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Incorporation of Different Types of n-3 Fatty Acids Into Tissue Lipids of Poultry12
Incorporation of Different Types of n-3 Fatty Acids Into Tissue Lipids of Poultry1'2 P. CHANMUGAM, M. BOUDREAU, T. BOUTTE, R. S. PARK, J. HEBERT, L. BERRIO, and D. H. HWANG3 Pennington Biomedical Research Center and Louisiana Agricultural Experiment Station, Louisiana State University, Baton Rouge, Louisiana 70808 (Received for publication April 15, 1991)
1992 Poultry Science 71516-521
INTRODUCTION
boxanes, but also the conversion of dietary linoleic acid (Ci8:2n-6) to C20:4n-6 (Lands et It has been suggested (Dyerberg and al., 1973). The reduction in the concentraBang, 1978; Kromhout et al, 1985; Herold tions of C20:4n-6 as well as its metabolites, and Kinsella, 1986) that the beneficial particularly thromboxane A2, which has a effect of seafood consumption in reducing potent proaggregatory effect on platelets, the risk of coronary heart disease is due is implicated as one of the reasons why primarily to the polyunsaturated fatty n-3 fatty acids reduce the risk of coronary acids (PUFA) of the n-3 series, which are heart disease (Leaf and Weber, 1988). abundant in the lipids of fish. Dietary n-3 These findings have led to the suggesfatty acids inhibit not only the metabolism tion that it may be desirable to increase of arachidonic acid (C20:4n-6)/ which is the the dietary n-3:n-6 fatty acid ratio (Lands, principal substrate for lipoxygenase and 1989), although such a change has not yet cyclooxygenase in the biosynthesis of been recommended to the general public. leukotrienes, prostaglandins, and thromHowever, Americans have traditionally preferred red meat and poultry to fish, except certain gourmet shellfish. Because 1 Supported by National Institutes of Health Grant poultry consumption in the United States (IROI DK 41868-01), USDA Competitive Research is high and continues to increase, there Grant (87CRCR-1-2513), and a grant from the Louis- have been attempts to increase the n-3 iana Board of Regents. fatty acid content of poultry by supple2 Approved for publication by the Director of the mentation of poultry diets with oils rich in Louisiana Agricultural Experiment Station as Manun-3 fatty acids (Hulan et al, 1988, 1989). script Number 91-25-5172. 3To whom correspondence should be addressed. These studies used fish oil, rich in the long 516
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ABSTRACT The objective of the present study was to compare the incorporation of different types of n-3 polyunsaturated fatty acids into thigh muscle lipids of poultry. Nine groups of broiler chicks were fed diets supplemented with three levels (1.0,2.5, and 5.0%) of either corn, linseed, or menhaden oil. Birds supplemented with linseed oil, rich in linolenic acid (Ci8:3n-3X had significantly higher levels of n-3 fatty acids and higher n-3:n-6 ratios than those supplemented with the same level of menhaden oil, primarily due to an accumulation of Ci8:3n-3- Levels of eicosapentaenoic acid (C20£n-3) were increased (P<.05), compared with the controls fed the same level of corn oil, in the groups fed the two higher levels of linseed oil, and in all the groups fed menhaden oil. Linolenic acid is less susceptible to auto-oxidation, and is less likely to impart an off-flavor to the muscle. Thus, if it is desirable to increase the n-3:n-6 ratio in poultry, seed meals or oils with a high content of Ci8:3n-3 could be used in poultry feeds. {Key words: n-3 fatty acids, n-3 to n-6 ratio, fish oil, linolenic acid, broiler chicks)
517
n-3 FATTY ACIDS IN POULTRY
MATERIALS AND METHODS One-day-old Cobb broiler chicks purchased from a commercial hatchery4 were individually wing-banded and randomly placed into 18 pens (five males and five females per pen, 2 pens per dietary treatment) of an electrically heated Petersime5 battery brooder. Birds in each pen were allowed ad libitum access to water and diets as described in Table 1. The diets were prepared by mixing 1.0, 2.5, or 5.0% c o n v linseed,7 or menhaden oil8 to a formulated basal diet and met National Research Council (1984) requirements. The fatty acid compositions of the diets are
4
Sanderson Farm, Hammond, LA 70401. ^etersime Incubator Co., Gettysburg, OH 45328. *ICN Biomedicals, Inc., Costa Mesa, CA 92628. 7 Harrison Paint Co., Baton Rouge, LA 70801. 8 Empire Menhaden Products, Empire, LA 70050. ^ u Chek Prep, Elysian, MN 56028.
