Study On Molecular Mechanism Of Genistein Regulating Lipid Metabolism And Immune Function Of Broiler Breeders And Their Offsprings

In our study,we used the laying hens,broiler breeder hens and their offspring broilers as the animal object.The four experiments were conducted to clarify the effects and molecular mechanism of genistein(GEN)on the lipid metabolism,immunity and bone status of broiler breeder hens and their offspring.Exp.l was designed to clarify the efficacy and regulatory mechanismof GEN on fatty liver syndrome(FLS)in laying hens during the late egg-laying period.Three treatments were set up with nine replicates each.Laying hens in the control group(CON)were fed with a high-energy and low-choline diet to establish the FLS model.The livers of hens from CON group were pale yellow,friable and swollen,presenting with haemorrhagic sign;hepatocyte steatosis and inflammatory cell infiltration exist around the blood vessels,which suggested the FLS model was established successfully.Hens in the low-genistein(LGE)and high-genistein(HGE)groups were fed GEN at 40 mg/kg and 400 mg/kg diet,respectively.40 mg/kg GEN significantly increased the mRNA expression of gonadotropin-releasing hormone(GnRH)in the hypothalamus,the serum level of estrogen,and the egg-laying rate;while 400 mg/kg GEN supplementation down-regulated the expression of GnRH and the laying rate.Both high-and low-dose GEN treatment could alleviate lipid metabolic disorders and inflammatory response in laying hens with FLS.GEN treatment significantly decreased the serum levels of glutamic-pyruvic transaminase,creatinine,triglyceride,total cholesterol and free fatty acids.Meanwhile,the levels of triglyceride,long-chain fatty acids(LCFAs),n-6/n3 family fatty acids in the livers of the LGE and HGE groups compared with the CON group.PCR analysis indicated that dietary GEN significantly down-regulated the expression of genes related to fatty acid synthesis(SREBP1c,LXRa,FAS and ACC),as well as the expression of fatty acid transporter(FAT).Furthermore,GEN treatment up-regulated the transcription of genes related to fatty acid oxidation(PPARa,PPARδ,ACOT8,ACAD8 and ACADs)in the liver and reduced the expression of PPARγ and AFABP in abdominal fat,in addition to decreasing the abdominal fat rate.Compared with the CON diet,GEN treatment alleviated inflammatory cell infiltration in the livers of the LGE and HGE groups,down-regulated the expression of TNF-α,IL-6 and IL-1β.Moreover,it significantly increased the activity of total superoxide dismutase and reduced malondialdehyde in the liver.In conclusion,GEN supplementation in feed can alleviate the lipid metaboliam disorder and the inflammatory response in laying hens with FLS.Exp.2 was to clarify the effects of dietary GEN on the growth development,lipid metabolism and imunne function of broiler breeder hens and their embryos.A total of 720 broiler breeder hens were randomly allocated into three groups and fed with GEN at 0,40 and 400 mg/kg respectively.Then,the eggs were collected and hatched according to breeder groups.Dietary GEN significantly increased the egg production and eggshell strength.It could deposit into the eggyolk,and decrease the content of malondialdehyde in the eggyolk and follicle.The levels of vitellogenin,progesterone and FSH in serum were elevated by dietary GEN.Furthermore,it down-regulated the mRNA expression of insulin-like growth factor-binding protein in fallopian tube,while 40 mg/kg GEN up-regulated estrogen receptor a expression.This additional level has advantage over the improvement of laying performenace.Transcriptome analysis showed that GEN 400 mg/kg promoted the process of bone mineralization and phosphorus metabolism in chickens,which had the advantage in the improvement of bone status.In short,GEN supplementation in the feed can improve the laying performance,egg quality and bone strength of breeder hens during the late egg-laying period.In the aspect of metabolism,GEN supplementation in the diet of breeder hens reduced total cholesterol and LDL-cholesterol in the serum,as well as triglyceride and free fatty acids in the liver.Gene Ontology(GO)clustering showed 400 mg/kg GEN enhanced the hepatic ATPase and ATP binding;40 mg/kg GEN treatment promoted lipid binding,carboxylic acid metabolism,and enhanced phospholipase activity.It also activiated PPAR signaling pathway with the up-regulated down-stream genes(APOAV,FABP3,FABP4,FABP7,PEPCK,PLIN,ANGPTL4).GEN treatment promoted glucose-6-phosphoric acid anabolism with the activiated insulin signaling pathway.As to immunity,40 mg/kg dietary GEN promoted antigen presentation process of MHC class I compex,anti-viral response and immune system function of breeder hens,as well as up-regulated the expression of IL16R,IL20R,IL22R