| Experiments were conducted to investigate biological effect of maternal Zn-Gly in broiler breeders and their offspring,and different sources of maternal Zn on mortality,anti-oxidative capacity and protection from oxidative stress injury in different developmental stages of embryos,for Zn-Gly optimum dosage in broiler breeders and molecular mechanism of different maternal Zn sources regulating chicken embryo development.Experiment 1:Effects of Zn-Gly on reproductive performance and anti-oxidative capacity in broiler breedersA total of 600 39-week-old broiler breeders were randomly assigned to 6 groups with 5 replicates,which included 20 birds each.The control group fed with basal diet,ZnSO4 and Zn-Gly groups fed with basal diet supplemented with 80 mg Zn/kgdiet as ZnSO4,or 20,40,60and 80 mg Zn/kg diet as Zn-Gly respectively.The Zn content of basal diet was 24 mg/kg.After 4 weeks of pre-experiment with basal diet to exhaust body Zn,the feeding experiment lasted for 8 weeks.Fertilized eggs were collected during the last week of experimental period,stored at 18℃ and incubated under normal hatchery management.At the end of the feeding experiment,3 birds per replicate of 6 groups were slaughtered,samples were collected for analysis.The results showed that the 80 mg Zn/kg Zn-Gly group was the best in all groups.(1)Compared with the control group,80 mg Zn/kg Zn-Gly group increased laying rate,egg weight,fertility,hatchability and healthy bird rate,decreased feed to egg ratio and broken egg rate(P<0.05).Compared with the ZnSO4 group,80 mg Zn/kg Zn-Gly group improved fertility,hatchability and healthy bird rate,reduced the broken egg rate(P<0.05).(2)In contrast with the control group,80 mg Zn/kg Zn-Gly group enhanced the Zn content of yolk,albumen,serum,liver and muscle(P<0.05).In contrast with the ZnSO4 group,80 mg Zn/kg Zn-Gly group increased the Zn content of albumen,liver and muscle(P<0.05).(3)In comparison with the control group,80 mg Zn/kg Zn-Gly group enhanced the T-AOC,T-SOD and CuZn-SOD activities in serum and liver,and the liver MT content(P<0.05).In comparison with the ZnSO4 group,80 mg Zn/kg Zn-Gly group increased serum T-SOD and CuZn-SOD activities,liver CuZn-SOD activity,MT content and MT,CuZn-SOD mRNA abundance(P<0.05).In addition,Zn supplementation in diet had no influence on eggshell strength,eggshell thickness and egg shape index(P>0.05).Results above indicated that Zn supplementation in diet elevates reproductive performance by increasing Zn contents in tissues and eggs and improving anti-oxidative capacity in broiler breeders,and the best group is 80 mg/kg Zn-Gly.Experiment 2 Effects of maternal Zn-Gly on development and anti-oxidative capacity in chick embryoDuring incubation of Experiment 1,eggs were candled on d7 and d17,and the dead embryo eggs were dissected on d22.The early(1-6d),middle(7-16d)and late(17-21d)embryo mortalities were analyzed and calculated according to the morphological development.On d9,d14,d19 and d21 of incubation,6 live chicken embryos per replicate of 6 groups were selected and dissected for liver and muscle.The samples were stored at-20℃ or-80℃ for analysis.Results showed that 80 mg Zn/kg Zn-Gly group was the best in all groups.(1)Compared with the control group,80 mg Zn/kg Zn-Gly group decreased the late and the whole embryo mortalities(P<0.05).Compared with the ZnSO4 group,80 mg Zn/kg Zn-Gly group reduced the late embryo mortality(P<0.05).(2)In contrast with the control group,80 mg Zn/kg Zn-Gly group improved liver and muscle Zn content on d9,d14,d19 and d21(P<0.05).In contrast with the ZnSO4 group,80 mg Zn/kg Zn-Gly group increased liver Zn content on d9,d19 and d21,and muscle Zn content on d9,d14 and d21(P<0.05).