The Effect Of Supplementing Cholamine On Production Performance, Physiological Parameters And Liver Lipid Metabolism In Laying Hens

In egg production,as the laying period extends,the physiological functions of hens gradually decline,leading to reduced production performance and egg quality,especially when the laying period is extended to 700 days.Hens often experience excessive fat deposition and fatty liver,which further reduces their production performance.Lipids are one of the main components of egg yolk,so factors affecting lipid metabolism may have a significant impact on the production performance and physiological functions of hens.The effects of cholamine,a raw material for synthesis of some active lipids,are unknown in poultry.Previous studies have shown that adding cholamine to feed can affect the composition of intestinal bacteria in livestock and the production performance of animals.However,it is not yet clear whether adding cholamine affects the production performance and physiological functions of hens and the related mechanisms.This experiment first clarified the effects of adding cholamine to feed on the production performance,blood biochemistry,lipid metabolism,intestinal morphology,and intestinal microbiota of hens through feeding trials,and then detected the effects of cholamine treatment on the expression of relevant genes in chicken primary liver cells at the cellular level to provide an explanation for the main observations in the feeding trials.Finally,the DNA polymorphism of the key enzyme ETNK1 involved in cholamine metabolism was screened in a chicken population,and the genes and pathways affected by ETNK1 were further examined by transcriptome sequencing of overexpressed cells to better understand the mechanism of cholamine supplementation on the physiological functions and production performance of hens.In the feeding trial,180 52-wk-old Hyline laying hens were randomly divided into three groups(20 replicates per group with three hens per replicate).The control group was fed the basal diet and the experimental group was fed the basal diet supplemented with 500 mg/kg and 1000 mg/kg low and high concentrations of cholamine.After 35 d of feeding,the results of the study were as follows.(1)The addition of cholamine significantly reduced egg production rate and daily feed intake.Supplementing 500 mg/kg of cholamine to the basal diet led to significant reduction of average daily egg production rate(P<0.001),and supplementing 500 or 1000 mg/kg of cholamine led to significant reduction of average daily feed intake(P<0.01).There was no significant effect of supplementing cholamine on average egg weight,the feed-to-egg ratio,eggshell color,egg shape index,egg weight,albumen height,yolk color,Haugh units,yolk weight,eggshell strength,and eggshell thickness(P>0.05).(2)Dietary supplementation of 500 and 1000 mg/kg of cholamine to the basal diet significantly lowered high-density lipoprotein cholesterol(HDL-C)level(P<0.05),supplementing 1000 mg/kg of cholamine lowered blood glucose level in a tendency(P=0.05),but had no significant effect on other indexes,including alanine aminotransferase(ALT)activity,aspartate transaminase(AST)activity,the ratio of aspartate aminotransferase to the activity of alanine aminotransferase(AST/ALT),Total protein(TP),Albumin,Globulin,the ratio of albumin level to globulin level(ALB/GLB),total cholesterol(TCH),Triacylglycerol and low density lipoprotein-cholesterol(LDL-C)(P>0.05).(3)Supplementing 500 mg/kg of cholamine to the basal diet significantly reduced the absolute weight of the liver and the relative weight to body weight(P<0.05),but supplementing 500 or 1000 mg/kg of cholamine had no significant effect on the absolute or relative weights of the spleen,jejunum,and abdominal fat to body weight(P>0.05).(4)Supplementing 500 mg/kg or 1000 mg/kg of cholamine to the basal diet had no significant effect on to total SOD(T-SOD)activity and MDA content in the liver(P>0.05).Supplementing 1000 mg/kg of cholamine significantly increased triglyceride content in the liver of laying hens(P<0.05).Supplementing 1000 mg/kg of cholamine significantly reduced the relative content of C15:0 in liver fat,but tended to increase the relative content of C20:0、C18:2n-6 and the ratio of n-6 polyunsaturated fatty acids(PUFA)to n-3 PUFA(P<0.10).(5)Supplementing 500 mg/kg of cholamine to the basal diet significantly reduced the crypt depth of the ileum and significantly increased the ratio of villus height to crypt depth(P<0.05),but had no significant effect on villus height.Supplementing 500 mg/kg of cholamine had no significant impact on the thickness of uterine muscular and mucosal layers(P>0.05).