Effect of Dietary Supplementation with Rosemary Extract and α-Tocopheryl Acetate on Lipid Oxidation in Eggs Enriched with ω3-Fatty Acids

Effect of Dietary Supplementation with Rosemary Extract and α-Tocopheryl Acetate on Lipid Oxidation in Eggs Enriched with ω3-Fatty Acids J. Galobart,* A. C. Barroeta,*,1 M. D. Baucells,* R. Codony,† and W. Ternes‡ *Unitat de Nutricio´ i Alimentacio´ Animals, Facultat de Veterina`ria, Universitat Auto`noma de Barcelona, E-08193 Bellaterra, Spain; †Departament de Nutricio´ i Bromatologia-CeRTA, Facultat de Farma`cia, Universitat de Barcelona, Avinguda Joan XXIII, E-08028 Barcelona, Spain; and ‡Zentrumsabteilung fu¨r Chemische Analytik und Endokrinologie, Tiera¨rztliche Hochschule, Bischofsholer Damm, 15, D-30173 Hannover, Germany malonaldehyde content. Stability to iron-induced lipid oxidation was also measured. Results showed the clear antioxidant effect of dietary α-TA supplementation on ω3-FA enriched eggs. In contrast, dietary supplementation with rosemary extract showed no effect on any of the lipid oxidation parameters evaluated.

(Key words: ω3-fatty acid-enriched eggs, dietary α-tocopherol, rosemary extract, carnosic acid, lipid oxidation) 2001 Poultry Science 80:460–467

Bote et al., 1997), and eggs (Cherian et al., 1996; Galobart et al., 1999). Researchers are seeking other natural antioxidant alternatives to α-tocopherol. Extracts from rosemary and sage have been shown to have strong antioxidant activity in foods and food model systems (Cuvelier et al., 1994). Rosemary extracts (RE) have a wide range of different phenolic compounds with biological activities, e.g., carnosic acid (CA), carnosol, rosmanol, epirosmanol. Carnosic acid is the most active antioxidant present in RE (Cuvelier et al., 1996; Richheimer et al., 1996; Offord et al., 1997) with an antioxidant activity approximately three times higher than carnosol and seven times higher than butylated hydroxytoluene and butylated hydroxyanisol (Richheimer et al., 1996). A synergistic antioxidant effect of RE with α-tocopherol has been reported in vitro (Wada and Fang, 1992). Because there are very few studies dealing with the antioxidant activity of RE in animal tissues when added to animal diets, the objective of this study was to evaluate the antioxidant effect of a commercial dietary RE and to compare it with that of α-tocopherol and its possible synergistic effect in ω3-FA enriched eggs.

INTRODUCTION Due to the health benefits associated with the consumption of ω3-fatty acids (FA), much research has been done in the last few years to enrich different foods of animal origin, such as broiler meat or eggs, with those FA (Van Elswyk et al., 1992; Ajuyah et al., 1993; Cherian et al., 1996; Lo´pez-Ferrer et al., 1999; Baucells et al., 2000). However, higher polyunsaturated FA (PUFA) content leads to an increase in the unsaturation of such products and, thus, to greater susceptibility to lipid oxidation. Lipid oxidation in foods is of major importance because it adversely affects the overall quality of foods, including flavor, taste, and nutritional value. Moreover, lipid oxidation products are related to the development of cardiovascular diseases and other diseases. In order to prevent such undesirable oxidative effects, antioxidants have been widely used by the food industry, by direct addition to the final product (usually synthetic antioxidants). Another more natural way is to increase the intrinsic antioxidant concentration through dietary supplementation with natural antioxidants, such as tocopherols. Dietary supplementation with α-tocopherol has been demonstrated to beneficially affect enhancement of lipid stability in foods from animal origin, such as poultry meat (Ajuyah et al., 1993), turkey meat (Ahn et al., 1998), pork (Kingston et al., 1998), rabbit meat (Lo´pez-

MATERIALS AND METHODS Animals and Diets A 3 × 2 factorial arrangement was replicated four times in order to study the influence of six dietary treatments Abbreviation Key: CA = carnosic acid; CHP = cumene hydroperoxide; FA = fatty acids; LHP = lipid hydroperoxides; MDA = malondialdehyde; PUFA = polyunsaturated fatty acids; RE = rosemary extract; TCA = trichloroacetic acid; α-TA = α-tocopheryl acetate.

