METABOLISM AND NUTRITION Genistein supplementation to the quail: Effects on egg production and egg yolk genistein, daidzein, and lipid peroxidation levels F. Akdemir and K. Sahin1 Department of Animal Nutrition, Faculty of Veterinary Medicine, Firat University, 23119 Elazig, Turkey ABSTRACT Genistein, a soy phytoestrogen, is a powerful antioxidant. In the present study, we investigated the effects of dietary genistein supplementation on Japanese quail (Coturnix coturnix japonica) laying performance and egg yolk contents of malondialdehyde (MDA), vitamin A, and vitamin E. Malondialdehyde is an indicator of lipid peroxidation, whereas vitamins A and E have antioxidant properties. Birds (n = 150; 5 wk old) were randomly assigned to 1 of 3 groups consisting of 50 birds (5 replicates of 10) and were fed a basal diet or the basal diet supplemented with either 400 or 800 mg of genistein/kg of diet. The experimental period lasted 90 d with a 17L:7D photo schedule. As antioxidant indices, yolk MDA and vitamin (A and E) concentrations were measured by HPLC. Dietary genistein supplementation (800 mg/kg) increased feed
intake, egg production, egg weight, Haugh unit, shell thickness, and shell weight and improved feed efficiency at a greater extent than the other levels (0 and 400 mg/ kg). Egg yolk genistein concentration was increased (P < 0.0001), whereas egg yolk MDA concentration was decreased (P < 0.0001) at the highest level of genistein supplementation. However, genistein supplementation did not affect egg yolk daidzein, vitamin A, and vitamin E levels. There was an inverse relationship between egg yolk genistein and MDA concentration (y = 0.02 × egenistein, R2 = 0.74, P < 0.0001). Results of the present study indicate that supplementing with dietary genistein (800 mg/kg) improved performance, egg quality, and egg yolk genistein content and decreased egg yolk MDA concentration in quail.
Key words: genistein, egg yolk, malondialdehyde, Japanese quail 2009 Poultry Science 88:2125–2131 doi:10.3382/ps.2009-00004
INTRODUCTION Phytoestrogens are phytochemicals that have properties similar to estrogens. The major groups of phytoestrogens are isoflavones, lignans, and coumestans (Setchell, 1998). Isoflavones are found in high concentrations in soybean and soy products (Adlercreutz, 1995). Isoflavones are diphenolic compounds and exist in conjugated or unconjugated (aglycone) forms. Aglycone forms of isoflavones include genistein, daidzein, and glycitein (Kudou et al., 1991). Soy isoflavones are structurally and functionally similar to natural estrogens and can weakly bind to estrogen receptors and may exert either estrogenic or antiestrogenic effects. Soy isoflavones are also shown to prevent certain types of cancer (Adlercreutz, 1995; Franke et al., 1995), reduce the risk of osteoporosis (Adlercreutz, 1995; Zhang et al., 2008), lower plasma cholesterol (Anderson et al.,
©2009 Poultry Science Association Inc. Received January 3, 2009. Accepted June 13, 2009. 1 Corresponding author:
[email protected]
1998; Arjmandi et al., 1998; Ho et al., 2000), and can act as antioxidant agents (Adlercreutz, 1995; Kirk et al., 1998; Ho et al., 2000; Munro et al., 2003; Onderci et al., 2004; Jiang et al., 2007) and immune enhancers (Adlercreutz, 1995) in humans and laboratory animals. A high proportion of protein in poultry diet comes from soybean meal. Isoflavones contained in soy can be passed to or accumulated in animal products (Lin et al., 2004; Jiang et al., 2007), which allows animal products to be enriched with isoflavones, leading to production of functional foods for humans. Genistein can be deposited in the egg (Lin et al., 2004; Jiang et al., 2007). Thus, adding antioxidants into poultry diets seems to be an efficient means for improving the oxidative stability of eggs as reflected by decreased egg yolk MDA levels (Cherian et al., 1996; Garcia et al., 2002; Sahin et al., 2008). There is an inverse relationship between malondialdehyde (MDA), an indicator of lipid peroxidation, and dietary antioxidant level in poultry products (Guo et al., 2001; Sahin et al., 2008). Antioxidants also improve animal origin food quality in terms of color, tenderness, and storage properties (Angelo, 1992; Flachowsky et al., 2002).
