Effect of dietary fats and vitamin E on fatty acid composition, vitamin A and E content and oxidative stability of egg yolk

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1 Arch. Geflügelk. 2002, 66 (6), , ISSN Verlag Eugen Ulmer GmbH & Co., Stuttgart Effect of dietary fats and vitamin E on fatty acid composition, vitamin A and E content and oxidative stability of egg yolk Einfluss von Futterfettquelle und Vitamin E-Gehalt auf das Fettsäuremuster, den Gehalt an Vitamin A und E sowie die Oxidationsstabilität des Eidotters L. Pál 1, K. Dublecz 1, F. Husvéth 1,L.Wágner 1,Á. Bartos 1 and G. Kovács 2 Manuskript eingegangen am 25. März 2002, angenommen am 26. Mai 2002 Introduction Recently, numerous studies have been focused on the enrichment of eggs with n-3 polyunsaturated fatty acids (PUFA), because these modified eggs can play a beneficial role in prevention of cardiovascular diseases (Lewis et al., 2000). The PUFA content of yolk can be increased effectively through the supplementation of hen diets with fish oil, marine algae, flax seed or linseed oil (Herber and Van Elswyk, 1996; Scheideler and Froning, 1996; Gao and Charter, 2000). However, the highly unsaturated nature of n-3 fatty acids predisposes the egg yolk to lipid oxidation (Marshall et al., 1994). The potentially toxic oxidation products are responsible for deterioration of nutritional and organoleptic values of animal products (Frankel, 1984). The oxidative damages of lipids can be prevented or limited by natural antioxidants such as tocopherols and carotenoids. Supplementation of hen s diets with a-tocopherol seems to be the most suitable choice to increase the content of this vitamin and to decrease the amount of the primary (lipid hydroperoxides) and secondary (thiobarbituric acid reactive substances; TBARS) oxidation products in eggs and egg products (Cherian et al., 1996a; Galobart et al., 2001a, b). Table egg is an important source of vitamin A and E for humans. These fat soluble vitamins and fatty acids can interfere in their absorption from the intestinal lumen, hepatic metabolism and transport to the yolk (Sklan, 1983; Surai, 1999). Studies reported by several authors have shown a negative correlation between dietary PUFA content and uptake of fat soluble vitamins, especially vitamin E(Weber, 1981; Hollander, 1981). On the other hand, contradictory results were published concerning the effects of vitamin E supplementation on the incorporation of n-3 fatty acids in the yolk (Cherian et al., 1996a; Qi and Sim, 1998; Galobart et al., 2001a). Free radical scavengers, including vitamin E and vitamin A can serve as an antioxidant or prooxidant depending on their concentrations and interactions (Tesoriere et al., 1994; Chen et al., 1998). Concentration of vitamin E in yolk was reduced by high dietary contents of vitamin A (Surai et al., 1998). However, vitamin A incorporation into the egg yolk seemed to be more effective when dietary retinyl acetate 1 Dept. of Animal Physiology and Animal Nutrition 2 Dept. of Animal Breeding, Georgikon Faculty, University of Veszprem, Keszthely, Hungary was combined with vitamin E (Bardos et al., 1996). Vitamin A and E take part in the second level of antioxidant defence as well. Data of Surai et al. (1998) indicated that vitamin A and carotenoid accumulation in the embryonic chick liver reflect their concentrations in the initial yolk. Thus, the amount of these antioxidants in the yolk influences the antioxidant defence system of the developing chick during embryonic and early postnatal life (Gaal et al., 1995; Surai et al., 1999). Although several experiments have revealed the role of additional dietary vitamin E and its effect on the oxidative stability of yolk lipids, the possible role of the yolk vitamin A in this process is not fully understood. On the other hand, the fatty acid profile of the supplemental fats (n-6 vs. n-3 fatty acids) can also affect the interactions between the two vitamins. Furthermore, there is a lack of information about the dietary use of excess vitamin E combined with cod liver oil, rich in vitamin A. Thus, the aim of the experiment was to investigate the effect of different types of