PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION Effect of Dietary Supplementation with Conjugated Linoleic Acid, with Oleic, Linoleic, or Linolenic Acid, on Egg Quality Characteristics and Fat Accumulation in the Egg Yolk1 J. H. Kim,* J. Hwangbo,† N.-J. Choi,‡ H. G. Park,* D.-H. Yoon,† E.-W. Park,† S.-H. Lee,† B.-K. Park,‡ and Y. J. Kim*2 *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 ABSTRACT The effects of conjugated linoleic acid (CLA) with other fatty acids on the fatty acid composition of egg yolk and on egg quality characteristics were studied in 5 groups: 1) CLA 0% (control), 2) CLA 2%, 3) CLA 2% + oleic acid (OA) 2% (CLA + OA), 4) CLA 2% + linoleic acid (LA) 2% (CLA + LA), and 5) CLA 2% + α-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, α-linolenic acid, egg quality 2007 Poultry Science 86:1180–1186
INTRODUCTION Conjugated linoleic acid (CLA) refers to positional and geometric isomers of linoleic acid (C18:2, LA) with various conjugated double bond arrangements. Among them, the cis-9, trans-11 isomer is the principal form of isomer, and trans-10, cis-12 CLA and other isomers are also present in nature. Conjugated linoleic acid is a natural food component that may serve as a health-promoting and therapeutic agent (Pariza et al., 2000; Belury, 2002). There is a great deal of evidence that CLA is effective in cancer chemoprevention (Ha et al., 1987; Eynard and Lopez, 2003; Lee et al., 2004; Lee et al., 2005) and adipose depletion (Lee et al., 1998; Hargrave et al., 2004), and numerous studies have focused on defining other physiological functions of CLA isomers (Lee et al., 1994; Butz et al., 2006; Noto et al., 2007). The CLA is known to be the only
©2007 Poultry Science Association Inc. Received December 5, 2006. Accepted February 16, 2007. 1 The first 2 authors contributed equally to this work. 2 Corresponding author:
[email protected].
fatty acid unequivocally shown to inhibit carcinogenesis in animal studies (NRC, 1996). However, the application of CLA as a functional food component is currently possible only with synthetic CLA containing various uncharacterized isomers, because the level of consumption of natural CLA is far below the level showing physiological activity (Ip et al., 1994). Dietary CLA can be assumed to balance the undesirable effects of large amounts of saturated fatty acids (SFA) and cholesterol derived from animal foods (Eynard and Lopez, 2003). Because of the various benefits of CLA to human health, extensive trials have been conducted to enrich the CLA content of food products and to maximize its physiological advantages (Kelly et al., 1998; Kim and Liu, 2002). 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
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CONJUGATED LINOLEIC ACID ACCUMULATION IN THE EGG YOLK Table 1. Composition (%) of experimental diets Dietary group1 Item Ingredient Corn Wheat hulls Soybean meal Corn-gluten meal Salt Vitamin-mineral mix2 L-Lys DL-Met Limestone Tricalcium phosphate Fatty acid CLA3 Oleic acid Linoleic acid α-Linoleic acid Total
Control
CLA
CLA + OA
CLA + LA
CLA + LNA
60.00 10.50 15.00 3.00 0.30 0.50 0.20 0.20 7.30 1.00
60.00 10.50 15.00 3.00 0.30 0.50 0.20 0.20 7.30 1.00
55.00 13.50 14.50 3.50 0.30 0.50 0.20 0.20 7.30 1.00
55.00 13.50 14.50 3.50 0.30 0.50 0.20 0.20 7.30 1.00
55.00 13.50 14.50 3.50 0.30 0.50 0.20 0.20 7.30 1.00
— — — — 98.00
2.00 — — — 100.00
2.00 2.00 — — 100.00
2.00 — 2.00 — 100.00
2.00 — — 2.00 100.00
1 Control = conjugated linoleic acid (CLA) 0%; CLA = CLA 2%; CLA + OA = CLA 2% + oleic acid 2%; CLA + LA = CLA 2% + linoleic acid 2%; CLA + LNA = CLA 2% + α-linolenic acid 2%. 2 Provided (per kg of diet): vitamin A, 5,500 IU; vitamin D3, 1,100 IU; vitamin E, 11 IU; vitamin B12, 0.0066 mg; riboflavin, 4.4 mg; niacin, 44 mg; pantothenic acid, 11 mg (Ca-pantothenate, 11.96 mg); choline, 190.96 mg (choline chloride, 220 mg); menadione, 1.1 mg (menadione sodium bisulfite complex, 3.33 mg); folic acid, 0.55 mg; pyridoxine, 2.2 mg (pyridoxine hydrochloride, 2.67 mg); biotin, 0.11 mg; thiamin, 2.2 mg (thiamine mononitrate, 2.40 mg); ethoxyquin, 125 mg; Cu, 30 mg; Zn, 60 mg; Mn, 90 mg; Co, 0.25 mg; I, 1.2 mg; Se, 0.3 mg. 3 Total CLA content used in the feeding trials was 80%.
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 cosupplemented 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.
MATERIALS AND METHODS
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% + α-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 animalbased procedures were in accordance with the “Guidelines for the Care and Use of Experimental Animals of Korea Universities.”
Sample Collections and Egg Quality 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).
Birds and Diets
Gas Chromatography Analysis
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)
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
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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 × 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 × 0.32 mm i.d., 0.25 m 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.
