Effect Of Daidzein On Hepatic Expression Of Vitellogenin In The Chicken Liver And The Mechanisms Involved

In order to investigate the effects of daidzein on the expression of vitellogenin gene in hen's liver, broiler breeders were employed in this study. Hens were raised with basal diet (control group) or basal diet with 10 mg per Kg food (Daidzein group). In order to further explore which estrogen receptors might be involved in this pathway, hepatocytes from the liver of embryo were cultured in vitro with or without daidzein treatment.1 Effects of Daidzein on Laying Performance, Egg Quality and the Expression of VTG mRNA in Liver of Broiler BreedersTo investigate the effects of daidzein in broiler breeders, 480 broiler breeders were allocated at randomly to blank control (C) and daidzein treated (Da) groups. Each group has 8 replicates. The C group hens were fed with basal diet and the Da groups were fed with basal diet supplemented with 10 mg/kg daidzein. The whole investigate time persisted for 8 weeks. Hens were kept in environmental conditions (22±1℃) with a 15h light: 9h dark cycle and were given access to water freely and reared on a restricted feeding program recommended by the breeder's management guide. At the end of the fifth week treated with daidzein, 30 eggs of each group produced in three days of fifth week were chosen randomly for egg quality analysis. At the end of sixth week, 10 hens from each group were decapitated, samples including blood, liver, ovary, follicles and oviduct tissues were collected for analysis. The results showed that the laying rate was significantly increased during the first two weeks with daidzein supplementation (p < 0.05) and maintained high laying performance during the whole investigation period. The rate of abnormal eggs was not changed with daidzein supplement (p > 0.05), while the rate of broken egg were significantly decreased during the whole investigation. Egg quality analysis showed that average egg weight were not affacted (60.67±0.75 g vs 59.56±0.68 g) (p < 0.05), while egg shell thickness(0.32±0.01mm vs 0.35±0.01 mm) and the ratio weight of eggshell to the whole egg mass was (12.12±0.13% vs 12.57±0.15%) significantly increased in daidzein group (p < 0.01, p < 0.05, respectively). Additionally, egg Haugh unit increased significantly in daidzein group (80.98±1.13 vs 76.98±1.33) (p < 0.05), while other parameters were not affected. Hens body weight weight (2.67±0.08 kg vs 2.75±0.05 kg), numbers of large follicles (5.6±0.24 vs 5.6±0.34), ovary weights (54.64±2.33 g vs 53.67±1.50 g) were not influenced with daidzein treatment (p > 0.05). RT-PCR results showed that the expression of VTG mRNA in liver was not changed by daidzein treatment (p > 0.05).2 Effects of Daidzein on Expression of VTG mRNA and Estrogen Receptors mRNA of Hepatocytes in VitroHepatocytes were cultured in 5% FBS medium for 24h. To avoid the interference caused by factors in serum, after 24h cluture, the medium was removed and changed to fresh serum-free medium with 10μg/mL insulin, 5μg/mL transferrin and 3×10^-8 mol/L sodium selenite. Daidzein, 17β-estradiol and 10μM tamoxifen were alone or combination added into the medium. After 48h treatment, cells were harvested for total RNA extraction. Quantitative changes in VTG and ERβmRNA levels were measured by semi-quantitative RT-PCR usingβ-actin as an internal standard. The results showed that 100μM Da induced the expression of VTG mRNA in hepatocytes with serum-free medium in vitro; which showed different impacts on the expression of VTG mRNA of hepatocytes under different concentrations of E₂, With 1μM E₂ basal condition, 1μM and 10μM daidzein showed no impacts on the expression of VTG mRNA, while 100μM daidzein has co-effect with E₂ on the VTG gene expression. When in 10μM E₂ condition, daidzein showed antagonist of E₂, and significantly decreased the expression of VTG mRNA, with 10μM E₂ basal condition, 100μM daidzein resulted in undetected level of the expression of VTG gene. As a selective antagonist of estrogen receptor, Tamoxifen (TAM) inhibited the expression of VTG mRNA when the cells were treated with daidzein or E₂ alone or combination together, while in the condition of 100μM daidzein with 10μM E₂, TAM did not inhibited VTG gene expression. The results also showed that daidzein has no effects on expression of ERαmRNA in culture condition of daidzein or E₂ alone or two combinations. However, daidzein, E₂, TAM alone induced the expression of ERβmRNA in hepatocytes. Additionally, daidzein with E₂ augmented these effects, while TAM inhibited the effect of up-regulated expression of ERβmRNA induced by daidzein.In conclusion, these results showed that daidzein improved the egg laying performance of broiler breeders during late periods of egg laying cycle, and significantly increased eggshell thickness and Haugh Unit. Daidzein can induce expression of VTG mRNA inserum-free medium in vitro, and has co-effect on expression of VTG mRNA with differentconcentration of E₂, which might be related with the up-regulation of the gene expression ofERβ.