Regulatory Mechanism Of In Ovo Feeding Of Creatine Pyruvate On Energy Metabolism And Muscle Development Of Broiler Chickens

In ovo feeding(IOF),the administrating exogenous nutrients into the amnion of late-term avian embryos,represents a feasible approach to provide early nutrition in poultry.Creatine pyruvate(CrPyr)is an organic compound that contains pyruvic acid and creatine at a ratio of 40:60.Previous studies have shown that CrPyr can provide two metabolites including pyruvate and creatine to ameliorate energy metabolism and promote the muscle development.However,the information about the impacts of IOF of CrPyr on energy metabolism and muscle development is generally limited.Therefore,the present study was conducted to evaluate the effects of IOF of CrPyr on hatchability,growth performance,energy status and muscle development of late-term embryos and broilers.In addition,research on protein abundance about LKBl/AMPK and Wnt/?-catenin pathway and differential protein profile in breast muscle were carried out to preliminarily explore the possible mechanism.1.The effects of IOF of CrPyr on hatchability,growth performance and energy status of embryos and broilersExperiment 1:A total of 1400 fertile broiler eggs(Arbor Acres)were randomly assigned in a microcomputer automatic incubator.At the end of embryonic day 16,1200 available eggs with similar weight(56.64 ± 0.51 g)were randomly divided into five treatment groups with eight replicates of 30 eggs each.The IOF procedures were performed on embryonic day 17.5.Treatment 1 was non-injected group(Control),treatment 2 was 0.6 mL physiological saline(0.75%)injected group(Saline),treatments 3 to 5 were injected with 0.6 mL physiological saline(0.75%)solution containing 3,6 or 12 mg CrPyr/egg(CrPyr3,CrPyr6 or CrPyr12),respectively.Upon hatch,all male hatched chicks from one treatment were pooled and weighted.Eighty male chicks with similar weight close to the average body weight(BW)of their pooled group were selected and randomly assigned into eight replicates of 10 chicks within each treatment.The duration of the experiment was 21 days.The results showed that there was no difference on hatchability among treatments(P>0.05).The hatching weight and BW of birds in CrPyr12 were significantly elevated compared with the control and saline groups at 3 and 7 days post-hatch(P<0.05).On 21 day post-hatch,the BW was greater in CrPyr6 and CrPyr12 than the control group(P<0.05),respectively.Birds showed similar growth performance among treatments during 1-7 days post-hatch(P>0.05).However,the chickens in CrPyr6 and CrPyr12 exhibited higher body weight gain and feed intake than the control and saline groups during 8-21 days post-hatch and the entire experiment period(P<0.05).All treatments had similar feed/gain ratio irrespective of the growth period(P>0.05).IOF of CrPyr linearly and quadratically increased the hatching weight,BW on 3,7 and 21 day post-hatch,body weight gain and feed intake during 8-21 days post-hatch and the entire experiment period.Compared with the control and saline groups,all IOF of CrPyr groups increased the yolk sac weight and relative yolk sac weight on 19 d of incubation(19 E),the day of hatch and 3 day post-hatch(Linear and Quadratic,P<0.05).Significant increases in total and relative weight of pectoral muscle were observed in CrPyr6 and CrPyr12 treatments compared with the control and saline groups at 19 E,hatch,3 and 21 days of age(P<0.05).Increasing levels of CrPyr linearly and quadratically(P<0.05)increased the total and relative weight of pectoral muscle of broilers on 19 E,the day of hatch,3,7 and 21 day post-hatch.Compared with the control and saline groups,IOF of CrPyr increased the concentration of pyruvate in plasma on the day of hatch and 3 day post-hatch(Linear and Quadratic,P<0.05),as well as the concentration of creatine in plasma on the day of hatch(Linear and Quadratic,P<0.05).The pyruvate concentration in liver of CrPyr-treated broilers was higher than those in other treatments on 19 E(P<0.05).The concentrations of glycogen were increased in CrPyr6 and CrPyr12 in comparison to the control,saline and CrPyr3 groups in liver at 19 E(P<0.05).Meanwhile,these two groups had higher concentration of glucose than other samples at 19 E and hatch in liver(P<0.05).At 3 day post-hatch,birds in CrPyr6 and CrPyr12 had higher concentration of glycogen in liver than other groups.There were significant positive effects on concentration of creatine in pectoral muscle compared with the control and saline groups in all IOF of CrPyr groups at 19 E and hatch(P<0.05).Additionally,the concentrations of phosphocreatine were higher in all CrPyr-treated groups than the control and saline groups at 19 E(P<0.05).IOF of CrPyr linearly and quadratically increased the concentrations of pyruvate and glucose in liver on 19E and the day of hatch,the concentrations of