sum total area percentage of C\^, CX&Q, and Cigfl. 5 ZMONO = Sum total area percentage of Ci6:i, Cj 8: i, and C ^ i ^ U F A = Sum total area percentage of C18:2n-6, C 18:3n _ 6 , C 1 8 : 3 n _ 3 , C20:2n-6> C2o:3n-6. C2o:4n-6. C 20:5n-3> C22:4n-6' C22:5n-6> C22:5n-3. C22:6n-37 En-6 = Sum total area percentage of C l g : 2 n ^, C 18:3n . 6> C20:2n-6. c20:3n-6> C20:4n-6- c22:4n-6> c22:5n-68 2n-3 = Sum total area percentage of C18:3n_3, C20:Sn-3. C 22:5n . 3> C 22:6n _ 3 .
These data do not support the hypothesis that serum lipids are a direct reflection of dietary lipids. For example, the levels of saturated fatty acids in the serum were similar regardless of a three-fold difference in the amounts provided in the diet. However, the levels of some individual fatty acids, particularly PUFA, could be predicted from the content of the diet. The relative contents of Ci8:i and C2n-6 m m e serum paralleled those in the diet. Some of the present data suggest that the chicken possesses relatively high levels of desaturase activity. For example, the fats fed contained little or no preformed C2o:4n-6> v e t levels of the fatty acid in the serum and immune cells contributed up to 22% of the total fatty
acids present. Second, significant elevations in C20:5n-3 ^ d C22:6n-3 levels are seen in chickens fed LO. These birds were consuming diets with large quantities of a-linolenic acid ( C i g ^ ^ ) , but no preformed C20:5n-3 OT C22:6n-3Immune Tissue Fatty Acid Profiles The effect of feeding chickens various sources of fat on splenocyte, bursal, and thymic fatty acid composition is shown in Tables 5 to 7. As with the serum, immune tissues seemed fairly resistant to modifications in the relative amount of total saturated fatty acids, monounsaturated fatty acids, or PUFA. There was no
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16:0 C 16:l C 18:0 C 18:l C 18:2n-« C 18:3n-6 C 18:3n-3 C 20:l C 20:2n-6 C 20:3n-6 C 20:4n-6 c 20:5n-3 C 22:4n^ c 22:5n-« C 22:5n-3 c 22:6n-3 JSAT 4 ZMONO5 ZPUFA6 Zn-67 Zn-3g
DIETARY FATS AND CHICKEN IMMUNE TISSUES
1219
TABLE 7. Distribution of total thymus fatty acids of 3- to 4-wk-old chickens fed various fat
sourcesr^
Fat source Fatty acids
LA
CO
CA
C
.9" 23.4 a 3.6b 8.7a 40.4 b 15.8C .1 .4 d .5" .2 .4 2.1 a .2° .6 a .la .2° .3 b 32.9b 44.6 b 20.5° 19.4° 1.2d
.3° 16.2b l.ld 6.6b 27.4 ed 40.7 a .1 .8 d .2° .4 .4 2.3 a .1° .7 a .l a .2° .3" 23.1° 28.7 d 46. l a 44.8 a 1.3d
.3C 14.6° 1.9° 5.8° 47.2" 21.1 b .1 2.0 b .8a 2 .4 1.4b .l c .4 b 0b 2C .4"
LO
FO
SEM3
.3C 15.8b 2.0° 6.8 b 28.9° 20.3 b .1 19.3a .2° .2 .6 .9° .8 b 2C 0b .8 b .5 b 22.9° 31.1 01 43.6 22.2 b 21.4 a
3.6a 23.6 a 7.4a 7.2 b 25.4 d 15.7* .1 12" .8" .4 .2 .5° 3.0" .l c
.1 .3 .2 2 .9 .6 .02 2 .1 .2 .1 .2 2 .1 .1 .1 .1 .3 1.0 .9 .8 .4
(% 14:0
c
20.7"1 49.9" 26.3 23.6b 2.8°
o"
1.2a 2.0 s 34.4a 33.6° 24.5 b 17.1c 7.4b
a-d»Means within rows with no common superscripts are significantly different (P<05).
'Chicks were fed diets containing 7% by weight lard (LA), corn oil (CO), canola oil (CA), linseed oil (LO), or a menhaden fish oil (FO) for 3 to 4 wk prior to sample collection and analysis. 2 Values are expressed as means (n = 4 to 5). 'Pooled SEM. 4 ESAT = Sum total area percentage of C^-o, Ci&o, and CH^Q. 5 2MONO = Sum total area percentage of C ^ i , Cig : i, and C ^ a 6 SPUFA = Sum total area percentage of C i 8 : 2 n ^ , Ci 8 : 3 n _ 6 , C 1 8 : 3 n _ 3 , C20:2n-6> C20:3n-6. C2o:4n-6. C
20:5n-3> C22:4n-«> C22:5n-6. C22:Sn-3. Q22:6n-37 8
2n-6 = Sum total area percentage of C l g : 2 n . 6 , C i 8 : 3 n ^ , C ^ ^ . 6 , C^j^, C20:4n-6. C ^ - s ^ . 2n-3 = Sum total area percentage of Ci 8:3n _ 3 , C 20: 5 n -3. C 22: 5 n _ 3 , C2 2 : 6 n . 3 .
effect of fat source on the level of total saturated fatty acids in isolated splenocytes or in the bursa. Contrary to those observations, the fatty acid composition of the thymus showed a remarkable similarity to that of the diet. The impact of dietary fat source on immune tissue profiles are most evident for individual fatty acids, particularly long-chain PUFA. For example, C20:4n-6 accounted for up to 21% of the total fatty acids present in the splenocytes from chicks fed LA, CO, or CA (Table 5). Feeding diets rich in n-3 fatty acids (i.e., LO or FO) lowered significantly (P<.001) the relative content of C20:4n6 in splenocytes and significantly elevated (P<001) n-3 levels 400 to 500%. Feeding the n-3 rich fats (LO or FO) also depressed significantly (P<.05) the levels of
C-20:4n-6 m m e bursa (Table 6) and thymus (Table 7) compared with levels in LA- and COfed chicks. Similar observations have been reported for liver, heart, brain, thigh, and breast muscle lipids in the chicken (Edwards and Marion, 1963; Miller and White, 1975; Anderson, et al., 1989; Huang et al, 1990). Incorporation of long-chain n-3 PUFA (i.e., Q20:5n-3 and C22:6n-3) w a s dependent on the source of the n-3 fatty acids. Feeding preformed sources of these n-3 PUFA (i.e., FO) resulted in a greater elevation of these fatty acids in the immune tissues compared with the Ci8:3n.3-rich LO. Others have shown that feeding FO to rats or mice results in a greater deposition of C20:5n-3 and C22:6n-3 in liver and immune tissues compared with LO, despite an
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16:0 Cl6:l c 18:0 C 1S:1 Cl8:2n-6 Cl8:3n-6 c 18:3n-3 c 20:l c 20:2n-6 C 20:3n-6 C20:4n-6 Q20:5n-3 c 22:4n-6 c 22:5n-6 C 22:5n-3 c 22:6n-3 2SAT 4 2MON0 5 EPUFA6 Zn-67 Zn-38
1220
FRITSCHE ET AL. BURSA
SERUM
b
H 20:4n-6
20:5n-3
22:6n-3
20:4n-6
25 "8
THYMUS
Lard Corn oil Canola oil Linseed oil Fish oil
20
<
1 H a
"o
22:6n-3
10 51 0
20:4n-6
20:5n-3
22:6n-3
20:4n-6
20:5n-3
22:6n-3
FIGURE 1. Effect of dietary fat source on selected fatty acid levels in chicken serum and immune tissues. Chicks were fed diets that contained 7% of the fat from the sources indicated for 3 to 4 wk prior to sample collection. Total lipids were extracted and the fatty acids were analyzed by gas-liquid chromatography. Data represent the mean (n = 4) percentage of the weight of the fatty acids indicated. Error bars on the fish oil data represent the pooled SEM. Bars with no common letters are significantly different (P<.05). Fatty acids are denoted by the number of carbons: number of double bonds, followed by the position of the first double bond relative to the methyl end (n-).