(3)In comparison with the control group,80 mg Zn/kg Zn-Gly group enhanced liver T-AOC and CuZn-SOD activity on d14,T-SOD and CuZn-SOD activities on d19,T-AOC and T-SOD,CuZn-SOD activities on d21,and liver MT content on d9,d14,d19 and d21(P<0.05),decreased liver MDA content on d9 and d19(P<0.05).In comparison with the ZnSO4 group,80 mg Zn/kg Zn-Gly group increased liver CuZn-SOD activity on d19,T-SOD and CuZn-SOD activities on d21,and liver MT content on d9,d14,d19 and d21(P<0.05).(4)Compared with the control group,80 mg Zn/kg Zn-Gly group increased muscle T-AOC and CuZn-SOD activity on d9,T-AOC and T-SOD,CuZn-SOD activities on d14,d19 and d21(P<0.05),decreased muscle MDA content on d9,d19 and d21(P<0.05).Compared with the ZnSO4 group,80 mg Zn/kg Zn-Gly group elevated muscle T-SOD and CuZn-SOD activities on d19 and d21(P<0.05).Results above indicated that Zn supplementation in diet reduces embryo mortality by increasing tissue Zn content and improving anti-oxidative capacity in chick embryos,and the best results is 80 mg/kg Zn-Gly supplementation in the maternal diet.Experiment3 Effects of maternal Zn on growth performance,tissue zinc deposition and anti-oxidative capacity in offspring broilersAccording to the results from Experiment 1 and Experiment 2,80 mg/kg Zn-Gly group was the optimum choice for broiler breeders.A total of 800 1-day-old chicks from 80 mg Zn/kg Zn-Gly group and ZnSO4 group of Experiment 1,400 1-day-old chicks per group,were used for a 2×2 completely random design trail,2 maternal Zn sources and 2 levels of Zn(0 or 80 mg Zn/kg offspring broiler diet).The total 1-day-old chicks were assigned to 4 groups with 5 replicates(40 birds per replicate).On d21 or d60 of experiment,a total of 60 broilers were slaughtered and samples were collected for analysis,3 broilers per replicate.The results showed that:(1)Compared with maternal ZnSO4 treatment,Zn-Gly treatment increased average daily gain and decreased mortalities in the late and the whole period.Additionally,maternal Zn-Gly treatment also enhanced the following indexes:①liver Zn content on d21 and the liver and muscle Zn content on d60(P<0.05),②serum T-AOC and T-SOD activities on d21 and d60,serum CuZn-SOD activity on d21,liver MT content and T-SOD,CuZn-SOD activities on d21 and d60,liver T-AOC on d60,and muscle T-AOC on d21 and d60(P<0.05),③ serum IgM and HSP70 levels on d60,and decreased the following indexes:① serum,liver and muscle MDA content on d21 and d60(P<0.05),② serum IL-1,TNF-αand CORT levels on d60(P<0.05).(2)In contrast with no Zn supplementation treatment in offspring diets,Zn supplementation treatment elevated average daily gain in different periods and reduced mortalities in the late and the whole periods.Moreover,Zn supplementation treatment also increased the following indexes:① serum and liver Zn contents on d21 and d60(P<0.05),② T-AOC and T-SOD,CuZn-SOD activities in serum,liver and muscle on d21 and serum and liver on d60,liver MT content on d21 and d60,muscle T-AOC on d60(P<0.05),③ serum IgA and IgM level on d21,and serum IgG,IgM and HSP70 levels on d60(P<0.05),and reduced the following indexes:①MDA content in serum,muscle and liver on d21d,and serum,liver on d60(P<0.05),② serum CORT on d2 land d60,serum IL-1 and TNF-alevels on d60(P<0.05).The interaction between maternal Zn treatment and Zn supplementation treatment in offspring diets had significantly influence on the following indexes:①mortalities in the late and the whole periods(P<0.05),②MDA content in serum,liver and muscle on d21(P<0.05),③ serum T-SOD activity and liver T-AOC,and T-SOD,CuZn-SOD activities on d60(P<0.05),④ serum IL-1,TNF-a,CORT and HSP70 levels on d60(P<0.05).Results above indicated that compared with maternal ZnSO4 treatment,Zn-Gly increases average daily gain and decreases mortality by improving Zn deposition in progeny tissues,enhancing anti-oxidative