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Table 1. Ingredient and chemical composition of the basal diet (%)1 Item
%
Corn Soybean meal2 Corn oil Limestone Dicalcium phosphate Vitamin-mineral premix3 Sodium chloride dl-Methionine Chemical analyses, DM basis CP, % Crude fat, % Crude fiber, % Ash, % ME, kcal/kg Daidzein, mg/kg
60.00 27.15 2.00 8.82 1.30 0.25 0.35 0.13 18.01 5.18 3.47 7.15 2,750 52.1
1 All analyses were conducted in triplicate. Genistein was added to the basal diet at the expense of corn in the amount of 0, 400, and 800 mg per kilogram. After reconstitution of the basal diet, respective experimental diets contained 69.1, 330.8, and 661.0 mg of genistein per kilogram. 2 Soybean meal contained the following: 48% protein, 0.8% fat, 5.9% ash, 6.6% fiber, 69.1 mg of genistein/kg, and 52.1 mg of daidzein/kg. 3 Supplied the following per kilogram of diet: retinyl acetate, 12,000 IU; cholecalciferol, 2,400 IU; dl-α-tocopheryl acetate, 30 mg; menadione sodium bisulfite, 2.5 mg; thiamine hydrochloride, 3 mg; riboflavin, 7 mg; niacin, 40 mg; d-pantothenic acid, 8 mg; pyridoxine hydrochloride, 4 mg; vitamin B12, 0.015 mg; vitamin C, 50 mg; folic acid, 1 mg; dbiotin, 0.045 mg; choline chloride, 125 mg; Mn (MnSO4·H2O), 80 mg; Fe (FeSO4·7H2O), 30 mg; Zn (ZnO), 60 mg; Cu (CuSO4·5H2O), 5 mg; Co (CoCl2·6H2O), 0.1 mg; I as KI, 0.4 mg; and Se (Na2SeO3), 0.15 mg.
A limited number of studies performed on avian species consistenly demonstrated functional nutriceutical effects of genistein on nutrient utilization (Sahin et al., 2006), antioxidant status (Onderci et al., 2004), and bone metabolism (Sahin et al., 2007). This experiment was conducted to ascertain responsiveness of quails to genistein supplementation with respect to performance and egg quality.
MATERIALS AND METHODS Production of Genistein-Enriched Eggs One hundred fifty Japanese quail (5 wk old; Coturnix coturnix japonica), provided by a commercial company (Insanay AY Kanatli Hayvan Uretim Paz. Tic. Inc., Elazig, Turkey), were used in accordance with animal welfare regulations at the Veterinary Control and Research Institute of Elazig, Turkey. After a 7-d adaptation period, the birds were randomly assigned to 3 groups, 50 birds each as 5 replicates. All quails were hatched from a large group of parent stock that was identical in age. Birds were housed in individual cages at dimensions of 20 × 20 cm, providing 400 cm2 per bird. Birds were fed either a basal diet containing 18% CP or 2,750 kcal/kg of ME and 69.1 mg of genistein/kg of diet or the basal diet reconstituted with addition of 400 mg or 800 mg of synthetic genistein per kilogram of diet at the expense of corn. This synthetic genistein contained 98% aglycone and 2% starches as a carrier (Bonistein,
DSM Nutritional Products, Istanbul, Turkey). Diets (Table 1) were prepared in batches and stored in black plastic containers at 4°C to avoid photooxidation. The birdhouse was set to a 17L:7D cycle. Water and diets were offered for ad libitum consumption throughout the experiment. The animal experiment lasted 90 d.
Performance Variables and Egg Quality Feed consumption was measured weekly, whereas egg production and egg weights were recorded daily. Egg quality measurements were egg weight, shell weight, shell thickness, and Haugh unit. Haugh unit, an indicator of albumen quality, was calculated using the following formula: Haugh unit = 100 × log (H + 7.57 − 1.7 × W0.37), where H = albumen height (mm) and W = egg weight (g) (Eisen et al., 1962) after determining albumen height by using a micrometer (TLM-N1010, Saginomiya, Tokyo, Japan) and egg weight.
Sample Collection and Laboratory Analyses Chemical analyses of the basal diet for CP (method 988.05), ether extract (method 932.06), crude fiber (method 962.09), crude ash (method 936.07), Ca (method 968.08), and P (method 965.17) were performed in triplicate using procedures described by the AOAC (1990). After reconstitution of the basal diet by adding genistein (0, 400, and 800 mg), the respective experimental diets were analyzed for genistein and daidzein (Lin et al., 2004). As indices of antioxidant status, egg yolk genistein, daidzein, vitamin A, vitamin E, and MDA were measured on eggs that were collected at the last 3 d of experiment. Five eggs were randomly collected from each replicate and pooled by replicate after weighing each and separating albumen and yolk. Pooled yolks for each replicate were subjected to analyses for isoflavone (Lin et al., 2004), vitamins (Franchini et al., 2002; Mori et al., 2003), and MDA (Karatepe, 2004) using HPLC (Shimadzu, Kyoto, Japan). Intra- and interassay CV were 3.6 and 5.1% for vitamin E, 4.2 and 5.8 for vitamin A, and 2.75 and 4.8% for MDA. Alltrans-retinol and α-tocopherol were used as standards (Sigma Chemical Co., St. Louis, MO). The equipment for HPLC consisted of a pump (LC-20AD), a UV-visible detector (SPD-20A) for MDA, a fluorescent detector (RF-10AXL) for isoflavones, a column oven (CTO10ASVP), an autosampler (SIL-20A), a degasser unit (DGU-20A5), and a computer system with LC Solution Software (Shimadzu). Inertsil ODS-3 C18 column (250 × 4.6 mm, 5 μm, GL Sciences Inc., Tokyo, Japan) was used as the HPLC column.