overall greater n-3 content in the latter oil (Croft ex al, 1984; Fritsche and Johnston, 1990). Furthermore, the level of Cig:3n_3 in the spleen (Table 5) and bursa (Table 6) was elevated (P<.005) only when LO was fed. The thymus (Table 7) showed a greater enrichment of Cig. 3n_3 compared with the spleen and bursa, and the relative content exceeded that found in the serum. This may be related to the high affinity die tfrymus demonstrated for Cig fatty acids, which generally accounted for over 50% of the total fatty acids present. Shown in Figure 1 are the levels of the three fatty acids (C20:4n6> C 2 0 : 5n-3.
C
22:6n-3)
mos
*
relevant to die eicosanoid-producing capacity of these immune tissues. Eicosanoids are the oxygenated metabolites of C20 fatty acids. Arachidonic acid (C2o:4n-6) is the predominate precursor in most mammalian tissues. Other precursors include C2o:3n-6 and C20:5n-3- ^ e former has been omitted from this figure for two
reasons. First, levels of this fatty acid in the spleen and thymus were not altered by fat source and were only modestly affected in the bursa. Second, this fatty acid is a minor constituent (<2%) of the tissue lipids examined. Serum and tissue C22;6n-3 have been included in this figure because this fatty acid is known to be a potent inhibitor of eicosanoid synmesis (Corey ex al., 1983). Figure 1 illustrates the impact of dietary fat source on the relative amount of eicosanoid precursors present in the immune tissues of the chicken. Marked differences between precursor levels are apparent in the immune tissues studied. The bursa contains only half as much C20:4n-6 a s splenocytes, and thymus contains even less than the bursa. Tissue-specific differences in PUFA content may reflect differences in desaturase activity or acyl-coenzyme A transferase activities (Willis, 1981). Such tissuespecific differences in C20:4n-6 content may have
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25-1 SPLENOCYTES
20:5n-3
DIETARY FATS AND CHICKEN IMMUNE TISSUES
ACKNOWLEDGMENTS
The authors are grateful to Zapata Haynie, Reedville, VA 22539; Best Foods, a division of CPC International Inc., Union, NJ 07083; Kraft, Inc., Glenview, EL 60025; and Cargill, Inc., Minneapolis, MN 55440 for supplying the dietary oils used in this research project. The authors appreciate Margaret Campen's assistance with the care and feeding of the animals used in these studies. REFERENCES Anderson, G. J., W. E. Conner, J. D. Corliss, and D. S. Lin, 1989. Rapid modulation of the n-3 docosahexaenoic acid levels in the brain and retina of the newly hatched chick. J. Lipid Res. 30:433-441. Chouaib, S., L. Chatenoud, D. Klatzmann, and D. Frazelizi, 1985. The mechanisms of inhibition of
human IL-2 production. PGE2 induction of suppressor T lymphocytes. J. Immunol. 132:1851-1857. Codde, J. P., L. J. Beilin, K. D. Croft, and R. Vandongen, 1985. Study of diet and drug interactions on prostanoid metabolism. Prostaglandins 29:895-910. Corey, E. J., C. Shin, and J. R. Cashman, 1983. Docosahexaenoic acid is a strong inhibitor of prostaglandin but not leukotriene biosynthesis. Proc. Natl. Acad. Sci. 80:3581-3584. Craig-Schmidt, M. C , S. A. Faircloth, and J. D. Weete, 1987. Modulation of avian lung eicosanoids by dietary omega-3 fatty acids. J. Nutr. 117:1197-1206. Croft, K. D., L. J. Beilin, R. Vandongen, and E. Mathews, 1984. Dietary modifications of fatty acid and prostaglandin synthesis in the rat: Effect of variations in the level of dietary fat. Biochim. Biophys. Acta 795:196-207. Culp, B. R„ B. G. Titus, and WJE.M Lands, 1979. Inhibition of prostaglandin biosynthesis by eicosapentaenoic acid. Prostaglandins Leukotrienes Med. 3:269-278. Edwards, H. M., Jr., and J. E. Marion, 1963. Influence of dietary menhaden oil on growth rate and tissue fatty acids of the chick. J. Nutr. 81:123-130. Fritsche, K. L. and P. V. Johnston, 1988. Rapid autoxidation of fish oil in diets without added antioxidants. J. Nutr. 118:425-426. Fritsche, K. L., and P. V. Johnston, 1990. Effect of dietary omega-3 fatty acids on cell-mediated cytotoxic activity in BALB/c mice. Nutr. Res. 10:577-588. Fritsche, K. L., N. A. Cassity, and S. C. Huang, 1991. Effect of dietary fat source on antibody production and lymphocyte proliferation in chickens. Poultry Sci. 70:611-617. Goldyne, M. E., and J. D. Stobo, 1981. Immunoregulation of prostaglandins and related lipids. CRC Crit Rev. Immunol. 2:189-223. Goodwin, J. S., A. D. Bankhurst, and R. P. Messner, 1977. Suppression of human T-cell mitogenesis by prostaglandin: Existence of a prostaglandin-producing suppressor cell. J. Exp. Med. 146:1719-1734. Huang, Z-B., H. Leibovitz, C. M Lee, and R. Miller, 1990. Effect of dietary fish oil on o-3 fatty acid levels in chicken eggs and thigh flesh. J. Agric. Food Chem. 38:743-747. Johnston, P. V., 1985. Dietary fat, eicosanoids, and immunity. Adv. Lipid Res. 21:37-86. Johnston, P. V., 1988. Lipid modulation of immune responses. Pages 37-86 in: Nutrition and Immunology. R. K. Chandra, ed. Alan R. Liss, Inc., New York, NY. Kates, M., 1986. Pages 123-128 in: Techniques of Lipidology. Isolation, Analysis, and Identification of Lipids. American Elsevier, New York, NY. Kinsella, J. E., B. Lokesh, S. Broughton, and J. Whelan, 1990. Dietary polyunsaturated fatty acids and eicosanoids: Potential effects on the modulation of inflammatory and immune cells: An overview. Nutrition 6:24-60. Likoff, R. O., D. R. Guptill, L. M. Lawrence, C. C. McKay, M. M. Mathias, C. F. Nockels, and R. P. Tengerdy, 1981. Vitamin E and aspirin depress prostaglandins in protection of chickens against Escherichia cott infection. Am. J. Clin. Nutr. 34: 245-251. Miller, D., and V. White, 1975. Chick tissues fatty acid composition as affected by selenium, DL-oc-tocoph-
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important consequences with regard to the role that eicosanoids play in immunoregulation at those tissue sites. Evidence is mounting that dietary PUFA may influence immune cell function and disease resistance through the modulation of eicosanoid production (Johnston, 1988). Although not measured in the present study, others have shown that changes in tissue fatty acid composition similar to those reported here can modulate eicosanoid production in chickens (Craig-Schmidt et ah, 1987). Recently, the present authors have demonstrated that feeding chicks an n-3-rich diet (7% FO) significantly enhanced antibody production and altered lymphocyte proliferation (Fritsche et ah, 1991). The present data indicates that the serum and immune tissues from FO-fed chicks are rich in n-3 PUFA. Two of the n-3 fatty acids abundant in fish oils, C20:5n-3 a n d Q22:6n-3> are potent inhibitors of PG synthesis (Culp et ah, 1979; Corey et ah, 1983; Codde et ah, 1985). In mammals, PGE2 has been shown to inhibit a variety of immunologic responses, including lymphocyte proliferation (Goodwin et ah, 1977), antibody formation (Plescia et ah, 1975), and lymphokine production (Chouaib et ah, 1985). Eicosanoids, such as PGE2, may also play an important role in regulating avian immune responses. Likoff et ah (1981) demonstrated that inhibition of PG production with high dietary vitamin E or aspirin enhanced chicken's resistance to bacterial infection. Fatty acid composition may play an important role in determining the eicosanoid-synthesizing capacity of immune cells or tissues.
