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Ghent University, Faculty of Agricultural and Applied Biological Science, Department of Animal Production, Proefhoevestraat, 10–9090 Melle, Belgium;
*
CLO-DVV, Burg. Van Gansberghelaan, 92–9820 Merelbeke, Belgium; and
INVE Technologies, Oeverstraat, 7–9200 Baasrode, Belgium
2To whom correspondence should be addressed. E-mail: Stefaan.Desmet{at}rug.ac.be.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • conjugated linoleic acid • egg yolk • fatty acids • laying hens
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Furthermore, in determining the quality of food products, consumer concerns are increasingly important. Within the framework of a general quality concept (2
), direct egg quality characteristics can be categorized into external appearance traits and internal quality traits. The latter characteristics include mainly properties related to albumen as well as yolk quality, e.g., nutritional value, physicochemical value, sensory properties and safety and health aspects (3
).
The role of dietary cholesterol and fatty acid composition in the etiology of cardiovascular disease and other ailments remains controversial. The many attempts to reduce egg cholesterol content have met little practical application (4
). An alternative way to reduce the cholesterolemic effects of eggs is by altering the yolk fatty acid composition. The cholesterol-lowering effects of (n-6) and (n-3) polyunsaturated fatty acids (PUFA)3
have been recognized for some years (5
–7
) and feeding layers diets rich in PUFA results in a large increase in the relative and absolute concentrations of PUFA in yolk total lipid (8
–13
).
Conjugated linoleic acid (CLA), a group of isomers of octadecadienes, has received much attention recently. Several beneficial effects are attributed to these isomers, e.g., anticarcinogenic and antiatherosclerotic effects, influencing both fat metabolism and protein deposition (14
). CLA 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 the ruminal production (15
,16
). Eggs and other products from monogastric animals contain negligible amounts of CLA (17
). If the results of animal studies are extrapolated, an intake of 3.0 g CLA/d would be required for a 70-kg person to ensure health benefits (18
). Such intake is 
3 and 10 times that of the daily estimated consumption of adults in the United States (19
) and in Germany, respectively (20
). It is therefore 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 30–35% fat. 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 (n-3) fatty acids, would lead to even greater health benefits.
The aim of this experiment was to study the effect of commercial CLA preparations in layers diets with different levels of fat from different sources on the zootechnical performance as well as on the fatty acid metabolism and incorporation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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A flock of medium weight laying hens (ISA-brown) in the second half of their production cycle (53–59 wk of age), was randomly allocated to three-tier battery pens of 4 laying hens each, under conventional conditions of ventilation, temperature (18–22°C) and lighting (16 h light/d). Each pen was previously controlled for its laying persistency to improve the homogeneity of the entire flock. The experimental period consisted of the following 3 subperiods: (1
) 14 d consuming one of 4 reference diets, i.e., a low fat (LF) diet and 3 high fat diets containing either soybean oil (SB), animal fat (AF) or flax seed oil (FSO) (each diet was fed to 9 pens); (2
) 14 d consuming one of 12 diets made from the combinations of the reference diets and 3 CLA treatments, i.e., without CLA (LF0, SB0, AF0, FSO0) and with either CLA premix I (containing commercial CLA oil I 
LF1, SB1, AF1, FSO1) or CLA premix II (containing commercial CLA oil II LF2, SB2, AF2, FSO2) (each diet was fed to 3 pens); and (3
) another 14 d as a steady-state period, assigned to the same 12 treatments.
The following conventional zootechnical data were recorded: daily feed intake (g/laying hen), laying rate (%, eggs/100 laying hens), egg weight (g), daily egg mass (g/laying hen) and feed efficiency (daily feed intake/daily egg mass: g/g).
Diet preparation.