capacity,anti-stress and immune function in offspring broilers.Experiment 4 Protective effects of maternal Zn on oxidative stress chick embryoThe experiment was conducted to investigate the protective effects of maternal Zn on oxidative stress chick embryo based on the oxidative stress models of chick embryo established by our research group.A total of 300 36-week-old Lingnan broiler breeders were randomly allocated to 3 groups with 5 replicates,which included 20 birds each.The control group fed with the basal diet without Zn supplementation,ZnSO4 group fed with the basal diet supplemented with 80 mg Zn/kg diet as ZnSO4,and Zn-Gly group fed with the basal diet supplemented with 80 mg Zn/kg diet as Zn-Gly.Zn content of the basal diet is 24 mg Zn/kg diet.The feeding experiment lasted for 8 weeks after 4 weeks of pre-experiment with the basal diet to exhaust body Zn.Fertilized eggs of each group were collected during the last week of the experimental period,stored at 18℃ and incubated under normal hatchery management.On embryonic d17,40 eggs of each group were randomly selected,placed in another incubator,and heated at 39.5℃ for 6h.Fourteen live embryo eggs per replicate were selected from the high temperature stimulation group and the normal temperature incubation group,dissected and collected for livers.Samples were fixed with 2.5%glutaraldehyde or 4%paraformaldehyde respectively.The remaining samples were stored in-20℃ or-80℃ for analysis.The results showed that:(1)Compared with the control and ZnSO4 groups,Zn-Gly group decreased the late and the whole period mortalities of the developing embryo(P<0.05).The interaction between oxidative stress and maternal Zn treatments had no significant influence on the above indexes(P>0.05).(2)Oxidative stress increased liver ROS,MDA,protein carbonyl,8-OHdG and HSP70 levels(P<0.05).In contrast with maternal ZnSO4 group,Zn-Gly group decreased the above indexes under oxidative stress condition and normal condition(P<0.05).The interaction between oxidative stress and maternal Zn treatments significantly influenced MDA content and HSP70 levels(P<0.05).(3)Oxidative stress reduced liver T-AOC,T-SOD,CuZn-SOD,GSH-Px and CAT activities,and MT content(P<0.05).In comparison with maternal ZnS04 group,Zn-Gly group improved the above indexes under oxidative stress condition and normal condition(P<0.05).The interaction between oxidative stress and maternal Zn treatments significantly influenced on the MT content(P<0.05).(4)Oxidative stress impaired mitochondrial morphology,decreased the mitochondrial membrane potential,and increased UCP mRNA abundance(P<0.05).The maternal Zn treatment reduced the mitochondrial morphology injure,and enhanced the mitochondrial membrane potential and UCP mRNA abundance(P<0.05).The effects of maternal Zn-Gly were significantly better than those of ZnSO4(P<0.05).The interaction between oxidative stress and maternal Zn treatment significantly influenced the UCP mRNA abundance(P<0.05).(5)Oxidative stress decreased CuZn-SOD,MT gene expression by improving gene expression of Nrf2 and Bax,and increased caspase3 activity and the apoptotic index(P<0.05).The maternal Zn treatment elevated gene expression of Nrf2,CuZn-SOD and MT,decreased Bax gene expression,caspase3 activity and the apoptotic index(P<0.05).The interaction between oxidative stress and maternal Zn treatment significantly influenced the gene expression of CuZn-SOD and Bax,and apoptotic index(P<0.05).Results above indicated that maternal Zn treatment can decrease chicken embryo mortality in development through activation of Keap1-Nrf2 signaling pathway,and then enhancing anti-oxidative capacity and reducing caspase-3 activity,further reducing oxidative stress damage and cell apoptosis. |
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