| Series of six trials were conducted to investigate effects of in ovo injection of zinc(Zn)or manganese(Mn)on embryonic development,antioxidation and anti-heat stress effects in offspring broilers from the aspects of hatchability,antioxidant ability and epigenetics.Experiment 1 was conducted to determine an optimal embryonic age for early in ovo injection.A total of 720 fertilized eggs with similar weights were randomly allotted to 4 treatments with 6 replicates per treatment and 30 eggs per replicate in a completely randomized design.The eggs were injected with 0.1 mL sterilized water at 3,6 and 9 embryonic days of incubation(E3,E6 and E9)or without injection(the control),respectively.The results showed that E3 and E6 injections increased(P?0.05)the embryonic mortalities,and decreased(P<0.05)hatchabilities compared to the non-injected control,but no differences(P>0.05)between E9 injection and the non-injected control were observed in either embryonic mortality or hatchability.The findings suggested that the E9 was the optimal embryonic age for early in ovo injection.Experiment 2 was conducted to investigate the effect of in ovo Zn injection on the embryonic development,tissue Zn contents,antioxidation and related gene expressions of fertilized eggs of Arbor Acres broiler breeders.A total of 672 fertilized eggs with similar weights were randomly allocated to 7 treatments with 6 replicates per treatment and 16 eggs per replicate in a completely randomized design.The eggs were injected with 0(the negative control),50,100,150,200,or 250 ?Zn/egg as reagent grade ZnS04-7H20 in a 0.1 mL solution,or without injection(the positive control),respectively at E9-10.The results demonstrated that no differences(P>0.05)among 50 and 100 ?g Zn/egg groups and the control were observed in the embryonic mortality and hatchability,however,the injection of 200 ?g Zn/egg increased(P<0.05)the embryonic mortality,and injections of 150 and 200 ?g Zn/egg decreased(P<0.05)hatchabilities compared with the controls.The embryonic tibia Zn contents at E20 were increased(P<0.05)by injections of 150,200 and 250 ?g Zn/egg.Zinc injection did not affect(P>0.05)malonaldehyde(MDA)contents,copper-and Zn-containing superoxide dismutase(CuZnSOD)activities and mRNA expression levels in the liver and heart of chick embryos at E15 and E20.Compared with the negative control,injections of 50,150 and 200?g Zn/egg up-regulated(P<0.05)the metallothionein ?(MT4)mRNA expression levels in the embryonic liver at E20.These results indicated that in ovo Zn injections increased Zn contents in the embryonic tibia and MT4 mRNA expression levels in the embryonic liver at E20,however,injections of 150-250 ug Zn/egg were harmful to the embryonic development.Experiment 3 was conducted to investigate the effect of in ovo Zn injection on the embryonic development,antioxidation and anti-heat stress in offspring broilers based on the results of experiments 1 and 2.During embryonic stage,a completely randomized design involving a 1(the non-injected control)+ 1(the injected control with sterilized water)+ 2(Zn source)× 2(Zn level)factorial arrangement of treatments was used.The two injected Zn sources were inorganic Zn sulfate and organic Zn-lysine chelate with a moderate chelation strength,and the two injected Zn levels were 50 and 100?g/egg.During progeny stage,offspring broilers from 6 embryonic treatments at 23 d of age were allotted to 2 rearing temperatures(220C vs 340C)on the basis of sex and body weight.The results showed that in ovo Zn injection decreased(P<0.05)embryonic mortality,and increased(P<0.05)hatchability and healthy chick ratio.In ovo Zn injection increased(P<0.05)embryonic tibia Zn content.In ovo Zn injection decreased(P<0.05)mortality at 1-22 d of age.Average daily feed intake(ADFI)and average daily gain(ADG)in the injection of 100 ?g Zn/egg were higher than in the 50 ?g Zn/egg.Hyperthermia decreased(P<0.05)ADFI and ADG,increased(P<0.05)feed/gain ratio(F/G);while in ovo Zn injection increased(P<0.05)increased ADFI and ADG,decreased(P<0.05)F/G at 23-42 d of age.Hyperthermia decreased(P<0.05)eviscerated yield and breast muscle,increased drip loss of breast and thigh muscle,and pH and shear force in the thigh muscle,and contents of triglyceride(TG)and thyroxin(T4)at 42 d of age.In ovo Zn injection increased(P<0.05)eviscerated yield.In ovo Zn injection had no effect(P>0.05)on CuZnSOD activities and MT4 or their mRNA expression levels and MDA levels in the embryonic liver.The in ovo Zn injection,hyperthermia and their interaction had no effects(P>0.05)on the CuZnSOD activities and MDA levels in the liver and pancreas at 42 d of age.Hyperthermia decreased(P<0.05)heat shock protein 90(HSP90)mRNA expression.The in ovo Zn injection,hyperthermia and their interaction had no effects(P>0.05)on the CuZnSOD,MT4,MT1,HSP70 and heat shock factor ?