1221
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FRITSCHE ET AL.
erol acetate, ethoxyquin and polyunsaturated oils. Nutr. Rep. Int. 12:245-255. National Research Council, 1984. Nutrient Requirements of Poultry. 8th ed. National Academy Press, Washington, DC. Plescia, D. J., A. H. Smith, and K. Grinwich, 1975. Subversion of the immune response by tumor cells and role of prostaglandins. Proc. Natl. Acad. Sci. 72: 1848-1851. Rola-Pleszczynski, M., 1985. Immunoregulation by leukotrienes and other lipoxygenase metabolites. Im-
munol. Today 6:302-307. Steel, R.G.D., and J. H. Torrie, 1980. Principles and Procedures of Statistics. A Biomedical Approach. McGraw-Hill, New York, NY. Stenson, W. F., and C. W. Parker, 1982. Prostaglandins and the immune response. Pages 39-89 in: Prostaglandins. J. B. Lee, ed. Elsevier, New York, NY. Traill, K. N., and G. Wick, 1984. Lipids and lymphocyte function. Immunol. Today 5:70-76. Willis, A. L., 1981. Unanswered questions in EFA and PG research. Prog. Lipid Res. 20:839-850.
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1991 Poultry Science 70:1213-1222 INTRODUCTION
Apart from storing and transporting forms of metabolic fuel, the fatty acid portion of dietary fats serves several other important functions in the body. These include providing for the essential fatty acid requirements, acting as structural components of cell membranes, and serving as precursors for eicosanoid production. Eicosanoids, such as the prostaglandins (PG) and leukotrienes, are recognized as important modulators of cell-mediated immunity and humoral immunity (Goldyne and Stobo, 1981; Stenson and Parker, 1982; RolaPleszczynski, 1985). Considerable evidence has accumulated that suggests that dietary fats may influence the immune response through their ability to modulate eicosanoid production (Johnston, 1985, Kinsella et ai, 1990). It is widely recognized that the fatty acid moieties of phospholipids determine many membrane functions. The possible influence of lipids on
receptor binding, signal transmission, and lymphocyte proliferation makes knowing the fatty acid composition of immune cells of particular interest (Traill and Wick, 1984). Little is known about the influence that dietary fat source has on the fatty acid composition of chicken immune cells. The objective of the present study was to determine the effect of feeding various fat sources on the fatty acid composition of immune cells in chickens. The fats tested came from both animal sources [lard (LA) and menhaden fish oil (FO)] and vegetable sources [corn (CO), canola (CA), and linseed (LO)]. A wide range of fatty acid compositions are represented, including fats rich in saturated (LA), monounsaturated (CA), and polyunsaturated (CO, LO, FO) fatty acids (PUFA). The PUFA could be further separated into n-6 fatty acid rich (CO) or n-3 fatty acid rich (LO and FO) oils. MATERIALS AND METHODS
Contribution from the Missouri Agriculture Experiment Station; Journal Series Number 11,280. ^To whom correspondence should be addressed. ^ e n Roy Hatchery, Berger, MO 63014.
Animals and Diets One hundred female Leghorn-type chickens (Babcock B-300)3 were used for these studies.
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ABSTRACT The purpose of the present study was to measure the effect of dietary fat source on the fatty acid composition of immune cells in chickens. One-day-old female chicks were fed corn and soybean meal-based diets containing 7% of either lard, corn oil, canola oil, linseed oil (LO), or menhaden fish oil (FO). After being fed experimental diets for 3 to 4 wk, samples of serum, thymus glands, bursa of Fabricius glands, and splenocytes were collected. All samples were frozen and stored at -80 C until lipid analysis. Results indicate that the fatty acid composition of the sera and immune tissues of chickens reflected the fat in the diet The relative content of long-chain polyunsaturated fatty acids varied considerably among immune tissues, with, from greatest to least, spleen, bursa, and thymus. The young chick demonstrated a substantial capacity to elongate and desaturate linoleic (Cjg^-fi) and a-linolenic acids (Cig:3n.3). Feeding chicks fats rich in n-3 fatty acids (e.g., LO or FO) decreased significantly (P<.05) the level of arachidonic acid (C2o:4n-6) present in the serum and immune tissues by 50 to 75%. The levels of eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C20:6n-3) w e r e substantially increased (P<05) by FO and LO feeding. However, LO, which is rich in Ci8:3n_3, was generally only one-half to one-quarter as effective as FO in elevating EPA and DHA levels in immune tissues. The implications for these changes in serum and immune tissue fatty acid profiles are discussed briefly. (Key words: fatty acid composition, spleen, thymus, bursa of Fabricius, dietary fat)
1214
FRTTSCHE ET AL.