Received for publication August 2, 2000. Accepted November 21, 2000. 1 To whom correspondence should be addressed: Ana.Barroeta@ uab.es.

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ABSTRACT The antioxidant effect of dietary supplementation with 500 or 1,000 mg/kg of a commercial rosemary extract vs. 200 mg/kg of α-tocopheryl acetate (αTA) on the lipid oxidative stability of ω3-fatty acid (FA)enriched eggs was compared. Lipid oxidation was measured in fresh eggs by the lipid hydroperoxide level and

LIPID OXIDATION IN ω3-PUFA ENRICHED EGGS TABLE 1. Composition of the basal diet Ingredients Corn Soybean meal (44% CP) Barley Wheat Linseed oil Calcium carbonate Dicalcium phosphate DL-methionine Salt Vitamin and mineral premix1

38.80 24.04 10.53 10.00 5.00 8.57 2.12 0.14 0.40 0.40 100.00 2,893.00 91.52 15.76 3.10 7.12 14.20 3.82 0.45 0.83 0.38 0.69

1 Supplied per kilogram of total diet: vitamin A, 8,000 IU; cholecalciferol, 1,600 IU; vitamin K3, 2 mg; vitamin B1, 1.5 mg; vitamin B2, 4 mg; vitamin B6, 3 mg; vitamin B12, 11.8 µg; folic acid, 0.35 mg; biotin, 150 µg; pantothenic acid, 10 mg; nicotinic acid, 20 mg; Mn, 30 mg; Zn, 50 mg; I, 0.3 mg; Fe, 50 mg; Cu, 6 mg; Se, 0.1 mg. 2 Calculated values.

on FA composition and lipid oxidation in fresh eggs. Lohmann laying hens (n = 144) were randomly distributed into the six dietary treatments (four replicates of six birds each per treatment). Diets were formulated to meet NRC (1994) requirements (Table 1). Experimental treatments resulted from the supplementation of a basal diet containing 5% linseed oil2 with 0 or 200 mg of α-tocopheryl acetate (α-TA)3/kg of feed; 0, 500 or 1,000 mg of a commercial powder RE4/kg of feed; or their combination. Feed and water were provided ad libitum. Egg production was recorded daily, and egg quality, including Haugh units, shell thickness, and yolk color, was measured three times during the experimental period. Feed consumption was measured for the whole period of the experiment. Feed samples were taken three times during the experiment for FA, α-tocopherol, and CA analyses. A complementary experiment was designed in order to evaluate the effect of dietary supplementation with a RE with a higher CA concentration on lipid oxidation of fresh eggs. For this experiment, 12 laying hens were randomly distributed into two dietary treatments (two

2 Linseed oil was provided by Caila´ -Pare´ s S.A., E-08040 Barcelona, Spain. 3 Rovimix威 E-50 Adsorbate. F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland. 4 RMS威 1100, Hausmann Aromatic SA, E-08740 Sant Andreu de la Barca, Spain. 5 Shimadzu Corporation, 604-8511 Kyoto, Japan. 6 SGE, 3134 Ringwood, Victoria, Australia. 7 Shimadzu Europe GmbH, D-47269 Duisburg, Germany. 8 Sigma, St. Louis, MO 63103.

replicates of three birds each per treatment) resulting from the supplementation of a basal diet containing 5% linseed oil with the amount necessary of a new RE to obtain 0 or 500 mg of CA/kg of diet.

Sample Collection After 25 d of feeding the experimental diets, four eggs from every experimental replicate were collected and pooled. The pooled eggs were stored at −80 C until their analyses for FA composition, α-tocopherol and CA contents and lipid oxidation (measured by TBA values), and susceptibility to iron-induced oxidation (induced TBA). Measurement of the lipid hydroperoxide (LHP) values was performed immediately after homogenization of the eggs. For the complementary experiment, after 25 d of feeding the experimental diets, all eggs produced from every experimental replicate were collected, cracked, and pooled. This pool of eggs was frozen at −80 C until their analyses for iron-induced oxidation (induced TBA).

