Mechanism Of Folic Acid To Regulate Production Performance Of Female Broiler Breeders And Its Offsprings Muscle Development

Poultry meat,as a high-quality and inexpensive protein,occupies the first place in the total global meat consumption.Genetic selection has laid the foundation for the improvement of broiler production performance.At present,the research on improving broiler production performance from the nutritional level mainly focuses on the breeding stage of commercial broiler chickens,and the regulation of offspring performance from parental nutrition has attracted increasing attention.Folic acid can participate in the process of one-carbon unit metabolism and protein metabolism.Preliminary studies by our group have shown that the addition of folic acid to commercial broiler diets can improve their production performance and promote muscle protein deposition."Fetal programming" suggests that maternal nutrition is crucial to the growth and development of offspring and lifelong health.This study intends to add folic acid to female broiler breeders diets to study its effects on female broiler breeders production performance and folic acid deposition in eggs.Furthermore,the effect of folic acid in hatching eggs mediated by "chick embryo programming" was explored to realize the regulation of offspring production performance,in order to exert the cascade amplification effect of breeding birds,thereby widely affecting offspring production performance.Based on this,in this experiment,by adding folic acid to the diet,the effect on the production performance of breeders and offspring was detected,and the mechanism of folic acid regulating ovarian function and offspring muscle development of breeders was explored by means of transcriptomic technology.To verify the promoting effect of folic acid on muscle proliferation and differentiation,it aims to provide a theoretical basis for the regulation of offspring traits by folic acid nutrition in poultry mothers,and to provide technical support for the implementation of folic acid nutritional interventions in breeder flocks to optimize offspring performance.Experiment 1 Effects of folic acid on performance,ovarian function and folic acid deposition in eggs of female broiler breedersThe purpose of this experiment was to investigate the effect of dietary folic acid supplementation on performance,ovarian function and folic acid deposition in eggs of female broiler breeders.In this experiment,50 30-week-old Arbor Acres female broiler breeders were selected and randomly divided into two treatments after two weeks of pre-feeding,namely the control group(Con: 0 mg/kg)and the folic acid group(FA: 4mg/kg).Groups of 5 replicates,5 mice per replicate,were treated for 16 weeks.The production performance of the female broiler breeders was counted,the folic acid content of eggs and plasma was determined,and the ovary transcriptome analysis of the breeder hen ovaries was performed.The results showed that folic acid in female broiler breeders diets did not affect their average egg weight,egg production rate,egg breaking rate and egg quality;folic acid significantly increased folic acid deposition in breeding eggs(P < 0.05),and decreased hen plasma levels.Homocysteine(Hcy)content(P < 0.05);folic acid significantly reduced proton-coupled folate transporter(PCFT),reduced folate transporter(PCFT)in ovary,liver and jejunum folate transporter(RFC),and folate receptor(FR)mRNA expression(P < 0.05),but significantly increased RFC mRNA expression in the duodenum.A total of 326 differentially expressed genes were identified in the ovary transcriptome of the female broiler breeders in the Con group and the FA group,of which 217 were significantly up-regulated and 109 were significantly down-regulated(P < 0.05).KEGG analysis of differential genes significantly enriched 10 differential metabolic pathways,among which up-regulated differential genes E2F1,CCNA1,PTTG2,CDK2,WIF1,CAMK2 A,WNT4,WNT6,CYP3A5 and CYP17A1 are involved in cell cycle,Wnt signaling pathway and steroid biosynthesis signal path.The above results show that folic acid supplementation in breeder hens’ diets does not affect production performance,but can increase the expression of genes related to ovarian reproductive function and promote the deposition of folic acid in eggs.Experiment 2 Effects of folic acid in female broiler breeders diets on offspring performance and muscle developmentExperiment 1 found that dietary folic acid improved hen ovarian function and increased folic acid deposition in eggs.The purpose of this experiment was to explore the regulatory mechanism of folic acid in female broiler breeders diets on offspring growth and development.In this experiment,the eggs of Con group and FA group in Experiment 1 were selected for incubation,and 48 chicks were selected from each group and randomly divided into 6 replicates,with 8 chicks in each replicate.The test results showed that: folic acid in female broiler breeders diet had no significant effect on hatching weight of offspring chicks,but increased the content of folic acid in plasma of chicks(P < 0.01)and decreased