and IL18R in the liver.We found that GEN treatment increased the white cells,B lymphocyte transformation rate and IgA in the peripheral blood.400 mg/kg dietary GEN significantly increased the levlel of IgM.Thus,dietary GEN can enhance the immune function of laying broiler breeder hens during the late egg-laying period.However,the different expressed genes between high-and low-GEN treatment were clustered into estrogen respond precess,carboxylic acid metabolism,immune response,myocyte development,JNK cascade and insulin receptor signaling pathway.40 mg/kg GEN supplementation for breeder hens increased embryonic length,weight and liver index,as well as the width of the proliferative zone in the tibial growth plate of chick embryos.Transcriptome GO cluster analysis showed that GEN treatment promoted system development and cell proliferation of embryos with the up-regulated expression of IGF2.40 mg/kg GEN treatments increased IGFBP3 mRNA expression in the embryonic liver,while 400 mg/kg GEN treatment increased IGFBP5 expression and activated the apoptosis process.Furthermore,martemal GEN effect influenced metabolic process in the liver of embryos:(1)It promoted the glycolysis process in the embryonic liver through the insulin signaling pathway with the up-regulated target genes(PGM2,HKDC1,ALDOC,PFKP,EN02).High-dose GEN treatment up-regulated the transcriptional level of hexokinase(HK).(2)It also enhanced the metabolism of carboxylic acids,and up-regulated the transcriptional level of solute carrier family(SLC),PNPLA8,LIPG,CETP,LPL and the genes relating to VLDL and HDL remodeling,which improved the tansport of carboxylic acids and cholesterol.(3)It promoted the synthesis of unsaturated fatty acid(arachidonic acid)in the liver through LXR,SREBP-1 and long chain fatty acid elongation factor(ELOVL4,ELOVL5).(4)It increased fatty acid β-oxidation and Na+/K+-ATPase activity in the embryonic liver through activation of PPARs(PPARα and PPARδ)and the AMPK signaling pathway,which could provide energy for embryonic development.In addition,GEN treatment increased superoxide dismutase activity and metallothionein expression in the chick embryonic liver,and promoted lymphocyte proliferation through CDKN1A,IL12RB1,Soxll,PRKAR1A,PRKCQ and TCF3.In conclusion,adding GEN to the diet of breeder hens can enhance the development and metabolism of chick embryos with the improved immunity and antioxidant capacity.Exp.3 aimed to clarify the effects of GEN supplementation in the diet of breedr hens on the growth,lipid metabolism and antioxidative capability of male offspring chicks.We chose the male offspring chicks of breeder hens supplementing with 0 and 40 mg/kg GEN from Exp.2.They were grouped according to the mother generation with 8 replicates each.Hepatic transcriptome data revealed 3,915 differentially expressed genes(DEGs,P-adjusted<0.05,fold change>1.5 or fold change<0.67)between chicks in the two groups.Adding 40 mg/kg GEN into the diet of breeder hens activated the GH-IGF1-IGFs receptor signalling pathway,which promoted developmental and cellular amino acid metabolic processes and inhibited the apoptotic process of male chicks.Maternal GEN effect significantly increased the weight gain,breast muscle percentage and liver index of male chicks.PANTHER clustering analysis suggested maternal GEN enhanced the antioxidant activity of offspring via the up-regulation of gene expression(SOD3,MT1 and MT4).Accordingly,T-AOC and T-SOD activity of chick livers were increased after GEN treatment on breeder hens.Overrepresentation tests revealed that maternal GEN effect enhanced the glycolysis,unsaturated fatty acid biosynthesis,acyl-coenzyme A metabolism,lipid transport and cholesterol metabolism into cholic acid in the liver of chicks.It increased the level of VLDL in the liver and HDL in the serum of chicks,and decreased the serum level of LDL,free fatty acids and chelosterol.CHIP-qPCR anylysis revealed that maternal GEN effect might induce histone H3-K36 trimethylation on the PPARδ gene(PPARD)promoter through Iws1 and methyltransferases,as well as induce histone H4-K12 acetylation at the PPARD promoter through MYST2,which activated PPAR signalling pathways and promoted fatty acid β-oxidation in the liver of chicks.Thus,GEN Treatment significantly decreased Liver cholesterol and long-chain fatty acid levels.However,the level of arachidonic acid in the livers of the GEN-treated group was higher than those of the CON group.In summary,40 mg/kg GEN supplementing for breeder hens could improve the antioxidative capability and growth performance of male chicks,as well as enhance glycolysis,cholesterol metabolism and intracellular amino acid metabolism in the liver.It also promoted fatty acid P-oxidation in offsprings through