Diets were least-cost formulated using a linear programming technique with an identical set of nutrient specifications, except for nutrient density in combination with either the low or high dietary fat concentration (Table 1
). Diets were formulated to include different fat contents and fatty acid profiles high in (n-6) and (n-3) PUFA. All feedstuffs were mixed homogeneously before introduction into the final diets. The high fat diets (SB, AF and FSO with 8.1% total fat) were based on the low fat feed formula LF0, by adding a supplementary 4.5% of either crude soybean oil (SB0), rendered animal fat (AF0) or crude flaxseed oil (FSO0), respectively. Commercial liquid CLA-mixtures [obtained as Selin-CLA from Grünau, Illertissen, Germany (CLA1), and from TrofoCell, Hamburg, Germany (CLA2)], containing 99% triglycerides, were premixed with silica to obtain a CLA concentration of 33% in CLA premix I and II (or 1% on a dietary base). The total fat concentration was maintained for all treatments by decreasing the fat content from the respective fat sources by an amount equivalent to the fat content of the CLA-supplemented premixes. Both CLA sources contained other fatty acids [16:0, 18:0, 18:1 and 18:2(n-6)] in addition to CLA. Also, they differed in the total amount of CLA and in the ratio of the different CLA isomers. According to the specifications of the producers, CLA premix I and II contained 60 and 70% CLA, respectively. These values corresponded well with the total CLA contents as measured by gas chromatography (GC), 57 and 74%, respectively. The two CLA sources also showed a different isomer profile by GC analysis, i.e., CLA I contained 11.4% c9, t11; 33.3% t10, c12 and 12.5% tt isomers, whereas CLA II contained 30.3% c9, t11; 41.6% t10, c12 and 2.1% tt isomers. The fatty acid profiles of the final diets, as analyzed by GC, are given in Table 2
. It is worthwhile to note that, after inclusion of CLA, the level (g/100 g of total fatty acids, Table 2
) of both linoleic and linolenic acid decreased slightly. This target allowed us to study the possible effects of CLA (isomers) on the fatty acid metabolism of essential (n-6) and (n-3) fatty acids.
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-tocopherol-acetate and 50% -tocopherol-acetate) and 10 mg vitamin E in the INVE-premix and 45 mg BHT/kg feed. Furthermore, all other vitamins and trace elements were provided by the INVE-premix according to layers feed recommendations. The diets were fed in mash form (i.e., not pelleted) and consumed ad libitum. Each treatment (4 basal fat sources x 3 CLA-treatments = 12 diets) consisted of 3 replicates with 4 laying hens per replicate. This trial was thus performed on 36 pens concurrently, or 144 laying hens in all. Egg preparation.
Uniform samples of 3 eggs per pen were collected just before the trial, at the end of the first subperiod and 3 times during the steady-state period (at 14, 21 and 28 d after the introduction of CLA) to determine the following: 1) the mass of the main egg components shell, yolk and albumen; 2) the yolk color in terms of Roche Color Fan numbers (RCF1993) (21
); 3) the dry matter of the yolk after freeze-drying; 4) the cholesterol content of the dry yolk material (22
); and 5) the yolk fat level (mg/g yolk) and fatty acid composition.
Fatty acid analysis.
Fatty acid analysis was carried out as described (23
, 24
). In brief, lipids were extracted from fresh yolk (pooled sample of 3 eggs/pen) using chloroform/methanol [2:1, v/v, adapted from Folch et al. (25
)]. Nonadecanoic acid (19:0) was added as an internal standard. To make sure that the methylation procedure did not cause further isomerization, different solvents were compared in earlier work. The solvents examined were tetramethylguanidine in methanol (1:4, v/v; 60 min at 100°C), H2SO4 in methanol (1:115, v/v; 60 min at 100°C), NaOH in methanol (0.5 mol/L; 30 min at 50°C) and NaOH in methanol (0.5 mol/L; 30 min at 50°C) followed by HCl in methanol (1/1, v/v; 10 min at 50°C). The two-step methylation procedure, using a basic reagent NaOH/methanol followed by an acid reagent HCl/methanol was finally chosen because it causes little if any further isomerization and its acid component was required to esterify the free fatty acids [for an extended description see (24
)]. The fatty acid methyl esters (FAME) were analyzed by GC (HP 6890, Hewlett-Packard, Brussels, Belgium) using a CP-Sil88 column for FAME (100 m x 250 µm x 0.25 µm) (Chrompack, Middelburg, The Netherlands). The GC conditions were as follows: injector temperature, 250°C; detector temperature, 280°C, carrier gas, H2; split ratio, 1/50; temperature program, 150°C for 2 min, followed by an increase of 1.5°C/min to 200°C, then 5°C/min to 215°C. Peaks were identified by comparison of retention times with those of the corresponding standards (Sigma, Botnew, Belgium; Nu-Chek-Prep, Elysian, MN). Identification of the peaks included fatty acids between 12:0 and 22:6 and 5 different CLA isomers, i.e., c9, t11; t10, c12; ct11,13; cc and tt CLA isomers. Other CLA isomers could not be detected by simple GC, e.g., t8, c10 CLA is probably present in a trace amount in CLA oil II according to the manufacturer, but its peak is supposed to overlap with that of c9, t11 CLA.
Calculations and statistics.