Sample Collection
TABLE 1. Composition of chick starter diet1 Ingredients
g/kg
Corn Soybean meal (48% CP) DL-methionine Fat Dicalcium phosphate Limestone (ground) Salt (NaCl) Mineral mix 2 Vitamin mix3 Se premix (200 mg Se/kg) Choline chloride (70% aqueous solution)
575.3 319.9 .3 70.0 17.4 9.1 4.0 1.0 .5 1.0 13
'Diet provided approximately 3,200 kcal MEJkg and 20.4% crude protein. 2 Mineral premix from Calcium Carbonate Co., Quincy, IL 62301. Provides (in milligrams per kilogram of diet): Mn, 120 (manganous oxide); Zn, 80 (zinc oxide); Fe, 60 (iron sulfate); Cu, 10 (copper sulfate); I, 1 (calcium iodate); Ca, 160 (calcium carbonate). 3 Provides in milligrams per kilogram of diet (except as noted): all-frani-retinylpalmitate, 8,818 IU; cholecalciferoL 3,307 ICU; d-alpha-tocopheryl acetate, 9 5 IU; vitamin Bi2, 8.8 |ig; riboflavin, 5.5; niacin, 55; calcium d-pantothenate, 16.8; folic acid, 1.1; pyridoxine HCL 1.3; thiamine mononitrate, .6; menadione, .66 (menadione sodium bisulfate, 2.0); ethoxyquin, 55.
bursa glands were immediately placed in glass vials and snap frozen in a dry-ice acetone bath. Spleens were placed in 10 mL of sterile-filtered ice-cold Hanks balanced salt solution without Ca 2+ and Mg 2+ . Single-cell suspensions of splenocytes were made by forcing each spleen through a tissue sieve* equipped with an 80-mesh stainless-steel screen. Using a 10-mL syringe without a needle, cell clumps were dispersed by several gentle washings through the sieve. Red blood cells and dead cells were removed by centrifugation of the spleen cell suspension over lymphocyte separation medium, density 1.077 to 1.080 at 20 C as described by the manufacturer.5 Cells at the interface (predominantly lymphocytes and monocytes) were collected, washed twice, and resuspended in 1 mL of 10-mM EDTA. All samples were stored at -80 C prior to lipid extraction.
Four chicks were selected randomly from each dietary group and were anesthetized by CO2 inhalation. This process was repeated on five separate days. Blood (5 to 10 mL), collected by cardiac puncture, was allowed to clot at room temperature. Serum was collected following centrifugation (450 X g, 10 min) of the clotted blood. Immediately following blood collection, spleens, thymus glands, and bursa of Fabricius glands were removed and weighed. Thymus and Fatty Acid Analysis Samples of serum (1 mL) and splenocytes (-2-5 x 107 cells) were diluted 1:1 with buffer 4 (50 vaM Trizma-HCl; 1 vaM EDTA; .32 M Sigma, St. Louis, MO 63178-9916. 5 Organon Teknika Corp., Durham, NC 27704-0969. sucrose; pH 7.4). Thymus and bursa glands were
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Upon receipt, 1-day-old chicks were weighed and randomly assigned to one of five treatment groups (20 chicks per group). Chicks were fed diets that contained 1%, (wt/wt) LA, CO, CA, LO, or FO. Chicks were housed in a standard battery. Electric heaters were set at 90 to 95 C. Following an acclimation period of 3 to 5 days, chicks were housed individually in stainlesssteel wire hanging cages; uiey continued to receive the experimental diet originally assigned on Day 1. Throughout the study, the room was maintained at 80 to 85 C, with a relative humidity of 45 to 50%. A diurnal light cycle of 12 h was maintained. Chicks had free access to food and water until the time of sample collection. The diets were formulated to meet or exceed all the nutritional requirements of the growing chick (National Research Council, 1984). Composition of the diets is shown in Table 1. Fatty acid composition of individual fats was analyzed, as was the etfier extractable portion of the dry diet ingredients prior to the addition of fat. All diets contained 10% total lipids (3% in the dry ingredients to which 7% was added). The fatty acid composition of the various diets was then calculated based on the relative contribution of each of these sources (i.e., endogenous and exogenous; Table 2). Fresh feed was provided every other day; any feed remaining being discarded. Autoxidation of the oils in the diet was prevented by following previously described guidelines (Fritsche and Johnston, 1988). Peroxide content, as measured by peroxide value, of diets stored at 20 C or kept at room temperature for 48 h did not significantly increase. Chicks were weighed weekly.
1215
DIETARY FATS AND CHICKEN IMMUNE TISSUES TABLE 2. Fatty acid composition of chick
1
Sets
Treatment2 LA
C
.9 20.6 2.0 10.1 37.8 25.1 .6 .6 .2
14:0 16:0 Cl6:l Cl8:0 C 18:l C 18:2n-6 C 18:3n-3 C 20:l C20:4n-6 C 20:5n-3 022:511-3 c
CO
.
.
9.4 .1 1.7 27.2 58.6 1.0
CA
LO
FO
5.7 .1 2.0 51.8 31.0 5.5 1.1
6.3 .1 3.0 22.7 28.7 37.8
5.3 15.3 8.1 2.8 16.3 18.5 .9
9.3 22.8 66.5
.7 10.7 1.8 62 23.4 24.5 38.8
•
C22:fa-3
ZSAT4 XMONO5 ZPUFA6
31.6 40.4 25.9
11.1 27.3 59.6
7.7 53.0 36.5
'Fatty acid composition of individual fats was analyzed, as was the ether attractable portion of the dry diet ingredients prior to the addition of fat. All diets contained 10% total lipids (3% in the dry ingredients to which 7% was added). The fatty acid composition of the various diets was then calculated based on the relative contribution of each of these sources (i.e., endogenous and exogenous). Because all diets contained the same amount of fat (10%), the amount of individual fatty acids in grams per kilogram of diet can be determined by multiplying the weight percentage by a factor of .1. treatment groups are based on the source of added fat in the diet with LA = lard, CO = corn oil; CA = canola oil; LO = linseed oil; and FO = menhaden fish oil. 3 Fatty acids are denoted by the number of carbons: number of double bonds, followed by the position of the first double bond relative to the methyl end (n-). 4 XSAT = Sum total area percentage of CM-Q, Ci&o and Cig-g. 5 2MONO = Sum total area percentage of Cis : i, Ci8 : i, C20:i^ U F A = Sum total area percentage of C 1 8 : 2 n ^, C 18 . 3n _ 3 , C20:4n-6. Q20:5n-3. C22:5n-3> C22:6Q-3-
quickly thawed and homogenized in 10 volumes of the Tris, EDTA, and sucrose buffer with a mechanical homogenizer.6 Lipids in the diluted serum, splenocytes, or tissue homogenate (2 mL) were extracted with four volumes of chloroform and methanol (2:1, vol/vol). The organic phase containing the lipid extract was removed and the aqueous phase re-extracted with four volumes of chloroform, methanol, and 12N HC1 (2:1:.013, vol/vol/vol). The organic layers were pooled, filtered, and reduced in volume under N2. Total lipids were transmethylated with 4% sulfuric acid in methanol for 1 h in a 60 C water bath. The mixture was saponified according to the procedure of Kates (1986) by the addition of 33% potassium hydroxide and subsequent heating at 60 C for 1
^issumizer, Tekmar, Cincinnati, OH 45222. 'Hewlett-Packard, Inc., Avondale, PA 19311. 8 Supelco, Inc., Bellefonte, PA 16823-0048.