epigenetic modification.In Exp.4,to further determine whether maternal GEN have different effect on the antioxidative capability and growth performance of female and male chicks.Chicks of each sex were respectively divided into 4 treatments.We supplied dietary genistein for breeder hens(F0)and their offsprings(FI).The broilers are divided into 4(male)+ 4(female)treatments.breeders and offspring from CC group were fed with the basal control diet(BD);breeders from CG group were fed with BD and their offspring fed with 40 mg/kg GEN;breeders from GC group were supplemented with 400 mg/kg GEN and offsprings fed with BD;breeders and offspring form GG group were all fed with GEN.The results showed that GEN supplementation for offsprings decreased FCR of female broilers during 1-21 d.GEN treatment on breeder hens and offsprings had interactive effects on body weight gain of female broilers(1-21 d),female chicks in GG groups had the most body weight gain.However,GEN supplementation for breeder hens significantly reduced the body weight gain and feed intake during the whole period(1-42 d)of female broilers with the increased FCR.GEN supplementation for offspring significantly increased the body weight gain of female chicks and decreased FCR during 1-42 d;it also significantly increased the carcass weight,eviscerated weight,semi-eviscerated weight and breast muscle rate of female broilers at 42 d,as well as decreased the abdominal fat percentage significantly.GEN treatment on breeder hens and offsprings might regulate the growth and development of female broilers through estrogen,triiodothyronine(T3)and growth hormone.During 1-21 d,supplementing offspring male chicks with 40 mg/kg GEN decreased the feed intake and PCR;GG group has the best effect on weight gain.During 1-21 d,The addition of GEN in F0 and F1 had an interaction effect on body weight gain of male offspring chicks;supplementation of GEN in feed at F1 stage had a tendency to reduce PCR of male chicks.The liver organ index of the GEN-treated groups was significantly increased.Adding GEN 400 mg/kg in the diet of breeder hens increased the serum growth hormone level,but it had effect on estrogen and thyroid hormone.We further study the effects of supplementing breeder hens and their offspring with dietary GEN on the growth performance and antioxidation of male chicks.We observed that GEN treatment increased the body weight gain,tibial length and tibial width of male broilers at 21 d with the reduced feed weight ratio.Transcriptome data revealed that GEN treatment affected the growth and development of male broilers significantly.Furthermore,the assembly of skeletal muscle myosin rosy filaments and the growth of skeletal muscle were enhanced after GEN treatment,with the up-regulated transcription level of MBNL1,PDZ and LIM domain protein.Compared with the control group,GEN treatment up-regulated the growth hormone level in the serum and the mRNA expression of IGF-I and IGFBP1 in the livers of male chicks,as well aincreased the liver index,slaughter rate,breast rate and leg muscle rate of male broilers significantly.Supplementing GEN for either breeder hens or their offsprings can up-regulated the mRNA expression of metallothionein(MT),and increased the activity of SOD and GSH-Px in the livers of male broilers on day 21d.It could improve the antioxidative capability of male and female chicks with additive effects.In summary,supplementing breeder hens and their offspring with dietary GEN could enhance the capability of male and female chicks,as well as improve the growth performance through GH-IGFs-IGFBPs system.It might inhibit the growth of female broilers at the late growth stage through estrogen and T3.In conclusion,adding GEN into the feed could allievate the metabolic disorder and inflammatory reaction in laying hens induced by FLS.It could also improve the laying performance,egg quality and bone strength of broiler breeder hens during the late egg-laying period.Maternal GEN effect promoted the growth as well as lipd and glucose metabolism,and also improved the antioxidative capability and growth performance of offspring embryos.There were differences in the above functions of chick embryos between high-dose and low-dose GEN treatment.Adding GEN in the brrder hens’ diet can improve the antioxidant capacity and growth performance of the male broilers,and promote glycolysis,cholesterol metabolism and Amino acid synthesis,as well as enhance the process of fatty acid beta oxidation through epigenetic modification;supplementing breeder hens and their offspring with dietary GEN could improve the growth performance of chicks through GH-IGFs-IGFBPs system.It might inhibit the growth of female broilers at the late growth stage through estrogen and T3.