Amounts of fatty acids were expressed as a percentage (g/100 g) of total fatty acids. The addition of an internal standard made it possible to quantify the fatty acids as concentrations (mg/g feed or yolk). The deposition rate of each fatty acid can be derived from its own, typical dose-response line, Y = a + bX, where Y is the yolk fatty acid (mg/kg); a is the intercept value (0 without any de novo synthesis) at X = 0; X is the dietary fatty acid level (mg/kg); b is the "deposition rate x FC/AYS", where the deposition rate (%) is the deposited/consumed fatty acid ratio x 100, FC is feed conversion, which is the daily feed intake/daily egg mass (g/g) and AYS is the average yolk share (% of average egg weight).
We first checked whether there was a significant effect of sampling time in the final sampling period (14, 21 and 28 d after the introduction of CLA). Because this was not the case, the three samplings were considered to be replicates. To examine the effect of CLA treatment and fat source, a two-factorial ANOVA was carried out with these two main factors and their interaction term. Mean values for the different CLA treatments were compared within each of the fat source groups using the Bonferroni multiple range test (SPSS software 9.0; Chicago, IL).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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During the preliminary period, the zootechnical performance was relatively homogeneous across pens with mean values for feed intake (g/d), laying rate (%), egg weight (g), daily egg mass (g) and feed efficiency (g/g) of 117.1, 80.0, 67.3, 53.9 and 2.17, respectively. In addition, the values for the percentage of yolk, albumen and shell (on average 27.1, 60.7 and 12.3, respectively) did not differ across pens. Results for the 12 diets during the steady-state period are given in Table 3
. The overall means for daily feed intake (g), laying rate (%), egg weight (g), daily egg mass (g) and feed efficiency (g/g) were 106.2, 78.1, 65.0, 50.7 and 2.09, respectively. The percentages of yolk, albumen and shell (on average 27.4, 60.2 and 12.4, respectively) did not differ from those of the preliminary period and within normal ranges.
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Fatty acid metabolism.
The fatty acid profile of the yolk fat was relatively unsaturated with unsaturated/saturated (U/S) ratios varying from 2.1 to 2.4 for the 4 reference diets without any CLA supplementation (Table 4
). The saturated fatty acid (SFA) concentration remained fairly constant (between 90 and 100 mg SFA/g yolk), whereas the major changes were within the 18-unsaturates with proportional and inverse changes between 18:1 and 18:2(n-6), or 18:3(n-3).
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) 22–26 and (2
) 10–13 g/100 g of total fatty acids in the low fat (LF) and in the high fat (SB, AF and FSO) diets, respectively. Because no significant effect of duration of feeding CLA on the fatty acid composition was found after 14 d of treatment, the data are presented as means of the three samplings at 14, 21 and 28 d after starting CLA feeding (Table 4)
. The inclusion of CLA in the respective diets did not affect total fatty acid content of the yolk (on average, 281 mg fatty acids/g yolk).
Dietary supplementation with CLA significantly enhanced the SFA concentration and significantly decreased the MUFA concentration of egg yolk in birds consuming all feeds. The response in PUFA concentration depended, however, on the dietary fat level: a significant decrease was observed in egg yolk of those fed diet LF but no effect was observed for those fed the high fat diets (Table 4)
.
The effect of CLA supplementation on the levels of PUFA of the (n-6) and (n-3) series varied. The different proposed modes of action of CLA on the fatty acid metabolism are presented in Figure 1
.
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In the (n-3) series, the amount of 18:3(n-3) increased slightly due to CLA in treatments LF, SB and AF. This was accompanied by an increase in 22:5(n-3) and a decrease in 22:6(n-3). However, at an excess of dietary 18:3(n-3) (treatment FSO), 20:5(n-3) accumulated in the egg yolk. When the FSO diet was supplemented with CLA premix, a decrease of 18:3(n-3) and 20:5(n-3) was observed in the egg yolk, whereas 22:5(n-3) and 22:6(n-3) increased.
Apparent deposition rate.