Statistical Analyses In sample size calculation, 10% improvement in egg genistein concentration was considered to be significant
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Table 2. Effects of genistein supplementation to quail on performance and egg quality Dietary genistein, mg/kg Variable
0
Feed intake, g/d Egg production,2 % Egg weight,2 g Feed conversion3 Shell weight, g Shell thickness, mm Haugh unit4 Egg yolk weight, g
400 c
29.50 81.92c 11.17c 3.23b 1.32c 0.20c 77.42c 2.92b
800 b
31.92 89.00b 11.49b 3.12ab 1.40b 0.24b 88.25b 3.17ab
a
33.00 92.67a 11.82a 3.01a 1.49a 0.28a 92.83a 3.47a
SEM
Treatment effect, P
0.05). Quails supplemented with 800 mg of genistein per kilogram of feed consumed the greatest amount of feed, produced the greatest number of eggs, had the heaviest egg weight, and had the most efficient feed conversion, followed by quails supplemented with 400 mg of genistein per kilogram of feed. The control quails achieved the poorest performance indices (Table 2; Figure 1A).
Egg Quality Egg and yolk weights were the greatest in quails supplemented with 800 mg of genistein per kilogram of feed, whereas they were the lowest in quails unsupplemented with genistein (Table 2; Figure 1B). Similar trends were also noted in shell weight, shell thickness, Haugh unit, and yolk weight in response to the dietary treatments.
Egg Yolk Antioxidants Initial egg yolk genistein and MDA contents (0.61, 0.57, and 0.59 µg/g of genistein; 0.095, 0.092, and 0.094 µg/g of MDA; P > 0.05) were similar across the treatments. Egg yolk genistein concentration and total yolk genistein amount for quails supplemented with 800 mg of genistein per kilogram of diet were greater than for those supplemented with 400 mg of genistein and those unsupplemented with genistein (Table 3). There were no differences in egg yolk daidzein, vitamin A, and vitamin E concentrations across the diets. Total yolk daidzein and vitamin E amounts were not different due to the diet, but total yolk vitamin A amount was the greatest at the highest level of genistein supplementation compared with the middle level and control diet. Egg yolk MDA concentration decreased with the genistein level in the diet, but total yolk MDA amount was the greatest in quails supplemented with the highest level of genistein than other groups, due to a greater magnitude increase in yolk weight. Egg yolk MDA level in eggs of quails fed diets supplemented with genistein was less than those fed the control diet (0.08 vs. 0.09, P < 0.0001) and it decreased linearly as dietary genistein supplementation increased (Table 3). However, dietary genistein supplementation did not affect egg yolk daidzein (0.126 µg/g), vitamin A (5.06 µg/g), and vitamin E (11.93 µg/g) levels (Table 3). Egg yolk genistein content was negatively correlated with yolk MDA concentration (r = −0.651, P < 0.0001). Moreover, egg yolk MDA content depressed exponentially (Y = 0.02 × egenistein, R2 = 0.74, P < 0.0001) as egg yolk genistein content increased (Figure 2).
DISCUSSION Isoflavones act as antioxidants in vitro and in vivo and exert antiproliferative activities (Messina and Barnes, 1991; Kurzer and Xu, 1997; Brouns, 2002; Zhang et al., 2008). Antioxidant function of genistein takes place
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Akdemir and Sahin Table 3. Effects of genistein supplementation to quail on egg yolk genistein, daidzein, vitamin A, vitamin E, and malondialdehyde levels1 Dietary genistein,2 mg/kg Variable Genistein, µg/g of yolk Genistein, µg/yolk Daidzein, µg/g of yolk Daidzein, µg/yolk Vitamin A, µg/g of yolk Vitamin A, µg/yolk Vitamin E, µg/g of yolk Vitamin E, µg/yolk Malondialdehyde, µg/g of yolk Malondialdehyde, µg/yolk
0
400 b
0.58 1.82b 0.13 0.39 0.33 1.04c 12.70 27.38 0.094a 0.26b
800 b
0.82 2.71b 0.12 0.41 0.60 1.97b 8.32 39.00 0.081b 0.27b
a
1.48 5.21a 0.13 0.44 0.87 3.06a 17.39 60.48 0.073c 0.29a
SEM
Treatment effect, P
