Effects of dietary conjugated linoleic acid and linoleic:linolenic acid ratio on polyunsaturated fatty acid status in laying hens1

METABOLISM AND NUTRITION Effects of Dietary Conjugated Linoleic Acid and Linoleic:Linolenic Acid Ratio on Polyunsaturated Fatty Acid Status in Laying Hens1 M. Du, D. U. Ahn,2 and J. L. Sell Department of Animal Science, Iowa State University, Ames, Iowa 50011-3150 CLA feeding. The amount of arachidonic acid was decreased after CLA feeding in linoleic acid- and linolenic acid-rich diets, but amounts of eicosapentaenoic acid and docosahexaenoic acid were increased in the linolenic-rich diet, indicating that the synthesis or deposition of longchain n-3 fatty acids was accelerated after CLA feeding. The increased docosahexaenoic acid and eicosapentaenoic acid contents in lipid may be compensation for the decreased arachidonic acid content. Dietary supplementation of linoleic acid increased n-6 fatty acid levels in lipids, whereas linolenic acid increased n-3 fatty acid levels. Results also suggest that CLA might not be elongated to synthesize long-chain fatty acids in significant amounts. The effect of CLA in reducing the level of n-6 fatty acids and promoting the level of n-3 fatty acids could be related to the biological effects of CLA.

(Key words: dietary conjugated linoleic acid, biosynthesis, arachidonic acid, docosahexaenoic acid, egg yolk) 2000 Poultry Science 79:1749–1756

INTRODUCTION Dietary conjugated linoleic acid (CLA) has anticarcinogenic, antiartherogenic effects and modulates immune responses (Lee et al., 1995; Ip et al., 1995; Belury et al., 1996; Ip, 1997). Ip et al. (1997) reported that CLA inhibited the postinitiation phase of carcinogenesis. Visonneau et al. (1997) found that CLA suppressed the growth of human breast adenocarcinoma cells. Nicolosi et al. (1997) showed that CLA reduced plasma lipoprotein content and early development of atherosclerosis in hamsters. Sugano et al. (1997, 1998) reported that CLA feeding lowered the concentration of prostaglandin E2 and leukotriene 4 in the serum and spleen of rats. Li and Watkins (1998) also suggested that CLA changed fatty acid composition and reduced the prostaglandin E2 production in rats. Prostaglandin E2 is suspected to have cancer-promoting effects. Lee et al. (1995) showed

Received for publication September 27, 1999. Accepted for publication June 30, 2000. 1 1Journal paper No. J-18617 of the Iowa Agriculture and Home Economics Experiment Station (Ames, IA 50011-3150); Project No. 3322, and supported by the Iowa Egg Council (Ames, IA 50010) and Center for Designing Foods to Improve Nutrition (CDFIN). 2 To whom correspondence should be addressed: [email protected].

that the content of monounsaturated fatty acids in tissues decreased after CLA feeding. Sugano et al. (1998) reported a decrease in the concentration of arachidonic acid and other polyunsaturated fatty acids (PUFA) in the total lipid of spleen lymphocytes and peritoneal exudate cells after feeding CLA to mice. Ahn et al. (1999) reported similar compositional changes in egg yolk lipids after feeding hens with diets containing CLA. Long-chain PUFA can be synthesized from either n-3 or n-6 precursors. Bretillon et al. (1999) reported that dietary cis9, trans11 CLA isomer inhibited the activity of ∆6-desaturase in rat liver microsomes, which indicates that the decreases in unsaturated fatty acids could be caused by the competitive inhibition of ∆6-desaturase by CLA (Belury and Kempa-Steczko, 1997; Du et al., 1999). If this desatarase inhibition is true, the biosynthesis of arachidonic acid and docosahexaenoic acid (DHA) should be inhibited, because in birds, ∆6-desaturase is needed to synthesize both of them. To test the influence of CLA on the synthesis of long-chain PUFA, linolenic

Abbreviation Key: CLA = conjugated linoleic acid; DHA = docosahexaenoic acid; EPA = eicosapentaenoic acid; GC = gas chromatography; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PUFA = polyunsaturated fatty acid; TG = triglyceride.