listed in Table 2. After 7 to 8 wk on the experimental diets, two males and two females, randomly selected from each pen, were killed by cervical dislocation. Throughout the experiment, care was taken to minimize pain and discomfort to the birds. Thighs were removed, frozen in liquid nitrogen, and stored at -20 C until assayed for fatty acid composition. Total lipids were extracted from samples of diet and thigh muscle, including skin, by the method of Folch et al. (1957) and saponified and methylated using boron trifluoride as the methylating agent (Morrison and Smith, 1964). Fatty acid composition of total lipids was determined by gas liquid chromatography as described by Hwang and Carroll (1980). Individual fatty acids were identified by comparing the retention times of the fatty acids with those of fatty acid methyl ester standard mixtures. 9 Data were analyzed by ANOVA using SAS Institute (1982) procedures. Duncan's multiple range test (Duncan, 1955) was used to determine whether there were significant (P<.05) differences among means obtained from the different treatments. All comparisons were made among groups supplemented with the same level of dietary oil, so that the dietary n-3:n-6 ratio was the only variable and all other dietary factors, such as the calorie:protein ratio, were constant. RESULTS Feed consumption or feed efficiencies were not significantly affected by n-3 fatty acid supplementation compared with controls fed the same level of corn oil (data not shown). There were also no significant differences in mean body weight at 54 days among the nine treatments (2.10, 2.05, 2.12, 2.04, 2.14, 2.02, 2.01, 2.04, and 2.10 kg, respectively). The fatty acid composition of thigh muscle lipids is listed in Table 3. These data show that dietary n-3 supplementation resulted in significantly increased levels of n-3 fatty acids in thigh muscle lipids compared with the controls fed the same level of corn oil. Birds fed linseed oil showed significantly higher levels of total n-3 fatty acids than those fed the same level of menhaden oil. This was primarily
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chain n-3 PUFA eicosapentaenoic acid (C20:5n-3) an<3 docosahexaenoic acid (C22:6n-3)/ as a source of n-3 fatty acids. Linolenic acid (Ci8:3n-3)/ a shorter chain n-3 PUFA that is abundant in some seed oils such as linseed oil and in chloroplast lipids, has generally been overlooked as a source of dietary n-3 fatty acids. Phetteplace and Watkins (1989) studied the effects of dietary linseed oil, menhaden oil, soybean oil, and chicken fat on the fatty acid composition of broiler lipids. They supplemented the diets with 5% of the dietary fats used. Previous studies (Miller et al, 1967; Dean et al, 1969), have reported an unacceptable flavor in poultry fed 1.5 to 5% fish oil. The objective of the present study was to determine the effects of diets supplemented with 1 to 5% of linseed or menhaden oil on the fatty acid composition of chicken lipids. The incorporation of n-3 fatty acids into lipids of broiler chicks fed 1, 2.5, or 5% linseed or menhaden oil was compared by determining the fatty acid composition of total lipids in samples of thigh muscle, including skin. Chicks fed three levels of corn oil served as controls for experimental groups fed the same level of oils rich in n-3 fatty acids.
518
CHANMUGAM ET AL. TABLE 1. Experimental design
Ingredients, %
A
Basal diet1*2 Corn oil Linseed oil Menhaden oil
99.0 1.0
Corn oil diets B C 975 25
95.0 5.0
D
Linseed oil diets E F
G
99.0
975
95.0
1.0
25
5.0
Fish oil diets H I
99.0
975
95.0
1.0
25
5.0
due to a considerable accumulation of linolenic acid (Ci8:3n_3) in the birds fed linseed oil. Levels of eicosapentaenoic acid (Q»)5n-3) w e r e higher in thigh muscle lipids of the groups fed linseed oil compared with those of controls fed the same level of corn oil with the differences being significant in the groups fed 2.5 or 5% linseed oil. Levels of C205n-3 m all the groups fed menhaden oil were significantly higher than the groups fed the same level of either corn oil or linseed oil. Similarly, levels of docosapentaenoic acid (C22:6n-3) were higher than controls fed the same level of corn oil in the groups fed linseed oil, but the differences were not statistically significant. All the groups fed menhaden oil had C22:6n-3 levels that were significantly higher than the groups fed the same level of either corn oil or linseed oil. The n-3:n-6 fatty acid ratio in thigh muscle lipids was significantly higher in all the birds supplemented with dietary n-3 fatty acids than in controls fed the same level of corn oil. Linseed oil supplementation resulted in significantly higher values compared with the same level of menhaden oil supplementation (Table 3). The C205n-3 to C20:4n-6 ratio was higher in all the birds fed linseed oil than controls fed the same level of corn oil, with the difference being statistically significant in those fed 2.5 or 5% linseed oil. The lipids of all the groups fed menhaden oil had significantly higher C20Sn-3 to C20:4n-6 ratios compared with the linseed and corn oil groups fed the same level of oil.
DISCUSSION The results demonstrate that the n-3 fatty acid content of broiler thigh muscle can be increased by dietary supplementation with either linseed or menhaden oil, although most of the increases in n-3 fatty acids in the groups fed linseed oil were due to the accumulation of linolenic acid (Ci8:3n-3)- The level of linolenic acid (21.86%) in thigh muscle lipids of chicks fed 5% linseed oil is similar to that (24.19%), of birds fed the same level of linseed oil, reported by Phetteplace and Watkins (1989). Previous work by Hwang ef al. (1988) has shown that Ci83n-3 can suppress levels of arachidonic acid and its metabolites synthesized via the cyclooxygenase pathway in rats, but does not do this as effectively as eicosapentaenoic acid (C20:5n-3>- However Cis^n-3 is a poor substrate for leukotriene synthesis, whereas C205n-3 is preferred over arachidonic acid (C20:4n-6) as a substrate for 5-lipoxygenase (Ochi, 1983). Thus, the total synthesis of lipoxygenase products, which can act as mediators of inflammation, may be reduced more by Ci8-.3n-3 than C2fl5n-3These results also demonstrate that either 2.5% linseed oil or 1% menhaden oil in the diets of broilers can significantly increase levels of C205n-3 in thigh muscle lipids above controls fed the same level of corn oil. Phetteplace and Watkins (1989) also reported increased levels of C205n-3 in breast (1.79%) and thigh (1.24%) muscle
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•'Basal ingredients include (as a percentage of the total): ground corn, 55.0; soybean meal, 36.0; limestone, .8; DL-methionine, .1; dicalcium phosphate, 25; choline chloride, .1; vitamin and mineral premix, 50; (supplied per kilogram of diet: vitamin A, 11,000 IU; cholecakiferoL 1,650 IU; vitamin E, 8.25 IU; menadione sodium bisulfite, .73 mg; thiamine, 1 mg; riboflavin, 4.4 mg; niacin, 33 mg; d-pantothenic acid, 8.1 mg; folic acid, .45 mg; biotin, .05 mg; pyridoxine, 12 mg; vitamin B12, .01 mg; choline, 400 mg; manganese, 60 mg; zinc, 44 mg; iron, 20 mg; copper, 2 mg; iodide, 1.2 mg; and cobalt, 20 mg). 2 Calculated nutrient composition: crude protein, 21%; metabolizable energy, 3,100 kcal/kg; calcium, 1.2%; crude fat, 25%; linoleic acid, 1.18%.
