The Effect Of Supplemental Betaine On Methionine-deficient Or Methionine-excessive Diets By Omics Analysis In Geese

Methionine(Met)is the first limiting amino acid in poultry.Betaine(Bet)can be used as methyl donor to promote the metabolism of nutrient.Bet plays an important role in Met cycle.The objective of this study was to investigate the molecular mechanism of effect of supplemental betaine on methionine-deficient or methionine-excessive diets by omics analysis in geese.1.Effects of dietary Bet replace Met on growth performance and hepatic protein synthesis of geeseSix methionine-deficient diets(Met:0.28%)were prepared according to a 2x3 interaction design,which included 3 methionine levels(0,0.06%and 0.12%),and 2 betaine levels(0 and 0.06%).Three hundred 21-days-old healthy male geese with similar body weight were randomly distributed into 6 groups with 5 replicates per treatment and 10 geese per replicate.All geese hadfree access to diets and water for 49 d.Growth performance,slaughter performance,serum biochemical indices and amino acid availability were determined.The results showed as follows:(1)The body weight of goslings in the 0.12%Met group was significantly higher than that of geese in the group that received no additional Met(P<0.05).Conversely,supplemental Bet did not significantly affect growth performance(P>0.05).Supplemental Bet significantly increased the percentage of eviscerated yield and percentage of half-eviscerated yield(P<0.05).Furthermore,significant interaction effects between Met and Bet were detected for dressing percentage,percentage of half-eviscerated yield,and eviscerated yield(P<0.05).Supplemental Bet significantly increased keel length and shank circumference(P<0.05).(2)Supplemental Met significantly increased serum TP,ALB and GLOB level(P<0.05).Supplemental Bet significantly decreased serum TG level(P<0.05).Supplemental Met significantly increased the coefficient of crude fat retention,and the digestibility of methionine and cysteine of 67-70 d goslings.(3)Supplemental Met significantly increased hepatic crude protein concentration(P<0.05).Supplemental Bet significantly decreased hepatic crude fat concentration(P<0.05).Met significantly increased the content of cysteine and phenylalanine in leg muscle(P<0.05).Bet significantly increased the content of aspartic acid,arginine,valine,isoleucine,leucine and phenylalanine in breast muscle(P<0.05).Furthermore,significant interaction effects between Met and Bet were detected for total amino acid(P<0.05).In conclusion,optimal Met dietary supplementation(0.34%-0.40%)could increase growth performance,hepatic protein synthesis in growing goslings.Furthermore,dietary supplementation with Bet could possibly replace Met for slaughter performance and skeletal development.2.Transcriptome analysis of hepatic gene expression and DNA methylation in Met-and Bet-supplemented geeseThe goal of this study was to identify the effects of dietary supplementation of Met and Bet on growth performance,transcriptomic gene expression,and epigenetic mechanisms in geese on a Met-deficient diet.At 70 d,three geese were randomly selected from Met-deficient group(Control group),Met group(Control+0.12%of Met),and Bet group(Control+0.06%Bet).Liver samples were extracted to obtain total RNA and DNA for RNA-seq and DNA methylation level detection.(1)The transcriptome sequenced 9 liver samples,obtaining 114 460 Transcript and 86 683 Unigene.The percentage of Q30 bases is above 95.34%.The expression of many genes critical for Met metabolism is increased in Met supplementation group.In the Bet-supplemented group,genes involved in energy production and conversion were up-regulated.(2)DNA methylation levels in the promoter regions of IL4I1 and LOC106032502 were quantified using bisulfite sequencing PCR(BSP).No significant difference was observed in the total methylation level in the IL4I1 promoter region as Met exposure increased.Dietary supplementation with Bet and Met altered DNA methylation.We observed changes in the methylation of the LOCI 06032502 promoter and corresponding changes in mRNA expression.In conclusion,Met and Bet supplementation in geese affects the transcriptional regulatory network and alters the hepatic DNA methylation of LOC106032502.3.Establishment of methionine-induced hyperhomocysteinemia model in geeseThe goal of this study was to establish a model of methionine-induced hyperhomocysteinemia geese.One hundred healthy male Yangzhou geese of similar body weight were randomly distributed into 2 groups with 5 replicates per treatment and 10 geese per replicate:Control diet(Control group),Control+1%of Met(Met toxicity group).All geese had free access to diets and water for 56 d.Clinical symptoms serum Hcy level were detected.The results showed as follows:In conclusion,serum Hcy level is a sensitive index to identify hyperhomocysteinemia geese.(hyperhomocysteinemia geese:higher than 30 ?mol/L vs Normal level:16-21 ?mol/L).1.35%DL-methionine could lead to hyperhomocysteinemia in geese.4.Effect of hyperhomocysteinemia on heart tissue in goslings by Whole geneme bisulfite sequencing and LncRNA transcriptome sequencingThe goal of this study was to identify the effects of hyperhomocysteinemia on heart tissue in goslings by Whole geneme bisulfite sequencing(WGBS)and LncRNA transcriptome sequencing.At 70 d,three geese were randomly selected from control group and Met toxicity group from trial 3.Heart samples were extracted to obtain total DNA and RNA for WGBS and LncRNA transcriptome sequencing.The results showed as follows:(1)There were significant differences in the whole genome methylation level between the hyperhomocysteinemia group and the control group.A total of 6 592 differential genes and 18023 differential regions were sequenced.Genome methylation occurs mainly at CG location,and these differentially expressed genes are mainly located in introns(INTRON),accounting for 56.44%of the total DMR in genome structure.