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ABSTRACT A study was conducted to determine the effects of dietary conjugated linoleic acid (CLA) and the ratio of linoleic:linolenic acid on long-chain polyunsaturated fatty acid status. Thirty-two 31-wk-old White Leghorn hens were randomly assigned to four diets containing 8.2% soy oil, 4.1% soy oil + 2.5% CLA (4.1% CLA source), 4.1% flax oil + 2.5% CLA, or 4.1% soy oil + 4.1% flax oil. Hens were fed the diets for 3 wk before eggs and tissues were collected for the study. Lipids were extracted from egg yolk and tissues, classes of egg yolk lipids were separated, and fatty acid concentrations of total lipids, triglyceride, phosphatidylethanolamine, and phosphatidylcholine were analyzed by gas chromatography. The concentrations of monounsaturated fatty acids and non-CLA polyunsaturated fatty acids were reduced after

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DU ET AL.

TABLE 1. Percentage composition of diets fed to laying hens Ingredients

Percentage 35.29 18.17 22.85 8.77 0.40 3.00 2.50 0.30 0.30 0.14 0.08 8.20–03 4.10–03 0–4.103 2,905 17.00 0.70 0.40 0.90 3.85 0.35 0.20 10.31

1 Mineral premix provides per kilogram of diet: Mn, 80 mg; Zn, 90 mg; Fe, 60 mg; Cu, 12 mg; Se, 0.147 mg; sodium chloride, 2.247 g. 2 Vitamin premix supplies per kilogram of diet: retinyl acetate, 8,065 IU; cholecalciferol, 1,580 IU; 25-hydroxy-vitamin D3, 31.5 µg; dl-α-tocopheryl acetate, 15 IU; vitamin B12, 16 µg; menadcre, 4 mg; riboflavin, 7.8 mg; pantothenic acid, 12.8 mg; niacin, 75 mg; Choline chloride, 509 mg; folic acid, 1.62 mg; biotin, 0.27 mg. 3 In A group, soy oil, 8.20%. In B group, soy oil, 4.10%; conjugated linoleic acid (CLA) source, 4.10% (equal to 2.5% CLA). In C group, flax oil, 4.10%; CLA source, 4.10%. In D group, soy oil, 4.10%; flax oil, 4.10%.

acid- or linoleic acid-rich diets were used to analyze the accumulation of arachidonic acid and DHA after CLA feeding. Polyunsaturated fatty acids are precursors of eicosanoids, which relate to the immune response, cancer promotion, and atherosclerosis. Analysis of the influence of CLA on PUFA composition will also help to illustrate the mechanism of CLA in modulating these processes. In this study, diets rich in linoleic or linolenic acid were formulated using soy oil and flax oil to assess the effects of dietary CLA on PUFA composition in vivo.

MATERIALS AND METHODS Hen Feeding and Sample Preparation Thirty-two 31-wk-old White Leghorn hens, kept in individual cages, were assigned to each of four dietary treatments that consisted of diets containing 8.2% soy oil, 4.1% soy oil + 2.5% CLA (4.1% CLA source), 4.1% flax oil + 2.5% CLA, or 4.1% soy oil + 4.1% flax oil (Table 1). The CLA source used in this study was obtained from a commercial company3 and contained 61% CLA. Therefore, 4.1% CLA source added in the diet is equiva-

3

Conlinco, Inc., Detroit Lakes, MN 56502. Brinkman Instruments, Inc., Westbury, NY 11590-0207. Sigma-Aldrich, 89552 Steinheim, Germany.

4 5

Lipid Extraction Two-gram (egg yolk and liver) or 4-g (muscle) samples were weighed into a test tube with 10 volumes of Folch 1 (chloroform:methanol = 2:1, wt/vol; Folch et al., 1957), and homogenized with a Brinkman polytron4 (Type PT 10/35) for 10 s at high speed. Twenty-five micrograms of butylated hydroxyanisole (10%) dissolved in 98% ethanol was added to each sample prior to homogenization. The homogenate was filtered through a Whatman #1 filter paper into a 100-mL graduated cylinder and 1/4 volume (on the basis of Folch 1) of 0.88% NaCl solution was added. After the cylinder was capped with a glass stopper, the filtrate was mixed well. The inside of the cylinder was washed twice with 10 mL of Folch 2 (3:47:48/CHCl3:CH3OH:H2O), and the contents were stored until the aqueous and organic layers clearly separated. The upper layer was siphoned off, and the lower layer was moved to a glass scintillation vial and dried at 50 C under nitrogen.