dietary fats and vitamin E supplementation on the vitamin E and vitamin A level, fatty acid profile and oxidative stability of egg yolk. Material and Methods Isabrown hens (n ¼ 144) at 59 weeks of age were randomly housed in laying cages (two birds per cage) in a windowed poultry house with a light regimen of 16 h light to 8 h dark periods. Six groups of 24 hens (twelve replicates per group) were randomly assigned to one of six dietary treatments. Diets were isocaloric and formulated according to the recommendations of the breeding company (Hubbard ISA S.A., 2000). A basal diet (Table 1) containing 4% pumpkin seed oil or cod liver oil was supplemented with 0, 30 or 60 mg of dl-a-tocopheryl acetate/kg of feed. The fatty acid composition and analysed content of vitamin A and vitamin E of the six diets are shown in Table 2. After three weeks of feeding twelve eggs per dietary group (one egg per replicate) were collected and yolks of two eggs were pooled to compose one sample. The samples were stored at 20 C for three months then they were analysed for fatty acid composition, content of vitamin E, vitamin A and TBARS values. Total fat content of diets was extracted and fatty acids were methylated by the method of Association of Official Analitical Chemists (Aoac, 1990a, b). Total lipids of yolk were extracted with chloroform : methanol (2 : 1 vol/vol)

2 252 PÁL et al., Effects of dietary fats and vitamin E on parameters of egg yolk Table 1. Composition and calculated nutrient content of the basal diet Zusammensetzung und kalkulierter Nährstoffgehalt der Basisration Ingredients and composition % Maize Wheat Soybean meal (44%) Added fat Monocalcium phosphate 1.10 Limestone 9.60 Salt 0.30 Premix Calculated nutrient content 3 AME n (MJ/kg) Crude protein Crude fibre 3.00 Calcium 3.70 Phosphorus (available) 0.37 Lysine 0.91 Methionine 0.32 Methionine þ cystine Pumpkin seed oil or cod liver oil. 2 Provided the following per kilogram of diet: vitamin A, 8,000 IU; vitamin D 3, 2,000 IU; vitamin E (dl-a-tocopheryl acetate), 15.0 mg; vitamin K 3, 1.0 mg; thiamine, 2.0 mg; riboflavin, 5.0 mg; pyridoxine, 3.0 mg; vitamin B 12, 15.0 mg; D-calcium-panthotenate, 8.0 mg; niacin, 20.0 mg; folic acid, 0.40 mg; choline chloride, 300 mg; BHT, 80 mg; zinc, 60 mg; iron, 50 mg; manganese, 60 mg; copper, 6 mg; cobalt, 0.15 mg; iodine, 0.8 mg; selenium, 0.15 mg. 3 Based on analyses of ingredients according to the procedure of Folch et al. (1957). Total lipid extracts were converted to fatty acid methyl esters by using sulphuric acid as a catalytic agent. A Carlo Erba HRGC 5300 Mega Series chromatograph (Carlo Erba Strumentazione SA, Italy), equipped with an Omegavax 320 capillary column (30 m length 0.32 mm I.D., 0.25 mm film catalogue Nr ; Supelco, Bellefonte, USA), was used to determine the fatty acid composition. A standard mixture of fatty acid methyl esters (PUFA-2 catalogue Nr U; Supelco, Bellefonte, USA) was used to identify individual fatty acids. Vitamin E (total content of all tocopherols and tocotrienols) and vitamin A were determined by the Hungarian standard (Hungarian Feed Code, 1990) in feed and by the modified method of McMurray et al. (1980) in yolk samples. Portions ( mg) of yolk samples were saponified by the addition of 2.5 ml of 60% (w/v in water) KOH and 10 ml 5% (w/v in ethanol) pyrogallol at 70 C for 30 min. After cooling to room temperature, 20 ml H 2 O and 10 ml petroleum ether (40 70 C fraction) were added and the samples were mixed and allowed to stand for 1 h until two phases were separated. Then the upper phase was dried under N 2 at 40 C and the residue was resolved in 1.0 ml methanol. Ten microlitre portions of the methanol extract were analyzed on a high performance liquid chromatograph (Spectra-Physics) equipped with BST Rutin 10 mm C8, 240 mm length 4 mm I.D. column (Bio Separation Technologies, Budapest, Hungary). Chromatography was performed using a mobile phase of methanol-water (98 : 2, v/v) at a flow rate of 1 ml/min. Fluorimetric detection of vitamin E used excitation and emission wave lengths of 295 and 330 nm, respectively. The relevant wave lengths for vitamin A detection were 325 and 510 nm. Calibration