Studies Of Selenomethionine Supplementation In Broiler Breeder On Decreasing Chicken Embryonic Mortality During Late Development Period

Earlier studies in our laboratory on the different selenium(Se)sources and levels(sodium selenite,selenium-enriched yeast,selenomethionine)on broiler breeder and its offsprings’ production performance indicated that overall effects of selenomethionine(SM)and selenium-enriched yeast(SY)are significantly better than sodium selenite(SS).Maternal SM and SY could increase laying rate of breeders,hatchability and Se concentrations of eggs.Compared with SS,SM significantly reduced embryonic mortality rate in late incubation period.The optimum dose of SM was 0.15 mg Se/kg.Therefore,in this experiment,through establishing the oxidative stress damage model of chicken embryo and LMH cells,we studied the difference between SS and SM on protective effects under oxidative stress,on regulating target of Keap1-Nrf2-ARE signaling pathway and difference on gene expression efficiency of antioxidant selenoenzyme.We studied the molecular mechanism of SM regulating nutrition which aim to provide a theoretical basis for its development and application.Experiment 1 The protective effect of maternal SM on acute oxidative damage of chicken embryo1.1 Establishment of the chicken embryo model of acute oxidative stressThe chicken embryos were injected with gradient concentrations of diquat(0,10,25,50,75 μg)on the 17th day of the late incubation period.Chicken embryo showed apparent phenomena of chorioallantoic redness and congestion 24 h after injection.The mortality of chicken embryo was dose-dependent,higher concentration of diquat showed significantly mortality.25 μg diquat significantly increased the number of chicken embryo deaths,T-AOC activity and MDA content in liver.These results indicated that the chicken embryo model of acute oxidative stress was successfully established and the appropriate dose of diquat injected was 25 μg.1.2 The protective effect of maternal SM on chicken embryos induced by diquatBased on the oxidative stress model of chicken embryo established in Experiment 1.1,a 3×2 completely random experimental design was applied in this trail.3,Se sources:basal diet(0.04 mg Se/kg),basal diet supplemented with 0.15 mg Se/kg of SS or SM;2 diquat(DIQ)levels:0 and 25 μg.A total of 180 Meihuang-4 broiler breeders(40 weeks old)were randomly assigned to 3 dietary treatments.The feeding experiment lasted for 8 weeks after 4 weeks of adaptation period.Eggs of each dietary treatments were collected and randomly assigned into 2 diquat-injected treatments on the 17th day of incubation.There were 6 treatments(CON,CON-DIQ,SS,SS-DIQ,SM,SM-DIQ)with 8 replicates(20 eggs per replicate).On the 18th day,eggs were dissected for analysis.And mortality statistics was conducted on the 21 th day.The results were as follows:1)Diquat significantly increased the free radical level(NO)and oxidation product content(protein carbonyls)and reduced total antioxidant capacity(T-AOC)in chicken embryo liver.By HE staining,microscopic observation showed that hepatocytes were vacuolated.Observations using electron microscope showed that electron density of hepatocytes decreasing,part of the mitochondrial inner ridges disappearing and the shedding of endoplasmic reticulum ribosome.Diquat significantly increased the apoptosis rate of hepatocytes and mortality of chicken embryo.2)Maternal dietary supplementation with Se significantly increased T-AOC activity by increasing antioxidant enzymes activities(GPx,TrxR,CAT,γ-GCS),oxidase activities(MPO,XOD,MAO)and phase Ⅱ detoxification enzyme contents(HO-1,NQO1).Maternal Se significantly decreased the chicken embryo mortality by reducing free radical level(ROS,NO)and oxidation product contents.What’s more,maternal Se significantly increased the cell mitochondrial membrane potential(MMP),anti-apoptotic protein(Bcl-2)content and reduced apoptotic protein(Caspase-3)content,which strongly inhibited cell apoptosis.These results indicated that maternal Se significantly reduced chicken embryonic mortality through alleviating the oxidative stress induced by diquat,and SM showed better results compared with SS.Experiment 2 The activation mechanism of Keapl-Nrf2-ARE signaling pathway by maternal SM in liver of chicken embryoBased on Experiment 1,this experiment further studied the activation mechanism of Keap1-Nrf2-ARE signaling pathway by maternal SM in liver of chicken embryo.The results showed