519
n-3 FATTY ACIDS IN POULTRY TABLE 2. Fatty add composition of diets1 Linseed oil diets
Corn oil diets Fatty acid 2
A .05 .13 .06 1527 23 2.17 21.04 5828 .15 1.78
C .06 .10
16.05 23 2.02 2Z17 57.41 .13 1.82
.03 .06 .02 1338 .18 2.11 23.41 58.92 .12 1.77
.17
ratio
58.43 5828 .15 0 0
D
E
.03 .10 .03 11.98 .18 2.79 21.10 4759 16.02 .03
.05 .09 .04 10.91 .17 2.92 21.81 38.81 25.07
.13
5753 57.41 .13 0 0
59.04 58.92 .12 0 0
63.62 4759 16.03 34 0
F .08 .03 932 .15 3.05 23.00 32.44 31.80 .12
63.88 38.81 25.07 .64 0
6424 3X44 31.80 .98 0
Menhaden oiil diets G
H
I
.11 537 .31 19.89 5.44 239 1734 4024 36 2.40 .95 .73 .17 25 3.14 .11
.14 7.04 .43 19.86 7.81 2.70 16.66 3221 .64 2.08 1.55 .08 34 .44 5.36 20 .08 51 1.82 43.18 32.85 1033 31 1553
.18 9.45 56 2136 1057 2.85 14.97 22.69 .73 1.91 Z13 .07 .47 .62 7.60 29 .11 .72 2.70 38.06 2356 1450 .62 15.98
30 1.10 46.65 4054 6.11 .15 1758
1
Values are weight percentages of total identified fatty acids. The first number indicates the number of carbon atoms, the second the number of double bonds, the third the position of the first double bond, counting from the methyl or omega end. 3pUFA = polyunsaturated fatty acids. 2
lipids of chickens fed 5% linseed oil compared with birds fed 5% soybean oil or 5% chicken fat. The present results also suggest that broiler chicks are capable of desaturating Ci83n-3 to C205n-3- High levels (7.25%) of C20:5n-3 in thigh muscle lipids of chickens fed 5% menhaden oil were reported by Phetteplace and Watkins (1989). Hulan et al (1989) reported C2o-5n-3 levels of 1.66% in breast and .87% in thigh muscle lipids of broilers fed 12% red fish meal. They calculated that consumption of 100 g of these birds could provide approximately 52 mg of C205n-3/ which is about 30% of the 150 mg/day associated with a significant reduction in cardiovascular mortality in human subjects studied by Kromhout et al. (1985). Currently broiler meat contains approximately 10 mg of C205n-3/100 g (USDA, 1990). Recent work by Boudreau et al. (1991) has indicated that the dietary n-3:n-6 fatty acid ratio may be more important than the absolute amount of dietary n-3 fatty acids
in the inhibition of arachidonic acid metabolism. In the present study, all birds supplemented with either linseed or fish oil showed significant increases in the n-3: n-6 fatty acid ratio in thigh muscle lipids compared with controls fed the same level of corn oil. Studies by Miller et al. (1967) and Dean et al. (1969) report an unacceptable or offflavor in taste panel tests of poultry fed 1.5 to 5% fish oil or 3 to 14% fish meal. Supplementation of poultry diets with lower levels of fish oil together with a source of linolenic acid may be a way to achieve an acceptable product with an increased n-3:n-6 fatty acid ratio. The data indicate that linseed oil supplementation of poultry diets results in n-3:n-6 ratios that are greater than those obtained by supplementation with similar levels of menhaden oil. Unlike C205n-3 in menhaden oil, Cig^n.3 in linseed oil is not a preferred substrate for lipoxygenases and is less susceptible to auto-oxidation.