(6)Supplementing 500 mg/kg of cholamine to the basal diet reduced the α-biodiversity of ileal microflora,which is indicated by the Chao1 index(from 1563 to 872,P=0.081)and the Observed_species index(from 1100 to 671,P=0.12).Other indexes including Faith_pd,Pielou_e,Shannon and Simpson also decreased but not significantly.Supplementing 500 mg/kg of cholamine had no obvious effect on the α-biodiversity of cecal microflora.The compositional differences were identified between treatment and control groups by principal coordinate analysis using the Bray Curtis distance parameter.In addition,beta diversity analysis indicated that most of the samples from each group were aggregated into the same group,and the samples between the groups could be differentiated.The analysis on the relative abundance of intestinal microflora showed that the dominant phyla in the ileum of the control and cholamine-treated groups were Firmicutes(71.4%,75.4%),Bacteroidetes(18.2%,5.2%),Actinobacteria(5.2%,9.3%)and Proteobacteria(4.1%,9.0%),the dominant genera were Lactobacillus(33.2%,51.7%),Aeriscardovia(3.7%,6.5%),Bacteroides(8.3%,1.7%)and Enterococcaceae_Enterococcus(0.36%,4.5%),while the dominant phyla in the cecum were Firmicutes(51.4%,48.8%),Bacteroidetes(42.5%,47.3%),Actinobacteria(1.6%,1.5%)and Proteobacteria(1.1%,1.0%),and the dominant genera were Bacteroides(12.6%,16.4%),Faecalibacterium(3.7%,7.7%),Lactobacillus(6.0%,4.9%),and Oscillospira(3.0%,2.7%).Supplementing 500 mg/kg of cholamine significantly altered the relative abundance of some intestinal bacteria,i.e.,supplemented cholamine decreased the relative abundance of the major phylum Bacteroidetes(from 18.2%to 5.2%,P=0.13),the major genus Bacteroides(from 8.3%to 1.7%,P=0.13)in the ileum and Ruminococcaceae_Ruminococcus in the cecum(from 1.4%to 0.93%,P<0.10),but increased the relative abundance of Cyanobacteria phylum in the ileum(from 0.051%to 0.15%,P<0.05)and Faecalibacterium genus in the cecum(from 3.7%to 7.7%,P=0.096).(7)After treating primary chicken hepatocytes with different concentrations of cholamine(0,1.5625,3.125,6.25μM)for 14 h,the mRNA expression levels of some genes related to lipid metabolism(APOA4,FABP1,FATP2,MTTP,PPARG,CD36,PCK1,FASN,SCD1,ACOX1)in these cells were determined.Data shows that cholamine increased the expression levels of APOA4,PCK1,MTTP,PPARG,and CD36 genes in primary chicken liver cells,but inhibits the expression level of FABP1,significantly(P<0.05).(8)Transcriptome sequencing analysis showed that supplementing 1000 mg/kg of cholamine significantly affected the mRNA expression levels of 2065 genes:1151 differentially expressed genes(DEGs)were induced whereas 914 DEGs were repressed compared to the control group.The KEGG pathway analysis revealed six pathways were significantly enriched with the up-regulated DEGs,including Ribosome,Oxidative phosphorylation,Proteasome,Protein export.Cardiac muscle contraction,and Terpenoid backbone biosynthesis,and the KEGG pathways significantly enriched with the downregulated DEGs were mainly the Notch signaling pathway.Ten DEGs were selected for validation,including apolipoprotein A4(APOA4),phosphoenolpyruvate carboxykinase 1(PCK1),metallothionein 3(MT3),metallothionein 4-like(MT4L),lactate dehydrogenase B(LDHB),coiled-coil-helix-coiled-coil-helix domain containing 10(CHCHD10),3-hydroxy3-methylglutaryl-CoA synthase 2(HMGCS2),hydroxy acyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta(HADHB),pyrophosphatase 1(PPA1),and avian beta-defensin 9(AvBD9).The quantitative PCR analysis indicated all the selected genes except for MT4L were validated,suggesting that the transcriptome sequencing analysis was reliable.(9)Transcriptome sequencing after overexpression of ETNK1 in chicken primary hepatocytes,and a total of 3406 differential genes were screened,of which 1737 genes were up-regulated and 1669 genes were down-regulated.GO and KEGG analysis showed that ETNK1 was mainly enriched in cytokine-cytokine receptor interaction,cell adhesion molecules(CAMs),and toll-like receptor signaling pathway,which could be related to cell immune response,proliferation and differentiation and inflammatory response.In conclusion,supplementing cholamine to the diet of laying hens decreased blood HDL-C level,the biodiversity of intestinal microflora,and the relative abundance of Bacteroidetes phylum,Bacteroides genus and Ruminococcaceae_Ruminococcus genus,increased the percentage of C18:2n-6 in liver fat,the n-6 to n-3 PUFA ratio,and the villus height to crypt depth ratio of the intestine,induced the expression of FABP1 and APOA4 genes in the liver,and significantly affected the pathways such as oxidative phosphorylation and Notch signaling pathway:cholamine could induce mRNA expression of lipid metabolism related genes such as APOA4,MTTP,and CD36 in primary chicken liver cells;the differentially expressed genes affected by ETNK1 were mainly enriched in cell cytokinecytokine receptor interation,cell adhesion and other pathways.