h. Heptane was used to remove nonsaponifiable material and the solution was acidified by addition of fW hydrochloric acid and the free fatty acids extracted with heptane. The free fatty acids were again transmethylated. Fatty acid methyl esters (FAME) were analyzed using a Hewlett-Packard gas-liquid chromatograph, Model 58907 equipped with a 30-m x .25-mm inside diameter fused silica capillary column (SUPELCOWAX 10).8 Helium was the carrier gas with the flow rate set at 1 mL/min. Oven temperature was 190 C (10 min) then raised 3 C/min to 230 C and held for 14 min. The FAME were identified by comparing relative retention times of commercially available standards (PUFA-1 and PUFA-2).8 Results, expressed as a percentage of total fatty acids, were determined using a Hewlett-Packard 3380A integrator.7 A flame ionization detector response of 1.000 was assumed for all FAME. Therefore, the relative amount of a given fatty acid is directly proportional to the area under the
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Fatty Acid J
1216
FRITSCHE EI AL. TABLE 3. Immune organ weights (as a percentage of body weight) of chickens fed different sources of far
Fat source
Spleen
Thymus
Bursa of Fabricius
Number of samples per group Lard Com oil Canola oil Linseed oil Fish oil SEM (pooled) Probability value
20 .150 .155 .148 .152 .170 .008 .09
13 .496 .493 .500 .408 .515 .025 .96
13 .665 .532 .563 .621 .611 .045 .24
TABLE 4. Distribution of total serum fatty acids of 3- to 4-wk-old chickens fed various
fin
sources^~
Fat source Fatty acids
LA
CO
CA
C14.0 C16.0 Clfrl C18.0 Cig.j Ci8-2n-6 Ci8:3n^ C 18 . 3n _ 3 C20:2n-6 C20:3n-6 C2o-4n^ C20:5n-3 C 22:4o -6 C22.5n_6 C22.Sn.3 C22:fe.3 XSAT4 EMONO5 HPUFA6 Zn-67 Zn-38
.2 b 17.5* 1.4b 13.7* 18.1* 23.4C -3 .4C -3 b l-4 a 14.5* -3° -8 a 1.1* .4 d 2.2C 31.4 19.5* 45.2° 41.8 b 3£|
.2 b 17.4* .3 d 12.4*b 12.9b 36.8* -2 .5C .4* 1.2* 12.8b -1° -8a 1.1* .3 d 1.0* 29.9 13.3° 55.4* 53.3* L9^
(% of total weightr .lb .2 b 14.7b 16.9* .9° .5 d 13.2* 13.3* 18.3* 14.1 b 27.5b 27.2 b .5 .2 1.5b 11.2" .3 b .2= 1.4* 1.0b b 12.0 3.9° -6" 4.3 b -5* .ld .4 b 0° .8° 1.5b 3.5b 3.3 b 28.0 29.1 19.3* 15.5b b 49.0 53.0* 42.6b 32.6C 64= 20.3 b
LO
FO
SEM3
.6* 17.8* 1.8* 10.3b 9.4C 16.9d .6 .5° .2C .5° 3.5C 14.8* .3" ,2 b 3.0 15.4* 28.8 ll.ld 55.9* 22.2 d 33/7*^
.1 .4 .1 .8 .5 1.0 .2 1.0 .1 .1 .5 .2 .1 .1 .1 .4 .9 .5 1.2 1.1 .6
Means within rows with no common superscripts are significantly different (P<.05). Chicks were fed diets containing 1% by weight of lard (LA), com oil (CO), canola oil (CA), linseed oil (LO), or a menhaden fish oil (FO) for 3 to 4 wk prior to sample collection and analysis. •'Values are expressed as means (n = 4 to 5). 3 Pooled SEM. 4 SSAT = Sum total area percentage of C^a, Ci&o, and C ^ . 5 ZMONO = Sum total area percentage of Cjg.i, Cig : i, and C2o:i. 1
6 2PUFA = Sum total C20:5n-3, C22:4n_6, C 2 2 : 5 n ^, 7 2n-6 = Sum total area 8 2n-3 = Sum total area
area percentage of Cig:2n_6, C ^ j ^ , Cig:3n_3, C20:2n-6. c20:3n-6. C2o:4n.6. C22:5n_3, C22:&i-3. percentage of Cig :2n ^, C 1 8 : 3 n ^, C ^ Q . ^ , Caoan* C^n-fr C a ^ , C 22: 5 n ^. percentage of C i g ^ j , C 20: 5 n . 3 , C22:5n-3. C22:6o-3-
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'Chicks were fed corn and soybean meal diets containing 1% by weight of the various fat sources as indicated for 3 to 4 wk before tissue collection. 2 Values are expressed as means; fat source did not significantly (P<05) affect organ weight
DIETARY FATS AND CHICKEN IMMUNE TISSUES
1217
TABLE 5. Distribution of total splenocyte fatty acids of 3- to 4-wk-old chickens fed various fat
sourcesr^
Fat source Fatty acids
LA
CO
CA
C
1.0* 25.3 2.2 a 10.7 22.2" 10.3C .1 0b .lb .5° 1.1 16.51* 3.8a 2° .9° 1.5d 37.0 24.5a 37.0b 32.3° 4.6C
.2° 19.9 .6 b 11.6 13.4b 17.7a .1 0b .lb 1.3" 1.9 21.7" 5.6a 0° 1.5*
.ic 17.8 .9 b 10.6 20.6 ab 13.8b .2 .4" .5 a 1.0b 1.9 21.l a 4.9 s .3C 1.9b S.l^ 28.4 22.0 ab 48.7a 42.9 ab 5.7°
LO
FO
SEM3
.4* 20.1 .9" 8.6 14.8b 18.1a .1 5.0" 0b .6° 1.9 12.7bc 1.3b 5.4b 5.4 a 3.7b 29.1 15.8b 54.1 a 34.7bc 19.5b
yab 23.3
.2 2.0 .4 1.1 2.4 .7 .1 .8 .1 .1 2 2.3 .7 .4 .5 .4 22 2.7 2.8 3.0 .9
n of
ZSAT 4 ZMONO5 SPUFA 6 Sn-6 7 £a-3 8
21cd
31.8 14.1b 51.9a 48.3 a 3.6C
19ab
7.8 13.9b 11.6° 0 .lb 0b .4C 1.2 7.0° .9 b 12.5" 6.1 a 9.3 a 31.8 15.7b 49.0 s 21.1 d 28.0 s
Means within rows with no common superscripts are significantly different (P<05). Chicks were fed diets containing 7% by weight lard (LA), corn oil (CO), canola oil (CA), linseed oil (LO), or a fish (menhaden) oil (FO) for 3 to 4 wk prior to sample collection and analysis. 2 Values are expressed as means (n = 4 to 5). 3 Pooled SEM. 4 ZSAT = Sum total area percentage of Ci4^>, Cig-o, and Ci 8 .o. 5 ZMONO = Sum total area percentage of C\^.\, C ^ i , and C20:i. 1
6 c
ZPUFA = Sum total area percentage of Ci g:2 n-6, C 1 8 : 3 n . 6 , C 1 8 : 3 n .3, C20:2n-6. C 2 o :3 n-6. C 20: 4 n .6,
20:5n-3.