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Cl2:0 c 14:0 C 14:l C 16:0 Cl6:ln-7 C 18:0 C 18:ln-9 Cl82n-6 Cl83n-3 C 20:ln-9 Cl8:4n-3 C22:0 C 20:4n-6 C20:4n-3 C205n-3 C22:4n-6 C22^n-6 C 22£n-3 C 22:6n-3 Total PUFA 3 Total n-6 Total n-3 n-3m-6 ratio ^^11-3^20:411-6
B
± ± ± ± ± ± ± ± ±
3.74 59 20.05 655 5.70 37.77 23.63 1.04 .08 .10 .71
.02 .17 .04 .01 .12 26.06 24.64 129 .05
Cl4:02 C 14:l C l&0 ^16:1 C 18:0 C 18:ln-9 C 18:2n-6 C 183n-3 C 18:4n-3 QHh3n-6 C 20:4n-6 C20:4n-3 C 20:5n-3 C 22:4n-6 C 22Sn-« C2i5n-3 C 22:6n-3
6.13 .58 18.90 4.81 559 31.83 29.51 1.19 .09 .13 1.00
.00* .24 ± .04 ± .03 ± 24 ± 32.46 ± 30.92 ± 154 ± .05 ±
.99 .12 58 .38 .30 1.16 1.13c .19* .03 .01 .13abc
.01ef .04 .02 .01 .03d 127* 1.17° 20* .01*
± ± ± ± ± ± ± ± ± ± ±
.04 .02 .01 .06d .81c .76b .08f .01f
1.34 .14 .49 .16 .32 .86 .74b .07d .02 .01 .10*
.01 .18 .05 .05 .16 40.38 38.66 1.72 .04
3.32 50 1639 334 5.64 30.44 3752 1.31 .19 .12 .79
Corn oil dietsi B C
± ± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ± .01rf .03 .02 .03 .05d \XJ* 132" 25** .01f
.71 .13 56 26 21 .85 1.33a .14d .06 .02 .10ab .13 .05 .01 .15 .39 2452 19.42 5.09 .26
± ± ± ± ± ± ± ± ±
5.43 ± .38 ± 2052 ± 5.96 ± 6.35 ± 36.83 ± 18.60 ± 4.37 ± .05 ± .06 ± .71 ±
D 5.13 .43 17.76 5.00 5.97 33.13 19.12 11.44 25 .07 59
±157 ± .06 ± 27 ± 28 ± .19 ± .89 ± .SO*e ± .80b ± .10 ± .01 ± 12b«i
2.46 .38 13.96 359 5.17 29.60 2138 21.86 .37 .05 30 .02 31
± ± ± ± ± ± ± ± ± ± ± ± ±
.35 .05 .26 .26 .20 .70 .45cd .46a .14 .01 d .04 .01 .02d
.02^ 59 ± .03de .02 .06 ± .02 .01 .04 ± .04 55 ± .03 .03 57 ± .06 58±.04d .05d .46 ± .12d d 1.16 3258 ± 1.38° 44.82 ± .87* .87** 19.88 ± .70* 21.73 ± .48* .34d 12.70 ± .71b 23.09 ± .43a 1.06 ± .01a .01d .64 ± .02b
.91 .03 .35 .34 26 .99 .86e .31° .03 .01 .15abc
Linseed oil diets E F
Total PUFA3 Total n-6 Total n-3 n-3ai-6 ratio ^•Sn^Wiia-e 1.09 ± .10d 56 ± .OS*1 .02 ± .01e 0e .02 ± .01e 20 ± .03e ratio '''Means within rows with no common superscripts are significantly different (P<.05). ^Values are weight percentages of total identified fatty acids (x ± SEM of eight birds). 2 The first number indicates the number of carbon atoms, the second the number of double bonds, the third the p methyl or omega end. 3 PUFA = polyunsaturated fatty acids.
± ± ± ± ± ± ± ± ± ± ±
A
Fatty acid
TABLE 3. Fatly acid composition of broiler thigh muscle lipid
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n-3 FATTY ACIDS IN POULTRY
Finally, linseed oil supplementation may not result in poultry with the off-flavors associated with fish oil supplementation. The current results suggest that seed meals and oils containing a high content of linolenic acid could be used to increase the n-3:n-6 fatty acid ratio in poultry. REFERENCES
1988. Dietary linolenic acid and longer chain N-3 fatty acids: Comparison of effects on arachidonic acid metabolism in rats. J. Nutr. 118: 427-437. Hwang, D. H., and A. E. Carroll, 1980. Decreased formation of prostaglandins derived from arachidonic acid by dietary linolenate in rats. Am. J. Clin. Nutr. 33590-597. Kromhout, D., E. B. Bosschieter, and C.D.L. Coulander, 1985. Inverse relationship between fish consumption and 20 year mortality from coronary heart disease. N. Engl. J. Med. 312: 1205-1209. Lands, W.EM., 1989. N-3 fatty adds as precursors for active metabolic substances:dissonance between expected and observed events. J. Int. Med. 225: 11-20. Lands, W.E.M., P. E. LeTellier, L. H. Rome, and J. Y. Vanderhoek, 1973. Inhibition of prostaglandin biosynthesis. Adv. Biosci. 9:15-28. Leaf, A., and P. C. Weber, 1988. Cardiovascular effects of N-3 fatty acids. N. Engl. J. Med. 318: 549-557. Miller, D., E. H. Gruger, K. C. Leong, and G. KnobL 1967. Effect of refined menhaden oils on the flavor and fatty acid composition of broiler flesh. J. Food Sci. 32:342-345. Morrison, W. R., and L. M. Smith, 1964. Preparation of fatty acid methyl esters and dimethyl acetals from lipids with boron trifluoride-methanol. J. Lipid Res. 5:600-608. National Research Council, 1984. Nutrient Requirements of Poultry. 8th rev. ed. National Academy Press, Washington, DC. Phetteplace, H. W., and B. A. Watkins, 1989. Effects of various n-3 lipid sources on fatty acid compositions in chicken tissues. J. Food Compos. Anal. 2:104-117. Ochi, K., T. Yohimoto, and S. Yamamoto, 1983. Arachidonate 5-Iipoxygenase of guinea pig peritoneal polymorphonuclear leukocytes: activation by adenosine 5'-triphosphate. J. BioL Chem. 258:5754-5758. SAS Institute, 1982. SAS® Users Guide: Statistics. SAS Institute Inc., Cary, NC. USDA, 1990. Nutrient Data Base for Standard Reference. Human Nutrition Information Service, Hyattsville, MD.