glycogen in liver on 19E,the day of hatch,3 and 7 day post-hatch,and the concentration of creatine in pectoral muscle on 19E,the day of hatch,and 3 day post-hatch,as well as the concentration of phosphocreatine in pectoral muscle on 19E.2.The effects of IOF of CrPyr on hatchability,growth performance,the activities and gene expression of keys enzymes involved in energy metabolismExperiment 2:A total of 1200 fertile broiler eggs(Arbor Acres)were randomly assigned in a microcomputer automatic incubator.On embryonic day 16,960 eggs(64.50 ± 0.38 g)with viable embryos were randomly divided into three treatments with eight replicates of 40 eggs each.The IOF procedures were performed on embryonic day 17.5.The three experimental treatments included the following:Treatment 1 was non-injected group(Control),treatment 2 was 0.6 mL physiological saline(0.75%)injected group(Saline),treatments 3 was injected with 0.6 mL physiological saline(0.75%)solution containing 12 mg CrPyr/egg(CrPyr).Then,120 healthy male chicks per treatment with similar weight close to the average BW of their pooled group were selected and randomly assigned into eight replicates of 15 birds each.The duration of the experiment was 42 days.No difference on hatchability was observed among treatments(P>0.05).The hatching weight and BW of birds in CrPyr were significantly elevated compared with the control and saline groups on 3,7,21 and 42 days post-hatch(P<0.05).The broilers in CrPyr treatment had increased body weight gain and feed intake in comparison to the control and saline groups during 1-21,22-42 and 1-42 days post-hatch(P<0.05).CrPyr-treated broilers showed significant increased concentration of ADP and decreased concentration of ATP on 19 E,as well as increased concentration of ATP and decreased concentration of ADP on the day of hatch(P<0.05).CrPyr supplementation increased the concentration of plasma insulin on the day of hatch and 3 day post-hatch,as well as the concentration of plasma T4 on the day of hatch.A notable increase in activity of creatine kinase in pectoral muscle was observed in IOF of CrPyr group on 19 E when compared with the control and saline groups(P<0.05).Simultaneously,the activity of glucose-6-phosphatase in liver in CrPyr group was higher than other treatments(P<0.05).The activities of hexokinase(HK)and pyruvate kinase(PK)of breast muscle in CrPyr group were found to be higher than those in the control and saline groups on the day of hacth(P<0.05).Compared with broilers in the control and saline groups,CrPyr-treated broilers showed significant up-regulation mRNA expressions of pyruvate carboxylase(PC),phosphoenolpyruvate carboxykinase(PEPCK)and glycogen synthase(GYS)in liver on 19 E and the day of hatch(P<0.05),as well as the mRNA expressions of glucose transporters(GLUT3 and GLUT8).IOF of CrPyr increased the protein abundance of phosphorylated LKB1 and AMPK in breast muscle of broilers on the day of hatch(P<0.05).However,neither LKB1 nor AMPK protein abundance was altered by CrPyr supplementation on the day of hatch(P>0.05).3.The effects of IOF of CrPyr on muscle development,meat quality and regulatory mechanism of broiler chickensThe experimental design was as same as experiment 2.The results showed that the embryos and broilers subjected to CrPyr treatment had higher total and relative breast muscle weights than those of the control and saline groups on 19 E,the day of hatch,3,7,21 and 42 day post-hatch(P<0.05).There were no differences on the muscle pH values(pH45min and PH24h),meat color(L*,a*and b*),drip loss,cooking loss and shearing force among the three treatments(P>0.05).On the 3,7,21 and 42 day of age,the myofiber diameters and cross-sectional areas of breast muscle in CrPyr group were found to be higher than those in the control and saline groups(P<0.05).Neither myofiber diameter nor cross-sectional area was altered by CrPyr supplementation on 19 E and the day of hatch(P>0.05).The index of SCMA in the CrPyr group was significantly higher than that of the control and saline groups on 3 day post-hatch(P<0.05).The mRNA expression of MyoD was observed to be significantly upregulated in breast muscle tissues of CrPyr-treated broilers on the day of hatch and 3 day post-hatch(P<0.05).Compared with broilers in the control and saline groups,CrPyr-treated broilers showed significant upregulation of myogenin and Pax7 mRNA(P<0.05)and downregulation of myostatin mRNA(P<0.05)on 3 day post-hatch.The broilers in CrPyr treatment showed significant upregulation of wnt3a mRNA(P<0.05)and downregulation of GSK-3?(P<0.05)on 3 day post-hatch compared with broilers in the control and saline groups.IOF of CrPyr decreased the protein abundance of phosphorylated GSK-3? and ?-catenin in breast muscle of broilers on 3 day of hatch(P<0.05),as well as the protein abundance of GSK-3?.CrPyr supplementation increased the protein abundance of ?