the content of Hcy(P < 0.01).Folic acid in female broiler breeders diets had no significant effect on ADG,ADFI,FCR and liver index of offspring at 14 days,but significantly increased their breast muscle rate and muscle fiber area(P < 0.05).A total of 181 differentially expressed genes were identified by transcriptome analysis of chick breast muscle,of which 111 were significantly up-regulated and 70 were significantly up-regulated(P < 0.05).KEGG analysis of differential genes enriched 58 signaling pathways,4 of which were significantly different(P < 0.05),among which SGK1,WNT16,RNF152,ATP6V1C1 and WNT2 B were involved in the m TOR signaling pathway related to muscle protein synthesis.The above results indicated that folic acid in female broiler breeders diets could affect the folic acid status of offspring,and folic acid deposition in hatching eggs may mediate the m TOR pathway to promote offspring muscle development.Experiment 3 The regulation of embryonic muscle development of offspring by folic acid in female broiler breeders based on blastoderm lncRNA sequencingBased on the dynamic expression rule of folic acid transporter in chicken embryos,this experiment explored the effect of folic acid in female broiler breeders diets on the development of offspring chicken embryos.First,the parental Arbor Acres broiler eggs were selected for incubation,and the expression levels of folic acid transporters in the intestinal and vitelline membranes of chick embryos were continuously detected from the 11 th day of hatching(E11)to the first day after the chicks hatched(D1).It was found that the expression of PCFT and RFC in embryonic duodenum and jejunum was relatively stable in E11-E17,and gradually increased in E17-D1,and the expression level of D1 reached the maximum.Then,the eggs of Con group and FA group in Experiment 1 were selected for incubation,and 8 eggs or chicks were taken from E15,E19 and D1 to measure the expression of myogenic regulators in breast muscle,to explore the effect of folic acid supplementation in breeder hens’ diets on embryonic development of offspring.The test results showed that: folic acid in female broiler breeders diet had no significant effect on embryo weight,liver index and residual yolk index of E15,E19 and D1.Addition of folic acid significantly increased the expression of Myogenic differentiation antigen(MYOD)in E15 pectoralis muscle.A total of 209 differentially expressed mRNAs and 145 differentially expressed lncRNAs were identified by blastoderm transcriptome analysis.KEGG analysis of differential mRNAs enriched a total of 50 signaling pathways,3 of which were significantly different(P < 0.05),among which SDSL,SCTR,GRIA1,HTR1 A,LHCGR,GRID2,GRIN1,PLA2G2 A and GAL3ST1 were involved in amino acid metabolism,signaling molecules and interactions and lipid metabolism.The cis-trans target genes of differential lncRNAs were predicted,and the expression of multiple genes related to muscle development was enriched.The above results indicated that folic acid in female broiler breeders changed the expression of MYOD at the embryonic stage of offspring and the lncRNA expression profile of offspring blastoderm tissue,and lncRNA may be involved in the regulation of muscle development in offspring.Experiment 4 Effects of folic acid on proliferation and differentiation of myoblastsThis experiment aims to demonstrate the effect of folic acid on the proliferation and differentiation of skeletal muscle myoblasts by cell culture.The experiment was divided into 5 treatment groups,with 6 replicates in each group.The cells were inoculated and cultured for 6 h,and the growth cultures containing different folic acid concentrations(4,10,20,40,and 80 mg/L)were replaced.Cell viability assays were performed after h and 48 h.In the differentiation test,when myoblasts grew to 80%-90% confluence,they were cultured for 5 days with different folic acid concentrations(4,10,20,40 and 80 mg/L)in differentiation medium,and observed with Giemsa staining.Myotube formation.The results showed that with the increase of folic acid concentration,10 and 20 mg/L folic acid concentrations could promote the proliferation of myoblasts,but 40 and 80 mg/L showed inhibition;gene expression analysis showed that 20 mg/L folic acid treatment significantly increased the expression of proliferating cell nuclear antigen(PCNA),CCND1(Cyclin D1)and MYOD,the representative markers of myoblasts proliferation,and significantly increased the differentiation process of MYOD and myogenesis The expression level of Myogenin(MYOG)(P < 0.05)significantly decreased the expression level of Myostatin(MSTN)during the differentiation process(P < 0.05).The above results indicated that folic acid could promote the proliferation and differentiation of myoblasts.In conclusion,folic acid in female broiler breeders diets can increase the deposition of folic acid in eggs,which in turn mediates the expression of mRNA and lncRNA in the blastoderm of eggs,regulates the muscle development of embryos and offspring after hatching,and promotes the increase of the breast muscle rate of chicks during the rearing stage.