Effect Of Dietary Methionine On Growth,development And Hepatic LncRNA Gene Expression In Lambs

This study was conducted to investigate the effects of dietary methionine on growth and development and hepatic LncRNA genes expression in Lambs.Twin lambs weaned at 8 days old were selected as the experimental model with a matched-pairs design,normal methionine level and low methionine level diets were fed from 8 to 56 d of age,then all lambs consumed the same methionine level diets from 57 to 84 days.The growth performance,slaughter performance,development of organs and the apparent digestibility of nutrients,and hepatic lncRNA genes expression were determined to explore the regulatory mechanism of methionine on lambs growth and development.The outcome of this study was expected to promote the development of young animal cultivation.The present study covered five trails as follows:Experiment 1: Effects of dietary methionine on growth performance,nutrient digestion and serum parameters of lambsThis study aimed to investigate the effects of dietary methionine on growth performance,nutrient digestion and serum parameters of lambs.Twelve pairs of male Hu twin lambs weaned at 8 days old were selected and divided into two groups(control,CON;low methionine level,LM)with a matched-pairs design.During 8~56 days,lambs were fed with milk replacer and basal starter in CON,while LM group were fed the milk replacer and starter deducting 0.70% and 0.4% Met compared on control group basis,respectively.During 57~84 days,all lambs were fed with basal starter.Two periods of digestion trials were undertaken at 46 to 55 and 74 to 83 days of age using 4 pairs of twin lambs,respectively.In addition,blood samples collected from jugular vein at 56 and 84 d of age before the lambs slaughtered.The results showed that: body weight,body length,heart girth,body length indexes,apparent digestibility of nutrients(CP,EE,NDF),serum GH and INS at 56 days,the average daily gain,feed conversion rate during 8~56 days of LM was lower than lambs in CON(P<0.05).But no significant differences were observed in growth performance,nutrients digestibility,and serum parameters between groups(P>0.05)at the 84 days.The results suggested that low dietary methionine decreased nutrients digestibility,feed conversion rate,and growth performance.But a subsequent recovery appeared after the compensation of dietary methionine for 28 days.Experiment 2: Effects of dietary methionine on the visceral organ development and morphological development of the gastrointestinal tract of lambsThis study aimed to investigate the effects of dietary methionine on the visceral organ development and morphological development of the gastrointestinal tract of lambs.The experiment design and animals were the same as Experiment 1.Six twins each time were slaughtered at 56 and 84 days for weighting visceral organs and separating gastrointestinal tracts,while rumen fluid sampling after the slaughter to measure pH.The results showed as follows: Live body weight before slaughter,empty body weight,carcass weight,rumen weight,ruminal papillary width of 56-day-old lambs in LM group were lower than that in CON group(P<0.05).But no significant differences were observed in slaughter performance,visceral organ development,gastrointestinal tract morphological development between groups(P>0.05)at the end of 84 days.The results showed that dietary low methionine suppressed rumen development and decresed slaughter performance.But a subsequent recovery appeared after the compensation of dietary methionine for 28 days.Experiment 3: Effects of dietary methionine on the hepatic methyl metabolism of lambsThis study aimed to investigate the effect of dietary methionine on the hepatic methyl metabolism of lambs.The experiment design and animals were the same as Experiment 1.Blood samples collected from jugular vein at 56 and 84 d of age before the lambs slaughtered,six twins each time were slaughtered at 56 and 84 days for separating hepatic and liquid nitrogen preservation.The results showed that: The hepatic Met content and ratio of SAM/SAH were higher,while the BHMT was lower in LM compared with CON lambs at 56 days(P<0.05).But the hepatic content of Met,SAM,SAH,the key enzyme(BHMT,MAT,GNMT)of one carbon metabolism in LM were lower than CON lambs at 84 days(P<0.05).The results