was performed using standard solutions of alltrans retinol and a-tocopherol in methanol. TBARS values (expressed as nmol of malondialdehyde (MDA) per gram of yolk) from egg yolk were determined by the method of Dorman et al. (1995) using 1,1,3,3-tetraethoxypropane as standard. Table 2. Fatty acid composition (weight% of total fatty acids) and content of vitamin A (IU/kg) and vitamin E (mg/kg) of experimental diets Fettsäurezusammensetzung (in Prozent der Gesamtfettsäuren) und Gehalt an Vitamin A (IU/kg) sowie Vitamin E (mg/kg) der Versuchsrationen Fatty acids 1 and vitamins Experimental diets 2 Pumpkin seed oil diet Cod liver oil diet þ0 TA þ30 TA þ60 TA þ0 TA þ30 TA þ60 TA C14 : C16 : C16 : 1n C18 : C18 : 1n C18 : 2n C18 : 3n C20 : 1n C20 : 4n-6 C20 : 5n C22 : 5n-3 C22 : 6n SAT MUFA Total n Total n PUFA n-6 to n-3 ratio Vitamin E Vitamin A 9,240 NM 3 NM 19,060 NM NM 1 SAT ¼ saturated fatty acids; MUFA ¼ monounsaturated fatty acids; PUFA ¼ polyunsaturated fatty acids; n-6 ¼ n-6 polyunsaturated fatty acids; n-3 ¼ n-3 polyunsaturated fatty acids. 2 þ0ta,þ30 TA and þ60 TA ¼ 0, 30 and 60 mg of supplemental dl-a-tocopheryl-acetate/kg of diet 3 NM ¼ not measured

3 PÁL et al., Effects of dietary fats and vitamin E on parameters of egg yolk 253 Statistical analysis was carried out by two way analysis of variance using dietary fat type and added dl-a-tocopheryl acetate as main effects. Significant differences were tested by the least significant difference test (Snedecor and Cochran, 1967). All statistical analyses were conducted using the Statistica 5.0 statistical package (Statsoft, USA). Results are expressed as means SEM. Results Total lipid content and fatty acid composition of egg yolk after three weeks of treatment with cod liver oil or pumpkin seed oil are shown in Table 3. The total lipid content of yolk was higher (P < 0.01) in eggs from hens fed on the pumpkin seed oil diets than on the cod liver oil diets, whereas no influence of a-tocopheryl acetate on total lipid content was observed (P > 0.1). After feeding cod liver oil diets in comparison with pumpkin seed oil diets, a significantly higher (P < 0.05) content of n-3 fatty acids was observed. Eicosapentanoic (EPA; C20 : 5n-3) and docosapentanoic acid (DPA; C22 : 5n-3) were not detectable and only low amounts of docosahexanoic acid (DHA; C22 : 6n-3) were measured in the egg yolk from hens fed on pumpkin seed oil diets. The DHA content in the yolk of hens fed on cod liver oil diets was approximately eightfold higher than that in the pumpkin seed oil groups. In contrast to the n-3 PUFA content, the amount of n-6 fatty acids of the yolk was significantly higher (P < 0.001) in the pumpkin seed oil groups than in the cod liver oil groups. After feeding cod liver oil diets, linoleic acid (LA; C18 : 2n-6) and arachidonic acid (AA; C20 : 4n-6) content of yolk were by about 50% and 75% lower, respectively, than the values of pumpkin seed oil groups. In the present study, the n-6 to n-3 fatty acid ratio in egg yolk of hens fed on cod liver oil diets ranged between 2.9 and 3.5, and were significantly (P < 0.001) lower than in the pumpkin seed oil groups. The saturated fatty acid (SAT) content in the yolk was not influenced by type of fat, whereas a significantly (P < 0.05) higher monounsaturated fatty acid (MUFA) content was measured in the egg yolk of hens fed on cod liver oil diets. The different levels of added a-tocopheryl acetate did not affect significantly the content of SAT, n-3 or n-6 PUFA and the ratio of n-6 to n-3 fatty acids in the yolk (Table 3). The MUFA content of yolk, however, was significantly (P < 0.05) influenced by a-tocopheryl acetate supplementations. Vitamin E concentrations in egg yolk of hens fed on diets containing different types of fats and different levels of added a-tocopheryl acetate are shown in Figure 1. Dietary fat type failed to affect the vitamin E concentrations of egg yolk in the a-tocopheryl acetate unsupplemented groups and in the groups receiving 30 mg/kg added a-tocopheryl acetate. In contrast, comparing the yolk vitamin E contents at the level of 60 mg/kg supplemental a-tocopheryl acetate, significantly higher (P < 0.05) values