FA Analysis Fatty acid composition was determined for feed (four samples per treatment) and fresh eggs (four samples per treatment). The lipid fractions of feed and egg samples were extracted according to Folch et al. (1957), with some modifications, and methylated with 20% boron trifluoride-methanol complex in methanolic solution (Morrison and Smith, 1964). The FA profile was determined by gas chromatography in a GC-14A Shimadzu chromatograph5 equipped with a flame ionization detector and a capillary column (30 m × 0.53 mm i.d.) with a film thickness of 0.5µm stationary phase of 30% methyl- + 70% cyanopropylpolysiloxane (BPX70).6 Helium was used as the carrier gas. Oven temperature was programmed as follows: from 75 to 148 C at 4 C/min; from 148 to 158 C at 2.5 C/min; and from 158 to 225 C at 5 C/min. Injector and detector temperatures were 280 C; head pressure was 8.7 psi, and sample volume injected was 0.4 µL for feed samples and 0.5 µL for egg samples. Peak areas were integrated and converted to FA percentages (direct area normalization) by means of the CLASS-UniPac program.7 Fatty acid identification was carried out by comparison of the retention times with their corresponding standard and by addition of standards8 when necessary.

α-Tocopherol Analysis α-Tocopherol from feeds (three samples per treatment) was analyzed by F. Hoffmann-La Roche Ltd. using the method of Manz and Philipp (1981). α-Tocopherol from fresh eggs (four samples per treatment) was extracted using the method described by Abdollahi et al. (1993), and HPLC determination was performed according to the conditions described by Drotleff and Ternes (1999).

CA Analysis Carnosic acid content in egg samples was analyzed at the Zentrumsabteilung fu¨ r Chemische Analytik und

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Analyzed composition ME, Kcal/kg2 DM CP Crude fiber Crude fat Ash Calcium2 Available phosphorus2 Lysine2 Methionine2 Methionine + cystine2

%

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Lipid Hydroperoxide Determination Lipid hydroperoxide values were determined as described by Hermes-Lima et al. (1995) with some modifications. Three grams of fresh egg was weighed into a 50mL centrifuge tube and homogenized with 15 mL of cold (−20 C) HPLC-grade methanol using an Omni 2000 homogenizer.10 Homogenates were centrifuged (3 min at 1,400 × g), and the supernatants were removed for assay. Five hundred microliters of 0.25 mM FeSO4, 200 µL of 25 mM H2SO4, 200 µL of 0.1 mM xylenol orange,8 1,025 µL of water (volume of water needed to make up the final volume to 2 mL), and 75 µL of the methanolic extract were sequentially added into 10-mL screw-capped tube. After incubation at room temperature under attenuated light for 5 h, absorbance at 560 nm was read. The LHP value of samples was expressed as micrograms per gram of cumene hydroperoxide (CHP),11 and it was determined with reference to a linear regression curve performed with CHP as standard.

TBA Determination Malonaldehyde (MDA), as secondary oxidation product, was measured according the TBA method described by Botsoglou et al. (1994) using third derivative spectrophotometry with some modifications. A 1.5-g sample of fresh egg was weighed into a 25-mL screw-capped centrifuge tube, and 5 mL of 0.8% butylated hydroxytoluene in hexane was immediately added. Just before homogenization, 8 mL of 5% aqueous trichloroacetic acid (TCA)

9

Machery-Nagel, D-52355 Du¨ ren, Germany. Omni International, Warrenton, VA 20187. Aldrich, Gillinham, BH124QH Dorset, UK.

10 11

was added to the tube. The mixture was vortexed for 40 s; the top hexane layer was discarded, and the bottom aqueous layer was filtered through Whatman no. 1 filter paper. The volume was increased to 10 mL with TCA. After filtration, a 3-mL aliquot was transferred to another tube and mixed with 2 mL of 0.8% TBA. The mixture was incubated for exactly 30 min at 70 C in a water bath under gentle agitation and, after this time, was cooled in an ice bath for 7 min. After the tube was tempered for 45 min at room temperature, the reaction mixture was used for third-order derivative spectrophotometry. The height of the third-order derivative peak that appeared at approximately 521.5 nm was used for calculation of the MDA concentration in the examined extract on the basis of slope and intercept data of the computed least-squares fit of a freshly prepared standard curve. Tetraethoxypropane8 was used as MDA precursor in the standard curve.

