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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION |
* Department of Food and Biotechnology, Korea University, Jochiwon, 339-700, Korea; National Livestock Research Institute, Rural Development Administration (RDA), Suwon, 441-350, Korea; and Hanwoo Experiment Station, National Livestock Research Institute, Pyongchang, 232-952, Korea
2 Corresponding author: yk46{at}korea.ac.kr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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-linolenic acid (LNA) 2% (CLA + LNA). Some parameters of egg quality such as shell thickness, shell strength, yolk color, yolk index, egg diameter, and Haugh units were aggravated when CLA was fed alone, but the quality was improved when CLA was combined with some other fatty acids. The egg production rate, which was decreased by feeding CLA alone, was improved by co-supplementation with LA or OA. An increase in CLA content was observed in all the dietary groups fed CLA for 2 wk. Feeding hens with CLA + LNA led to a linear increase in CLA content in the egg yolk after the fourth week of the feeding trial. Egg yolks from hens given CLA had considerably higher amounts of saturated fatty acids and lower amounts of monounsaturated fatty acids than egg yolks from the control group. The pattern of change in CLA concentration during the feeding trial was similar to the level of C18:0, which was inversely correlated with the level of C18:1. The unsaturated fatty acid co-supplementation strategy applied in this study offers insight into the mechanism of CLA accumulation in the egg yolk without apparent adverse effects on egg quality and egg production.
Key Words: conjugated linoleic acid • oleic acid • linoleic acid • -linolenic acid • egg quality
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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In ruminants, CLA is synthesized as an intermediate product of biohydrogenation by rumen bacteria as a mechanism of detoxifying polyunsaturated fatty acids (PUFA; Chin et al., 1992) and is accumulated in the body tissues and their products to a greater degree than in monogastric animals and poultry, which have no such activity (Kepler and Tove, 1967; Adlof et al., 2000; Kim et al., 2000). Therefore, the CLA content in poultry relies mostly on the carryover of CLA from the diet or on desaturation of C18:1. However, the delivery of dietary fatty acids to the egg yolk has been limited because of the tendency to maintain the homeostasis of lipid metabolism (Watkins, 2003). Moreover, efficient accumulation of CLA in natural foods has not been feasible because excessive fatty acid intake by animals produces a variety of adverse effects due to the change in physiological membrane constituents, especially on the reproduction processes, as well as changes in the egg quality in birds (Chin et al., 1994; Aydin et al., 1999). This is thought to be attributable to the decrease in yolk oleic acid (OA) and increased SFA by CLA feeding as well as the shift in mineral exchange between the yolk and albumen (Aydin et al., 2001). Takahashi et al. (2003) showed that CLA feeding enhanced hepatic desaturation and fat synthesis in mice and that co-supplemented unsaturated fatty acids (UFA) may have affected the enzyme activity to a different degree. However, a clear explanation of the mechanism has yet to be determined.
Therefore, this study was performed to characterize the effects of a variety of dietary fatty acids or their combinations on CLA accumulation in the egg yolk, and thus to establish a strategy to increase the CLA content without adverse effects on egg quality induced by changes in the fatty acid profile.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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A total of 105 White Leghorn laying hens (30 wk old) were randomly distributed into 5 groups of 21 hens each and were maintained in individual laying cages for 4 wk. The hens were assigned to 5 dietary treatment groups: 1) no CLA (control), 2) CLA 2%, 3) CLA 2% + OA 2% (CLA + OA), 4) CLA 2% + linoleic acid (LA) 2% (CLA + LA), and 5) CLA 2% + -linolenic acid (LNA) 2% (CLA + LNA) (Lipozen Inc., Pyongtaek, Korea). The ingredients and chemical compositions of the experimental diets are shown in Table 1
. Feed and water were available ad libitum in each dietary group. The photoperiod was set at 17L:7D during the experiment. Eggs were collected and counted daily to obtain data on egg production, and feed consumption for each replicate was recorded daily for the entire study. Collected eggs were broken open to determine the egg quality twice per week, and contents were then frozen at –50°C for further analyses. All animal-based procedures were in accordance with the "Guidelines for the Care and Use of Experimental Animals of Korea Universities."