C22:4n-6> C22:5n-6. c22:5n-3> c 22:6n-3-
7
Zn-6 = Sum total area percentage Of C18:2n_<;, C 1 8 : 3 l l ^ , C20:2n-6. C20 :3n -6. C20:4n-6. C22:4n-6.
8
2n-3 = Sum total area percentage of C 1 8 : 3 n . 3 , C 2 0 : 5 n . 3 , C22:5i,.3, C22:6n-3-
gas-chromatographic peak relative to the sum of all the FAME peaks. Statistical Analysis Fatty acid data were subjected to one-way ANOVA to test for an effect of dietary fat source (Steel and Tonie, 1980). When significant differences occurred (P<.05), treatment mean differences were identified by Fisher's least significant difference and Scheffe's F test. All analyses were conducted on a Macintosh n® computer using version 1.03 of StatView H 9
C22:5n-6-
RESULTS A N D DISCUSSION
Immune Organ Weight Feeding chickens various sources of fat did not significantly affect the weight of the spleen, thymus, and bursa (Table 3). Data are presented as a percentage of body weight to account for differences in body weight. Fat source did significantly affect body weight (Fritsche et al., 1991). The FO-fed chicks tended to be heavier and LO-fed chicks lighter compared with chicks fed LA, CO, and CA. The differences in body weights were also reflected by differences in feed intake. Serum Fatty Acid Profile
9
Abacus Concepts, Inc., Berkeley, CA 94701.
The effect of feeding various sources of fat on serum fatty acids of chicks is shown in Table 4.
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14:0 C 16:0 Cl6:l Cl8:0 C 18:l C 18:2n-6 c 18:3n-« C 18:3n-3 ^20:1 C20:2n-6 C20:3n-6 C 20:4n-6 C 22:4n-6 C 20:5n-3 C 22:5n-3 C 22:6n-3
1218
FRTTSCHE ET AL.
TABLE 6. Distribution of total bursa fatty acids of 3- to 4-wk-old chickens fed various fat sources1^ Fat source Fatty acids
LA
CO
CA
C
1.0 21.4 6.6 6.5 29.5" 14.3b .2" .3"
.8 21.4 4.6 7.9 22.2 b 20.7a
>
i!i a 1.8a 11.6a .4C 3.3 a 2 1.0° 1.2° 30.1 27.2° 41.9* 39.2a 2.9b
1.2 20.4 5.7 7.4 31.6a 15.7b .5 a l.l" .8 a .7" 1.0b 5.9b 1.3C 1.6b .6 1.4° 2.0 bc 29.0 38.1 a 31.9 b 26.0 b 5.8b
LO
FO
SEM3
.8 20.4 5.0 7.5 22.0 b 14.2b 2h 8.3 a 0" Jte 1.2b 4.7 b 6.0b
1.5 19.6 6.8 12 23.4 b 10.1° .3 b .8" .3 b
.4 1.3 .9 1.0 1.3 1.0 .1 .5 .2 .1 .1 .7 .6 .3 .2 2 .3 1.4 .8 1.7 1.2 1.0
f"n C
14:0
4 ab
l.l" 9.8 a
.6°
2.4 b .3 1.5C 2.0*° 29.0 36.5a 33.2b 28.7b 4.4 b
4 ab
b :s .4
ab
.6° .3 4.4 a 2.4 b 28.7 27.0° 42.7 a 21.7° 21.1"
.3°
.5C 22c 10.9 s Jf> .6 4.8 a 7.5 a 28.4 30.5 b 38.5 a 14.5d 24.0*
Means within rows with no common superscripts are significantly different (P<05). 'Chicks were fed diets containing 7% by weight lard (LA), corn oil (CO), canola oil (CA), linseed oil (LO), or a menhaden fish oil (FO) for 3 to 4 wk prior to sample collection and analysis. Values are expressed as means (n = 4 to 5). 3 Pooled SEM. 4 ZSAT = Sum total area percentage of C\^, CX&Q, and Cigfl. 5 ZMONO = Sum total area percentage of Ci6:i, Cj 8: i, and C ^ i ^ U F A = Sum total area percentage of C18:2n-6, C 18:3n _ 6 , C 1 8 : 3 n _ 3 , C20:2n-6> C2o:3n-6. C2o:4n-6. C 20:5n-3> C22:4n-6' C22:5n-6> C22:5n-3. C22:6n-37 En-6 = Sum total area percentage of C l g : 2 n ^, C 18:3n . 6> C20:2n-6. c20:3n-6> C20:4n-6- c22:4n-6> c22:5n-68 2n-3 = Sum total area percentage of C18:3n_3, C20:Sn-3. C 22:5n . 3> C 22:6n _ 3 .
These data do not support the hypothesis that serum lipids are a direct reflection of dietary lipids. For example, the levels of saturated fatty acids in the serum were similar regardless of a three-fold difference in the amounts provided in the diet. However, the levels of some individual fatty acids, particularly PUFA, could be predicted from the content of the diet. The relative contents of Ci8:i and C2n-6 m m e serum paralleled those in the diet. Some of the present data suggest that the chicken possesses relatively high levels of desaturase activity. For example, the fats fed contained little or no preformed C2o:4n-6> v e t levels of the fatty acid in the serum and immune cells contributed up to 22% of the total fatty
acids present. Second, significant elevations in C20:5n-3 ^ d C22:6n-3 levels are seen in chickens fed LO. These birds were consuming diets with large quantities of a-linolenic acid ( C i g ^ ^ ) , but no preformed C20:5n-3 OT C22:6n-3Immune Tissue Fatty Acid Profiles The effect of feeding chickens various sources of fat on splenocyte, bursal, and thymic fatty acid composition is shown in Tables 5 to 7. As with the serum, immune tissues seemed fairly resistant to modifications in the relative amount of total saturated fatty acids, monounsaturated fatty acids, or PUFA. There was no
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16:0 C 16:l C 18:0 C 18:l C 18:2n-« C 18:3n-6 C 18:3n-3 C 20:l C 20:2n-6 C 20:3n-6 C 20:4n-6 c 20:5n-3 C 22:4n^ c 22:5n-« C 22:5n-3 c 22:6n-3 JSAT 4 ZMONO5 ZPUFA6 Zn-67 Zn-3g
DIETARY FATS AND CHICKEN IMMUNE TISSUES
1219
TABLE 7. Distribution of total thymus fatty acids of 3- to 4-wk-old chickens fed various fat
sourcesr^
Fat source Fatty acids
LA
CO
CA
C
.9" 23.4 a 3.6b 8.7a 40.4 b 15.8C .1 .4 d .5" .2 .4 2.1 a .2° .6 a .la .2° .3 b 32.9b 44.6 b 20.5° 19.4° 1.2d
.3° 16.2b l.ld 6.6b 27.4 ed 40.7 a .1 .8 d .2° .4 .4 2.3 a .1° .7 a .l a .2° .3" 23.1° 28.7 d 46. l a 44.8 a 1.3d
.3C 14.6° 1.9° 5.8° 47.2" 21.1 b .1 2.0 b .8a 2 .4 1.4b .l c .4 b 0b 2C .4"
LO
FO
SEM3
.3C 15.8b 2.0° 6.8 b 28.9° 20.3 b .1 19.3a .2° .2 .6 .9° .8 b 2C 0b .8 b .5 b 22.9° 31.1 01 43.6 22.2 b 21.4 a
3.6a 23.6 a 7.4a 7.2 b 25.4 d 15.7* .1 12" .8" .4 .2 .5° 3.0" .l c
.1 .3 .2 2 .9 .6 .02 2 .1 .2 .1 .2 2 .1 .1 .1 .1 .3 1.0 .9 .8 .4
(% 14:0
c
20.7"1 49.9" 26.3 23.6b 2.8°
o"
1.2a 2.0 s 34.4a 33.6° 24.5 b 17.1c 7.4b
a-d»Means within rows with no common superscripts are significantly different (P<05).
