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METABOLISM AND NUTRITION |
School of Animal Production Technology, Institute of Agricultural Technology, Suranaree University of Technology, Muang, Nakhon Ratchasima 30000, Thailand
1 Corresponding author: wisitpor{at}sut.ac.th
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Key Words: conjugated linoleic acid • egg yolk • fatty acid • layer • egg quality
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Recently, conjugated linoleic acid (CLA), a group of isomers of octadecadienes, has received much attention. Several health-beneficial effects are attributed to these isomers (e.g., anticarcinogenic and antiatherosclerotic effects), influencing both fat metabolism and protein deposition (Ha et al., 1987; Ip et al., 1991; Pariza et al., 2000). However, if the antiatherogenic activity of CLA found in rabbits (Lee et al., 1994), hamsters (Nicolosi et al., 1997), and mice (Munday et al., 1999) could be measured in humans, the benefit of CLA to human health might be of interest. Consumption of CLA by humans has been shown to elicit many favorable health benefits, such as the modulation of immune function, weight reduction, and protection against diseases such as cancer and arteriosclerosis (Chin et al., 1992; Lee et al., 1994; Nicolosi et al., 1997). Conjugated linoleic acid is naturally present in products originating from ruminants as a result of the specific metabolism of the rumen producing, in particular, c9, t11 CLA as the predominant isomer. However, recent studies have suggested that the endogenous synthesis of CLA by the action of 
9-desaturase on trans 18:1 fatty acids is probably more important than that of the ruminal production (Griinari et al., 2000; Santora et al., 2000). Eggs and other products from monogastric animals contain negligible amounts of CLA (Chin et al., 1992). It is worthwhile to consider the enrichment of food with CLA, because CLA is readily incorporated into the fat fraction of animal foods. The egg yolk can be considered to be a good carrier, because it contains 30 to 35% fat. Many studies have suggested that eggs from CLA-fed hens are a good source of CLA in the human diet (Chamruspollert and Sell, 1999; Du et al., 1999; Raes et al., 2002; Sun et al., 2003). One way to increase the CLA content in eggs is through supplementation of the layer diet with CLA. A combined incorporation of CLA into egg yolk, together with that of PUFA, would lead to even greater health benefits. The aim of this experiment was to study the effects of various CLA levels upon laying performance, egg yolk fatty acid compositions, and egg quality.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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All experiments were conducted in accordance with the principles and guidelines approved by the Suranaree University of Technology Animal Care and Use Committee, which followed Guidelines for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (Federation of Animal Science Societies, 1999). Three hundred laying hens (Hisex Brown; 27 wk old) were randomly divided into 5 groups. Each group (60 hens) was further randomly divided into 5 replicates of 12 hens. In each replicate, hens were then randomly allocated to 4 metal cages of 3 laying hens, under conditions of an evaporative cooling system and lighting program (16L:8D) throughout the entire experiment. Hens were fed diets and water ad libitum during the entire experimental period.
Experimental Diets
Each group of hens was randomly fed an experimental diet containing 0, 1, 2, 3, and 4% CLA. All experimental diets were isonitrogenous and isocaloric and formulated to meet the NRC (1994) requirements. Hens were fed experimental diets containing 2,862 kcal of ME/kg, 16.5% CP, 0.91% Lys, 0.65% Met + Cys, 3.78% Ca, and 0.41% P. Feed ingredients and chemical compositions of the experimental diets are presented in Table 1
. Commercial liquid CLA [BASF (Thai) Ltd., Bangkok, Thailand], containing 60% CLA (30% c9, t11; 30% t10, c12), 22% oleic acid, 6% palmitic acid, 4% stearic acid, 2% linoleic acid, and 6% of other isomers of fatty acids, was pre-mixed with silica to obtain a CLA concentration of 30% (15% c9, t11; 15% t10, c12). To equalize the concentration of total fat, the CLA source was substituted for soybean oil. Silica was also thoroughly mixed into the diets to maintain equivalent silica content in the diets. Each of the 5 experimental diets was fed to each group of laying hens for five 28-d periods, giving a total of 140 d for the entire experimental periods. Chemical analysis of the diets was made for CP, crude fiber, ether extract, and ash (Association of Official Analytical Chemists, 1998).
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Egg production and egg weight were recorded daily, whereas feed consumed was recorded weekly. Every 14 d throughout the experimental period, uniform samples of 4 eggs from each replication from each treatment were used to determine the mass of the main egg components (shell, yolk, and albumen). Yolk color was determined by comparing yolk color to the Roche color fan.