(2)Differential methylation level genes were enriched in 126 KEGG pathways,and the differential genes were mainly concentrated in amino acid metabolism,myocardial metabolism and cell apoptosis related pathways.These pathways are closely related to amino acid metabolism,muscle growth and development,and myocardial tissue growth.Among them,p53 signaling pathway and apoptosis pathway are involved in apoptosis.(3)The results of LncRNA transcriptome sequencing showed that 352 differentially expressed mRNAs(192 up-regulated genes and 160 down-regulated genes)were sequenced,and 3 427 differentially expressed LncRNA were screened(1 873 up-regulated genes and 1 554 down-regulated genes).(4)KEGG pathway enrichment analysis showed that 232 mRNAs were enriched in 88 pathways in each group.Apoptosis,vascular smooth muscle contraction,alanine and alanine.and alanine,aspartate and glutamate metabolismin methionine pathway of the geese in toxicity group were significantly up-regulated compared with control group.Phenylalanine metabolism,synthesis and degradation of ketone bodies of the geese in toxicity group were significantly down-regulated compared with control group(5)The results of genomic DNA methylation and LncRNA transcriptome sequencing showed that 5 of 51 GO terms were enriched in genes with negative correlation between methylation level and gene expression level.Among these DMEs,the methylation level of gene body region were negatively regulated with expression level in 26 genes and positively regulated in 25 genes.The methylation level of promoter region was negatively regulated in 26 promoters and positively regulated in 3 promoters.In conclusion,Met toxicity could significantly change the methylation level of gene related in apoptosis,myocardial metabolism and amino acid metabolism pathway,and alter apoptosis and vascular smooth muscle contraction pathway,resulting in hyperhomocysteinemia.It is suggested that the expression level and methylation of gene in promoter region occured mainly by negative regulation,but both of them in genebody region occured by positive and negative regulation.5.Detoxification of betaine on Met-induced hyperhomocysteinemia geeseThe goal of this study was to identify the effects of dietary Bet on Met-induced hyperhomocysteinemia geese.One hundred and fifty 14-day-old healthy male Yangzhou geese of similar body weight were randomly distributed into 3 groups with 5 replicates per treatment and 10 geese per replicate:Control diet(Control group),Control+1%of Met(Met toxicity group),Control+ 0.2%Bet(Bet detoxify group).All geese had free access to diets and water for 56 d.Growth performance,slaughter performance,serum biochemical indices and amino acid availability were determined.The results showed as follows:(1)Hcy for the geese at 28 d,49 d and 70 d in Met toxicity group was significantly higher than those in control group(P<0.05).Hcy for the geese at 70 d in detoxify group was significantly lower than that in Met toxicity group(P<0.05),but has no significant difference with control group(P>0.05).(2)The body weight for the geese at 28 d,49 d and 70 d in Met toxicity group was significantly lower than those in control group(P<0.05).There was no significant difference for the body weight at 70 d between toxicity group and detoxify group(P>0.05).The average daily feed intake and average daily gain for 14-28 d geese in toxicity group and detoxify group were significantly higher than those in control group(P<0.05).(3)The toxicity of Met had bad effect on the feather growth and physicochemical property of feathers on geese.Additional Bet could decrease the feather oxygen consumption index and increase feather quality.(4)Supplemental betaine could promote the synthesis of methionine by increasing the expression of BHMT and MS genes,and alleviate hyperhomocysteinemia in geese.In conclusion,supplemental 1%DL-methionine to basal diet(14-28 d Met:0.35%;28-70 d Met:0.36%)could lead to hyperhomocysteinemia in geese.The growth performance,slaughter performance and nutrient utilization of the geese were reduced.Adding Bet significantly could significantly reduce serum Hcy level,percentage of abdominal fat and percentage of eviscerated yield,and increase the utilization of crude fat,crude ash and amino acid,alleviating hyperhomocysteinemia in geese.6.Confirmation of pathway of key genes and apoptotic pathway in hyperhomocysteinemia geeseThe aim of this experiment was to identify the apoptosis rate of cardiomyocytes in order to verify that the molecular mechanism of hyperhomocysteinemia induced myocardial injury.At 70 d,the heart tissues of 5 goslings were collected from the control group,Met toxicity group and Bet detoxify group.Heart tissue were prepared for TUNEL and immunohistochemistry.In conclusion,Met-induced hyperhomocysteinemia could significantly increase the apoptosis rate of myocardial cells.The molecular mechanism was that the expression of B-cell lymphoma/leukemia-2(Bcl-2)protein was decreased to induce apoptosis of myocardial cells.Betaine could alleviate the apoptosis rate of myocardial cells,but had no significant effect on the p53 apoptosis pathway.Bcl-2 gene is an important gene that inhibits apoptosis in apoptosis pathway.The increase of Bcl-2 methylation level in heart tissue of hyperhomocysteinemia group inhibits Bcl-2 gene expression by negative regulation,leading to cardiomyocyte apoptosis and hyperhomocysteinemia.