Separation of Lipid Classes The dried lipids of egg yolk and tissue were redissolved with chloroform to set the final concentration of lipid at 0.2 g/mL. The lipid-chloroform solution (150 µL) was loaded onto an activated (120 C for 2 h) silica gel plate5 (20 × 20 cm). The plate was developed first in Solvent I, composed of chloroform:methanol:water (65:25:4, vol/vol/vol), until the solvent line reached the middle of the plate. The plate was air dried and then redeveloped in Solvent II, composed of hexane:diethyl ether (4:1, vol/vol), until the solvent front reached 1" below the top of the plate. After air drying for 10 min at room temperature (22 C), the plates were sprayed with 0.1% 2′,7′-dichlorofluororescein in ethanol. Lipid classes were identified under UV light, and the lanes corresponding to triglyceride (TG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) were scraped into separate test tubes (Ahn et al., 1995), and methylated.

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Corn Soy meal Wheat middlings Limestone Dicalcium phosphate Meat and bone meat Dehydrated alfalfa meal Mineral premix1 Vitamin premix2 DL-methionine Sodium chloride (iodized) Soybean oil Flax oil CLA source Calculated analysis Metabolizable energy, kcal/kg Protein TSAA Methionine Lysine Calcium Nonphytate Sodium Ether extract

lent to 2.5% CLA, and the actual amount of CLA (2.5%) instead of the CLA source (4.1%) was used in the text. Soybean oil, flax oil, and the CLA source were substituted on a weight:weight basis in different diets. Compositions of experimental diets are presented in Table 1. After feeding hens for 3 wk, eggs were collected for 4 consecutive d, and four eggs (from different hens) per treatment were randomly selected and analyzed. After their eggs were collected, four hens from each group were sacrificed, and liver and leg muscles were sampled for fatty acid composition analysis. Tissues were frozen in liquid nitrogen immediately after sampling. Lipid classes of egg yolk were separated by thin-layer chromatography. Fatty acid compositions of total egg yolk lipid and lipid classes were analyzed by gas chromatography (GC).

CONJUGATED LINOLEIC ACID AND POLYUNSATURATED FATTY ACIDS

Analysis of Fatty Acid Composition

Statistical Analysis The effect of dietary CLA on the fatty acid composition of egg yolk and tissue lipids was analyzed using SAS software (SAS Institute Inc., 1985). Student-NewmanKeul’s multiple range test was used to compare differences among mean values (P < 0.05). Mean values and standard errors of the mean are reported.

RESULTS AND DISCUSSION The control diet (8.2% soy oil) was rich in linoleic acid, the 4.1% soy oil + 2.5% CLA diet was high in CLA and

6

Sigma-Aldrich, St. Louis, MO 63178. Hewlett Packard Co., Wilmington, DE 16808-1610. 8 Matreya, Inc., Pleasant Gap, PA 16823. 9 Nuchek, Elysian, MN 56028. 7

linoleic acid, the 4.1% flax oil + 2.5% CLA diet contained CLA and was supplemented with linolenic acid, and the 4.1% soy oil + 4.1% flax oil treatment was rich in linoleic and linolenic acids (Tables 1 and 2). Calculated contributions of the supplemented oils to the dietary fatty acid levels for each diet are shown in Table 2. The control diet had 40.22% linoleic acid, the 4.1% soy oil + 2.5% CLA diet had 21.32% CLA and 22.76% linoleic acid, the 4.1% flax oil + 2.5% CLA diet had 21.32% CLA and 20.28% linolenic acid, and the 4.1% soy oil + 4.1% flax oil diet contained 26.02% linoleic and 23.50% linolenic acids. After 3 wk of feeding, there were significant differences in yolk lipid composition among the four groups (Table 3). The percentage content of arachidonic acid in egg yolk lipids from hens fed diets containing CLA was lower than that from the control diet, which may be due to the reduced linoleic acid level in CLA diets after soy oil was substituted with flax or the CLA source (Du et al., 1999). Sugano et al. (1998) also reported a decrease in the concentration of arachidonic acid after feeding CLA to mice. The concentration of linoleic acid in chicken yolk and tissue lipids was, in order: 8.2% soy oil > 4.1% soy oil + 4.1% flax oil > 4.1% soy oil + 2.5% CLA > 4.1% flax oil + 2.5% CLA group, which appeared to be proportional to the content of dietary linoleic acid (Tables 2 and 3). The concentrations of linolenic acid in egg yolk lipids from hens fed diets containing 4.1% flax oil + 2.5% CLA or 4.1% soy oil + 4.1% flax oil were significantly (P < 0.01) higher than those of the 8.2% soy oil and 4.1% soy oil + 2.5% CLA treatments, which may be due to the addition of flax oil. Egg yolk lipids from hens fed diets containing 4.1% soy oil + 2.5% CLA and 4.1% flax oil + 2.5% CLA had high amounts of stearic and palmitic acids, indicating that feeding CLA reduced the overall deposition of unsaturated fatty acid. There were significant differences in the arachidonic acid concentrations in egg yolk and tissues from the four dietary groups; the amount of arachidonic acid was highest with the 8.2% soy oil diet and lowest with the 4.1% flax oil + 2.5% CLA treatment. This concentration difference was related to the dietary linoleic acid that is a substrate for arachidonic acid synthesis, and could also be related to the inhibiting effects of CLA on ∆6desaturase activity (Bretillon et al., 1999). Egg yolk lipids from hens fed a diet containing 4.1% flax oil + 2.5% CLA contained significantly higher amounts of DHA than the 4.1% soy oil + 4.1% flax oil treatment, in spite of the same dietary content of linolenic acid (Table 3). Because the DHA content in the diet should be very low and about the same for all four groups (Tables 1 and 2), the increase of DHA content in egg yolk in CLA feeding groups indicated that CLA promoted synthesis of n-3 long-chain PUFA. Alternatively, CLA could alter DHA metabolism to increase its use for egg yolk accretion. Turek et al. (1998) fed a diet containing CLA or soybean oil (control) to rats and found that DHA content in rat tissue from the CLA diet was significantly higher than that from the soybean diet; Li and Watkins (1998) also