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Boudreau, M., P. Chanmugam, S. Hart, S. H. Lee, and D. H. Hwang, 1991. Lack of dose response by dietary n-3 fatty acids at a constant n-3m-6 fatty acid ratio in suppressing eicosanoid biosynthesis from arachidonic acid. Am. J. din. Nutr. 54: 111-117. Dean, P., W. F. Lamoreux, J. R. Aitkin, and F. G. Proudfoot, 1969. Flavor associated with fish meal in diets fed to broiler chickens. Can. J. Anim. Sci. 49:11-15. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics 11:1-6. Dyerberg, J., and H. O. Bang, 1978. Dietary fat and thrombosis. Lancet 1:152. Folch, J., M. Lees, and G.H.S. Sloane Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. Herold, P. M., and J. E. Kinsella, 1986. Fish oil consumption and decreased risk of cardiovascular disease: a comparison of findings from animal and human feeding trials. Am. J. Clin. Nutr. 43566-598. Hulan, H. W., R. G. Ackman, WAIN. Ratnayake, and F. G. Proudfoot, 1988. Omega-3 fatty acid levels and performance of broiler chickens fed redfish meal or redfish oil. Can. J. Anim. Sci. 68: 543-547. Hulan, H. W., R. G. Ackman, W.M.N. Ratnayake, and F. G. Proudfoot, 1989. Omega-3 fatty acid levels and general performance of commercial broilers fed practical levels of redfish meal. Poultry Sci. 68:153-162. Hwang, D. H., M. Boudreau, and P. Chanmugam,
521
1992 Poultry Science 71516-521
INTRODUCTION
boxanes, but also the conversion of dietary linoleic acid (Ci8:2n-6) to C20:4n-6 (Lands et It has been suggested (Dyerberg and al., 1973). The reduction in the concentraBang, 1978; Kromhout et al, 1985; Herold tions of C20:4n-6 as well as its metabolites, and Kinsella, 1986) that the beneficial particularly thromboxane A2, which has a effect of seafood consumption in reducing potent proaggregatory effect on platelets, the risk of coronary heart disease is due is implicated as one of the reasons why primarily to the polyunsaturated fatty n-3 fatty acids reduce the risk of coronary acids (PUFA) of the n-3 series, which are heart disease (Leaf and Weber, 1988). abundant in the lipids of fish. Dietary n-3 These findings have led to the suggesfatty acids inhibit not only the metabolism tion that it may be desirable to increase of arachidonic acid (C20:4n-6)/ which is the the dietary n-3:n-6 fatty acid ratio (Lands, principal substrate for lipoxygenase and 1989), although such a change has not yet cyclooxygenase in the biosynthesis of been recommended to the general public. leukotrienes, prostaglandins, and thromHowever, Americans have traditionally preferred red meat and poultry to fish, except certain gourmet shellfish. Because 1 Supported by National Institutes of Health Grant poultry consumption in the United States (IROI DK 41868-01), USDA Competitive Research is high and continues to increase, there Grant (87CRCR-1-2513), and a grant from the Louis- have been attempts to increase the n-3 iana Board of Regents. fatty acid content of poultry by supple2 Approved for publication by the Director of the mentation of poultry diets with oils rich in Louisiana Agricultural Experiment Station as Manun-3 fatty acids (Hulan et al, 1988, 1989). script Number 91-25-5172. 3To whom correspondence should be addressed. These studies used fish oil, rich in the long 516
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ABSTRACT The objective of the present study was to compare the incorporation of different types of n-3 polyunsaturated fatty acids into thigh muscle lipids of poultry. Nine groups of broiler chicks were fed diets supplemented with three levels (1.0,2.5, and 5.0%) of either corn, linseed, or menhaden oil. Birds supplemented with linseed oil, rich in linolenic acid (Ci8:3n-3X had significantly higher levels of n-3 fatty acids and higher n-3:n-6 ratios than those supplemented with the same level of menhaden oil, primarily due to an accumulation of Ci8:3n-3- Levels of eicosapentaenoic acid (C20£n-3) were increased (P<.05), compared with the controls fed the same level of corn oil, in the groups fed the two higher levels of linseed oil, and in all the groups fed menhaden oil. Linolenic acid is less susceptible to auto-oxidation, and is less likely to impart an off-flavor to the muscle. Thus, if it is desirable to increase the n-3:n-6 ratio in poultry, seed meals or oils with a high content of Ci8:3n-3 could be used in poultry feeds. {Key words: n-3 fatty acids, n-3 to n-6 ratio, fish oil, linolenic acid, broiler chicks)
517
n-3 FATTY ACIDS IN POULTRY
MATERIALS AND METHODS One-day-old Cobb broiler chicks purchased from a commercial hatchery4 were individually wing-banded and randomly placed into 18 pens (five males and five females per pen, 2 pens per dietary treatment) of an electrically heated Petersime5 battery brooder. Birds in each pen were allowed ad libitum access to water and diets as described in Table 1. The diets were prepared by mixing 1.0, 2.5, or 5.0% c o n v linseed,7 or menhaden oil8 to a formulated basal diet and met National Research Council (1984) requirements. The fatty acid compositions of the diets are
4
Sanderson Farm, Hammond, LA 70401. ^etersime Incubator Co., Gettysburg, OH 45328. *ICN Biomedicals, Inc., Costa Mesa, CA 92628. 7 Harrison Paint Co., Baton Rouge, LA 70801. 8 Empire Menhaden Products, Empire, LA 70050. ^ u Chek Prep, Elysian, MN 56028.