suggested that low dietary methionine levels significantly decreased the content of BHMT and improved the level of methionine in liver of lamb,and significantly affect the ratio of SAM/SAH,decreased levels of methyl donor,affect normal lamb methyl metabolism;recovery of dietary methionine,the liver metabolism of methionine cycle related indicators failed to fully restore with methionine supplementation,and show the delay effect.Experiment 4: Effects of dietary methionine on the hepatic glucose and lipid metabolism of lambsThis study aimed to investigate the effect of dietary methionine on the hepatic glucose and lipid metabolism of lambs.The experiment design and animals were the same as Experiment 1.Six twins each time were slaughtered at 56 and 84 days for separating the liver and liquid nitrogen preservation.The results showed that: the hepatic glucose and glycogen,TG,TC,TBA,the lipid metabolism key enzymes(ACC,FAS,SCD),and the cholesterol metabolism key enzymes(HMG-CoA)were lower in LM than CON at 56 days(P<0.05).The hepatic glucose,glycogen,LEP,HDL,LDL,TBA,the gluconeogenic key enzymes(PEPCK2,FBP1,G6 PD,and the cholesterol metabolism key enzymes(HMG-CoA,HMG-CoA reductase)of 84-day-old lambs in LM group were lower than that in CON group(P<0.05).The results suggested that dietary low methionine inhibited glucose and lipid metabolisms and their substrats production.These effects were failed to restore with methionine supplementation.Experiment 5: Effects of dietary methionine on hepatic LncRNA genes expression in lambsThis study aimed to investigate the effects of dietary methionine on hepatic LncRNA genes expression in lambs.The experiment design and animals were the same as Experiment 1.the hepatic tissue samples were collocted at 56 and 84 days for the determination of gene exression.The results showed that: low dietary methionine caused 1473 differential expressed mRNAs with 953 up regulated and 520 down regulated,and caused 28 differential expressed LncRNAs with 16 up regulated and 12 down regulated at 56 days.GO enrichment histogram showed that differential mRNAs were mainly reflected in the cell metabolism and binding functions,Differential LncRNAs were mainly reflected in the inorganic anion transport and binding functions.The KEGG enrichment analysis showed that dietary low methionine regulated the both mRNA and LncRNA target genes over 20 pathways.mRNA KEGG enrichment is mainly reflected in the sulfur metabolism,inositol phosphate metabolism,cytoplasmic DNA sensing pathway and nod receptors signaling pathways.LncRNA KEGG enrichment is mainly reflected in the polysaccharide biosynthesis,sheath sugar lipid biosynthesis and metabolism of nicotinic acid and nicotinamide and glyceride metabolism.Low dietary methionine caused 898 differential expressed mRNAs with 249 up regulated and 649 down regulated,and caused 23 differential expressed LncRNAs with 7 up regulated and 16 down regulated at 84 days.GO enrichment histogram showed that differential mRNAs were mainly reflected in the nucleotide sugar metabolism,phosphorylation,activate the enzyme activity,catalytic activity and binding functions,Differential LncRNAs were mainly reflected in the chromate salt transportation role,chromate across the plasma membrane functions.The KEGG enrichment analysis showed that dietary low methionine regulated the both mRNA and LncRNA target genes over 20 pathways.mRNA KEGG enrichment is mainly reflected in the folic acid metabolism,primary immunodeficiency,ABC transporters and glycerol phosphate metabolism.LncRNA KEGG enrichment is mainly reflected in the ketone body synthesis and degradation and metabolism of amino acid metabolism,sulfur and selenium metabolism.The results showed that dietary low methionine changed the hepatic mRNA and LncRNA gene expression pattern,and these effects were not fully restored with methionine supplementation.In conclusion,the hepatic LncRNA and mRNA gene expression patterns are greatly influenced by dietary methionine,which inhibited the glucose and lipid metabolism,changed the methyl production,decreased rumen development and nutrients diegestibiliy,and finaly retated the growth and slaughter performance.The phenotypes,e.g.growth and slaughter performance,were recovered while some metabolic and genotype were failed to fully restore with methionine supplementation.