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One milliliter of methylating reagent (anhydrous methanolic-HCl-3N) was added to the test tube containing total lipid, TG, PE, or PC, capped tightly, and incubated in a water bath at 60 C for 40 min. After cooling to room temperature, 2 mL of hexane and 5 mL of water were added, mixed thoroughly, and left at room temperature overnight for phase separation. The top hexane layer containing methylated fatty acids was used for GC analysis (Chin et al., 1992). Analysis of fatty acid composition was performed with a GC6 (HP 6890) equipped with an autosample injector6 and flame ionization detector. A capillary column7 (HP-5, 0.32 mm inside diameter, 30 m, 0.25 µm film thickness) was used. A splitless inlet was used to inject samples (1 µL) into the capillary column. Ramped oven temperature conditions (180 C for 2.5 min, increased to 230 C at 2.5 C/min, then held at 230 C for 7.5 min) were used. Temperatures of both inlet and detector were 280 C. Helium was used as a carrier gas, and a constant column flow of 1.1 mL/min was used. Flame ionization detector air, H2, and make-up gas (helium) flows were 350 mL/ min, 35 mL/min, and 43 mL/min, respectively. Fatty acids were identified using a Mass Selective detector6 (Model 5973). The GC-Mass Selective detector procedure was performed with the same column and oven temperature conditions described previously. The ionization potential of the Mass Selective detector was 70 eV, and the scan range was 45 to 450 m/z. Identification of fatty acids was achieved by comparing mass spectral data with those of the Wiley library.7 The CLA isomers in egg yolk lipids were identified by comparing against CLA standards purchased from Matreya8 and Nuchek9, and CLA standards according to the report of Christie et al. (1997). The compositions of CLA isomers and fatty acids were reported as percentages of composition of total lipids, and total peak area (pA*s) was used to calculate fatty acid composition.

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DU ET AL. TABLE 2. The fatty acid (FA) composition of soy oil, flax oil, and oil and conjugated linoleic acid (CLA) source, and the calculated contribution of oil sources to the fatty acid composition of diets FA composition of oil sources

Calculated FA contribution to diets

CLA source

Flax oil

Soybean oil

Soy

Soy + CLA1

Flax + CLA1

Soy + flax1

Palmitic Stearic Oleic Linoleic Linolenic Others CLA (cis9, trans11) CLA (cis10, trans12) CLA (trans9, trans11) Other CLA isomers CLA total

4.16 2.10 16.97 6.65 ND2 8.60 17.94 20.27 15.34 7.97 61.52

7.15 2.24 15.30 14.83 50.96 0.52 ... ... ... ... ...

14.64 4.56 21.56 50.54 8.07 0.63 ... ... ... ... ...

11.64 3.63 17.15 40.19 6.41 0.50 ... ... ... ... ...

7.47 2.65 15.32 22.74 3.20 3.67 7.14 8.06 6.10 3.17 24.47

4.50 1.72 12.83 8.54 20.26 3.63 7.13 8.06 6.11 3.17 24.47

8.67 2.70 14.66 25.99 23.47 1.45 ... ... ... ... ...

