Study On Egg Production Performance Of FLHS Laying Hens And Their Ovarian H3K27ac Modification Induced By High Energy And Low Protein Diets

Fatty liver hemorrhage syndrome(FLHS)is a commonly chronic nutritional metabolic disease in the poultry industry characterized by hepatic hemorrhage,steatosis,and decreased egg production,which causes serious economic losses to layer breeding industry.It has shown that acetylation(Acetylation,ac)of lysine 27(Lys,K)on histone H3 protein subunit--H3K27 ac,as a marker of activity enhancer and promoter,it plays an important role in mammalian reproductive health.To investigate the epigenetic regulation mechanism of FLHS in laying hens affecting egg production performance induced by high-energy and low-protein diet,90 150-day-old Hy-Line Brown laying hens were randomly divided into two groups of 45 hens each;the control group(CON)was fed corn-soybean meal basal diet and the experimental group(HELP)was fed high-energy and low-protein diet to induce FLHS disease model;on the80 th day of the feeding trial,the index of egg production performance of each group were measured,and the ovarian tissues were collected for RNA-seq and ChIP-seq.The main results were listed as follows.1.The results showed that compared with the control group,the number of dominant follicles(small yellow follicles and hierarchical follicles),average egg production rate,average egg weight,egg specific gravity,egg shell thickness,egg white weight,egg yolk weight and yolk ratio in HELP group were highly significantly decreased(P < 0.01);Albumen height and Haugh unit(HU)were highly significantly increased(P < 0.01).2.The results of egg white proteomics analysis showed that compared with the control group,the down-regulated proteins were mainly some intrinsic proteins in egg whites with antibacterial and protease inhibitory abilities(TF,OIH,OVM),and antioxidant protein(PDIA6);the up-regulated proteins were mainly some(metal)hydrolases(ANPEP)and certain exogenous proteins involved in the immune response,inflammatory response,transport and the structural composition(RARRES2,APOB,VTG2,GOLM1).In addition,MUC6,a key protein affecting HU,was significantly upregulated;GO analysis showed that differential proteins were mainly involved in biological processes,including lipid transport/localization,transport and response to(external)biological stimulis;KEGG analysis showed that differential proteins were mainly enriched in signaling pathways related to the immune system,digestive system,endocrine system,and cell growth and death;it was hypothesized that the elevation of HU may be associated with increased expression of MUC6 and CLU.3.The results of RNA-seq analysis showed that compared with the control group,377 differential genes were screened;GO enrichment analysis showed that the up-regulated differential genes were mainly involved in biological processes related to protein hydrolysis,biomolecule synthesis,biomolecule metabolism and its regulation,while the down-regulated differential genes were mainly enriched in biological processes related to biomolecular transport including lipid,amino acid and ion.The analysis of KEGG results revealed that differential genes were significantly enriched in signaling pathways associated with follicle development such as steroid hormone biosynthesis and neural ligand-receptor interactions,suggesting that changes in the expression of genes such as HSD3B1,SRD5A2,NPY,NPBWR1,INHA on these pathways may be directly responsible for affecting egg production performance.4.The results of H3K27 ac ChIP-seq analysis showed that the differential peaks were associated with 4074 genes(peak-genes).KEGG enrichment analysis showed that peakgenes were significantly enriched in signaling pathways related to egg production performance,including Gn RH signaling pathway,m TOR signaling pathway,steroid hormone biosynthesis,neuroactive ligand-receptor interactions,Fox O signaling pathways,TGF-β signaling pathway,apoptosis,and cell aging/cycle.The acetylation of key peakgenes such as CYP17A1,EGFR,TGFB1,IGF1 R were significantly decreased.It was hypothesized that the high-energy and low-protein diet in this experiment might reduce the synthesis and secretion of reproductive hormones,cell proliferation,differentiation,and promote apoptosis in the ovary,resulting in fewer oocytes and granulosa cells,more atretic follicles,and impairing follicular development,which in turn affected the egg production performance.Further motif analysis showed that differential peaks were significantly enriched in transcription factors(TFs)such as ZFX,Zac1,and GATA6,indicating that these TFs may be potential regulators affecting egg production performance.5.The results of combined analysis of ChIP-seq and RNA-seq showed that 35 of the314 down-regulated genes were regulated by H3K27 ac,including SYT1,INHA,and NPBWR1,genes directly related to ovarian function.Further protein interaction network analysis(PPI),the top 20 core genes were screened,including SLC2A2(core node of protein interaction network),PCK1,APOB,APOA4,ACSM3 and CYP3A7,among which the expression of SLC2A2,CL2,APOA4 and PCK1 were regulated by H3K27ac;it was speculated that these genes may serve as key candidate genes for high-energy and lowprotein diets affecting egg production performance.In conclusion,this experiment clarified that high-energy low-protein diets can seriously affect the egg production performance,and we revealed the changes in the expression of different functional genes and the level of H3K27 ac modification in the ovary under FLHS,which provide a theoretical basis for analyzing the intrinsic causes of FLHS affecting egg production performance at the transcriptomic and epigenetic levels.