Susceptibility to Iron Induced Lipid Oxidation Susceptibility to iron induced lipid oxidation (induced TBA) was measured using the methodology described by Kornsbrust and Mavis (1980) with some modifications. Nine g of egg was weighed into a 50-mL centrifuge tube and mixed with 21 mL of 1.15% KCl; 2.5 mL of this homogenate was mixed with 12.5 mL of 80 mM Tris-malate buffer (pH = 7.4), 5 mL of 5 mM FeSO4ⴢ7H2O, and 5 mL of 2 mM ascorbic acid and was incubated at 37 C. At the specified times, a 2-mL aliquot of the incubate was mixed with 4 mL TBA-TCA-HCl reagent, vortexed, and heated at 100 C for 15 min. After this time, the reaction mixture was cooled under tap water, vortexed, centrifuged at 3,300 rpm for 15 min, and absorbance at 531 nm was read against the blank. In all lipid oxidation determinations, the spectrophotometer used was a Shimadzu 1203 UV.5

Statistics ANOVA (n = 24) was performed to determine whether the level of supplementation with α-TA and RE affected the FA composition and lipid oxidation in fresh eggs. In all cases, P-values ≤ 0.05 were considered significant.

RESULTS AND DISCUSSION Diet Composition The FA composition and α-tocopherol and CA contents of the experimental diets are described in Tables 2 and 3. Diets showed a mean value for fat content of 7.12 % ± 0.51 (n = 48). These diets contained 35.93% of total ω3-FA, and linolenic acid was the predominant ω3-FA (35.83%). When the feeding experiment was planned and performed, no information about the concentration of CA in the extract was available. When the methodology for its determination was ready, CA contents of the RE and the feed were analyzed at the Zentrumsabteilung fu¨ r Chemische Analytik und Endokrinologie of the Tiera¨ rztliche

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Endokrinologie of the Tiera¨ rztliche Hochschule, Hannover, Germany, using the method described by Krause and Ternes (2000). The CA content was also determined in RE and feed samples. Fifty milligrams of the RE was mixed with 25 mL of HPLC-grade methanol in a volumetric flask. Two dilutions of this homogenate were made and injected directly to HPLC. The CA from feed samples was determined as follows: 2.5 g of feed were weighed into 50-mL flask and homogenized for 15 s with 6 to 8 mL of hot (50 C) HPLC-grade methanol. The extract was filtered through a Whatman no. 1 filter paper into a 25-mL volumetric flask. Extraction with methanol was repeated three times. Methanol was used to make up the volume to 25 mL. An aliquot of 8 mL of the filtrate was diluted with 24 mL distilled water, and the resulting mixture was purified with a Chromabond CP18 cartridge.9 The cartridge was conditioned with 6 mL of methanol and 6 mL of water. After loading the sample, the cartridge was dried with air, and CA was eluted with 3 mL methanol and collected in a 3-mL volumetric flask. This sample solution was analyzed by HPLC with the same conditions as for the egg samples.

LIPID OXIDATION IN ω3-PUFA ENRICHED EGGS TABLE 2. Analyzed fatty acid composition of feed, expressed as direct area normalization (%) Feed, %

C14:0 C15:0 C16:0 C16:1 ω7 C17:0 C18:0 C18:1 ω9 C18:1 ω7 C18:2 ω6 C18:3 ω6 C18:3 ω3 C18:4 ω3 C20:0 C20:1 ω9 C20:2 ω6 C20:3 ω6 C20:4 ω6 C20:4 ω3 C22:0 C20:5 ω3 C22:1 ω9 C22:4 ω6 C24:1 ω9 C22:5 ω3 C22:6 ω3 SFA MUFA PUFA ω3 ω6

0.09 0.03 9.53 0.13 0.09 3.15 18.65 1.02 30.06 0.11 35.83 0.00 0.44 0.16 0.05 0.00 0.01 0.02 0.13 0.03 0.07 0.24 0.00 0.00 0.04 13.47 20.04 66.40 35.93 30.47

1 SFA = total saturated fatty acids; MUFA = total monounsaturated fatty acids; PUFA = total polyunsaturated fatty acids; ω3 = total ω3 polyunsaturated fatty acids; ω6 = total ω6 polyunsaturated fatty acids.