The apparent deposition rate differed widely for the different fatty acids, and depended on the fat source of the diet (Table 5
). The apparent deposition rate decreased at higher dietary fat content and was also influenced by the presence of CLA in the feed. The apparent deposition rates for both linoleic and linolenic acids were lower than the average for the total fat, with linolenic acid at 50% of the apparent deposition rate of linoleic acid. The variation in the deposition rates was less influenced by the different dietary fat sources than by the addition of CLA. A clearly higher apparent deposition rate was found for the c9, t11 isomer than for the t10, c12 isomer.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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In general, the zootechnical responses were not clearly related to dietary composition, with the exception of both feed intake and feed efficiency, which were inversely related to nutrient density (LF as SB, AF, FSO). These zootechnical results are similar to those of Chamruspollert and Sell (26
) for the same dietary range of CLA. The latter authors observed an adverse effect of CLA only at the higher dietary concentration of 5.0%. On the other hand, Jones et al. (27
) observed an adverse effect on egg production of dietary CLA concentrations of 0.5 and 1.0 g/kg diet.
Although there were also some differences in the relative mass of the main egg components (yolk, albumen and shell) as well as in the yolk cholesterol content, these differences were apparently not related to dietary factors. Also, the egg yolk color changed relatively slightly throughout the entire steady-state period. This means that a period of 14 d was sufficient for the adaptation of the egg yolk color. This observation agrees with the findings of Huyghebaert et al. (unpublished data) demonstrating that 10 d are required for an entire shift in egg yolk color. Also, Marusich et al. (28
) and Williams et al. (29
) concluded that 9–11 d are required for a uniform maximal color deposition throughout the yolk, coinciding with the period needed for follicle maturation (as yolk at ovulation).
Fatty acid metabolism.
When only the reference diets (LF0, SB0, AF0 and FSO0) are considered, the yolk fat was relatively unsaturated. The amount of SFA did not vary greatly among the reference diets, yet major differences were present in the 18-unsaturates. The yolk fatty acid profile clearly reflected the dietary fatty acid composition. However, the U/S ratio was more resistant to dietary manipulations than were individual fatty acids. Analogous findings were reported by Huyghebaert et al. (11
) and Vahl et al. (13
).
Because no effect of duration of CLA feeding on the yolk fatty acid profile was found, the adaptation period of 14 d was proven to be long enough to obtain a steady state. This finding confirms the observation of Huyghebaert et al. (unpublished data) that 10 d is required for an entire shift in fatty acid profile throughout the yolk.
In egg yolk of hens fed the CLA-supplemented diets, a significant increase in SFA was observed at the expense of MUFA, compared with hens fed the reference diets. Increased levels of SFA and decreased levels of MUFA as a result of CLA supplementation were also found by Ahn et al. (30
) and Chamruspollert and Sell (26
) in eggs, and by Lee et al. (31
) in rabbit meat. These results may indicate that CLA inhibits 9-desaturase, the enzyme responsible for the conversion of 16:0 and 18:0 to their corresponding MUFA. In this study, the inhibition of the conversion from saturated to the corresponding monounsaturated fatty acids was slightly stronger in hens fed the diets containing the CLA premix II (with a relatively higher amount of the c9, t11-isomer) compared with the CLA premix I. Higher levels of SFA in yolk lipids due to CLA supplementation may be of concern because increased consumption of SFA, especially 14:0 and 16:0, is associated with increasing plasma total and LDL cholesterol concentrations in blood plasma, changes related to cardiovascular diseases (32
). However, if the antiatherogenic activity of CLA found in rabbits (33
), hamsters (34
) and mice (35
) could be extrapolated to humans, the adverse consequences of increased SFA levels could be counteracted by CLA.