(HSF3)mRNA expression in the liver at 42 d of age.In ovo Zn injection had no effect(P>0.05)on the global level of DNA methylation or DNA methylation and histone 3 lysine 9(H3K9)acetylation levels of the MT4 promoter in the embryonic liver.However,the organic Zn had higher(P<0.05)levels of DNA methylation and H3K9 acetylation than inorganic Zn.The in ovo Zn injection,hyperthermia and their interaction had no effects(P>0.05)on the DNA methylation and H3K9 acetylation levels of the MT4 promoter in the liver at the 42 d of age.The results indicated that in ovo Zn injection improved the embryonic development,decreased mortality at 1-22 d of age.Hyperthermia decreased offsprings' growth performance,carcass traits and meat quality,disrupted the HSP90 mRNA expression.In ovo Zn injection improved the offsprings' growth performance and carcass trait,but had no effect on the embryonic and offsprings' antioxidant ability and epigenetic indices.Experiment 4 was conducted to investigate the effect of in ovo Mn injection on the embryonic hatchability of fertilized eggs of Arbor Acres broiler breeders.A total of 800 fertilized eggs with similar weights were randomly allocated to 6 treatments with 6 replicates per treatment in a completely randomized design.The eggs were injected with 0(the negative control),6.25,12.5,25 and 50?g Mn/egg as reagent grade MnSO4·H2O in a 0.1 mL solution,or without injection(the positive control),respectively at E9-10.In ovo Mn injection had no effect(P>0.05)on the mortality and chick hatch weight,but affected(P<0.05)hatchability.Compared to the non-injected control,in ovo injection of 50?g Mn/egg decreased(P<0.05)the hatchability about 14.3%.There were no differences(P>0.05)in the hatchability between the 6.25,12.5 and 25 ?g Mn/egg groups.The results indicated that the maximum level of in ovo Mn injection was 25 ?g Mn/egg.Experiment 5 was conducted to investigate the effect of in ovo Mn injection on the embryonic development,antioxidation and anti-heat stress in offspring broilers based on the results of experiment 4.During embryonic stage,a completely randomized design involving a 1(the non-injected control)+ 1(the injected control with sterilized water)+ 2(Mn source)× 2(Mn level)factorial arrangement of treatments was used.The two injected Mn sources were inorganic Mn sulfate and organic Mn-proteinate chelate with a moderate chelation strength,and the two injected Mn levels were 12.5 and 25 ?g/egg.During progeny stage,offspring broilers from 6 embryonic treatments at 29 d of age were allotted to 2 rearing temperatures(22? vs 34?)on the basis of sex and body weight.The results showed that in ovo Mn injection had no effect(P>0.05)on mortality,hatchability,and healthy chick ratio.In ovo Mn injection increased(P<0.05)MnSOD mRNA expression in the embryonic heart.In ovo Mn injection had no effect(P>0.05)on CuZnSOD activities and MDA levels in the embryonic heart and ADFI,ADG,and F/G at 1-21 and 22-28 d of age.Hyperthermia decreased(P<0.05)ADG and ADFI,increased F/G at 29-42 d of age.Hyperthermia increased(P<0.05)AST and LDH activities,UA and T3 contents,abdominal fat,L value of breast and thigh muscle's color and shear force,decreased(P<0.05)breast muscle,alkaline phosphatase(ALP)activity,and red color of breast and thigh muscle.The results indicated in ovo Mn injection increased MnSOD mRNA expression in the embryonic liver,but had no effect on embryonic hatchability and growth performance at 1-21 and 22-28 d of age.Hyperthermia increased cell injure,decreased carcass traits and meat quality.Experiment 6 was conducted to investigate the effect of in ovo Mn injection on the embryonic development and anti-heat stress in offspring broilers based on the results of above experiments.During embryonic stage,a completely randomized design involving the non-injected control,0(injected control),Zn(100 ?g Zn/egg as reagent grade ZnSO4·7H2O),Mn(25 ?g Mn/egg as reagent grade MnSO4·H2O),and Zn-Mn(100 ?g Zn/egg as reagent grade ZnSO4·7H2O plus 25 ?g Mn/egg as reagent grade MnSO4·H2O)groups.During progeny stage,offspring broilers from 6 embryonic treatments at 29 d of age were allotted to 2 rearing temperatures(22 0C vs 340C)on the basis of sex and body weight.The results showed in oivo injection of Zn and Mn had no effect(P>0.05)on mortality,hatchability,and healthy chick ratio.In ovo Zn and Mn injection had no effect(P>0.05)on MT4 and CuZnSOD mRNA expression in the liver and MnSOD mRNA expression in the embryonic heart.The MnSOD activity in the Zn-Mn group was higher(P<0.05)than the Mn group,but as same as the injected control(P>0.05).In ovo Zn and Mn injection had no effect(P>0.05)on the ADFI,ADG,and F/G at 1-21 and 22-42 d of age.Hyperthermia decreased(P<0.05)ADFI and ADG at the 22-42 d of age,breast muscle and abdominal fat,and red color of breast and thigh muscle,increased(P<0.05)F/G,mortality,TC and T3 contents,L value of breast and thigh muscle,and shear force of thigh muscle.In ovo injection of both Zn and Mn increased(P<0.05)T3 content,but had no effect(P>0.05)on dressing,eviscerated yield,breast and thigh muscle,and abdominal fat.In ovo injection of both Zn and Mn decreased(P<0.05)drip loss.The results indicated in ovo Zn and Mn injection had no effect on embryonic hatchability and offsprings' growth performance.Hyperthermia decreased offsprings'growth performance and meat quality at 22-42 d of age.In ovo Zn and Mn injection increased T3 content and meat quality.The above new research achievements from the aspects of embryonic development,growth performance and antioxidant ability provide scientific evidence to promote the development of microelement nutrition-epigenetics and increase the growth and carcass performance of broilers in the production of broilers. |
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