Hochschule, Hannover, Germany. The CA concentration of the extract appeared to be 1.68%. The CA contents of the experimental diets are shown in Table 3. Levels found in the diets (a mean of 3.8 and 7.7 mg CA/Kg feed, for 500 and 1,000 mg/kg of RE supplementation) were lower than those expected (8.4 and 16.8 mg CA/kg feed). The differences found between the values obtained and those expected might have been due to the fact that CA is readily oxidized and transformed into other compounds that cannot be detected by the analytical method used. Although feed samples were stored at −20 C, CA could be oxidized during the production and storage of the feed, giving final CA concentrations lower than those expected.

TABLE 3. α-Tocopherol and carnosic acid content in the experimental diets Treatment1

α-Tocopherol (mg/kg diet)

Carnosic acid (mg/kg diet)

Control 500R 1,000R 200E 200E + 500R 200E + 1,000R

<5 <5 <5 225 217 217

ND2 3.83 7.69 ND 3.61 6.37

1 500R = 500 mg rosemary extract/kg; 1,000R = 1,000 mg rosemary extract/kg; 200E = 200 mg α-tocopheryl acetate/mg. 2 ND = not determined.

Egg Composition Table 4 shows the results obtained for the FA composition of fresh eggs. A total ω3-FA content of 11.97% was obtained, with linolenic acid being predominant (9.81%). Levels of eicosapentanoic acid, docosapentanoic acid, and docosahexanoic acid were 0.17, 0.27, and 1.62%, respectively. These results are in agreement with previous reports using linseed oil or flaxseed to obtain eggs enriched with ω3-FA (Cherian et al., 1996; Qi and Sim, 1998; Baucells et al., 2000; Galobart et al., 2001). α-Tocopheryl acetate and RE supplementation had no significant effect on FA composition of fresh eggs (Table 4). For α-tocopherol content of fresh eggs, those obtained from 200 mg/kg α-TA-supplemented diets had α-tocopherol levels 16-fold greater than those from nonsupplemented groups (115.01 vs. 6.86 µg/g respectively). Some authors have demonstrated that the amount of α-tocopherol deposited in eggs depends upon dietary level (Jiang et al., 1994). α-Tocopherol levels found in eggs were comparable to those obtained by other authors using similar levels of α-tocopherol in the diets (Jiang et al., 1994; Meluzzi et al., 1999; Galobart et al., 2001). No CA was detected in fresh eggs at any of the dietary supplementation levels. These results will be discussed later.

Lipid Oxidation Lipid hydroperoxide and TBA values in fresh eggs are shown in Table 5. In fresh eggs, LHP values were significantly reduced by dietary supplementation with 200 mg/ kg of α-TA but not by RE at any of the dietary levels. For TBA values of fresh eggs, no effect was found for αTA or RE supplementation, with values oscillating between 16 and 19 ng MDA/g egg. These values were very low and comparable to those obtained in previous works (Galobart et al., 2001). In the literature there are some reports dealing with lipid oxidation in PUFA-enriched eggs, but most of them measure MDA content, i.e., secondary oxidation products. To our knowledge, no data has been published regarding levels of lipid hydroperoxides, the first step in lipid oxidation, in PUFA-enriched eggs. Although the TBA values obtained were very low and confirm the oxidative stability of fresh egg, as has been previously described by some authors (Pike and Peng, 1985; Marshall et al., 1994; Cherian et al., 1996; Galobart et al., 1999), differences that we found in LHP values showed the antioxidant effect of α-TA supplementation. Because of this stability of the fresh egg to oxidation, eggs were subjected to prooxidant conditions by the ironinduced lipid oxidation method, in order to find differences in the susceptibility to lipid oxidation due to antioxidant supplementation. The evolution of oxidation, dependent on the main factors studied, is shown in Figures 1 and 2. During the whole incubation time, eggs from α-TAsupplemented treatments had lower TBA values than those from the nonsupplemented ones (Figure 1), which

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Fatty acid1

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GALOBART ET AL. TABLE 4. Effect of dietary supplementation with α-tocopheryl acetate and rosemary extract on the fatty acid composition of fresh eggs, expressed as direct area normalization (%)1 α-Tocopheryl acetate (mg/kg) 2