46.60 46.60 6.27

50.41 25.94 7.11

53.27 28.80 5.58

49.46 49.46 1.11

PUFA Non-CLA PUFA n6/n3 fatty acid ratio 1

Based on the calculated value of oils added in diet. ND = not detected.

2

showed that the concentrations of n-3 fatty acids eicosapentaenoic acid (EPA) and DHA increased after CLA feeding. The DHA contents of egg yolk from hens fed a diet containing 4.1% flax oil + 2.5% CLA and 4.1% soy oil + 4.1% flax oil were much higher than those of the hens fed diets containing 8.2% soy oil and 4.1% soy oil + 2.5% CLA, indicating that dietary linolenic acid enhanced the biosynthesis of DHA. Another possibility is that CLA influenced the partitioning or transport of DHA to the uptake of VLDL by the follicle. Arachidonic acid contents in diets with 8.2% soy oil and 4.1% soy oil +2.5% CLA were higher than those of diets without soy oil, indicating that dietary linoleic acid increased arachidonic acid level in yolk lipid. The concentration of oleic acid in yolk lipid from hens fed a diet containing 4.1% soy oil + 2.5% CLA was much

lower than that of hens fed the 4.1% flax oil + 2.5% CLA treatment, although the 4.1% soy oil + 2.5% CLA diet contained more oleic acid than the 4.1% flax oil + 2.5% CLA treatment (Tables 2, 3, 4, and 5). The reason for the discrepancy between diet and deposition is not clear. Li and Watkins (1998) speculated that CLA reduced the concentration of oleic acid by inhibiting liver ∆9-desaturase activity and found that dietary CLA decreased the concentrations of palmitoleic and oleic acid, which agrees with the present study. Lee et al. (1998) also showed that CLA inhibited stearoyl-coenzyme A desaturase mRNA expression. Phospholipids, which mainly exist in the membrane system, were expected to have a more important biofunction than neutral lipids. Therefore, egg yolk lipid was further separated into PC, PE, and TG parts using thin-layer chromatography. The concentration of arachi-

TABLE 3. Influence of dietary fat on the fatty acid composition of egg yolk lipid Fatty acid

Soy oil

Soy oil + CLA

Flax oil + CLA

Flax oil + soy

SEM

Palmitic Palmitoleic Stearic Oleic Linoleic Linolenic CLA1 (cis9, trans11) CLA (trans10, cis12) CLA (trans9, trans11) Other CLA isomers Arachidonic EPA2 DHA2 SAFA2 MUFA2 PUFA2 Non-CLA PUFA n6/n3 fatty acid ratio

22.82b 1.08b 11.84c 31.05a 23.56a 1.21b 0.00b 0.00b 0.00b 0.00c 3.95a 0.22b 2.72c 34.66 32.13 31.66 31.66 6.63

24.80a 0.49d 16.25a 23.22c 18.30c 0.82c 2.91a 2.31a 1.29a 0.99b 2.96b 0.21b 1.37d 41.05 23.71 31.16 23.66 8.86

25.11a 0.60c 14.57b 27.19b 11.98d 4.89a 2.80a 2.33a 1.30a 1.18a 2.06c 0.32a 3.36a 39.68 27.79 30.22 22.61 1.64

22.88b 1.13a 11.78c 31.86a 20.76b 4.87a 0.00b 0.00b 0.00b 0.00c 2.97b 0.30a 3.18b 34.66 32.99 32.08 32.08 2.77

0.256 0.019 0.271 0.324 0.321 0.060 0.040 0.065 0.060 0.018 0.047 0.010 0.076

Means within a row with no common superscript differ significantly (P < 0.05); n = 4. CLA = conjugated linoleic acid. 2 EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid; SAFA = saturated fatty acid; MUFA = monounsaturated fatty acid; PUFA = polyunsaturated fatty acid. a–d 1

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Fatty acid

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CONJUGATED LINOLEIC ACID AND POLYUNSATURATED FATTY ACIDS TABLE 4. Influence of dietary fat on the fatty acid composition of phosphatidylcholine of egg yolk lipid Soy oil

Soy oil + CLA

Flax oil + CLA

Flax oil + soy

SEM

Palmitic Palmitoleic Stearic Oleic Linoleic Linolenic CLA1 (cis9, trans11) CLA (cis10, trans12) CLA (trans11, trans11) Other CLA isomers Arachidonic DHA2 SAFA2 MUFA2 PUFA2 Non-CLA PUFA n6/n3 fatty acid ratio

