| It has become the restrictive factors of genetic potential and growth performance developing of broilers that nutrition regulation and feeding management are out of line with genetic improvement for decades.The period of early embryogenesis,which are vulnerable to the environment(such as nutrition,climate and so on),is critical for the establishment of the epigenome,and become the focus of epigenetics studies.Unlike in mammals,in poultry,embryonic development is mainly separated from the matrix.It is therefore difficult to estimate the effect of nutrients on embryogenesis if nutrients exhibit little or no accumulation(such as vitamin C)in eggs.Currently,in ovo feeding(IOF)is gradually operational and can thus be used to evaluate the effect of specific nutrients on epigenome reprogramming,providing an effective approach to the study of nutri-epigenetics.We try therefore to obtain DNA methylation variation trends during embryogenesis of chicken firstly,which will lay a good foundation for suitable time choosing of in ovo vitamin C feeding.Then we explore the effect of in ovo feeding of vitamin C on embryonic development,growth performance and immune function of broilers via in ovo experiments and next generation sequencing.Experiment 1This experiment was conducted to develop the HPLC for genomic DNA methylation levels detection,with which we can explore the genomic DNA methylation pattern of chick embryo tissues during embryogenesis.Firstly,we improved the HPLC on DNA extraction,DNA enzymolysis system and detection condition.Then we detected the DNA methylation levels of heart,liver and muscle during the whole stage of embryonic development of AA broiler breeder eggs respectively with HPLC.Results showed that DNA methylation levels of all the tissues increased with embryonic development and the whole stage of embryogenesis can be divided into three phases: embryonic age(E)2~4,E5~14,E15~19.Moreover,the methylation level of liver was significantly higher than that of heart and muscle in later embryonic development(P<0.05).It is concluded that genomic DNA methylation level rises gradually with chick embryonic development.Otherwise,the developed HPLC laid a good foundation for genome DNA methylation determination of tissues rapidly and accurately.Experiment 2Experiment 2 was conducted to reveal the DNA methylation patternof specific genes during the embryonic developmentof chicken.We firstly identified the degree of DNA methylation of the promoters involved in two specific genes(IGF2 and TNF-?)using the bisulfite sequencing polymerase chain reaction method,and then went deep into the analysis of gene bodiesmethylation status of IGF2 and TNF-?.In addition,we measured the expression levels of IGF2,TNF-? and DNA methyltransferase(DNMT)1,3a and 3b.Our results showed that in both the muscle and liver,the promoter methylation levels of TNF-? first increased and then decreased,whereas the gene body methylation levels remained lower at E8,E11 and E14 before increasing notably at E17.The promoter methylation level of IGF2 decreased persistently,whereas the methylation levels in the gene body showed a continuous increase.No differences in the expression of TNF-? were found among E8,E11 and E14,whereas a significant increase was observed at E17.IGF2 showed increasing expression level during the examined embryonic stages.In addition,the mRNA and protein levels of DNMTs increased with increasing embryonic ages.These results suggest that the genomic DNA methylation levels in tissues are closely related to the genes expression levels,and gene expression may be simultaneously regulated by promoter hypomethylation and gene body hypermethylation.Experiment 3This study was conducted to investigate the effect of in ovo feeding of vitamin C at embryonic age 11 on growth performance,oxidation resistance and immune function of broilers.A total of 240 hatching eggs were randomly divided into 2 groups(0 and 3 mg injection of vitamin C at embryonic age 11),and chicks hatched from each treatment were randomly divided into 6 replicates with 10 broilers per replicate after incubation.The results indicated that in ovo vitamin C injection improved the hatchability,the average daily feed intake during the late and the whole growth stage,and feed conversion ratio as well as average daily gain during the early growth stage.Simultaneously,in vitamin C group,an increase(d 1)and an ascending tendency(d 21)was observed in plasma ascorbic acid content.Stimulation index(d 35),IgA and IgM concentrations(d 1 or/and 21),as well as plasmic lysozyme activity(d 21)and T-AOC(d 42)of broilers showed a dramatic increase in vitamin C-exposed group.In ovo vitamin C injection also improved villus height(VH)and the ratio of villus height to crypt depth(V/C)of duodenum(d 21)and V/C of ileum(d 42)of broilers.On day 21,the splenic expressions of IL-4,GCN5,DNMT1 and DNMT3 awere up-regulated,whereas IL-1?,Tet2,Tet3 and Gadd45?weredown-regulated,in 3 mg vitamin C treatment group.On day 42,the splenic expressions of IL-4,GCN5,Tip60,DNMT3 a and Gadd45?were up-regulated,whereas IFN-?,Tet3,MBD4 and TDGwere down-regulated,in vitamin C group.In addition,mRNA levels of SVCT1 and SVCT2 showed a decreasing tendency in the spleen(d 42)of broilers.Our findings suggest that in ovo feeding of 3 mg vitamin C at embryonic age 11 can improve,to some extent,the growth performance,antioxidant activity,immune function and intestine morphology,and the enhance of immunity may associate with the rising of DNA methylation and histone acetylation