were obtained in the cod liver oil than in the pumpkin seed oil group. Significantly positive correlation was observed between yolk vitamin E content and supplemental a-tocopheyl acetate in the cod liver oil group (r ¼ 0.99, P < 0.01). However, the correlation between the two parameters was not significant in the pumpkin seed oil group (r ¼ 0.91, P > 0.05). Similarly, the difference in the yolk vitamin E concentrations between the hens fed on a diet supplemented by either 30 or 60 mg/kg a-tocopheryl acetate was significant (P < 0.05) only in the cod liver oil group. Despite the different amounts of vitamin A in the pumpkin seed oil and cod liver oil diets, no differences in the vitamin A concentration of egg yolk were established between the a-tocopheryl acetate unsupplemented groups (Figure 2). However, dietary fat type significantly (P < 0.05) affected the yolk vitamin A content when a-tocopheryl acetate was added to the diet. The vitamin A contents of yolk were higher (P < 0.05) in the cod liver oil groups than in the pumpkin seed oil groups at both sup- Table 3. Effect of supplemental fats and a-tocopheryl acetate on total lipid content (weight%) and fatty acid composition (weight% of total fatty acids) of egg yolk 1 Einfluss der Fettquelle und der Zulage an a-tocopherol auf die Gesamtlipide (Gewichtsprozent) und das Fettsäuremuster (in Prozent der Gesamtfettsäuren) des Eidotters Fatty acids 2 Supplemental fat 3 a-tocopheryl Acetate 4 Fat TA 5 PO CO P 6 þ0 þ30 þ60 P SAT NS NS NS MUFA *** b c a *** *** C18 : 2n *** NS NS C20 : 4n *** NS NS Total n *** NS NS C18 : 3n *** NS NS C20 : 5n-3 ND *** NS NS C22 : 5n-3 ND *** NS NS C22 : 6n *** NS NS Total n *** NS NS PUFA *** NS NS n-6 to n-3 ratio *** NS NS Total lipid ** NS * 1 Values are means SEM (n ¼ 15, 18 for PO and CO groups of supplemental fat, respectively; n ¼ 9, 12, 12 for treatments of 0, 30 and 60 mg a-tocopheryl acetate/kg, respectively). 2 SAT ¼ saturated fatty acids; MUFA ¼ monounsaturated fatty acids; PUFA ¼ polyunsaturated fatty acids; n-6 ¼ n-6 polyunsaturated fatty acids; n-3 ¼ n-3 polyunsaturated fatty acids. 3 PO ¼ pumpkin seed oil; CO ¼ cod liver oil. 4 Supplementation of 0, 30 and 60 mg of a-tocopheryl acetate/kg of feed. 5 Interaction between dietary fats and a-tocopheryl acetate (TA). 6 NS ¼ P > 0.05; * ¼ P < 0.05; ** ¼ P < 0.01; *** ¼ P < ND ¼ not detected. a c Means with different superscripts within a row are significantly (P < 0.05) different. P

4 254 PÁL et al., Effects of dietary fats and vitamin E on parameters of egg yolk Fig. 1. Vitamin E content of egg yolk as influenced by fat and a-tocopheryl acetate (TA) supplementation of the diet. Columns with different letters are significantly (P < 0.05) different Vitamin E-Gehalt im Dotter in Abhängigkeit von der Fettquelle und von der Zulage an a-tocopherol (TA) zur Ration. Spalten mit unterschiedlichen lateinischen Buchstaben unterscheiden sich signifikant (P < 0,05) Fig. 3. TBARS values of egg yolk as influenced by fat and a-tocopheryl acetate (TA) supplementation of the diet. Columns with different letters are significantly (P < 0.05) different TBARS-Werte des Dotters in Abhängigkeit von der Fettquelle und von der Zulage an a-tocopherol zur Ration. Spalten mit unterschiedlichen lateinischen Buchstaben unterscheiden sich signifikant (P < 0,05) Fig. 2. Vitamin A content of egg yolk as influenced by fat and a-tocopheryl acetate (TA) supplementation of the diet. Columns with different letters are significantly (P < 0.05) different Vitamin A-Gehalt im Dotter in Abhängigkeit von der Fettquelle und von der Zulage an a-tocopherol (TA) zur Ration. Spalten mit unterschiedlichen lateinischen Buchstaben unterscheiden sich signifikant (P < 0,05) plementary levels of 30 and 60 mg/kg a-tocopheryl acetate. As compared to the unsupplemented groups, supplementation of 30 mg/kg a-tocopheryl acetate resulted in a significant higher (P < 0.05) vitamin A content in egg yolk from hens fed on the cod liver oil diet, but yolk vitamin A content was not influenced in the pumpkin seed oil group. Yolk vitamin A concentrations were significantly lower (P < 0.05) in both oil groups at level of 60 mg/kg than at 30 mg/kg