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Eggs were collected during the experiment and stored at –50°C for subsequent analyses. Egg parameters, including egg weight, Haugh units, and egg yolk color, were measured with a QCM+ device (Technical Services and Supplies, York, UK), and eggshell thickness and strength were measured with an FHK device (Fujihara Co. Ltd., Saitama, Japan).
Gas Chromatography Analysis
Lipids from egg yolks were extracted with hexane:isopropanol (3:2, vol/vol). Fatty acids were converted into methyl esters as described in our previous report, with some modifications (Kim et al., 2003). Briefly, 0.5 mL of toluene and 2 mL of 5% KOH-MeOH were added to the lipids, and the samples were vortex-mixed and heated at 70°C for 8 min. The samples were then cooled in cold water, 2 mL of 14% BF3-MeOH was added, and they were heated to 70°C for another 8 min. The samples were cooled, 3 mL of 5% NaCl was then added and mixed, and 5 mL of distilled water and 0.5 mL of hexane were added to extract the fatty acid methyl esters. The mixture was vortexed and centrifuged at 3,000 x g for 5 min, and the upper phase was then collected and dried with sodium sulfate. Samples were analyzed for total fatty acids, including CLA isomers, using an HP5890 gas chromatograph with a flame-ionization detector (5890 Series II, Hewlett-Packard, Palo Alto, CA). The fatty acid methyl esters were separated using a Supelcowax-10 fused-silica capillary column (100 m x 0.32 mm i.d., 0.25 µm film thickness; Supelco, Inc., Bellefonte, PA) with 1.2 mL/min of helium flow. The oven temperature was increased from 220 to 240°C at the rate of 2°C/min. Injector and detector temperatures were 240 and 250°C, respectively. One microliter of sample was injected into the column in the split mode (50:1). The peak of each CLA isomer (cis-9, trans-11; trans-10, cis-12; cis, cis; and trans, trans isomers) and other fatty acids were identified and quantified by comparison with the retention time and peak area of each fatty acid standard (Sigma). Fatty acid content was expressed as the percentage of total fatty acids. Heptadecanoic acid (17:0) was included as an internal reference before the extraction of lipids to determine the recovery of fatty acids in each sample. The recovery of methylated fatty acids, calculated in comparison with the internal standard, was higher than 80%.
Statistical Analysis
Statistical differences were determined by ANOVA, with mean separations performed by the Duncan multiple range test using PROC GLM of the SAS statistical software package (SAS Institute, 1996). Egg yolk samples were analyzed in triplicate, and the variation between samples is expressed as the pooled standard error of the mean or mean ± standard error of the mean, where applicable.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The fatty acid compositions of experimental diets including UFA are shown in Table 1
. To determine the effect of fatty acid supplementation, CLA was fed alone or with other fatty acids (OA, LA, and LNA). The control group was not fed any supplemental fatty acids. The CLA was given at 2% of the total feed to the study groups, and an additional 2% of other fatty acids (OA for CLA + OA; LA for CLA + LA; LNA for CLA + LNA) were given to test groups as indicated. When dietary CLA was given alone, most of the parameters of egg quality were negatively affected. However, co-supplementation with other fatty acids reduced the degree of changes in egg weight, strength and thickness of the eggshell, albumen index, yolk index, yolk color, and yolk diameter as shown in Table 2. Shang et al. (2004) fed up to 7% CLA for 4 wk in a laying hen diet and found significant decreases in the egg weight, egg production, and feed conversion ratio. In the present study, no detrimental effects were found in any of the egg quality parameters with supplemented diets compared with the control diet. However, there was a change in eggshell color with the CLA group after 4 wk. Aydin et al. (2001) found discoloration of the egg yolk and albumen when CLA-enriched shell eggs were stored at 4°C, but their effect was not evident in the CLA + LA and CLA + LNA groups in the present trial. Moreover, Aydin et al. (1999) suggested that the change in egg quality from hens fed UFA was related to a change in the yolk water content and the movement of ions through the vitelline membrane, which would have been affected by shifts in the fat composition of the membrane; this effect may have been minimized by our co-supplementation strategies.