levels of chick embryo spleen.Experiment 4Experiment 4 was conducted to investigate the effect of in ovo feeding of vitamin C at embryonic age 15 on growth performance,oxidation resistanceand immune function of broilers.A total of 240 hatching eggs were randomly divided into 2 groups(0 and 3 mg injection of vitamin C at embryonic age 15),and chicks hatched from each treatment were randomly divided into 6 replicates with 10 broilers per replicate after incubation.The results indicated that in ovo vitamin C injection improved the hatchability.Simultaneously,in vitamin C group,an increase(d 1 and 42)was observed in thymus index.IgA(d 1),IgG and IgM(d 21)concentrations,as well as plasmic lysozyme activity(d 21)and T-AOC(d 42)of broilers,showed a dramatic increase in vitamin C-exposed group.In ovo vitamin C injection also improved VH(d 42),crypt depth(d 21)and V/C(d 42)of duodenum.Simultaneously a decrease in V/C of ileum(d 21)and a decreasing tendency in VH of ileum,VH and V/C of jejunum(d 42)was observed in vitamin C treatment.On day 21,the splenic expressions ofDNMT1 were up-regulated,whereas IL-6,IFN-?,Tet1 and TDGweredown-regulated,in 3 mg vitamin C treatment groups.On day 42,the splenic expressions of GCN5,DNMT1,DNMT3 B and Gadd45?were up-regulated,whereas IL-6,TNF-? and MBD4weredown-regulated,in vitamin C group.Our findings suggest that in ovo feeding of 3 mg vitamin C at embryonic age 15 can improve,to some extent,the antioxidant activity,immune function and intestine morphology,and the enhance of immunity may associate with the rising of DNA methylation and histone acetylation levels of chick embryo spleen.Experiment 5This study was conducted to reveal differentially expressed genes(DEG)and related regulatory pathways in brephic vitamin C-exposed spleen by virtue of transcriptome sequencing(RNA-seq).A total of 120 hatching eggs were randomly divided into 2 groups(0 and 3 mg injection of vitamin C at embryonic age 11).15 well-developed whole spleen were collected and mixed from each treatments at embryonic age 19,then tissue RNA was extracted and RNA-seq was carried out.Results showed that RNA-seq generated 12 Gb effective and high-quality data and the efficiency of alignment with reference genome were 90.76% and 91.41% respectively.Simultaneously,we got 184 DEGs,among which 117 genes were up-regulated and 67 genes were down-regulated,in the aggregate.With GO and KEGG Pathway enrichment analysis,we found that these DEGs were involved in immune adjustment,embryonic development,histone modification and reproduction.These results suggest that in ovo feeding of vitamin C may take part in the regulation of embryonic development,immunity and epigenetic modification.In addition,we discover 1719 new genes and 1601 were annotated successfully,which further perfecting Gallus gallus genome.Experiment 6This study was conducted to reveal differentially methylated genes(DMG)and related regulatory pathways in brephic vitamin C-exposed spleen by virtue of whole genome bisulfate sequencing(WGBS).A total of 120 hatching eggs were randomly divided into 2 groups(0 and 3 mg injection of vitamin C at embryonic age 11).15 well-developed whole spleen were collected and mixed from each treatments at embryonic age 19,then tissue DNA was extracted and WGBS was carried out.Results showed that WGBS generated 98 Gb effective and high-quality data and average sequencing depth was 32 X.The results of DNA methylation analysis showed that DNA methylation pattern of spleen was changed with brephic vitamin C supplementation,and vitamin C's function of DNA demethylation mainly play in CHG and CHH sites,as well as protein-coding regions.Simultaneously,we got 5514 DMGs in the aggregate.With GO and KEGG Pathway enrichment analysis,we found that these DMGs were involved in immune adjustment,embryonic development,epigenetic modification,reproduction and oxidoreduction.These results suggest that in ovo feeding of vitamin C may change the methylation status of related genes in the process of embryonic development,immune adjustment and epigenome reprogramming.In addition,we reveal whole-genome-levels methylation profile of chick embryo spleen,which may lay a good foundation for following related research,for the first time.Experiment 7In this study,we aim to carry out a combinatory analysis of the data got from RNA-seq and WGBS,and try to find common genes between DEGs and DMGs,as well as to conduct related functional analysis.Results showed that 59 common genes,among which the rate of DNA demethylation was 98.3%(VC vs NS),involved in immune adjustment,embryonic development and epigenetic modification were found.Simultaneously,we found that vitamin C supplemented via IOF had played widespread demethylation function and may participate in the regulation of embryonic development,immunity and epigenetic modification.Our findings reveal the relationship between DNA methylation and gene expression in the case of brephic vitamin C supplementation,which will offer some thoughts about related research.We also found that vitamin C can played widespread demethylation function in vivo and regulate gene expression for the first time.In addition,results suggest that DNA demethylation of gene bodies might not only up-regulate gene expression but also down-regulate gene expression.In summary,in ovo feeding of vitamin C might affect embryonic development via widespread DNA demethylation,and enhance the relationship between immune function and epigenetic regulation. |
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