Rosemary extract (mg/kg)

0

200

P

0

500

1,000

P

SEM

C14:0 C15:0 C16:0 C16:1 ω7t C16:1 ω7 C17:0 C18:0 C18:1 ω9 C18:1 ω7 C18:2 tt C18:2 ω6 C18:3 ω6 C18:3 ω3 C20:0 C20:1 ω9 C20:2 ω6 C20:3 ω6 C20:4 ω6 C20:4 ω3 C20:5 ω3 C22:5 ω3 C22:6 ω3 SFA MUFA PUFA Trans ω3 ω6

0.22 0.04 18.49 0.71 2.46 0.13 7.52 37.63 1.73 0.15 16.98 0.12 10.32 0.11 0.10 0.09 0.12 0.82 0.12 0.18 0.29 1.71 26.47 41.91 30.73 0.81 12.62 18.12

0.21 0.03 20.97 0.70 2.24 0.18 8.12 37.92 1.51 0.16 15.33 0.11 9.30 0.13 0.09 0.12 0.10 0.73 0.11 0.15 0.24 1.53 29.63 41.77 27.71 0.85 11.32 16.39

NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS

0.21 0.04 18.75 0.74 2.41 0.15 7.63 37.90 1.70 0.17 16.81 0.14 9.96 0.12 0.09 0.10 0.12 0.80 0.11 0.16 0.26 1.61 26.89 42.10 30.04 0.89 12.09 17.95

0.22 0.03 20.73 0.65 2.39 0.15 7.79 37.26 1.55 0.16 15.76 0.11 9.80 0.13 0.10 0.05 0.11 0.75 0.12 0.17 0.27 1.61 29.20 41.28 28.71 0.77 11.95 16.77

0.21 0.03 19.70 0.72 2.26 0.18 7.86 38.18 1.60 0.14 15.88 0.11 9.66 0.12 0.10 0.16 0.11 0.78 0.12 0.17 0.28 1.64 28.06 42.13 28.91 0.83 11.86 17.04

NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS NS

0.010 0.011 1.384 0.074 0.211 0.024 0.563 0.846 0.077 0.020 1.175 0.015 0.771 0.017 0.004 0.051 0.010 0.055 0.008 0.018 0.023 0.112 1.931 0.666 2.158 0.078 0.907 1.270

1 Values given in this table correspond to least-squares means obtained from ANOVA (n = 24) and their pooled SEM. 2 SFA = total saturated fatty acids; MUFA = total monounsaturated fatty acids; PUFA = total polyunsaturated fatty acids; Trans = total trans fatty acids; ω3 = total ω3 polyunsaturated fatty acids; ω6 = total ω6 polyunsaturated fatty acids.

increased very quickly (at 150 min, 121.6 vs. 17.7 ng MDA/g egg for the 0-mg and 200-mg α-TA supplementations, respectively). On the other hand, supplementation with RE (Figure 2) had no significant effect on reducing the TBA values during the whole incubation time (at 150 min, 69.2, 71.2,

and 68.6 ng MDA/g egg for control, 500 mg RE/kg feed, and 1,000 mg RE/kg feed, respectively). No synergistic effect between α-TA and RE was observed for any of the lipid oxidation parameters deter-

TABLE 5. Effect of α-tocopheryl acetate and rosemary extract supplementation on lipid hydroperoxide values, expressed as micrograms of cumene hydroperoxide (CHP) per gram of egg, and TBA values, expressed as nanograms of malonaldehyde (MDA) per gram of egg, of fresh egg Lipid hydroperoxides TBA values Dietary level (mg/kg feed) (µg CHP/g egg) (ng MDA/g egg) α-Tocopheryl acetate 0 200 P Rosemary extract 0 500 1,000 P SEM

411.7 7.3 ***

19.02 16.58 NS

234.9 214.9 177.9 NS 15.89

18.16 18.01 17.26 NS 1.923

Values given in this table correspond to least-squares means obtained from ANOVA (n = 24) and their pooled SEM. ***P ≤ 0.001

FIGURE 1. Effect of α-tocopheryl acetate (α-TA) supplementation on the evolution of induced TBA values, expressed as nanograms of malonaldehyde (MDA) per gram of egg. Control = samples from nonsupplemented treatments; 200 α-TA = samples from treatments supplemented with 200 mg α-tocopheryl acetate/kg feed.

