Effects Of In Ovo Equol Injection On Posthatch Growth And Meat Quality Of Broilers And Mechanism Research

In this study, long-term effects of equol (Eq) injected in ovo during embryonic stage on growth, carcass and meat quality of broilers were studied. In order to further investigate the relationship between the decreasement of drip loss and cooking loss of muscle caused by Eq injection and the anti-oxidant capacity, muscle cells were isolated from broiler embryonic muscle tissue and pre-cultured with different dosage of Eq for 1 h before 1 mM H2O2 treatment. Muscle cells were cultured in serum free medium to avoid the interference of estrogen in serum.1 Effects of in ovo equol injection on posthatch growth, meat quality and anti-oxidant status of broilersIn order to investigate the long-term effects of Eq on growth and meat quality in broilers,0μg (control, C),20μg (low dose, L) and 100μg (high dose, H) Eq dissolved in 100μL mixture of white mineral oil and ethanol, were injected into the albumen of the fertile eggs on 7 days of embryonic age, respectively. After hatching, broilers were fed under the same conditions following the standard procedure. On 49 days of age, chickens were slaughtered and samples were collected for analysis. The results showed that, after hatch, the body weight did not show any difference among 3 groups, albeit the hatching weight of chickens in L group was significantly lower than that in control group (P< 0.01). The total body fat and the ratio of the abdomen fat weight (AFW) to body weight were not significantly affected by equol treatment (P> 0.05). In addition, the weight of liver, leg muscle, breast muscle, and the breadth of abdomen fat, breast muscle area, the relative weight of liver to body weight were not significantly affected by equol treatment (P> 0.05).Compared with male broilers, the parameters of meat quality were changed significantly in female broilers after equol treatment. The redness (a*) of meat color in L and H group of female broilers were significantly decreased by 24.10% and 21.50%(P< 0.01), respectively. Cooking loss decreased by 12.11% and 16.82% in L and H group of female chickens broilers (P< 0.01), respectively. In H but not L group of female chickens, 24h and 48h drip loss were significantly decreased by 60.27% and 45.72%(P< 0.05), respectively. For male broilers, only cooking loss was significantly decreased by high dosage of equol treatment (P< 0.05).The antioxidative status was analyzed for further discovering the mechanism behind the improvement of water-holding capacity caused by equol in female broilers. The results showed that, the activity of glutathione peroxidase (GSH-Px) in plasma was greatly increased by 15.94% in L group (P< 0.01), while the total superoxide dismutase activity (T-SOD) and the content of malondialdehyde (MDA) in plasma were not changed (P> 0.05). The T-SOD activity in breast muscle of L and H groups were significantly improved by 23.14% and 18.82%(P< 0.05), respectively. GSH-Px in breast muscle of H group showed a tendency to increase (P= 0.06< 0.1). These results indicate that, equol injection in ovo do not affect the growth of broilers, but significantly improve muscle water-holding capacity, especially on female broilers, which is related to the improvement of antioxidative status.2 The protective effects of equol the damage of the muscle cells induced by H2O2Muscle cells were separated from leg muscle tissue of Sanhuang D broiler embryos incubated on 11 days of age. The culture medium with 10% fetal bovine serum was used to pre-culture the cells for 24 h, then shifted to the completed cell culture medium containing 1%ITS but serum free to stabilize muscle cells for another 24 h, then the cells were subjected to different solution treatment.1,10 and 100μM Eq were used to pre-treat the cells for 1 h, then the cells were treated with 10 mM H2O2 for 1 h, the medium containing solvent,10μM Eq alone and 10 mM H2O2 alone were served as the normal control,negative control and positive control group, respectively. After that, the cell viability was analyzed and the muscle cells were collected for analysis. The results showed that 10μM Eq alone did not change the cells viability (p> 0.05),1 mM H2O2 resulted in a significant decrease on the cell viability (p< 0.05). Subsequent treatment with 1μM Eq did not affect the reduction of the MTT value induced by 1 mM H2O2. There was no inhibitory effect of 10μM and 100μM Eq on the reduction of muscle cell viability that induced by H2O2, on the contrary, high dosage of Eq presented a certain damaged function synergized with H2O2 (p< 0.05).The LDH, SOD, GSH-Px activity and MDA content in cells and the suspension were measured by the commercial kits. The results showed that,10μM Eq alone did not change the MDA content,1 mM H2O2 increased the MDA content but without the significant difference, compared with the positive control (1 mM H2O2 damaged group),1μM and 100μM Eq pretreatment caused a significant decrease of MDA content, however, there was no difference of MDA content among the solvent control, positive control and 10μM Eq pretreatment group. With respect to the activity of LDH, there was no significant difference among the solvent control,10μM Eq alone group and 1μM Eq pretreatment plus 1 mM H2O2 treatment group (p> 0.05), however,1 mM H2O2 induced a significant increase of LDH activity. In addition,10 and 100μM Eq pretreatment could further decrease the activity of LDH in cultured muscle cells (p< 0.05). As to the activities of SOD and GSH-Px in muscle cells,10μM Eq alone had no significant effect on SOD activity in muscle cells, after 1μM Eq pre-treating, the damaged decrease induced by H2O2 in cells was notably improved (p< 0.05). Pre-treatment with 10μM and 100μM Eq could improve enzyme activity damaged by H2O2 in cells, however, this positive effect did not reach the statistical significance.10μM Eq alone had no significant effect on GSH-Px activity in muscle cells, after 10μM Eq pretreating, compared to H2O2 induced damaged treatment, the GSH-Px activity in cells was notably elevated (p< 0.05), while 1μM and 100μM equol had no significant protective effect on the reduction of GSH-Px activity in cells which damaged by H2O2.In order to investigate the damaged extent of cell histology, single cell gel electrophoresis experiment (comet assay) was employed in the present study. The results showed that the pre-culture with 10μM equol and subsequent administration with 1 mM H2O2 could significantly reduce the comet tail length (p< 0.05), siminarly,100μM Eq pre-culture had a tendency to reduce the comet tail length (p=0.067). Pre-culture with 1. 10,100μM Eq and subsequent treated with 1 mM H2O2 significantly decreased Olive Tail Moment (p<0.05); and the combined treatments of 10,100μM Eq with H2O2 obviously reduced the proportion of Tail Length to Head Length (p< 0.05). In addition, the combined treatments of 1.10.100μM Eq with H2O2 significantly increased the Head %DNA (p< 0.05) and significantly decreased the Tail%DNA (p< 0.05).The expression of apoptosis related gene Bcl-2 and Bax in cultured muscle cells was determined with relative quantitative real time RT-PCR. The results indicated that the Bcl-2 mRNA expression in 10μM equol alone pre-culture group was significantly higher than that in H2O2 alone treatment group (p< 0.05), however, the combined administration of different concentrations of equol with H2O2 did not change Bcl-2 mRNA expression in muscle cells. Additionally, there was no significant difference of Bax mRNA expression in muscle cells between 10μM Eq alone pre-culture,H2O2 treatment and the combined treatments of different concentrations of Eq with subsequent H2O2 treatment Similarity, the proportion of the mRNA expression abundance of Bax gene to Bcl-2 gene was not different among all treatment groups (p> 0.05).