RESULTS AND DISCUSSION Egg Quality and Productivity 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. In our previous study, CLA supplementation of the diets of laying hens decreased the egg production rate, in agreement with other PUFA feeding studies. The reason was thought to be similar to that of the change in egg quality (Shang et al., 2004). In the present study, however, egg production was not significantly altered in the CLA + OA and CLA + LA groups. This suggested that co-supplementation with OA or LA could reasonably support egg production, which has long been a problem in CLA enrichment trials. Increased SFA and decreased monounsaturated fatty acids resulting from CLA feeding were likely normalized by OA, and LA may have competed with CLA for incorporation into the membrane. Fatty acid co-supplementation may have led to homeostasis of lipid metabolism in the liver and thereby helped maintain egg quality during CLA supplementation. In fact, the major fatty acids in olive oil (which was used in a previous study to enhance the CLA content) were OA and LA, and chick hatchability and egg quality were improved, possibly because of a similar C18:1 to C18:0 ratio (Aydin et al., 2001). Thus, this ratio should be considered as an important factor affecting yolk vitelline membrane characteristics, as well as egg quality parameters. The incorporation of additional PUFA into the plasma membrane could lead to a change in molecular interactions in the vitelline membrane, and thus result in metabolic disturbances (Watkins, 2003). Therefore, fat supplementation for accumulation in the egg should be carefully designed to minimize side effects that may affect the reproduction capacity of the poultry.
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
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CONJUGATED LINOLEIC ACID ACCUMULATION IN THE EGG YOLK Table 2. Effect of different dietary oil supplementation on performance and egg quality of hens during the experiment Dietary group1 Quality parameter
Control
CLA
CLA + OA
CLA + LA
CLA + LNA
SEM
P-value
Feed intake (g/d) Rate of egg production (%) Eggshell color Egg weight (g/egg) Haugh units Albumen index Egg height Eggshell thickness (mm) Eggshell strength (kg/m2) Egg yolk color Egg yolk index Egg yolk diameter
104.8 86.88a 75.92a 60.25 84.13a 0.09 18.59 0.36 4.94 8.00ab 0.42b 41.23abc
105.6 77.74b 79.73a 56.34 82.89b 0.07 18.59 0.32 3.75 8.91a 0.50a 38.13d
105.5 80.47ab 77.36ab 59.29 83.43a 0.08 17.44 0.34 4.78 8.18ab 0.42b 41.72ab
106.9 86.67a 76.75b 59.12 84.70a 0.08 17.29 0.34 4.27 8.33ab 0.41b 42.49a
110.8 76.35b 76.46b 57.87 84.90a 0.08 17.00 0.34 4.23 8.36ab 0.42b 40.21bcd
7.03 4.153 2.966 5.911 6.319 0.021 2.073 0.035 1.147 1.010 0.067 2.396
NS 0.001 0.047 0.122 0.155 0.081 0.510 0.174 0.133 0.066 0.044 0.001
Means with different superscripts within the same row are different (P < 0.05). Control = conjugated linoleic acid (CLA) 0%; CLA = CLA 2%; CLA + OA = CLA 2% + oleic acid 2%; CLA + LA = CLA 2% + linoleic acid 2%; CLA + LNA = CLA 2% + α-linolenic acid 2%. a–d 1
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 shortterm 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
Table 3. Fatty acid composition of egg yolks after 4 wk of feeding the experimental diets (g/100 g of egg yolk fat) Dietary group2 Fatty acid1 Palmitic acid (C16:0) Palmitoleic acid (C16:1) Stearic acid (C18:0) Oleic acid (C18:1) Linoleic acid (C18:2) r-Linolenic acid (C18:3) α-Linolenic acid (C18:3) CLA Cis-9, trans-11 Trans-10, cis-12 Cis, cis Trans, trans Arachidonic acid (C20:4) Eicosapentaenoic acid (C20:5) SFA MUFA PUFA 18:0/18:1
Control
CLA
CLA + OA
CLA + LA
CLA + LNA
SEM
P-value
26.45c 2.80a 11.62c 43.59a 9.51d 0.12 0.14b ND3 NDc NDc NDc NDc 1.78a ND 38.07c 46.39a 11.43c 0.27d
31.51ab 1.33b 18.52ab 26.53c 12.23bc 0.18 0.25b 5.28ab 2.94ab 1.59ab 0.48a 0.27a 1.39bc ND 50.03ab 27.86c 13.87b 0.70b
30.30b 1.28b 16.69b 30.55b 12.62bc 0.07 0.20b 4.28b 2.47b 1.27b 0.26b 0.28a 1.38bc ND 46.99b 31.83b 14.21b 0.55c
31.66ab 0.83b 19.52a 21.99d 17.00a 0.05 0.20b 4.94ab 2.92ab 1.44ab 0.36ab 0.22ab 1.44b ND 51.18a 22.82d 18.64a 0.89a
31.54ab 1.09b 20.33a 22.87cd 12.94b 0.05 3.27a 4.56ab 2.86ab 1.32b 0.25b 0.13b 1.19c 0.14 51.87a 23.96cd 17.45a 0.89a
1.893 0.585 2.597 4.290 1.756 0.154 0.452 1.315 0.779 0.398 0.204 0.131 0.242 0.323 3.797 4.567 1.711 0.178