The effects of CLA supplementation on the (n-6) and (n-3) PUFA contents varied for the different fatty acids and was also diet dependent for some fatty acids. No effect of CLA was observed on the amount of 18:2(n-6), whereas it clearly decreased the 20:4(n-6) level in the egg yolk. The other (n-6) fatty acids showed a tendency to decrease by supplementing CLA, irrespective of the basal fat sources. On the other hand, 18:3(n-3) and 22:5(n-3) tended to increase or increased and 22:6(n-3) decreased as a result of supplementing CLA to the diets containing LF, SB and AF as the basal fat source. However, when an excess of 18:3(n-3) was provided by the FSO diet, the effect of CLA on the egg yolk (n-3) fatty acids was different. In the latter case, 22:5(n-3) and 22:6(n-3) increased, whereas 18:3(n-3) and 20:5(n-3) decreased. A reduction in 20:4(n-6) and in 20:5(n-3) (the latter only in treatment FSO) by CLA supplementation could be due to an inhibition of 6-desaturase. Belury and Kempa-Steczko (36
) found that CLA was able to act as a substrate for 6-desaturase, the rate-limiting step in the conversion of linoleic and linolenic acid into 20:4(n-6) and 20:5(n-3), respectively. However, if CLA is a competitive inhibitor of 6-desaturase, an accumulation of linoleic and linolenic acids should be observed. This was generally not the case, with the exception of treatment AF. A decrease in 22:6(n-3) concentration in the yolk lipids due to feeding CLA was also found by Chamruspollert and Sell (26
), 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 when a high amount of flaxseed oil (yielding an excess of linolenic acid) was fed to laying hens. In this case, the amount of 22:6(n-3) remained at the same level or tended to increase when CLA was fed. The reason why the incorporation of (n-3) fatty acids into the egg yolk is higher when CLA is fed is not clear. One possible explanation is based on the stereospecificity of the molecules. The structure of CLA is more comparable to linoleic acid than to linolenic acid. It is also known that the enzymes for desaturation and elongation of the (n-6) and (n-3) fatty acids are the same and that 6-desaturase acts preferentially on the (n-3) fatty acids. It is therefore possible that CLA is able to make a complex with 6-desaturase in the same way as linoleic acid does, thus preventing linoleic acid from being further converted to its long-chain metabolites. In addition, the incorporation of long-chain (n-3) fatty acids 20:5(n-3), 22:5(n-3) and 22:6(n-3) into cell membranes and phospholipids is in competition with the (n-6) long-chain PUFA. Because linoleic acid would be in competition with CLA for 6-desaturase in CLA-supplemented diets, relatively more (n-3) long-chain PUFA could be incorporated into the cell membranes of laying hens fed diets supplemented with CLA.
Changes in the concentrations of the end-products as well as intermediates in the (n-6) and (n-3) PUFA series are consistent with a competitive inhibition of the different desaturase activities (Fig. 1)
, except for the diet enriched in (n-3) fatty acids.
Apparent deposition rate.
Lower apparent deposition rates for 18:2(n-6) and 18:3(n-3) were observed than for total fat. These are essential fatty acids that cannot be synthesized de novo by the organism. Also, these two fatty acids undergo elongation to longer-chain fatty acids, which are partly incorporated into the egg lipids and partly used for the production of prostaglandins, eicosanoids and leucotrienes.
A higher incorporation rate was observed for c9, t11 CLA than for t10, c12 CLA. This finding is in agreement with results observed by Jones et al. (27
). Also Bee (37
) found that the c9, t11 isomer was incorporated into adipose tissue of sows at a higher rate than the other isomers. Park et al. (38
) noted that t10, c12 CLA appears to be metabolized more rapidly than c9, t11 CLA, particularly in skeletal muscle. Whether this is due to enhanced elongation/desaturation, enhanced ß-oxidation or both is not yet known.
Nutritional aspects of CLA and (n-3) enriched eggs.
On the basis of the data for the CLA concentrations in yolk lipids and the lipid content per egg yolk, the amount of CLA per egg can be estimated. On average, when 1% of CLA is included in the diet, the CLA amount per egg varied between 130 and 250 mg, depending on the fat level of the diet. Taking into account the current average consumption of eggs in the Western diet, the daily intake of CLA from eggs could range from 100 to 200 mg/d. This does not cover the total nutritional requirements for CLA. However, eggs containing substantial amounts of CLA could be a valuable dietary CLA source in combination with, for example, ruminant products that are naturally rich in CLA.
In addition to the positive health effects of CLA, the antiatherogenic, anti-inflammatory, antithrombotic and immunosuppressive effects of long-chain (n-3) PUFA are also provided (39
). These fatty acids are found mainly in fish and fish oil. However, the intake of these fatty acids in a northern European diet is 20% of what is required to elicit positive effects (40
). Therefore, eggs enriched with (n-3) fatty acids, especially with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are very interesting from a consumer’s health perspective. Considering the recommended daily intake requirements of linoleic, linolenic and EPA + DHA (41
), eggs produced by layers fed diets rich in flaxseed oil and supplemented with CLA are a promising food source to increase the intake of both (n-3) fatty acids and CLA.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: AF, animal fat; CLA, conjugated linoleic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FAME, fatty acid methyl esters; FSO, flaxseed oil; GC, gas chromatography; LF, low fat; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; S, saturated; SB, soybean oil; SFA, saturated fatty acid; U, unsaturated.
Manuscript received 25 May 2001. Initial review completed 25 July 2001. Revision accepted 17 October 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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effect of CLA in treatment LF, SB and AF; 
effect of CLA in treatment FSO); (arrow up means increase, arrow down means decrease). LF, low fat diet: SB, soybean oil diet; AF, animal fat diet; FSO, flaxseed oil diet.