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Fatty Acid Profile of the Egg Yolk
To investigate the effects of supplementation of UFA and CLA on the fatty acid profile of the egg yolk, egg samples were taken daily and major long-chain fatty acids in the egg yolk were analyzed (Table 3). In all dietary groups except CLA + OA, the total CLA content was enhanced by as much as 4% of total fat in the first week, but no further increase was evident thereafter (Figure 1). The CLA content in egg yolks from the CLA + OA group did not change in the first week but increased to 5.5% of total fat in the third week. The overall CLA content was increased in all dietary CLA groups up to the third week. In the CLA + LNA group, the CLA level was not significantly changed until the fourth week. These results indicated that CLA could accumulate in a relatively short-term period (4 wk), and some adverse effects that could be caused by fatty acid supplementation could be minimized. In fact, other researchers (Ahn et al., 1999; Hwangbo et al., 2005) have found little increase in the CLA content of the yolk even after prolonged feedings. The changes in the pattern of CLA accumulation at each trial may be ascribed to the effects of supplemented fatty acids on the desaturation of C18:1 fatty acids. Takahashi et al. (2003) showed that CLA feeding enhanced hepatic desaturation and fat synthesis in mice, whereas other UFA affected enzyme activity to a different degree. However, a clear explanation of this mechanism is not yet available. Further enzymatic studies in relation to gene expression are necessary to explain the different fatty acid profiles resulting from feeding CLA along with UFA. In a previous study, CLA supplementation at 2.5% of the dietary level led to CLA accumulation as much as 8% of total fat in chicken muscles after 6 wk of feeding (Lee et al., 1999). In the present trial, however, the increase in the total CLA content of the egg yolk reached as high as 7% of total fat by CLA feeding alone, and this was maximized in 3 wk, indicating that PUFA supplementation with CLA could be an efficient method of accumulating CLA in the egg yolk.
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). This indicated that homeostasis in fat metabolism could be efficiently maintained by PUFA supplementation, as indicated by the finding that the C18:0 to C18:1 ratio at the end of the test period was similar to that of the first week of the trial. The C18:0 to C18:1 ratio has been used as an important parameter determining membrane integrity and homeostasis in fat metabolism (Aydin et al., 2001). In fact, OA is one of the major fatty acids in the egg yolk, accounting for as much as 40% of the total fatty acids in the egg (Lee et al., 1999). The inverse relationship between C18:0 and C18:1 could be partially explained by the fact that CLA-PUFA feeding enhanced the desaturation of C18:1 to CLA while maintaining the balance with the degree of saturation of UFA for utilization as an energy source. Aydin et al. (2001) found that supplementing CLA with olive oil (10% of the diet), which is rich in OA, decreased the yolk CLA content (both cis-9, trans-11 and trans-10, cis-12), but there was no significant decrease in our study when the yolk ratio of CLA to OA was 1:1. The substantial amount of OA in the previous study may have inhibited the incorporation of CLA into chicken tissue. This effect would be partially ascribable to the esterification of supplemented fatty acids. These results confirmed that OA co-supplementation could be a good strategy to enhance the CLA level in the egg yolk.
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). Indeed, palmitic acid (C16:0) was increased in the CLA + OA group, which indicates that C18:1 may also induce C16:1 saturation to C16:0. No apparent change was observed in the C16:1 profile of other dietary groups. Desaturase activity for the formation of n-7 double bonds may have been less affected by PUFA supplementation compared with desaturase activity for n-9 double bond formation. Further studies warrant elucidation of the site-specific effects of PUFA supplementation of animal feeds.
On the other hand, CLA + LNA supplementation increased the LNA content in the yolk to 3% of total fat during the feeding (Figure 3). Compared with >4% increase in the CLA content in the CLA group and other study groups, LNA accumulation was not as efficient as CLA accumulation. The LA supplementation also resulted in a 5% higher LA accumulation in the yolk compared with other supplementation groups after 4 wk of feeding (Table 3), almost twice the amount for the control group. The increase in OA was not as evident as that of other supplemented fatty acids, but the CLA + OA group had a 20 to 30% higher OA content than the other supplementation test groups. The content of PUFA supplemented with CLA was not substantially altered during the feeding trial, which indicated that PUFA supplementation neither induced a further increase in the CLA content nor affected the desaturation of C18:2 to C18:3 fatty acids.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication December 5, 2006. Accepted for publication February 16, 2007.
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