listed in Table 2. After 7 to 8 wk on the experimental diets, two males and two females, randomly selected from each pen, were killed by cervical dislocation. Throughout the experiment, care was taken to minimize pain and discomfort to the birds. Thighs were removed, frozen in liquid nitrogen, and stored at -20 C until assayed for fatty acid composition. Total lipids were extracted from samples of diet and thigh muscle, including skin, by the method of Folch et al. (1957) and saponified and methylated using boron trifluoride as the methylating agent (Morrison and Smith, 1964). Fatty acid composition of total lipids was determined by gas liquid chromatography as described by Hwang and Carroll (1980). Individual fatty acids were identified by comparing the retention times of the fatty acids with those of fatty acid methyl ester standard mixtures. 9 Data were analyzed by ANOVA using SAS Institute (1982) procedures. Duncan's multiple range test (Duncan, 1955) was used to determine whether there were significant (P<.05) differences among means obtained from the different treatments. All comparisons were made among groups supplemented with the same level of dietary oil, so that the dietary n-3:n-6 ratio was the only variable and all other dietary factors, such as the calorie:protein ratio, were constant. RESULTS Feed consumption or feed efficiencies were not significantly affected by n-3 fatty acid supplementation compared with controls fed the same level of corn oil (data not shown). There were also no significant differences in mean body weight at 54 days among the nine treatments (2.10, 2.05, 2.12, 2.04, 2.14, 2.02, 2.01, 2.04, and 2.10 kg, respectively). The fatty acid composition of thigh muscle lipids is listed in Table 3. These data show that dietary n-3 supplementation resulted in significantly increased levels of n-3 fatty acids in thigh muscle lipids compared with the controls fed the same level of corn oil. Birds fed linseed oil showed significantly higher levels of total n-3 fatty acids than those fed the same level of menhaden oil. This was primarily
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chain n-3 PUFA eicosapentaenoic acid (C20:5n-3) an<3 docosahexaenoic acid (C22:6n-3)/ as a source of n-3 fatty acids. Linolenic acid (Ci8:3n-3)/ a shorter chain n-3 PUFA that is abundant in some seed oils such as linseed oil and in chloroplast lipids, has generally been overlooked as a source of dietary n-3 fatty acids. Phetteplace and Watkins (1989) studied the effects of dietary linseed oil, menhaden oil, soybean oil, and chicken fat on the fatty acid composition of broiler lipids. They supplemented the diets with 5% of the dietary fats used. Previous studies (Miller et al, 1967; Dean et al, 1969), have reported an unacceptable flavor in poultry fed 1.5 to 5% fish oil. The objective of the present study was to determine the effects of diets supplemented with 1 to 5% of linseed or menhaden oil on the fatty acid composition of chicken lipids. The incorporation of n-3 fatty acids into lipids of broiler chicks fed 1, 2.5, or 5% linseed or menhaden oil was compared by determining the fatty acid composition of total lipids in samples of thigh muscle, including skin. Chicks fed three levels of corn oil served as controls for experimental groups fed the same level of oils rich in n-3 fatty acids.
518
CHANMUGAM ET AL. TABLE 1. Experimental design
Ingredients, %
A
Basal diet1*2 Corn oil Linseed oil Menhaden oil
99.0 1.0
Corn oil diets B C 975 25
95.0 5.0
D
Linseed oil diets E F
G
99.0
975
95.0
1.0
25
5.0
Fish oil diets H I
99.0
975
95.0
1.0
25
5.0
due to a considerable accumulation of linolenic acid (Ci8:3n_3) in the birds fed linseed oil. Levels of eicosapentaenoic acid (Q»)5n-3) w e r e higher in thigh muscle lipids of the groups fed linseed oil compared with those of controls fed the same level of corn oil with the differences being significant in the groups fed 2.5 or 5% linseed oil. Levels of C205n-3 m all the groups fed menhaden oil were significantly higher than the groups fed the same level of either corn oil or linseed oil. Similarly, levels of docosapentaenoic acid (C22:6n-3) were higher than controls fed the same level of corn oil in the groups fed linseed oil, but the differences were not statistically significant. All the groups fed menhaden oil had C22:6n-3 levels that were significantly higher than the groups fed the same level of either corn oil or linseed oil. The n-3:n-6 fatty acid ratio in thigh muscle lipids was significantly higher in all the birds supplemented with dietary n-3 fatty acids than in controls fed the same level of corn oil. Linseed oil supplementation resulted in significantly higher values compared with the same level of menhaden oil supplementation (Table 3). The C205n-3 to C20:4n-6 ratio was higher in all the birds fed linseed oil than controls fed the same level of corn oil, with the difference being statistically significant in those fed 2.5 or 5% linseed oil. The lipids of all the groups fed menhaden oil had significantly higher C20Sn-3 to C20:4n-6 ratios compared with the linseed and corn oil groups fed the same level of oil.