added a-tocopheryl acetate. The value obtained in the pumpkin seed oil group with 60 mg/kg added a-tocopheryl acetate was significantly (P < 0.05) lower even than that obtained without tocopherol supplementation. The influence of experimental diets on TBARS values of egg yolks are shown in Figure 3. The effect of dietary oils on TBARS values was highly significant (P < 0.001). Feeding diets containing cod liver oil showed higher TBARS values than pumpkin seed oil diets when compared at any level of supplemental a-tocopheryl acetate. The yolk total n-3 PUFA content but not the n-6 PUFA level correlated positively with the concentration of MDA in the yolk (r ¼ 0.78, P < 0.001). Both dietary a-tocopheryl acetate (P < 0.05) and interaction of a-tocopheryl acetate and fat type (P < 0.01) showed significant effect on the yolk MDA content. Although no correlation was found between the yolk vitamin E and MDA values in the pumpkin seed oil groups (r ¼ 0.07, P > 0.05), a strong negative correlation was observed between the two factors in the cod liver oil groups (r ¼ 0.68, P < 0.01). In contrast, the level of vitamin A in the yolk did not show any significant relationship with the TBARS values in either groups. Discussion The differences in lipid content of egg yolk may relate to the influence of n-3 PUFA on hepatic lipid metabolism, resulting in reduced hepatic lipid biosynthesis, secretion and plasma lipid levels (Phetteplace and Watkins, 1989; Van Elswyk et al., 1991). The fatty acid composition of total yolk lipids reflected well the type of the supplemental dietary fats in our experiment. Eggs from hens fed on pumpkin seed oil diets can be considered as commercial in view of their fatty acid profile. Cod liver oil was effectively used for incorporation of n-3 PUFA into eggs. The values obtained in our experiment were in the range measured by other authors using fats rich in n-3 PUFA. Baucells et al. (2000) measured 3.18% DHA, 5.06% total n-3 fatty acids and 2.12 n-6 to n-3 ratio in egg yolk of hens fed on a diet supplemented with 4% fish oil for 14 weeks. The supplementation of laying hen diets with 5% linseed oil (Galobart et al., 2001b) or 5 15% flaxseed (Jiang et al., 1991; Scheideler and Froning, 1996), which are the most frequently used vegetable sources for the production of n-3 PUFA-rich eggs, yielded higher linolenic acid and total n-3 PUFA, but lower levels of EPA and DHA than those observed in the present study using cod liver oil. In agreement with other observations, much lower EPA to DHA ratio was found in the yolk than the ratio present in the feed (Cherian et al., 1996a, Baucells et al., 2000). It suggests an effective elongation and desaturation of EPA to DHA in the liver of hens. Taking into account that humans seem to have a more limited ability to perform this last step of DHA formation from LNA (Sanders, 1993), table eggs can be considered as an important source of DHA. In general, diets with supplemen-

5 PÁL et al., Effects of dietary fats and vitamin E on parameters of egg yolk 255 tal fats rich in LA such as pumpkin seed oil result in higher AA content than diets rich in n-3 PUFA (Cherian et al., 1996a, Baucells et al., 2000).The more pronounced reduction in the yolk AA content comparing to LA after feeding cod liver oil diets, is probably due to the greater utilisation of D6 desaturase in the n-3 than in the n-6 fatty acid pathway (Bezard et al., 1994). This enzyme acts in both pathways so the high concentrations of EPA and DHA seem to restrain the biosynthesis of AA generated from LA (Bezard et al., 1994). Higher levels of dietary vitamin E (200 IU/kg feed) are suggested to associate with lower proportions of total n-3 PUFA or certain n-3 fatty acids in the yolk (Meluzzi et al., 1999; Galobart et al., 2001ab) Although the contents of DHA and total n-3 fatty acids were reduced in eggs due to a-tocopheryl acetate supplementation, these reductions in the present experiment were statistically not significant (P > 0.05). Qi and Sim (1998) failed to find any effect of a-tocopheryl acetate supplementation on yolk n-3 PUFA concentration as well. On the other hand, Cherian et al. (1996a) measured higher content