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METABOLISM AND NUTRITION |

* Guangdong Public Laboratory of Animal Breeding and Nutrition, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, 510640, China; and
College of Science, Zhejiang University, Hangzhou 310027, China
1 Corresponding author: jiangZ38{at}hotmail.com
| ABSTRACT |
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Key Words: broiler isoflavone growth meat quality antioxidation
| INTRODUCTION |
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Soybean isoflavones (ISF) are potential additives in improving meat quality. They have been shown to possess antioxidant activity (Wei et al., 1995; Ruiz-Larrea et al., 1997), which might be related to their anticancer, antiinflammatory, and cardioprotective effects. It has been demonstrated that ISF decrease the production of free radicals in plasma, liver, brain, testes, and kidney of male rabbits (Yousef et al., 2004). The antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSHPx; McCord, 1979; Aebi, 1984; Ursini et al., 1995). The CAT convert H2O2 to H2O, and the SOD catalyze the dismutation of the superoxide radical anion. Diets containing isoflavone (150 and 250 mg/kg) obviously elevated antioxidant enzymatic levels in various organs of rats fed diets containing partially oxidized oil (Liu et al., 2005). Cai and Wei (1996) suggested that dietary genistein, 1 of the 2 major component of ISF, enhances the activities of antioxidant enzymes in various organs in SENCAR mice. These observations led us to speculate that dietary ISF may improve growth performance or meat quality through improving antioxidative status in animals. However, Payne et al. (2001a) reported that ISF supplementation in a typical C-SBM diet did not affect growth performance or meat quality in growing-finishing gilts.
It remains to be elucidated whether and how dietary ISF may affect growth and meat quality in poultry. Therefore, the objectives of the present study were A) to examine if exposure to ISF could affect the growth and meat quality; and B) to elucidate antioxidative mechanisms underlying the effects of ISF by determining lipid peroxidation and relative biochemical parameters in broiler chickens.
| MATERIALS AND METHODS |
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Male broiler chickens (Lingnan yellow broiler, a quality meat-type chicken, coming into the market at 63 d of age) were obtained at 1-d-old from a commercial hatchery and raised at our local facility under standard conditions with free access to water and feed. Chickens received corn-low soybean meal diet (10.4% soybean meal from d 1 to 21 and 4.3% soybean meal from d 22 to 42); until 42 d of age, 1,500 male broiler chickens were weighed and allotted to 5 treatment groups, each of which included 6 replicates of 50 birds. Broilers were randomly placed in floor pens (1.3 m x 3.5 m). Over a period of 3 wk the chickens were offered the same soybean meal-free basal diet that was supplemented with 0 (control), 10, 20, 40, or 80 mg of ISF (a synthetic soybean isoflavone, containing 98.4% glycitein)/kg of diet, respectively. The ISF was substituted at the expense of zeolite. Nutrient levels of the diets were based on the National Research Council (1994) recommended nutrient requirements of broiler chickens (Table 1
). Mortalities and feed consumption per pen were recorded daily during the 3-wk experiment. At 63 d of age, birds were deprived of feed for 12 h and weighed just prior to slaughter per pen, cumulative weight gain and feed intake were determined, whereas cumulative feed:gain ratios were calculated. Feed intake and feed:gain were adjusted for mortalities when appropriate. Twelve broilers per treatment group (2 birds per replicate) were killed by cervical dislocation for meat analyses. The birds were bled via brachial vein for a plasma sample and then slaughtered and dissected by a trained team. Plasma and breast muscle were collected and snap-frozen in liquid nitrogen. Frozen tissues were stored at 70°C prior to analysis.
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The pH value was measured 45 min postmortem in the right pectoralis major with a portable pH meter (HI8424, Beijing Hanna Instruments Science & Technology Co. Ltd., Beijing, China) equipped with an insertion glass electrode calibrated in buffers at pH 4.01 and 7.007 at ambient temperature.
Water-Holding Capacity
Water-holding capacity (WHC) was estimated by determining expressible juice using a modification of the filter paper press method described by Wierbicki and Deatherage (1958) as follows. A raw meat sample weighing about 1,000 mg was placed between 18 pieces of 11-cm-diameter filter paper and pressed at 35 kg for 5 min. Expressed juice was defined as the loss in weight after pressing and presented as a percentage of the initial weight of the original sample (Bouton et al., 1971). Total moisture content was determined in duplicate according to AOAC procedures (AOAC, 1984). The WHC was calculated as the fraction of water retained by the meat [1 (expressible juice/total moisture content)] (Allen et al., 1998).