DISCUSSION The results demonstrate that the n-3 fatty acid content of broiler thigh muscle can be increased by dietary supplementation with either linseed or menhaden oil, although most of the increases in n-3 fatty acids in the groups fed linseed oil were due to the accumulation of linolenic acid (Ci8:3n-3)- The level of linolenic acid (21.86%) in thigh muscle lipids of chicks fed 5% linseed oil is similar to that (24.19%), of birds fed the same level of linseed oil, reported by Phetteplace and Watkins (1989). Previous work by Hwang ef al. (1988) has shown that Ci83n-3 can suppress levels of arachidonic acid and its metabolites synthesized via the cyclooxygenase pathway in rats, but does not do this as effectively as eicosapentaenoic acid (C20:5n-3>- However Cis^n-3 is a poor substrate for leukotriene synthesis, whereas C205n-3 is preferred over arachidonic acid (C20:4n-6) as a substrate for 5-lipoxygenase (Ochi, 1983). Thus, the total synthesis of lipoxygenase products, which can act as mediators of inflammation, may be reduced more by Ci8-.3n-3 than C2fl5n-3These results also demonstrate that either 2.5% linseed oil or 1% menhaden oil in the diets of broilers can significantly increase levels of C205n-3 in thigh muscle lipids above controls fed the same level of corn oil. Phetteplace and Watkins (1989) also reported increased levels of C205n-3 in breast (1.79%) and thigh (1.24%) muscle
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•'Basal ingredients include (as a percentage of the total): ground corn, 55.0; soybean meal, 36.0; limestone, .8; DL-methionine, .1; dicalcium phosphate, 25; choline chloride, .1; vitamin and mineral premix, 50; (supplied per kilogram of diet: vitamin A, 11,000 IU; cholecakiferoL 1,650 IU; vitamin E, 8.25 IU; menadione sodium bisulfite, .73 mg; thiamine, 1 mg; riboflavin, 4.4 mg; niacin, 33 mg; d-pantothenic acid, 8.1 mg; folic acid, .45 mg; biotin, .05 mg; pyridoxine, 12 mg; vitamin B12, .01 mg; choline, 400 mg; manganese, 60 mg; zinc, 44 mg; iron, 20 mg; copper, 2 mg; iodide, 1.2 mg; and cobalt, 20 mg). 2 Calculated nutrient composition: crude protein, 21%; metabolizable energy, 3,100 kcal/kg; calcium, 1.2%; crude fat, 25%; linoleic acid, 1.18%.
519
n-3 FATTY ACIDS IN POULTRY TABLE 2. Fatty add composition of diets1 Linseed oil diets
Corn oil diets Fatty acid 2
A .05 .13 .06 1527 23 2.17 21.04 5828 .15 1.78
C .06 .10
16.05 23 2.02 2Z17 57.41 .13 1.82
.03 .06 .02 1338 .18 2.11 23.41 58.92 .12 1.77
.17
ratio
58.43 5828 .15 0 0
D
E
.03 .10 .03 11.98 .18 2.79 21.10 4759 16.02 .03
.05 .09 .04 10.91 .17 2.92 21.81 38.81 25.07
.13
5753 57.41 .13 0 0
59.04 58.92 .12 0 0
63.62 4759 16.03 34 0
F .08 .03 932 .15 3.05 23.00 32.44 31.80 .12
63.88 38.81 25.07 .64 0
6424 3X44 31.80 .98 0
Menhaden oiil diets G
H
I
.11 537 .31 19.89 5.44 239 1734 4024 36 2.40 .95 .73 .17 25 3.14 .11
.14 7.04 .43 19.86 7.81 2.70 16.66 3221 .64 2.08 1.55 .08 34 .44 5.36 20 .08 51 1.82 43.18 32.85 1033 31 1553
.18 9.45 56 2136 1057 2.85 14.97 22.69 .73 1.91 Z13 .07 .47 .62 7.60 29 .11 .72 2.70 38.06 2356 1450 .62 15.98
30 1.10 46.65 4054 6.11 .15 1758
1
Values are weight percentages of total identified fatty acids. The first number indicates the number of carbon atoms, the second the number of double bonds, the third the position of the first double bond, counting from the methyl or omega end. 3pUFA = polyunsaturated fatty acids. 2
lipids of chickens fed 5% linseed oil compared with birds fed 5% soybean oil or 5% chicken fat. The present results also suggest that broiler chicks are capable of desaturating Ci83n-3 to C205n-3- High levels (7.25%) of C20:5n-3 in thigh muscle lipids of chickens fed 5% menhaden oil were reported by Phetteplace and Watkins (1989). Hulan et al (1989) reported C2o-5n-3 levels of 1.66% in breast and .87% in thigh muscle lipids of broilers fed 12% red fish meal. They calculated that consumption of 100 g of these birds could provide approximately 52 mg of C205n-3/ which is about 30% of the 150 mg/day associated with a significant reduction in cardiovascular mortality in human subjects studied by Kromhout et al. (1985). Currently broiler meat contains approximately 10 mg of C205n-3/100 g (USDA, 1990). Recent work by Boudreau et al. (1991) has indicated that the dietary n-3:n-6 fatty acid ratio may be more important than the absolute amount of dietary n-3 fatty acids
in the inhibition of arachidonic acid metabolism. In the present study, all birds supplemented with either linseed or fish oil showed significant increases in the n-3: n-6 fatty acid ratio in thigh muscle lipids compared with controls fed the same level of corn oil. Studies by Miller et al. (1967) and Dean et al. (1969) report an unacceptable or offflavor in taste panel tests of poultry fed 1.5 to 5% fish oil or 3 to 14% fish meal. Supplementation of poultry diets with lower levels of fish oil together with a source of linolenic acid may be a way to achieve an acceptable product with an increased n-3:n-6 fatty acid ratio. The data indicate that linseed oil supplementation of poultry diets results in n-3:n-6 ratios that are greater than those obtained by supplementation with similar levels of menhaden oil. Unlike C205n-3 in menhaden oil, Cig^n.3 in linseed oil is not a preferred substrate for lipoxygenases and is less susceptible to auto-oxidation.