of EPA and DHA in the egg yolk after supplementation of mixed tocopherols in the diet. In our study, vitamin E deposition in yolk was not affected by the type of supplemental fats in the groups receiving 0 or 30 mg/kg added a-tocopheryl acetate in the feed. Similarly, lard and fish oil supplementation (Meluzzi et al., 2000) as well as linseed and sunflower oil (Galobart et al., 2001a) had no effect on a-tocopeherol deposition in eggs. Cherian et al. (1996a) also failed to find significant differences in the total (a-, g- and d-tocopherol) and a-tocopherol content in egg yolk of hens fed on diets supplemented with 3.5% menhaden, flax or sunflower oil without added tocopherols. However, studies reported by others generally agree on the negative impact of PUFA on tocopherol absorption from the intestine (Hollander, 1981; Meluzzi et al., 1999). The breast and thigh vitamin E content of broiler chickens were decreased by dietary fish oil in the study of Miller and Huang (1993). In our study, vitamin E content in egg yolk was significantly lower (P < 0.05) in the pumpkin seed oil than in the cod liver oil group at the level of 60 mg/kg added a-tocopheryl acetate. In addition, the significantly (P < 0.05) lowest amount of yolk vitamin A was observed also in the pumpkin seed oil group with 60 mg/kg supplemental a-tocopheryl acetate. These results might be explained either by the different effects of n-6 and n-3 fatty acids on vitamin E deposition in yolk at the level of 60 mg/kg added a-tocopheryl acetate or by the disproportional amounts of dietary vitamin A and E in this group. An interesting complex relationship exists between the metabolism of vitamins A and E in laying hens. At the beginning of egg production the vitamin A accumulation in the hen liver is sufficient to supply more than one hundred eggs (Surai et al., 1998). Furthermore, the first signs of vitamin A deficiency appear after eleven weeks when a diet without vitamin A is fed (West et al., 1992). Conversely, the vitamin E content in each egg is more than two times greater than the reserve of this vitamin in the liver (Surai et al., 1998). Hence, laying hens rely on continuous dietary vitamin E supplementation, whereas moderate alterations in dietary vitamin A, lasting for 3 4 weeks, probably do not influence yolk vitamin A accumulation because of the extent of liver storage of this vitamin. This may be the explanation for the lack of any difference in the vitamin A concentration of egg yolk of hens fed on pumpkin seed oil or cod liver oil diets differing in vitamin A content for a relatively short period in our experiment. On the other hand, the three weeks experimental period may be in the limit to obtain the stabilization of vitamin E concentration in yolk (Galobart et al., 2001a, b), the moderate supplementation of 30 mg/kg a-tocopheryl acetate in our experiment resulted in a sixfold higher content of yolk vitamin E than feeding diets without added tocopherol. The results of other experiments indicate a dose-dependent relationship between dietary vitamin E and plasma vitamin A. Abawi et al. (1985) found that dietary vitamin E at low to moderate levels enhanced plasma vitamin A concentration. Vitamin A accumulation in the egg yolk of Japanese quail was also more effective when dietary retinol acetate was combined with moderate amount of a-tocopheryl acetate (37.8 IU/kg; Bardos et al., 1996). The further increase in dietary vitamin E up to disproportional ratios of the two vitamins had no effect (Surai, 2000) or depressed vitamin A in the plasma and in the yolk (Abawi et al., 1985). On the basis of the present results both effects may depend on the dietary vitamin A level. Taking into account the high reserve of vitamin A in the liver of hens it is feasible to suggest that this synergism and competition can take place at the level of hepatic vitamin incorporation into carrier molecules. The results of this trial have revealed that despite their lower total PUFA content, eggs rich in n-3 fatty acids appeared to be more susceptible to lipid peroxidation than commercial eggs rich in n-6 fatty acids. A similar observation was previously reported by other authors (Cherian et al. 1996a). In addition, a-tocopheryl acetate supplementation resulted in a significant (P < 0.05) reduction in MDA values only in the cod liver oil diet. The antioxidant effect was shown at both levels of a-tocopheryl acetate and it was proportional to the amount of a-tocopheryl acetate added to the feed. Galobart et al. (2001b) reported that a higher a-tocopherol concentration was accompanied by a higher reduction of TBARS values, and the antioxidant effectiveness of a-tocopheryl acetate increased with increasing storage time of spray-dried eggs. Similar to our results, ineffectiveness of supplementation of mixed tocopherols in reducing yolk TBARS values was observed by Cherian et al. (1996a) who fed diets supplemented with palm oil and sunflower oil (rich in n-6 PUFA), whereas dietary tocopherols significantly decreased TBARS values when menhaden oil was added to the diet. Gebert et al. (1998) attributed a prooxidant effect to a-tocopherol at supplementary levels of 100 and 200 mg/kg in the diet, increasing the TBARS values of eggs stored for 6 months. The antioxidant properties of a-tocopherol was shown up to 50 mg/g egg yolk and the concentration of 75 mg a-tocopherol/g yolk was proved to be prooxidant level in the experiment of Chen et al. (1998). In our experiment, no prooxidant effect was observed at any level of dietary a-tocopheryl acetate. Our experiment is a pioneer work in studying the relationship between the yolk vitamin A content and its oxidative stability. Vitamin A is suggested to be a physiological antioxidant (Olson, 1993). In the light of the present findings, yolk vitamin A may not play any important role in the protection against lipid peroxidation compared to vitamin E. Fresh egg shows high stability to oxidation because of the presence of its natural antioxidants and phospholipidprotein complexes in the yolk LDL (Pike and Peng, 1985). However, Marshall et al. (1994) showed that fresh eggs of layers fed on a diet supplemented with 1.5% menhaden oil contained significantly higher yolk TBARS than those of layers fed on a typical maize-soybean based diet. Cherian et al. (1996b) observed that TBARS values in n-3 PUFA enriched eggs did not increase during the storage at 4 C for 5 to 6 weeks. Nevertheless, oxidative

6 256 PÁL et al., Effects of dietary fats and vitamin E on parameters of egg yolk stability of eggs decreased by processing at high temperature such as spray-drying (Galobart et al., 2001ab), cooking and scrambling (Cortinas et al., 2001). TBARS values have been reported to increase until the second month of storage in spray-dried eggs and then declined thereafter (Galobart et al., 2001b). MDA is supposed to react with a wide range of compounds and polimerises to form dimers or trimers (Aubourg, 1993). As a result, the amount of MDA available to react with TBA may decrease. In conclusion, the cod liver oil used in our experiment seems to be appropriate to increase the n-3 PUFA content of table egg. The n-3 PUFA content is considered mainly responsible for the susceptibility of egg yolk to lipid peroxidation during storage. Dietary supplementation of 30 or 60 mg/kg a-tocopheryl acetate results in higher yolk vitamin E content, but its effectiveness may be influenced by the type of dietary fat. Vitamin E enhances the oxidative stability of eggs rich in n-3 PUFA, however, it is ineffective at the levels used in this trial in reducing TBARS values of commercial eggs. In contrast to vitamin E, vitamin A is not related to the antioxidative status of table egg. Summary The effects of cod liver oil (rich in n-3 fatty acids and vitamin A) and pumpkin seed oil (rich in n-6 fatty acids) with a-tocopheryl acetate supplementation on fatty acid composition, vitamin A and vitamin E concentration and lipid peroxidation of egg yolk were studied. Laying hens (n ¼ 144) were fed on six experimental diets supplemented with 4% fat and dl-a-tocopheryl acetate (0, 30 and 60 mg/kg) for three weeks. Feeding cod liver oil diets resulted in higher (P < 0.05) concentration of n-3 polyunsaturated fatty acids (PUFA) in yolk compared to feeding pumpkin seed oil diets. Vitamin E content of diets did not