Fatty Acid Analysis
Fatty acid analysis was determined as previously described (Raes et al., 2000, 2001). In brief, lipids were extracted from fresh yolk using chloroform, methanol, or both (2:1, vol/vol; Folch et al., 1957). Nonadecanoic acid (19:0) was added as an internal standard. The fatty acid methyl esters (FAME) were analyzed by gas chromatography (GC; model 6890, Hewlett-Packard Co., Palo Alto, CA) using a CP-Sil88 column (Chrompack, Middelburg, The Netherlands) for FAME (100 m x 0.25 mm). The GC conditions were as follows: injected temperature, 240°C; detector temperature, 260°C; carrier gas, He; split ratio, 1:30; temperature program, 70°C for 4 min, followed by an increase of 13°C/min to 175°C then 4°C/min to 215°C. Peaks were identified by comparison of retention times with those of the corresponding standards (Supelco 37 component FAME Mix, Sigma-Aldrich Co., St. Louis, MO). Identification of the peak included fatty acids from 14:0 to 22:6 and the following CLA isomers: c9, t11; t10, c12; c9, c11; t9, t11.
Statistical Analysis
Because the CLA treatment caused similar responses in each of the five 28-d periods, the 5 observations from each replicate were averaged. The averaged observed effects between treatment groups were then statistically analyzed by ANOVA in a completely randomized designed (Steel and Torrie, 1986), and significant differences among means were compared by Duncan’s new multiple range test, according to the methods previously described by the SAS institute (1994). The effects of increasing CLA were partitioned into linear and quadratic components using orthogonal polynomial contrasts (SAS Institute, 1994).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The fatty acid profiles of the diets, as analyzed by GC, are given in Table 2. Inclusion of CLA, together with replacing CLA with soybean oil, decreased the level (g/ 100g of total fatty acids, Table 2) of both linoleic and linolenic acids. This can be attributed to the high concentration of linoleic and linolenic acids in soybean oil when soybean oil was replaced by CLA; thus, the level of linoleic and linolenic acids decreased.
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). Body weight changes and mortality rates across the entire experimental period were also similar (P > 0.05). However, hens fed 4, 2, and 1% CLA diets produced fewer eggs than that of hens fed unsupplemented control diet (P < 0.01; Table 3). All hens showed similar albumen height, Haugh units, and eggshell thickness, whereas hens fed a 4% CLA diet had lower egg weight (P < 0.05), yolk color (P < 0.01), yolk weight, and albumen weight (P < 0.05) than that of other hens.
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Egg Yolk Fatty Acid Content
Conjugated linoleic acid supplementation in the diets significantly increased (P < 0.001) saturated fatty acids (SFA) and CLA content of egg yolk but significantly reduced monounsaturated and polyunsaturated fatty acid content of egg yolk. Linoleic acid content of egg yolk markedly decreased (P < 0.001) with increasing level of CLA addition in the diets. The dietary fatty acid composition affected the yolk fatty acid profile. Raes et al. (2002) found that when diets containing only plant oil without CLA addition were considered, the yolk fat was relatively unsaturated. The amount of SFA did not vary greatly among these diets, yet major differences were present in the 18 unsaturates, particularly 18:2. In contrast to the above research, the present study found significant increases in C14:0, C16:0, and C18:0 but found decreases in C18:2.
Because of the putative benefits of CLA to human health, food products rich in CLA have been extensively investigated in recent years. It has been found that egg yolk is a good carrier of fatty acids (Chamruspollert and Sell, 1999). Chamruspollert and Sell (1999) found that the CLA concentration of egg yolk lipids increased linearly with increasing dietary CLA. Other studies have achieved similar results (Ahn et al., 1999; Cherian et al., 2002; Szymczyk and Pisulewski, 2003; Shang et al., 2004). It has been found that the greater the supplementation of dietary CLA, the more CLA is deposited in yolk lipids. In this study, the total CLA and its isomers in yolk lipids were increased linearly and quadratically.
The findings of the present study are comparable to the previous study, which found that the c9, t11 CLA isomer accounted for 50 to 65% of the total CLA in yolk lipids (Chamruspollert and Sell, 1999). The concentration of individual CLA isomers in egg yolk lipids does not completely reflect those in diet. As shown in Table 3
, the percentages of c9, t11 and t10, c12 in the CLA source were similar at 15 and 15%, respectively, however, their percentages in the yolk lipids differed appreciably at 75 and 25%, respectively. Szymczyk and Pisulewski (2003) reported that the relative proportions of the c9, t11 CLA isomer in egg yolk lipids exceeded those found in the CLA product fed. However, the reason for the c9, t11 isomer being deposited at higher levels in yolk lipids is poorly understood, although it may be connected with the fact that some isomers are more effectively metabolized compared with others (Park et al., 1999). Raes et al. (2002) reported that the deposition rate of the c9, t11 isomer in yolk lipids was higher than that of the t10, c12 isomer.