Color Measurements
Meat color was measured 45 min postmortem with a chroma meter (CR-410, Minolta Co. Ltd, Suitashi, Osaka, Japan) to measure CIE LAB values (L* measures relative lightness, a* relative redness, and b* relative yellowness). A reading was made from the surface of the sample, representing the whole surface of the muscle. A white tile (L* 92.30, a* 0.32, and b* 0.33) was used as standard.
Measurement of Shear Force
The breast muscles were refrigerated overnight at 4°C and then brought to room temperature before cooking. The breast muscle from each bird was cooked to an internal temperature of 70°C on a digital thermostat water bath (HH-4, Jiangbo instrument, Jiangsu, China). End point internal temperature was monitored with a thermometer. Cooked muscle was cooled to room temperature. Slices of 1 cm x 1 cm were cut perpendicular to the fiber orientation of the muscle. Ten 1 cm x 1 cm cores about 3 cm thick were removed parallel to the fiber orientation through the thickest portion of the cooked muscle. Warner-Bratzler shear force was determined by using an Instron Universal Mechanical Machine (Instron model 4411, Instron Corp., Canton, MA). A Warner-Bratzler apparatus was attached to a 50-kg load cell, and tests were performed at a cross head speed of 127 mm/min. Signals were processed with the Instron Series ninth software package.
Biochemical Determinations
Forty milligrams of frozen tissue in 4 mL of homogenization buffer (0.05 M Tris-HCl, pH 7.4, 1 mM EDTA, 0.25 M sucrose) was homogenized on ice with an Ultra-Turrax homogenizer (T8, IKA-Labortechnik, Staufen, Germany) for 5 s at 13,500 rpm. The homogenate was centrifuged at 3,000 rpm for 10 min at 4°C, and the supernatant was stored at 70°C until analysis. The activities of total SOD (T-SOD), GSHPx, CAT, total antioxidant capability (T-AOC), the contents of malondialdehyde (MDA), a lipid oxidation product, and lactic acid (LD) were assayed using colorimetric methods with a spectrophotometer (Biomate 5, Thermo Electron Corporation, Rochester, NY). The assays were conducted using the assay kits purchased from Nanjing Jiancheng Insititute of Bioengineering (Nanjing, Jiangsu, China) and the procedures accordingly. The activities of creatine kinase and lactate dehydrogenase in plasma were determined in a Beckman spectrophotometer (model CX5, Beckman Instruments, Fullerton, CA) at 340 nm using the assay kits from Beckman Coulter Inc. (Fullerton, CA). All samples were measured in triplicate, at appropriate dilutions, to give activities of the enzymes in the linear range of standard curves constructed with pure enzymes. Protein content of supernatants was determined using the Coomassie Brilliant Blue G250 (Sigma Chemical, St. Louis, MO) assay with bovine serum albumin.
Statistical Analysis
All statistical analyses were computed using the GLM procedures of SAS software (SAS Institute, 1996). A software program using Duncans multiple range test to compare treatment means was applied. A P < 0.05 was considered statistically significant. Replicate was considered as the experimental unit for performance determined. The experimental unit was a bird for the other parameters. Numbers used for statistics are noted in the tables. All data were expressed as means ± SE.
| RESULTS |
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As shown in Table 2
, final BW and weight gain of birds significantly increased by supplementation of 10 or 20 mg of ISF/kg (P < 0.01). Feed intake was increased by 7.37% (P < 0.05) and 11.2% (P < 0.01) by 10 or 20 mg of ISF/kg supplementation, respectively. The feed:gain ratio from 43 to 63 d of age was significantly improved by the supplemental ISF at the 10-mg level (P < 0.05).
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The results of meat quality of breast muscles are presented in Table 3
. The data clearly show that the difference in shear force of breast muscles was not significant between groups (P > 0.05). The WHC was slightly but not significantly improved by 17.24% with the 40 mg of ISF/kg addition (P = 0.0555). Meanwhile, adding 20 or 40 mg of ISF/kg to the diet significantly increased the pH value of meat (P < 0.01). However, the L* of meat color significantly increased by supplemental 40 or 80 mg of ISF/kg (P < 0.05).
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Lipid peroxidation, determined as the concentration of MDA in plasma and breast muscle, is showed in Figures 1
and 2
. The MDA production was slightly reduced in plasma of 20 mg of ISF/kg supplemented chickens (P < 0.1) and significantly decreased in breast muscles of 20, 40, or 80 mg of ISF/kg supplemented chickens (P < 0.01).