27.62a 0.45b 15.76b 25.07c 20.76a 0.70b 0.00b 0.00b 0.00b 0.00b 4.98a 3.20b

25.69a 0.24d 18.69a 22.55d 17.80c 0.10c 1.93a 1.19a 1.59a 1.03a 3.58b 1.49c

26.65a 0.33cd 18.26a 26.02b 12.42d 2.15a 1.96a 1.27a 1.62a 1.10a 2.40c 3.88a

26.64a 0.70a 15.42b 28.57a 19.27b 2.09a 0.00b 0.00b 0.00b 0.00b 3.28b 3.30b

0.614 0.030 0.621 0.219 0.383 0.031 0.042 0.038 0.063 0.017 0.203 0.188

43.38 25.52 29.64 29.64 6.60

44.38 22.79 28.71 22.97 13.45

44.91 26.35 26.80 20.85 2.46

42.06 29.27 27.94 27.94 4.18

Means within a row with no common superscript differ significantly (P < 0.05); n = 4. CLA = conjugated linoleic acid. 2 DHA = docosahexaenoic acid; SAFA = saturated fatty acid; MUFA = monounsaturated fatty acid; PUFA = polyunsaturated fatty acid. a–d 1

donic acid in PC was closely related to the dietary content of linoleic acid, which was highest with 8.2% soy oil and lowest with the 4.1% flax oil + 2.5% CLA treatment, and was consistent with the result of total lipids (Table 4). The DHA content in PC was higher in eggs from hens fed a 4.1% flax oil + 2.5% CLA diet than in eggs from hens fed a 4.1% soy oil + 4.1% flax oil diet. Phosphatidylethanolamine of egg yolk contained a much higher amount of arachidonic acid and DHA than other lipid classes (Table 5). However, their relative changes were still similar to total lipid and PC. The concentration of eicosapentaenoic acid in PE of egg yolk from hens fed a 4.1% flax oil + 2.5% CLA diet was very high compared with other groups. The concentration changes of arachidonic acid, eicosapentaenoic acid, and

DHA in phospholipids may have significant physiological effects in vivo. Because these PUFA are the precursors for the biosynthesis of eicosanoids, they could be closely related to immune response and carcinogenesis in animals. The fatty acid composition of TG of egg yolk was different from those of the PC and PE (Table 6). Very low levels of arachidonic acid and DHA in TG indicate that arachidonic acid and DHA are mainly deposited to PC and PE. The concentrations of linolenic acid in TG from the 4.1% flax oil + 2.5% CLA and 4.1% soy oil + 4.1% flax oil diets were high compared with those from the 8.2% soy oil and 4.1% flax oil + 2.5% CLA diets. The linoleic acid concentration and other fatty acid compositions of TG were similar to those of the dietary sources.

TABLE 5. Influence of dietary fat on the fatty acid composition of phosphatidylethanolamine of egg yolk lipid Fatty acid Palmitic Stearic Oleic Linoleic Linolenic CLA1 (cis9, trans11) CLA (cis10, trans12) CLA (trans9, trans11) Other CLA isomers Arachidonic EPA2 DHA2 SAFA2 MUFA2 PUFA2 Non-CLA PUFA n6/n3 fatty acid ratio

Soy oil b

12.41 30.27a 15.71b 14.37b 1.28cd 0.00b 0.00c 0.00b 0.00b 14.02a 0.69b 9.39b 42.68 15.71 39.75 39.75 2.50

Soy oil + CLA a

14.70 26.88b 15.64b 16.17a 1.19d 2.06a 1.32a 1.76a 1.25a 10.26b 0.88b 4.85c 41.58 15.64 39.74 33.35 3.82

Flax oil + CLA a

15.54 26.63b 18.37a 9.16c 1.90a 1.95a 1.15b 1.64a 1.26a 7.10c 3.49a 11.24a 42.17 18.37 38.89 32.89 0.98

Flax oil + soy b

11.60 30.26a 19.16a 13.14b 1.46b 0.00b 0.00c 0.00b 0.00b 11.54b 0.84b 10.76a 41.86 19.16 37.74 37.74 1.89

SEM 0.472 0.661 0.423 0.433 0.029 0.085 0.040 0.064 0.033 0.412 0.186 0.260

Means within a row with no common superscript differ significantly (P < 0.05); n = 4. CLA = conjugated linoleic acid. 2 EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid; SAFA = saturated fatty acid; MUFA = monounsaturated fatty acid; PUFA = polyunsaturated fatty acid. a–d 1