'Chicks were fed diets containing 7% by weight lard (LA), corn oil (CO), canola oil (CA), linseed oil (LO), or a menhaden fish oil (FO) for 3 to 4 wk prior to sample collection and analysis. 2 Values are expressed as means (n = 4 to 5). 'Pooled SEM. 4 ESAT = Sum total area percentage of C^-o, Ci&o, and CH^Q. 5 2MONO = Sum total area percentage of C ^ i , Cig : i, and C ^ a 6 SPUFA = Sum total area percentage of C i 8 : 2 n ^ , Ci 8 : 3 n _ 6 , C 1 8 : 3 n _ 3 , C20:2n-6> C20:3n-6. C2o:4n-6. C
20:5n-3> C22:4n-«> C22:5n-6. C22:Sn-3. Q22:6n-37 8
2n-6 = Sum total area percentage of C l g : 2 n . 6 , C i 8 : 3 n ^ , C ^ ^ . 6 , C^j^, C20:4n-6.
effect of fat source on the level of total saturated fatty acids in isolated splenocytes or in the bursa. Contrary to those observations, the fatty acid composition of the thymus showed a remarkable similarity to that of the diet. The impact of dietary fat source on immune tissue profiles are most evident for individual fatty acids, particularly long-chain PUFA. For example, C20:4n-6 accounted for up to 21% of the total fatty acids present in the splenocytes from chicks fed LA, CO, or CA (Table 5). Feeding diets rich in n-3 fatty acids (i.e., LO or FO) lowered significantly (P<.001) the relative content of C20:4n6 in splenocytes and significantly elevated (P<001) n-3 levels 400 to 500%. Feeding the n-3 rich fats (LO or FO) also depressed significantly (P<.05) the levels of
C-20:4n-6 m m e bursa (Table 6) and thymus (Table 7) compared with levels in LA- and COfed chicks. Similar observations have been reported for liver, heart, brain, thigh, and breast muscle lipids in the chicken (Edwards and Marion, 1963; Miller and White, 1975; Anderson, et al., 1989; Huang et al, 1990). Incorporation of long-chain n-3 PUFA (i.e., Q20:5n-3 and C22:6n-3) w a s dependent on the source of the n-3 fatty acids. Feeding preformed sources of these n-3 PUFA (i.e., FO) resulted in a greater elevation of these fatty acids in the immune tissues compared with the Ci8:3n.3-rich LO. Others have shown that feeding FO to rats or mice results in a greater deposition of C20:5n-3 and C22:6n-3 in liver and immune tissues compared with LO, despite an
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16:0 Cl6:l c 18:0 C 1S:1 Cl8:2n-6 Cl8:3n-6 c 18:3n-3 c 20:l c 20:2n-6 C 20:3n-6 C20:4n-6 Q20:5n-3 c 22:4n-6 c 22:5n-6 C 22:5n-3 c 22:6n-3 2SAT 4 2MON0 5 EPUFA6 Zn-67 Zn-38
1220
FRITSCHE ET AL. BURSA
SERUM
b
H 20:4n-6
20:5n-3
22:6n-3
20:4n-6
25 "8
THYMUS
Lard Corn oil Canola oil Linseed oil Fish oil
20
<
1 H a
"o
22:6n-3
10 51 0
20:4n-6
20:5n-3
22:6n-3
20:4n-6
20:5n-3
22:6n-3
FIGURE 1. Effect of dietary fat source on selected fatty acid levels in chicken serum and immune tissues. Chicks were fed diets that contained 7% of the fat from the sources indicated for 3 to 4 wk prior to sample collection. Total lipids were extracted and the fatty acids were analyzed by gas-liquid chromatography. Data represent the mean (n = 4) percentage of the weight of the fatty acids indicated. Error bars on the fish oil data represent the pooled SEM. Bars with no common letters are significantly different (P<.05). Fatty acids are denoted by the number of carbons: number of double bonds, followed by the position of the first double bond relative to the methyl end (n-).
overall greater n-3 content in the latter oil (Croft ex al, 1984; Fritsche and Johnston, 1990). Furthermore, the level of Cig:3n_3 in the spleen (Table 5) and bursa (Table 6) was elevated (P<.005) only when LO was fed. The thymus (Table 7) showed a greater enrichment of Cig. 3n_3 compared with the spleen and bursa, and the relative content exceeded that found in the serum. This may be related to the high affinity die tfrymus demonstrated for Cig fatty acids, which generally accounted for over 50% of the total fatty acids present. Shown in Figure 1 are the levels of the three fatty acids (C20:4n6> C 2 0 : 5n-3.