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Fatty acid

LIPID OXIDATION IN ω3-PUFA ENRICHED EGGS

FIGURE 2. Effect of rosemary extract supplementation on the evolution of induced TBA values, expressed as nanograms of malonaldehyde (MDA) per gram of egg. Control = samples from nonsupplemented treatments; 500R = samples from treatments supplemented with 500 mg rosemary extract/kg feed; 1,000R = samples from treatments supplemented with 1,000 mg rosemary extract/kg feed.

are very few reports of in vivo antioxidant effects of RE, given that a standard RE is not available. Lee and Choi (1989) reported the in vitro antioxidant effect of 1,000 mg/kg of a RE on sardine oil. But when this sardine oil with RE was fed to rats, no antioxidant effect was found on plasma or on liver tissue (Choi, 1990). On the other hand, Lo´ pez-Bote et al. (1998) observed a reduction of MDA formation in broiler meat when 500 mg/kg of a rosemary oleoresin was fed to animals. However the same authors did not find any antioxidant effect of the rosemary oleoresin when it was fed to pigs and attributed this difference to the different digestive capacities of poultry and mammals. Kuzmenko et al. (1999), fed rats for 14 d with a nutritional supplement containing 6% CA and found a reduction in the liver mitochondrial lipid peroxides as measured by chemiluminescence. There is very little information about the absorption, metabolism, and deposition of CA and other phenolic diterpenes in avians or mammals. Botsoglou et al. (1997) fed a thyme extract to hens, found some protective effect on yolk lipid oxidation, and attributed this effect to the transfer of some of the phenolic antioxidants of thyme from feed to egg. In recent work parallel to our study, Krause and Ternes (2000) studied the transfer of CA from feed to egg. These authors, supplementing hen diets with 500 or 1,000 mg of CA/kg diet, established that the transfer rate of CA from feed to eggs was of 0.0025%. Consequently, by using this transfer rate to calculate the CA transferred to the egg in our experiment, the highest amount of CA expected to be deposed should be of 0.19 ng/g egg yolk (corresponding to the supplementation with 1,000 mg/kg of RE), which is far below the detection limit of the analytical procedure (19.5 ng/g egg yolk). Thus, the main reason for the lack of detected effect of the RE used in our study was probably the low CA concentration present in the extract. When the experiment was planned and performed, we did not know the exact amount of CA present in the extract. Moreover, most of the works in the literature did not describe the amount of CA used in the in vivo experiments but only the amount of RE used. Thus, we assayed doses of RE according to the data published. In a complementary experiment using another RE extract with higher CA content, we only found traces of CA in egg when more than 500 mg/kg of CA was added to the diet. An improvement of lipid oxidation in such eggs was observed (Figure 3). Eggs from the CA-supplemented treatment showed a delay in the iron-induced lipid oxidation. Thus, these results indicate that CA may effectively act as an antioxidant in eggs when high enough doses are used in the hen diets. Another possible explanation for the lack of effect of the RE could be the transformation of CA in vivo to other derivatives with lower antioxidant activity. Kuzmenko et al. (1999) found that the activity of the same dose of CA was different when it was studied in vitro or in vivo. Those authors attributed this difference to the fact that CA could be transformed in the organism into other less