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Fatty acid

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DU ET AL. TABLE 6. Influence of dietary fat on the fatty acid composition of triglycerides of egg yolk lipid Soy oil

Soy oil + CLA

Flax oil + CLA

Flax oil + soy

SEM

Palmitic Palmitoleic Stearic Oleic Linoleic Linolenic CLA1 (cis9, trans11) CLA (cis10, trans12) CLA (trans9, trans11) Other CLA isomers Arachidonic DHA2 SAFA2 MUFA2 PUFA2 Non-CLA PUFA n6/n3 fatty acid ratio

21.03b 2.28b 4.61c 38.18a 31.37a 3.05c 0.00c 0.00b 0.00c 0.00c 0.35a 0.23b

26.43a 0.95d 9.08a 23.99d 26.89b 1.22d 3.45a 3.07a 1.36b 1.50b 0.12c 0.00c

21.89b 1.20c 8.37b 30.23c 13.71d 12.88b 3.24b 2.94a 1.63a 1.64a 0.12c 0.45a

18.82c 2.99a 3.61d 32.70b 24.89c 15.15a 0.00c 0.00b 0.00c 0.00c 0.21b 0.22b

0.326 0.050 0.035 0.456 0.475 0.209 0.043 0.073 0.025 0.021 0.021 0.017

25.64 40.46 35.00 35.00 6.97

35.51 24.94 37.61 28.23 22.14

30.26 31.43 36.61 27.16 1.04

22.43 35.69 40.47 40.47 1.63

Means within a row with no common superscript differ significantly (P < 0.05); n = 4. CLA = conjugated linoleic acid. 2 DHA = docosahexaenoic acid; SAFA = saturated fatty acid; MUFA = monounsaturated fatty acid; PUFA = polyunsaturated fatty acid. a–d 1

This similarity indicated that fatty acid composition of TG in yolk lipid mainly reflected the fatty acid composition of the diet. Both the 4.1% soy oil + 2.5% CLA and 4.1% flax oil + 2.5% CLA diets were supplemented with the same level of CLA, but the amounts of linoleic and linolenic acids in the diets were different. The hens fed a linoleic acidrich diet (4.1% soy oil + 2.5% CLA) had higher arachidonic acid concentrations in egg yolk lipids than those fed a linolenic acid-rich diet (4.1% flax oil + 2.5% CLA), but the concentration of DHA was higher in linolenic acid-rich diets than in linoleic acid-rich diets (Tables 3, 4, and 5). These results indicate that the fractional content of arachidonic acid, EPA, and DHA of egg yolk varies directly with the dietary concentration of their

precursor fatty acids under the dietary conditions employed in this study. Liu and Belury (1998) reported that adding linoleic acid to a CLA diet enhanced arachidonic acid synthesis. In the 4.1% soy oil + 2.5% CLA and 4.1% flax oil + 2.5% CLA treatments (Tables 3, 4, and 5), high amounts of CLA isomers were incorporated into lipid. If CLA could be elongated to synthesize long-chain unsaturated fatty acids in significant amounts, there would be more long-chain fatty acids detected by the GC-Mass Selective detector procedure. However, GC results did not indicate that there were significant amounts of long-chain fatty acids available, except arachidonic acid, DHA, and EPA. Therefore, CLA may not be a favorable substrate for enzymes (desaturase and elongase) involved in the

TABLE 7. Influence of dietary fat on the fatty acid composition of liver tissues Fatty acid

Soy oil

Soy oil + CLA

Flax oil + CLA

Flax oil + soy

SEM

Palmitic Palmitoleic Stearic Oleic Linoleic Linolenic CLA1 (cis9, trans11) CLA (cis10, trans12) CLA (trans9, trans11) Other CLA isomers Arachidonic EPA2 DHA2 SAFA2 MUFA2 PUFA2 Non-CLA PUFA n6/n3 fatty acid ratio

20.79ab 0.73a 18.12b 29.80a 19.43a 1.03c 0.00b 0.00c 0.00b 0.00b 8.09a 0.17b 2.25c 38.91 30.53 30.97 30.97 7.98

22.32a 0.29c 19.85a 26.96b 15.49c 0.72c 1.78a 2.25a 0.91a 0.83a 6.13c 0.20b 2.01c 42.71 27.25 30.32 24.55 7.38

21.90a 0.57b 17.06b 26.89b 12.78d 2.73b 1.86a 1.99b 0.75a 0.61a 5.19d 0.52a 5.93a 38.96 27.46 32.36 27.15 1.96