affect the fatty acid composition of egg yolk. No relationships were observed either between dietary PUFA and deposition of vitamin A and E in the egg. Supplementation of diets with 30 mg/kg a-tocopheryl acetate resulted in higher yolk vitamin E contents regardless of the type of added fat compared to the unsupplemented groups. However, the level of 60 mg/kg added a-tocopheryl acetate was more effective in the cod liver oil than in the pumpkin seed oil group. Concentration of vitamin A in yolk was not affected by different vitamin A contents in the cod liver oil and pumpkin seed oil diets without added tocopherol, whereas the effect of dietary a-tocopheryl acetate on the vitamin A content in yolk was dose-dependent. The rate of lipid peroxidation, measuring thiobarbituric acid reactive substance (TBARS) values in yolk was significantly (P < 0.001) related to the concentration of n-3 fatty acids in yolk lipids, however, no relationship was found between TBARS values and total PUFA content. Oxidative stability of yolk was proportionably enhanced by dietary a-tocopheryl acetate treatments in the cod liver oil group, whereas TBARS values in the pumpkin seed oil group remained constant after a-tocopheryl acetate supplementation. In contrast to vitamin E concentrations, vitamin A values in yolk were not related to TBARS values in either group. Keywords Layer, pumpkin seed oil, cod liver oil, vitamin A, vitamin E, oxidative stability, egg yolk Zusammenfassung Einfluss von Futterfettquelle und Vitamin E-Gehalt auf das Fettsäuremuster, den Gehalt an Vitamin A und E sowie die Oxidationsstabilität des Eidotters Der Einfluss von Droschöl (reich an n-3 Fettsäuren und Vitamin A) und Kürbiskernöl (reich an n-6 Fettsäuren), in Kombination mit einem Zusatz von a-tocopherol-acetat, wurde auf das Fettsäuremuster, den Gehalt an Vitamin A und E sowie auf die Lipidperoxidation im Eidotter untersucht. Die Legehennen (n ¼ 144) wurden mit 6 Futterrationen über drei Wochen gefüttert, die 4% Fettzusatz und dl-a-tocopherol-acetat in verschiedenen Dosierungen (0, 30, 60 mg/kg) enthielten. Die Fütterung von Dorschöl führte zu höheren Konzentrationen an langkettigen, mehrfach ungesättigten Fettsäuren (PUFA) des Typs n-3 im Dotter im Vergleich zu den Futterrationen mit Kürbiskernöl (P < 0,05). Der Vitamin E-Gehalt der Rationen beeinflusste das Fettsäuremuster nicht. Ferner wurden keine Beziehungen zwischen dem PUFA- Gehalt und der Einlagerung von Vitamin A und E in den Dotter beobachtet. Die Zulage von 30 mg/kg a-tocopherol-acetat führte unabhängig vom verwendeten Futterfett zu höheren Vitamin E- Gehalten im Dotter im Vergleich zu den unsupplementierten Gruppen. Die Zulage von 60 mg/kg a-tocopherol-acetat war aber bei Dorschöl effektiver als bei Kürbiskernöl. Die Gehalte an Vitamin A im Dotter hingen ohne Zulage von Tocopherol nicht von den unterschiedlichen Vitamin A-Gehalten in den Dorschölund Kürbiskernöl-Rationen ab, während die Zulage von a-tocopherol-acetat den Vitamin A-Gehalt dosisabhängig erhöhte. Die Höhe der Lipidoxidation im Dotter, gemessen anhand der Reaktionsprodukte (TBARS), hing signifikant (P < 0,001) von der Konzentration an n-3 Fettsäuren in den Dotterlipiden ab. Es wurden jedoch keine Beziehungen zwischen den TBARS-Werten und dem Gesamtgehalt an PUFAs gefunden. Die Oxidationsstabilität des Dotters war proportional zur Zulage von a-tocopherol-acetat in der Dorschölgruppe verbessert, während in der Kürbiskernölgruppe die Zulage von a-tocopherol-acetat keine Auswirkungen hatte. Im Gegensatz zu den Vitamin E-Gehalten lagen für die Vitamin A-Gehalte keine Beziehung zu den TBARS in den Behandlungen vor. Stichworte Legehenne, Kürbiskernöl, Dorschöl, Vitamin A, Vitamin E, Oxidationsstabilität, Dotter Acknowledgements This research was supported by the Hungarian Scientific Research Fund (OTKA F029350). The authors wish to thank Mária Csucska, Erika Kulcsár, Judit Varga and Csaba Ertsey for their excellent technical assistance during this study. References Abawi, F. G., T. W. Sullivan and S. E. Scheideler, 1985: Interaction of dietary fat with levels of vitamins A and E in broiler chicks. 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