C
22:6n-3)
mos
*
relevant to die eicosanoid-producing capacity of these immune tissues. Eicosanoids are the oxygenated metabolites of C20 fatty acids. Arachidonic acid (C2o:4n-6) is the predominate precursor in most mammalian tissues. Other precursors include C2o:3n-6 and C20:5n-3- ^ e former has been omitted from this figure for two
reasons. First, levels of this fatty acid in the spleen and thymus were not altered by fat source and were only modestly affected in the bursa. Second, this fatty acid is a minor constituent (<2%) of the tissue lipids examined. Serum and tissue C22;6n-3 have been included in this figure because this fatty acid is known to be a potent inhibitor of eicosanoid synmesis (Corey ex al., 1983). Figure 1 illustrates the impact of dietary fat source on the relative amount of eicosanoid precursors present in the immune tissues of the chicken. Marked differences between precursor levels are apparent in the immune tissues studied. The bursa contains only half as much C20:4n-6 a s splenocytes, and thymus contains even less than the bursa. Tissue-specific differences in PUFA content may reflect differences in desaturase activity or acyl-coenzyme A transferase activities (Willis, 1981). Such tissuespecific differences in C20:4n-6 content may have
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25-1 SPLENOCYTES
20:5n-3
DIETARY FATS AND CHICKEN IMMUNE TISSUES
ACKNOWLEDGMENTS
The authors are grateful to Zapata Haynie, Reedville, VA 22539; Best Foods, a division of CPC International Inc., Union, NJ 07083; Kraft, Inc., Glenview, EL 60025; and Cargill, Inc., Minneapolis, MN 55440 for supplying the dietary oils used in this research project. The authors appreciate Margaret Campen's assistance with the care and feeding of the animals used in these studies. REFERENCES Anderson, G. J., W. E. Conner, J. D. Corliss, and D. S. Lin, 1989. Rapid modulation of the n-3 docosahexaenoic acid levels in the brain and retina of the newly hatched chick. J. Lipid Res. 30:433-441. Chouaib, S., L. Chatenoud, D. Klatzmann, and D. Frazelizi, 1985. The mechanisms of inhibition of
human IL-2 production. PGE2 induction of suppressor T lymphocytes. J. Immunol. 132:1851-1857. Codde, J. P., L. J. Beilin, K. D. Croft, and R. Vandongen, 1985. Study of diet and drug interactions on prostanoid metabolism. Prostaglandins 29:895-910. Corey, E. J., C. Shin, and J. R. Cashman, 1983. Docosahexaenoic acid is a strong inhibitor of prostaglandin but not leukotriene biosynthesis. Proc. Natl. Acad. Sci. 80:3581-3584. Craig-Schmidt, M. C , S. A. Faircloth, and J. D. Weete, 1987. Modulation of avian lung eicosanoids by dietary omega-3 fatty acids. J. Nutr. 117:1197-1206. Croft, K. D., L. J. Beilin, R. Vandongen, and E. Mathews, 1984. Dietary modifications of fatty acid and prostaglandin synthesis in the rat: Effect of variations in the level of dietary fat. Biochim. Biophys. Acta 795:196-207. Culp, B. R„ B. G. Titus, and WJE.M Lands, 1979. Inhibition of prostaglandin biosynthesis by eicosapentaenoic acid. Prostaglandins Leukotrienes Med. 3:269-278. Edwards, H. M., Jr., and J. E. Marion, 1963. Influence of dietary menhaden oil on growth rate and tissue fatty acids of the chick. J. Nutr. 81:123-130. Fritsche, K. L. and P. V. Johnston, 1988. Rapid autoxidation of fish oil in diets without added antioxidants. J. Nutr. 118:425-426. Fritsche, K. L., and P. V. Johnston, 1990. Effect of dietary omega-3 fatty acids on cell-mediated cytotoxic activity in BALB/c mice. Nutr. Res. 10:577-588. Fritsche, K. L., N. A. Cassity, and S. C. Huang, 1991. Effect of dietary fat source on antibody production and lymphocyte proliferation in chickens. Poultry Sci. 70:611-617. Goldyne, M. E., and J. D. Stobo, 1981. Immunoregulation of prostaglandins and related lipids. CRC Crit Rev. Immunol. 2:189-223. Goodwin, J. S., A. D. Bankhurst, and R. P. Messner, 1977. Suppression of human T-cell mitogenesis by prostaglandin: Existence of a prostaglandin-producing suppressor cell. J. Exp. Med. 146:1719-1734. Huang, Z-B., H. Leibovitz, C. M Lee, and R. Miller, 1990. Effect of dietary fish oil on o-3 fatty acid levels in chicken eggs and thigh flesh. J. Agric. Food Chem. 38:743-747. Johnston, P. V., 1985. Dietary fat, eicosanoids, and immunity. Adv. Lipid Res. 21:37-86. Johnston, P. V., 1988. Lipid modulation of immune responses. Pages 37-86 in: Nutrition and Immunology. R. K. Chandra, ed. Alan R. Liss, Inc., New York, NY. Kates, M., 1986. Pages 123-128 in: Techniques of Lipidology. Isolation, Analysis, and Identification of Lipids. American Elsevier, New York, NY. Kinsella, J. E., B. Lokesh, S. Broughton, and J. Whelan, 1990. Dietary polyunsaturated fatty acids and eicosanoids: Potential effects on the modulation of inflammatory and immune cells: An overview. Nutrition 6:24-60. Likoff, R. O., D. R. Guptill, L. M. Lawrence, C. C. McKay, M. M. Mathias, C. F. Nockels, and R. P. Tengerdy, 1981. Vitamin E and aspirin depress prostaglandins in protection of chickens against Escherichia cott infection. Am. J. Clin. Nutr. 34: 245-251. Miller, D., and V. White, 1975. Chick tissues fatty acid composition as affected by selenium, DL-oc-tocoph-
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important consequences with regard to the role that eicosanoids play in immunoregulation at those tissue sites. Evidence is mounting that dietary PUFA may influence immune cell function and disease resistance through the modulation of eicosanoid production (Johnston, 1988). Although not measured in the present study, others have shown that changes in tissue fatty acid composition similar to those reported here can modulate eicosanoid production in chickens (Craig-Schmidt et ah, 1987). Recently, the present authors have demonstrated that feeding chicks an n-3-rich diet (7% FO) significantly enhanced antibody production and altered lymphocyte proliferation (Fritsche et ah, 1991). The present data indicates that the serum and immune tissues from FO-fed chicks are rich in n-3 PUFA. Two of the n-3 fatty acids abundant in fish oils, C20:5n-3 a n d Q22:6n-3> are potent inhibitors of PG synthesis (Culp et ah, 1979; Corey et ah, 1983; Codde et ah, 1985). In mammals, PGE2 has been shown to inhibit a variety of immunologic responses, including lymphocyte proliferation (Goodwin et ah, 1977), antibody formation (Plescia et ah, 1975), and lymphokine production (Chouaib et ah, 1985). Eicosanoids, such as PGE2, may also play an important role in regulating avian immune responses. Likoff et ah (1981) demonstrated that inhibition of PG production with high dietary vitamin E or aspirin enhanced chicken's resistance to bacterial infection. Fatty acid composition may play an important role in determining the eicosanoid-synthesizing capacity of immune cells or tissues.
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erol acetate, ethoxyquin and polyunsaturated oils. Nutr. Rep. Int. 12:245-255. National Research Council, 1984. Nutrient Requirements of Poultry. 8th ed. National Academy Press, Washington, DC. Plescia, D. J., A. H. Smith, and K. Grinwich, 1975. Subversion of the immune response by tumor cells and role of prostaglandins. Proc. Natl. Acad. Sci. 72: 1848-1851. Rola-Pleszczynski, M., 1985. Immunoregulation by leukotrienes and other lipoxygenase metabolites. Im-
munol. Today 6:302-307. Steel, R.G.D., and J. H. Torrie, 1980. Principles and Procedures of Statistics. A Biomedical Approach. McGraw-Hill, New York, NY. Stenson, W. F., and C. W. Parker, 1982. Prostaglandins and the immune response. Pages 39-89 in: Prostaglandins. J. B. Lee, ed. Elsevier, New York, NY. Traill, K. N., and G. Wick, 1984. Lipids and lymphocyte function. Immunol. Today 5:70-76. Willis, A. L., 1981. Unanswered questions in EFA and PG research. Prog. Lipid Res. 20:839-850.
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