-catenin on 3 day of hatch(P<0.05).4.The effects of IOF of CrPyr on difference of protein profiles in breast muscle of broiler chickensThe experimental design was as same as experiment 2.The differences of protein profiles in breast muscle of broilers on the day of hatch and 3 day post-hatch were identified with iTRAQ proteomic technique.Compared with the control group,IOF of showed up-regulated of 22 different proteins and down-regulated of 11 different proteins on the day of hacth.In addition,IOF of CrPyr showed up-regulated 11 different proteins and down-regulated of 9 different proteins on 3 day post-hacth.After Gene Ontology(GO)analysis,the differential proteins in CrPyr group on the day of hatch were related to several biological processes as follows:muscle development,system development,anatomical structure development,organ development,muscle filament sliding,actin-myosin filament sliding,ventricular cardiac muscle tissue morphogenesis,cell differentiation,muscle tissue morphogenesis,regulation of ATPase activity,muscle fiber development and so on.The differential proteins in CrPyr group on the day of hatch were related to several cell components as follows:muscle myosin complex,myosin II complex,myosin filament,sarcomere,contractile fiber part,actin cytoskeleton and so on.The differential proteins in CrPyr group on the day of hatch were related to several molecular functions as follows:actin-dependent ATPase activity,calcium-dependent ATPase activity,lipase binding,enzyme inhibitor activity,structural constituent of muscle and so on.After Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis,the differential proteins in CrPyr group on the day of hatch were related to tight junction and thyroid hormone signaling pathway.After Gene Ontology(GO)analysis,the differential proteins in CrPyr group on 3 day post-hatch were related to several biological processes as follows:organonitrogen compound metabolic process,small molecule metabolic process,oxoacid metabolic process,organic acid metabolic process,cell development,carboxylic acid metabolic process and so on.The differential proteins in CrPyr group on 3 day post-hatch were related to several cell components as follows:cytoplasmic part,cytosol,collagen type I trimer and so on.The differential proteins in CrPyr group on 3 day post-hatch were related to several molecular functions as follows:extracellular matrix structural constituent,fatty acid binding,ADP binding,acetoacetyl-CoA reductase activity,tRNA adenylyltransferase activity and so on.After Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis,the differential proteins in CrPyr group on 3 day post-hatch were related to several pathways as follows:PPAR signaling pathway,tight junction,PI3K-Akt signaling pathway,pyruvate metabolism,AMPK signaling pathway and glutathione metabolism.As mentioned above,the conclusions are as follows:(1)IOF of CrPyr increased the body weight of broilers on 42 day post-hatch.It is suggested that IOF of CrPyr might be an effective technique to stimulate avian embryo development and increase the market weight of growing chickens.The appropriate injection level of CrPyr was recommended at 12 mg/egg in the present study.(2)IOF of CrPyr enhanced the concentrations of glucose and glycogen in liver of embryos and neonatal broilers,possibly through increasing the activity of liver glucose 6-phosphatase enzyme and up-regulating the mRNA expressions of pyruvate carboxylase(PC),phosphoenolpyruvate carboxykinase(PEPCK)and glycogen synthase(GYS).(3)IOF of CrPyr increased the total and relative weight of breast muscle on 42 day post-hatch,and improved the breast muscle growth of broilers.Nevertheless,IOF of CrPyr did not affect the meat quality of breast muscle.IOF of CrPyr increased satellite cell mitotic activity of broilers on 3 d post-hatch,possibly through up-regulating MyoD,myogenin,and Pax7 mRNA expression and down-regulating myostatin mRNA expression.The effect of CrPyr on satellite cells may be associated with the Wnt/?-catenin signaling pathway.(4)This study investigated the effects of IOF of CrPyr on protein profiles of breast muscle on the day of hatch and 3 day post-hatch with iTRAQ proteomic technique.Compared with the control group,IOF of showed up-regulated of 22 different proteins and down-regulated of 11 different proteins on the day of hacth.In addition,IOF of CrPyr showed up-regulated 11 different proteins and down-regulated of 9 different proteins on 3 day post-hacth.After GO and KEGG analysis,IOF of CrPyr could regulate the energy metabolism of neonatal broilers by regulate the ATPase activity,carbohydrate metabolism,Lipid metabolism,thyroid hormone signaling pathway and AMPK signaling pathway.In addition,IOF of CrPyr could regulate the breast muscle development by regulate muscle fiber development,proliferation and differentiation of cells and protein synthesis.