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Cl2:0 c 14:0 C 14:l C 16:0 Cl6:ln-7 C 18:0 C 18:ln-9 Cl82n-6 Cl83n-3 C 20:ln-9 Cl8:4n-3 C22:0 C 20:4n-6 C20:4n-3 C205n-3 C22:4n-6 C22^n-6 C 22£n-3 C 22:6n-3 Total PUFA 3 Total n-6 Total n-3 n-3m-6 ratio ^^11-3^20:411-6
B
± ± ± ± ± ± ± ± ±
3.74 59 20.05 655 5.70 37.77 23.63 1.04 .08 .10 .71
.02 .17 .04 .01 .12 26.06 24.64 129 .05
Cl4:02 C 14:l C l&0 ^16:1 C 18:0 C 18:ln-9 C 18:2n-6 C 183n-3 C 18:4n-3 QHh3n-6 C 20:4n-6 C20:4n-3 C 20:5n-3 C 22:4n-6 C 22Sn-« C2i5n-3 C 22:6n-3
6.13 .58 18.90 4.81 559 31.83 29.51 1.19 .09 .13 1.00
.00* .24 ± .04 ± .03 ± 24 ± 32.46 ± 30.92 ± 154 ± .05 ±
.99 .12 58 .38 .30 1.16 1.13c .19* .03 .01 .13abc
.01ef .04 .02 .01 .03d 127* 1.17° 20* .01*
± ± ± ± ± ± ± ± ± ± ±
.04 .02 .01 .06d .81c .76b .08f .01f
1.34 .14 .49 .16 .32 .86 .74b .07d .02 .01 .10*
.01 .18 .05 .05 .16 40.38 38.66 1.72 .04
3.32 50 1639 334 5.64 30.44 3752 1.31 .19 .12 .79
Corn oil dietsi B C
± ± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ± .01rf .03 .02 .03 .05d \XJ* 132" 25** .01f
.71 .13 56 26 21 .85 1.33a .14d .06 .02 .10ab .13 .05 .01 .15 .39 2452 19.42 5.09 .26
± ± ± ± ± ± ± ± ±
5.43 ± .38 ± 2052 ± 5.96 ± 6.35 ± 36.83 ± 18.60 ± 4.37 ± .05 ± .06 ± .71 ±
D 5.13 .43 17.76 5.00 5.97 33.13 19.12 11.44 25 .07 59
±157 ± .06 ± 27 ± 28 ± .19 ± .89 ± .SO*e ± .80b ± .10 ± .01 ± 12b«i
2.46 .38 13.96 359 5.17 29.60 2138 21.86 .37 .05 30 .02 31
± ± ± ± ± ± ± ± ± ± ± ± ±
.35 .05 .26 .26 .20 .70 .45cd .46a .14 .01 d .04 .01 .02d
.02^ 59 ± .03de .02 .06 ± .02 .01 .04 ± .04 55 ± .03 .03 57 ± .06 58±.04d .05d .46 ± .12d d 1.16 3258 ± 1.38° 44.82 ± .87* .87** 19.88 ± .70* 21.73 ± .48* .34d 12.70 ± .71b 23.09 ± .43a 1.06 ± .01a .01d .64 ± .02b
.91 .03 .35 .34 26 .99 .86e .31° .03 .01 .15abc
Linseed oil diets E F
Total PUFA3 Total n-6 Total n-3 n-3ai-6 ratio ^•Sn^Wiia-e 1.09 ± .10d 56 ± .OS*1 .02 ± .01e 0e .02 ± .01e 20 ± .03e ratio '''Means within rows with no common superscripts are significantly different (P<.05). ^Values are weight percentages of total identified fatty acids (x ± SEM of eight birds). 2 The first number indicates the number of carbon atoms, the second the number of double bonds, the third the p methyl or omega end. 3 PUFA = polyunsaturated fatty acids.
± ± ± ± ± ± ± ± ± ± ±
A
Fatty acid
TABLE 3. Fatly acid composition of broiler thigh muscle lipid
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n-3 FATTY ACIDS IN POULTRY
Finally, linseed oil supplementation may not result in poultry with the off-flavors associated with fish oil supplementation. The current results suggest that seed meals and oils containing a high content of linolenic acid could be used to increase the n-3:n-6 fatty acid ratio in poultry. REFERENCES
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Boudreau, M., P. Chanmugam, S. Hart, S. H. Lee, and D. H. Hwang, 1991. Lack of dose response by dietary n-3 fatty acids at a constant n-3m-6 fatty acid ratio in suppressing eicosanoid biosynthesis from arachidonic acid. Am. J. din. Nutr. 54: 111-117. Dean, P., W. F. Lamoreux, J. R. Aitkin, and F. G. Proudfoot, 1969. Flavor associated with fish meal in diets fed to broiler chickens. Can. J. Anim. Sci. 49:11-15. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics 11:1-6. Dyerberg, J., and H. O. Bang, 1978. Dietary fat and thrombosis. Lancet 1:152. Folch, J., M. Lees, and G.H.S. Sloane Stanley, 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. Herold, P. M., and J. E. Kinsella, 1986. Fish oil consumption and decreased risk of cardiovascular disease: a comparison of findings from animal and human feeding trials. Am. J. Clin. Nutr. 43566-598. Hulan, H. W., R. G. Ackman, WAIN. Ratnayake, and F. G. Proudfoot, 1988. Omega-3 fatty acid levels and performance of broiler chickens fed redfish meal or redfish oil. Can. J. Anim. Sci. 68: 543-547. Hulan, H. W., R. G. Ackman, W.M.N. Ratnayake, and F. G. Proudfoot, 1989. Omega-3 fatty acid levels and general performance of commercial broilers fed practical levels of redfish meal. Poultry Sci. 68:153-162. Hwang, D. H., M. Boudreau, and P. Chanmugam,
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