19.98b 0.76a 14.56c 28.85ab 17.83b 3.58a 0.00b 0.00c 0.00b 0.00b 7.29b 0.49a 5.19b 34.54 29.61 34.38 34.38 2.71

0.464 0.046 0.368 0.542 0.350 0.101 0.044 0.069 0.060 0.031 0.219 0.025 0.110

Means within a row with no common superscript differ significantly (P < 0.05); n = 4. CLA = conjugated linoleic acid. 2 EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid; SAFA = saturated fatty acid; MUFA = monounsaturated fatty acid; PUFA = polyunsaturated fatty acid. a–d 1

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CONJUGATED LINOLEIC ACID AND POLYUNSATURATED FATTY ACIDS TABLE 8. Influence of dietary fat on the fatty acid composition of muscle tissues Soy oil

Soy oil + CLA

Flax oil + CLA

Flax oil + soy

SEM

Palmitic Palmitoleic Stearic Oleic Linoleic Linolenic CLA1 (cis9, trans11) CLA (cis10, trans12) CLA (cis11, trans13) Other CLA isomers Arachidonic EPA2 DHA2

19.39 1.56bc 10.25 34.24a 28.38a 1.40c 0.00c 0.00c 0.00c 0.00c 6.40a 0.19b 0.56c 29.64 35.80 36.93 36.93 16.18

19.94 1.02c 12.20 31.42b 23.22c 1.03d 1.84a 2.20a 0.93a 0.96a 4.79b 0.14b 0.46c 32.14 32.44 35.57 29.64 17.18

18.36 1.45a 10.60 30.50b 22.00c 2.79a 1.13b 1.22b 0.55b 0.51b 2.87c 0.39a 1.63a 28.96 31.95 33.09 29.68 5.17

19.24 2.06ab 9.96 31.44b 25.89b 2.29b 0.00c 0.00c 0.00c 0.00c 3.58c 0.33a 1.06b 29.20 33.50 33.15 33.15 8.01

0.537 0.189 0.580 0.587 0.518 0.109 0.044 0.068 0.044 0.029 0.264 0.030 0.094

SAFA2 MUFA2 PUFA2 Non-CLA PUFA n6/n3 fatty acid ratio

Means within a row with no common superscript differ significantly (P < 0.05); n = 4. CLA = conjugated linoleic acid. 2 EPA = eicosapentaenoic acid; DHA = docosahexaenoic acid; SAFA = saturated fatty acid; MUFA = monounsaturated fatty acid; PUFA = polyunsaturated fatty acid. a–d 1

synthesis of long-chain fatty acids. However, Sebedio et al. (1997) showed that CLA isomers could be elongated to C20:4 because there were higher quantities of C20:4 ∆-5,8,12,14, and C20:4 ∆-5,8,11,13 in liver lipids of rats fed CLA than in liver lipids of controls. In liver (Table 7) and muscle (Table 8) lipids, the concentration of arachidonic acid was much higher in 8.2% soy oil and 4.1% soy oil + 2.5% CLA than in 4.1% soy oil + 2.5% CLA and 4.1% flax oil + 2.5% CLA. This result could be caused by the high amount of linoleic acid in the diets with added soy oil. The concentrations of EPA and DHA in the liver and muscle of hens fed 4.1% flax oil + 2.5% CLA and 4.1% soy oil + 4.1% flax oil were higher than those of hens fed the 8.2% soy oil and 4.1% soy oil + 2.5% CLA diets. Significantly higher amounts of DHA in the liver and muscle of hens fed 4.1% flax oil + 2.5% CLA than in those of hens fed the 4.1% soy oil + 4.1% flax oil diet indicate that CLA promoted the synthesis or deposition of DHA and EPA. This finding illustrates that CLA increases the level of n-3 long-chain PUFA. Ward et al. (1998) showed that arachidonic acid and DHA could affect each other’s levels. The increased DHA level could be caused by the reduced arachidonic acid concentration, which might have some feedback effects and induce more DHA synthesis or incorporation in compensation (Ward et al., 1998). This study indicated that CLA feeding reduced monounsaturated fatty acid and non-CLA PUFA content in egg yolk and tissue lipids. The concentration of DHA in lipid was increased by dietary CLA, which could be related to the decreased arachidonic acid content. Results show that dietary supplementation of linoleic or linolenic acid can enhance the level of arachidonic acid or DHA in yolk and tissue lipids dramatically.

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