Effect Of In Ovo Feeding Of Vitamin C On Splenic Purine Nucleotide Metabolism And Apoptosis Of Broilers

With the shortening of the market cycle of broilers,the proportion of the incubation period in the whole growth period is increased correspondingly.Therefore,the nutritional state of embryonic period is more important for regulating the development of tissues and organs in broilers.Meanwhile,the epigenetic reprogramming process occurs in the early stage of embryonic development,which makes the epigenome more sensitive to the environment.By this time,the change of nutritional status of embryo can affect the growth performance by regulating metabolism and gene expression in chicken.It has become a research trend for nutritionists,which control the growth of chicken by adding exogenous nutrients to the eggs through in ovo feeding(IOF)technology.Our previous study found that in ovo feeding of vitamin C could improve the immune response and anti-stress ability of broilers.And the growth and maturity of immune organs were the basis for immune functions.Then,whether the regulation of vitamin C on immune functions was related to the regulation of metabolism and development of immune organs during pre-and post-hatch period in broilers.In this study,the potential mechanism of vitamin C regulating spleen growth and maturation in broilers was explored at embryonic period,adult period and cell level respectively by means of in ovo feeding and primary culture of spleen cells in vitro.EXP.1 The potential pathway of in ovo feeding of vitamin C to regulate spleen development in broilersA total of 120 fertilized eggs of AA broiler were randomly divided into 2 treatments.The eggs were injected of vitamin C at embryonic age 11.The injection procedure is as follows: 1)the control group(CON)was injected with normal saline into the yolk,and the injection volume was 0.1 m L;2)the experimental group(VC)was injected with the same volume of vitamin C solution with concentration of 30 mg/m L.On the embryonic age 19,20well-developed embryo eggs were selected from each treatment and spleen tissues were collected.Spleen samples from CON and VC group were mixed,respectively.Then,RNA was extracted for transcriptome sequencing.The results showed that: 1)Pathway enrichment analysis of the Gene Ontology(GO)based on differentially expressed genes showed that in ovo feeding of vitamin C regulated the various biological processes of spleen development in embryonic period,such as phosphorus metabolism,nucleotide metabolism and biosynthetic,purine nucleotide metabolism and multicellular biological reproduction,etc;2)KEGGpathway which involved in purine metabolic pathway,fatty acid metabolism and degradation pathways,propionate metabolic pathway and vascular smooth muscle contraction pathway,ect.Among them,The most significantly level was occurred in purine metabolic pathway,followed by fatty acid metabolic pathway.The results of this experiment showed that in ovo feeding of vitamin C could extensively regulate splenic metabolic pathways and biological processes in embryonic period.Combined with the result of GO and KEGG analysis,it was suggested that purine nucleotide metabolism might be a potential way for vitamin C to regulate spleen development.EXP.2 The effects of in ovo feeding of vitamin C on splenic purine nucleotide metabolism and cell apoptosis in broilersA total of 240 fertilized eggs of AA broiler were randomly divided into two treatments.The control group and vitamin C group were injected with normal saline and vitamin C solution at a concentration of 30 mg/m L into the yolk at embryonic age 11,respectively,with the injection volume of 0.1 m L.The well-developed chicks were selected from each group after incubation and randomly divided into 6 replicates with 10 birds per replicate.The spleen,thymus and bursa samples of broilers were collected at 21 and 42 days.The results showed that: 1)In ovo feeeding of vitamin C significantly increased the splenic vitamin C content on d 21(P<0.05),whereas had no significant effect on vitamin C content in d 42 spleen,also in thymus and bursa.2)In vitamin C group,the splenic expression of adenosine deaminase(ADA)was significantly decreased on d 21(P<0.05),and had no significant effect on the expression of adenine phosphoribosyltransferase(APRT)in d 21 and 42 spleen;3)In ovo feeding of vitamin C significantly down-regulated the splenic expression of AKT-1,PCNA and Bcl-2 on d 21,whereas the expression of Caspase-9 was up-regulated on d 21 and d 42(P<0.05).Our finding suggested that in ovo feeding of vitamin C could specifically increased the splenic vitamin C content in the adult period of broilers,and maintained the regulation effect of vitamin C on purine nucleotide metabolic pathway until the late period of growth.At the same time,to some extent,the apoptosis of spleen cells is promoted by vitamin C supplementation,may associated with splenic maturation.EXP.3 Study of vitamin C regulate metabolism and development of spleen cells in vitroPrimary spleen lymphocytes were isolated from healthy broilers of 21 days.The experiment was designed with control group(CON)and treatment groups with four vitamin C concentration,vitamin C concentrations were set as follows: 250 mg/L(VC-1),500 mg/L(VC-2),1000 mg/L(VC-3)and 2000 mg/L(VC-4),cultured for 24,48 h,respectively.Afterthe treatment,cells were collected and total RNA was extracted.Relative gene expression was detected by RT-q PCR,cell proliferation was detected by MTT,and apoptosis rate was detected by Annexin v-FITC/PI double staining.The results showed that: 1)After 24 h for cultured,the treatment concentration of VC-2 group was taken as the inflection point.With the increased of vitamin C concentration,the regulatory effect of vitamin C on the cell proliferation showed a tendency of first enhancement and then inhibition.After 48 h for cultured,the proliferation of spleen cells was gradually inhibited with the increase of vitamin C concentration.2)The relative expression of APRT,Caspase-9,Cytc were significantly up-regulated in VC-3 and VC-4 groups(P<0.05),whereas the expression of Caspase-3 were significantly down-regulated(P<0.05).3)vitamin C significantly increased the apoptosis rate of spleen cells(P<0.05).4)The expression APRT was positively correlated with the expression of apoptosis-related gene.The results of this experiment showed that vitamin C promoted the apoptosis of spleen cells in a time and concentration-dependent manner,and vitamin C concentration of 1000 mg/L and above could promote the occurrence of apoptosis.Moreover,APRT expression was positively correlated with the expression of apoptosis-related genes Caspase-9,Cytc and Caspase-3,suggesting that the occurrence of apoptosis may be related to the reaction pathway catalyzed by APRT.In conclusion,in ovo vitamin C injection could regulate the splenic metabolic process in the embryonic period,extensively,and it suggested that purine nucleotide metabolic pathway may be a potential way for vitamin C to regulate the growth of spleen.Simultaneously,the regulatory effects of in ovo feeding could continue to the late growth period of broilers.To some extent,vitamin C promoted the apoptosis of spleen cells,thus regulating splenic maturation.Moreover,the apoptosis-promoted of vitamin C may associated with the purine nucleotide metabolic pathway mediated by APRT.