The results also demonstrated that all fatty acids in yolk lipids were significantly altered by dietary CLA supplementation (Table 4). The concentrations of C14:0, C16:0, and C18:0 in the egg yolk lipids were increased linearly and quadratically, whereas other fatty acids were decreased linearly and quadratically, except for C20:3, C20:4, and PUFA, which were reduced linearly. The results of this study, which combined increased dietary CLA and decreased dietary soybean oil, were significantly enhanced SFA concentration and significantly decreased monounsaturated fatty acid (MUFA) and PUFA concentration of the yolk.
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The yolk fatty acid profile clearly reflected the dietary fatty acid composition. However, the unsaturated fatty acids:SFA ratio was more resistant to dietary manipulations than individual fatty acids. Analogous findings were reported by Huyghebaert et al. (1991) and Vahl et al. (1991). The effect of dietary CLA on the fatty acid composition of egg yolks has been extensively studied. Ahn et al. (1999) found that the concentration of SFA increased, whereas MUFA tended to decrease in yolk lipids of hens fed CLA compared with yolks from hens fed a control diet. Similar findings were also reported (Schafer et al., 2001; Cherian et al., 2002; Yang et al., 2002; Szymczyk and Pisulewski, 2003). Changes in SFA and MUFA may be due to the inhibition of the 9-desaturase (stearoyl-coenzyme A desaturase) enzyme system in the liver caused by CLA. Park et al. (1999) observed that hepatic stearoyl-coenzyme A desaturase was inhibited by t10, c12 CLA and its derivatives in mice. Supportive evidence for this concept can be found in the study of Choi et al. (2000), which found the t10, c12 isomers of CLA downregulated the activity of stearoyl-coenzyme A desaturase enzyme of mice. Furthermore, Shang et al. (2004) observed that supplementation of CLA in diets resulted in a dose-dependent decrease in the mRNA transcription and activity of the stearoyl-coenzyme A desaturase in the livers of laying hens. The 9-desaturase enzyme catalyzes the addition of a double bond at the ninth position of a SFA. If this enzyme is inhibited by CLA, MUFA would be decreased, and SFA would be increased.
The increase of SFA concentration in egg yolk is a potential human health concern, because it may increase blood cholesterol content and increase the incidence of atherosclerosis. Humans eating CLA-supplemented eggs would consume increased amounts of SFA. Moreover, increased SFA intake increases cholesterol synthesis and plasma cholesterol concentrations, whereas the effect of dietary cholesterol is equivocal and dependent on dietary SFA intake. Reductions in dietary cholesterol intake do not remove health risks due to increased dietary SFA intake. However, if the antiatherogenic activity of CLA found in rabbits (Lee et al., 1994), hamsters (Nicolosi et al., 1997), and mice (Munday et al., 1999) could be measured in humans, the adverse consequences of increased SFA level could be counteracted by CLA. In addition, it has been reported that the cholesterol content in egg yolk was significantly reduced by dietary CLA (Sun et al., 2003). Therefore, the concerns about the increase of SFA appear to be unfounded.
Raes et al. (2002) found that the effects of CLA supplementation on the PUFA contents varied for the different fatty acids and was also diet-dependent for some fatty acids. They also found that no effect of CLA was observed on the amount of linoleic acid, whereas it clearly decreased the arachinodic acid (C20:4) level in the egg yolk. The other fatty acids showed a tendency to decrease by supplementing CLA. However, the present study found reduction in all PUFA present in egg yolk. A reduction in PUFA could be due to inhibition of 6-desaturase, the rate-limiting step in the conversion of linoleic and linolenic into C20:4. However, if CLA is a competitive inhibitor of 6-desaturase, an accumulation of linoleic and linolenic acids should be observed. A decrease in docosahexaenoic acid (C22:6) concentration in the yolk lipids due to feeding CLA was also found by Chamruspollert and Sell (1999), although in combination with a much wider change in dietary linolenic acid levels. This suggests that the CLA isomers may also inhibit the enzyme responsible for this conversion (i.e., 4-desaturase). The possible mechanism behind this effect was not clear.
In the present study, replacing soybean oil in the basal diet with the CLA source linearly decreased the concentration of linoleic acid in egg yolk lipids. This result was expected, because fatty acid composition of yolk lipids generally reflected the different combinations of CLA and soybean oil concentrations included in the diet (Ahn et al., 1999; Chamruspollert and Sell, 1999; Sun et al., 2003). In summary, dietary CLA supplementation was shown to increase the relative amount of CLA present in egg yolk fatty acids. This incorporation of CLA isomers into egg yolk lipids implies that CLA supplementation of layer diet could provide value-added healthful animal products for human consumption.
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ACKNOWLEDGMENTS |
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Received for publication December 8, 2005. Accepted for publication June 5, 2006.
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