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Table 4
shows the biochemical indices in plasma and breast muscle of male broilers. In plasma, no differences were observed in the activities of GSHPx, creatine kinase, and lactate dehydrogenase. However, the addition of 40 or 80 mg of ISF/kg to the diet significantly increased T-AOC of chickens (P < 0.01), and slightly elevated T-SOD activity (P = 0.074). The CAT activity slightly increased by 80 mg of ISF/kg supplementation (P = 0.0595). In breast muscle, treatment of birds with ISF caused an increase of T-SOD activity by 7.87% (P > 0.05), 6.58% (P > 0.05), 25.36% (P < 0.05), or 63.93% (P < 0.01), respectively. The CAT activity was significantly improved by the supplemental ISF at the 40-mg level (P < 0.05). Also, 10, 20, or 40 mg doses of ISF/kg decreased LD contents (P < 0.05).
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| DISCUSSION |
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The extent of lipid peroxidation by reactive oxygen species can be monitored by MDA levels (Sumida et al., 1989). Young et al. (2003) reported that MDA production was decreased in pectoralis major of ascorbic acid-
-tocopherol-supplemented chickens. Decreased lipid oxidation in chicken muscle following supplements with a high content of antioxidants originating from plants, e.g., tea catechins (100 to 300 mg/kg; Tang et al., 2000) and rosemary-sage extracts (500 mg/kg; Lopez et al., 1998), has previously been shown. Soybean isoflavones are capable of suppressing formation of plasma lipid oxidation products in vivo (Tikkanen et al., 1998; Wiseman et al., 2000; Chen, 2001). The concentrations of MDA in plasma and tissue are significantly decreased by ISF treatment in male rabbits (Yousef et al., 2004). In the present study, MDA production of the breast muscle decreased by adding ISF in diet (above 10 mg/kg) in male broilers. This finding suggested that ISF supplementation in broilers may be needed to protect tissues against attack by lipid oxidation products.
There has been very little research conducted on the effect of ISF on antioxidative status in broilers. In the study presented here, the activity of T-AOC in plasma and T-SOD activity in breast muscles significantly increased by adding 40 or 80 mg of ISF/kg to diets; the supplemental ISF at 40 mg level significantly improved CAT activity in breast muscles. The present results had the implication that ISF improved antioxidative status of male broilers by elevating the activity of antioxidant enzymes.
The rate of discoloration of meat is believed to be related to the effectiveness of oxidation processes and enzymic reducing systems in controlling metmyoglobin levels in meat (Faustman and Cassens, 1989). The L* value of meat color was significantly increased by supplemental 40 and 80 mg of ISF/kg in the present study. Payne et al. (2001a) demonstrated that the a* and b* color scores were decreased linearly as ISF increased. Supplemental daidzein in the maternal diet during late gestation did not affect meat color of the progeny (Rehfeldt et al., 2007). These results suggested that different mechanisms or factors possibly influence oxidative damage to lipids and proteins (Sen et al., 1997).
This study showed that WHC of breast muscles was slightly improved by 17.24% with the 40 mg of ISF/kg treatment. Meanwhile, the addition of 20 and 40 mg of ISF/kg diet significantly increased the pH value of breast muscles. This result may be attributed to a decreased LD production in muscles postmortem (Lee et al., 1979; Raj et al., 1990, 1992). The inability of muscle cells to rid themselves of metabolic by-products such as LD cause a decrease in pH (Judge et al., 1989). This decrease in pH can affect WHC (Ferket and Foegeding, 1994; Pearson, 1994). The present results showed that ISF addition reduced the concentration of LD in breast muscles. This finding suggested that improved antioxidative status of male broilers by ISF supplementation may protect skeletal muscle cells against metabolic by-products such as LD. The previous study showed that improvements in WHC and pH development postmortem were due to increased antioxidative status in the chickens (Young et al., 2003). Therefore, the presence of supplemental ISF would be expected to reduce meat oxidation and improve meat quality.
In conclusion, the antioxidative status of chickens determined as antioxidative enzymes and the oxidative stability of lipids, increased after supplementing ISF to the diet. Increased antioxidative status obtained through supplementation improve growth performance, WHC, and pH value. In addition, the improved antioxidative status protected against lipid oxidation in breast muscles of ISF supplemented chickens. These results demonstrate that ISF shows good potential as an antioxidant in male broilers.
| ACKNOWLEDGMENTS |
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Received for publication December 1, 2006. Accepted for publication April 1, 2007.
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