that:1)Maternal SM significantly up-regulated Nrf2 mRNA abundance,Nrf2 and p-Nrf2 protein content and promoted nuclear translocation of Nrf2 protein.2)Maternal dietary supplementation with SM down-regulated the 20S proteasome activity of chicken embryo hepatocyte.SM significantly up-regulated the mRNA abundance and protein expression of antioxidant selenoenzymes(GPx,TrxR)and phase Ⅱ detoxification enzymes(HO-1,NQO1).And SM was more effective than SS.These results indicated that SM can increase gene expression of Nrf2 and p-Nrf2 by activating Keap 1-Nrf2-ARE signaling pathway.Besides,SM also inhibited ubiquitination degradation of Nrf2 protein to up-regulate the downstream gene expression of antioxidant enzymes and phase Ⅱ detoxification enzymes.Experiment 3 The protective effect of SM on LMH cell under oxidative stress and the regulation mechanism of Keapl-Nrf2-ARE signaling pathwayBased on in vivo results,we further studied the protective effect of SM on oxidative damage and the regulation mechanism of Keap 1-Nrf2-ARE signaling pathway in vitro of LMH cell.3.1 Establishment of the LMH cell model of acute oxidative stress1)The LMH cells were treated with H2O2 for 24h.The results showed that the LDH activity of LMH cell supernatant gradually increased and cell viability gradually decreased with the increase of H2O2 concentrations within 0-1000 μmol/L.The LDH activities and cell viability were significantly correlated in a dose-dependent manner,and the dose of 1000 μmol/L H2O2 could inhibit cell viability to 50%.Therefore,the dose of 1000μmol/L H2O2 was chosen for the following experiments.2)SS and SM significantly increased cell viability and GPx activity of LMH cells within 10-100 ng/mL.1000 ng/mL SS treatment group significantly reduced cell viability due to cytotoxicity,while SM did not show similar result.Therefore,the dose of 100 ng/mL Se was chosen for the following experiments.The results indicated that the LMH cell model of acute oxidative stress induced by H2O2 was successfully established.The appropriate dose of H2O2 was 1000μmol/L.The appropriate dose of Se was 100 ng/mL.3.2 The protective effect of SM on LMH cell under oxidative stressThe experiment further studied that the protective effects of SM on oxidative damage base on the LMH model and Se treatment dose established and optimized in Experiment 3.1.The results showed that:1)H2O2 significantly increased ROS,NO and MDA contents in LMH cells.H2O2 significantly increased the protein content of Caspase-3,which strongly promoted cell apoptosis.2)SM significantly inhibited ROS,NO and MDA content by increasing T-SOD activity and T-AOC level.SM significantly inhibited LMH cell apoptotic by enhancing MMP and reducing caspase-3 content.The results indicated that SM alleviated the oxidative damage induced by H2O2,and SM showed better results than SS.3.3 The regulation mechanism of SM on Keapl-Nrf2-ARE signaling pathway in LMH cell.On the basis of Experiment 3.2,we also studied the regulation mechanism of SM on Keap1-Nrf2-ARE signaling pathway in LMH cell.The results showed:SM significantly stimulated the mRNA expression of GPx,TrxR and HO-1 gene,increased GPx activity and Trxr content through up-regulating Nrf2 mRNA level and down-regulating 20S proteasome activity,SM had better effect compared with SS.These results further indicated that SM alleviated oxidative stress through activating Keapl-Nrf2-ARE signaling pathway,inhibiting ubiquitination degradation of Nrf2 protein and regulating the expression of antioxidant selenoprotein and phase Ⅱ detoxification enzyme genes.3.4 The effect of SM on expression efficiency of antioxidant selenoenzyme gene in LMH cellsFurther research was done based on Experiments 3.1 and 3.3.The results showed that:Se significantly increased the mRNA stability and protein synthesis rate of downstream selenoenzyme(GPx,TrxR,SEPP)of Keap1-Nrf2-ARE signaling pathway in LMH cell,and SM showed better results than SS.The results indicated that SM can effectively increase the gene expression efficiency of antioxidant selenoenzymes.In summary,maternal SM can activate Keapl-Nrf2-ARE signaling pathway to increase gene expression of Nrf2 and p-Nrf2.Also,SM further up-regulated downstream genes of antioxidant selenoenzyme and phase II detoxification enzyme by inhibiting ubiquitination degradation of Nrf2 protein.Therefore,maternal SM can alleviate oxidative stress and reduce mortality of chicken embryo by enhancing the antioxidant capacity.