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mined. For a better understanding, as no interactions were found, discussion of lipid oxidation results will be divided according to the main factors studied. Effect of α-TA Supplementation on Lipid Oxidation in Fresh Eggs. For α-tocopherol supplementation, our results confirm the antioxidant effect of tocopherols on preventing lipid oxidation in ω3 FA-enriched eggs, as previously reported by several authors. Galobart et al. (2001) found that dietary supplementation with 200 mg/ kg α-TA significantly reduced LHP content in fresh PUFA-enriched eggs. Moreover, α-TA supplementation also reduced LHP and TBA values in spray-dried eggs after 12 mo of storage. Cherian et al. (1996) found a protective effect of a blend of different tocopherol isomers in PUFA-enriched eggs. Qi and Sim (1998) showed that egg TBA values decreased with increasing levels of tocopherols in hen diets from 0 to 400 mg/kg diet. In spray-dried eggs, Wahle et al. (1993), using increasing levels of α-TA, showed that TBA values were inversely related to αtocopherol content. In contrast to our results and those reported above, Gebert et al. (1998) found that the addition of 100 or 200 mg/kg feed of α-TA to hen diets increased the TBA values of eggs stored for 6 mo and attributed a prooxidant effect to α-tocopherol at these doses. Similarly, Chen et al. (1998) found that α-tocopherol has an antioxidant activity at concentrations below 50 µg/g egg yolk and a prooxidant effect at 75 µg/g egg yolk, values corresponding to 60 and 120 mg/kg diet, respectively. In our case, eggs from α-TA-supplemented treatments showed a mean concentration of α-tocopherol of 115 µg/g of egg (approximately 350 µg/g egg yolk). Effect of RE Supplementation on Lipid Oxidation in Fresh Eggs. Supplementation with RE had no antioxidant effect in preventing lipid oxidation in ω3 FA-enriched eggs at any of the dietary levels. Direct comparison of our results with other studies is difficult because there

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those eggs at the doses used. However, it cannot be concluded that CA is not an effective antioxidant when supplemented in hen diets, because some antioxidant activity was found when higher doses of CA were used.

ACKNOWLEDGMENTS

active compounds, or by its incomplete adsorption into the tissues. Some authors have found that the in vitro antioxidant activity of RE was higher, or was comparable with, αtocopherol (Lee and Choi, 1989; Baratta et al., 1998; Kuzmenko et al., 1999), whereas other researchers have shown that α-tocopherol has a higher activity in vitro (Wong et al., 1995) or in vivo (Lo´ pez-Bote et al., 1998). In our study, no synergism between RE and α-tocopherol was found, which is in concordance with results reported by Wong et al. (1995). This lack of synergism between RE and αtocopherol may be due to the low CA content in the egg. The lack of concordance in the results of previous reports, in vitro or in vivo, can be attributed, in part, to the different composition of the extracts used. As previously mentioned, the antioxidant activity of a RE is mainly due to CA (Arouma et al., 1992; Cuvelier et al., 1994, 1996). The composition and thus the antioxidant activity of an RE depends upon different factors such as the quality of the original plant, its geographic origin, the climatic conditions, the harvesting date, its storage, the commercial formula (powder or liquid), and the extraction parameters (Cuvelier et al., 1996). Moreover, CA is readily oxidized and transformed to carnosol and other derivatives, with lower antioxidant activity, thus reducing the amount of CA available to be absorbed and deposited in the egg yolk. Thus, there is a great variation in the composition of the extracts and, consequently, in the range of their antioxidant activities. In order to homogenize and facilitate the comparison of results, it would be of great importance to know the CA content of the RE and oleoresins used. More research is needed in order to elucidate the absorption, fate, and antioxidant effect of CA and other phenolic diterpenes on eggs. From our results we can conclude that dietary supplementation with α-TA is an effective way to prevent lipid oxidation in ω3 FA-enriched eggs. In our conditions, the RE used did not show an effective antioxidant effect in

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FIGURE 3. Effect of supplementation with carnosic acid from rosemary extract on the evolution of induced TBA values, expressed as nanograms of malonaldehyde (MDA) per gram of egg. Control = samples from nonsupplemented treatments; 500CA = samples from treatments supplemented with 500 mg carnosic acid/kg feed.

This work was supported in part by research grants from the Comissio´ Interdepartamental de Recerca i Innovacio´ Tecnolo`gica (CIRIT) and the Comisio´ n Interministerial de Ciencia y Tecnologı´a (CICYT). The authors are grateful to F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland, for the donation of α-tocopheryl acetate used and for the α-tocopherol analyses performed and to Haussman Aromatic S.A., E-08740 Sant Andreu de la Barca, Spain, for the kind donation of the rosemary extract used. The authors wish to acknowledge the skilled technical support of Olga Ban˜ os, Unitat de Nutricio´ i Alimentacio´ Animals, Universitat Auto`noma de Barcelona, E-08193 Bellaterra, Spain, as well as all the staff at the Zentrumsabteilung fu¨ r Chemische Analytik und Endokrinologie of the Tiera¨ rztliche Hochschule, Hannover, Germany.

LIPID OXIDATION IN ω3-PUFA ENRICHED EGGS

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