Effect And Mechanism Of Betaine On Growth, Meat Quality, And Protein Metabolism Of Partridge Shank Broiler Chickens

The studies on the effect of betaine in broilers production had been reported,but these researches mainly focused on fast-growing broilers,while there were few studies in highquality chickens(partridge shank broiler chickens).Moreover,most of these studies were on the regulation of lipid metabolism,and there was still a lack of in-depth research on the effect of protein metabolism.Muscle growth are closely related to protein anabolism.A systematic study of the effect and mechanism of betaine on protein metabolism is great important to understand how betaine promotes muscle growth of broilers.At present,chemically synthesized anhydrous betaine and hydrochloride betaine are mainly used in feed.However,the molecular structure,physicochemical property,and function of these two betaines may be different due to diverse synthetic processes,and it is still unclear whether these two betaines have different effects in broiler production.Therefore,this study firstly compared the effects of anhydrous betaine and hydrochloride betaine on growth performance,digestive function,and meat quality of partridge shank broiler chickens,and then studied the molecular mechanisms of betaine regulating muscle growth from the perspective of methyl,amino acids,and protein metabolism.The purpose of this study was to provide a theoretical basis for the rational application of betaine in partridge shank broiler chickens diets,and to provide a new nutritional strategy for improving the muscle yield and quality of high-quality broilers.This study consisted of the following five parts:Trial 1 Effects of different levels of anhydrous betaine on growth and meat quality of partridge shank broiler chickensThis trial was aimed to investigate the effects of different levels of anhydrous betaine on growth performance,carcass characteristics,meat quality,myogenic genes expression,and insulin like growth factor-1(IGF-1)signaling pathway in partridge shank broiler chickens.A total of 192 one-day-old partridge shank broiler chickens were randomly divided into 4 groups with 6 replicates of 8 chickens.Broilers were fed a basal diet supplemented with 0,250,500,and 1 000 mg/kg anhydrous betaine(96%),respectively,for a 52-d feeding trial.The results showed that broilers average daily gain(ADG)and breast muscle yield were linearly increased(P < 0.05),and feed gain ratio(F/G)and abdominal fat rate were linearly decreased(P < 0.05)by dietary betaine supplementation.Incremental levels of betaine linearly increased(P < 0.05)total protein(TP)content,and linearly decreased(P < 0.05)the contents of blood urea nitrogen(BUN),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C),and high density lipoprotein cholesterol(HDL-C)in serum of broilers.Dietary betaine supplementation could linearly increase(P < 0.05)breast muscle p H45 min,p H24 h,and crude protein content,and thigh muscle a*45 min,and linearly decrease(P < 0.05)24 h drip loss and cooking loss of breast muscle,and L*45 min,b*45 min,24 h drip loss,and cooking loss of thigh muscle.In addition,with increasing betaine levels,the m RNA expressions of myogenic differentiation factor 1(Myo D1),myogenin(Myo G),and myocyte enhancer 2B(MEF2B)in breast muscle were linearly increased(P < 0.05),myocyte enhancer 2D(MEF2D)in breast muscle was quadratically increased(P < 0.05),and the m RNA levels of Myo G and MEF2 B in thigh muscle were linearly increased(P < 0.05).Incremental levels of betaine linearly increased(P < 0.05)the content of IGF-1,the m RNA levels of IGF-1 and mechanistic target of rapamycin(m TOR),and the protein phosphorylation level of m TOR in breast muscle,and quadratically increased(P < 0.05)the concentration of IGF-1,linearly increased(P < 0.05)the m RNA levels of phosphoinositide 3-kinase(PI3K),protein kinase B1(Akt1),m TOR and ribosomal protein S6 kinase 1(S6K1),and the protein phosphorylation level of m TOR in thigh muscle.Trial 2 Comparison of anhydrous betaine and hydrochloride betaine on growth performance and digestive function of partridge shank broiler chickensThis trial was conducted to compare the effects of anhydrous betaine and hydrochloride betaine on growth performance,carcass characteristics,intestinal morphology,apparent nutrient utilization,and digestive enzyme activity of partridge shank broiler chickens,and to explore the mechanism of betaine affecting digestive enzyme activity through researching digestive enzyme activity and kinetic test in vitro.In vivo test: a total of 400 newly hatched partridge shank broiler chickens were randomly divided into 5 groups with 8 replicates of 10 broilers each.Broilers in the control group were fed a basal diet,and those in the other four experimental groups were fed the basal diet supplemented with 500 or 1 000 mg/kg anhydrous betaine(96%),and 642.23 or 1 284.46 mg/kg hydrochloride betaine(98%),respectively,for a 52-d feeding trial.In vitro test: adding low(0.5 mol/L)and high(1 mol/L)concentrations of anhydrous betaine and hydrochloride betaine to trypsin,amylase,and lipase solutions,respectively,to measure the changes in digestive enzyme activities.The kinetic parameters of the digestive enzymes were determined by changing the substrate concentration,according to Michaelis equation.In vivo,compared with the control group,diets supplemented with low and high doses of anhydrous betaine significantly improved(P < 0.05)the whole-term ADG and 52 d ileum villus crypt ratio,and reduced(P < 0.05)52 d serum D-lactic acid(D-LA)content of broilers.The high-dose anhydrous betaine group further increased(P < 0.05)52 d breast muscle yield,52 d jejunum villus height,49~51 d apparent utilization of organic matter(OM),21 d pancreatic lipase activity,52 d jejunal chyme trypsin activity,and decreased(P < 0.05)whole-term F/G,21 d abdominal fat rate,52 d abdominal fat rate and subcutaneous fat thickness,52 d serum diamine oxidase(DAO)activity.Compared with the control group,diet supplemented with high-dose of hydrochloride betaine decreased(P < 0.05)22~52 d broilers F/G,52 d serum DAO activity and D-LA content,and increased(P < 0.05)52 d jejunum and ileum villus crypt ratio,49~51 d apparent utilization of OM.In terms of main effect of variety,compared with hydrochloride betaine,anhydrous betaine significantly increased(P < 0.05)broilers ADG,average daily feed intake(ADFI),21 d jejunum villus height and villus crypt ratio,52 d jejunum crypt depth,18~20 d apparent utilization of dry matter and OM,21 d pancreatic amylase and lipase activities,21 d jejunal chyme amylase and trypsin activities,and decreased(P < 0.05)21 d broilers abdominal fat rate and subcutaneous fat thickness,21 d serum DAO activity,52 d serum D-LA content,52 d jejunum villus crypt ratio.In vitro,anhydrous betaine had no significant effects on the activities of trypsin,amylase,and lipase,but hydrochloride betaine reduced(P < 0.05)the activity,maximum reaction rate(Vm),and catalytic efficiency,and increased(P < 0.05)the Michaelis constant(Km)of these three digestive enzymes when compared with the control group.In terms of main effect of variety,compared with hydrochloride betaine,anhydrous betaine significantly increased(P < 0.05)the activity,Vm,and catalytic efficiency of trypsin,amylase,and lipase,and decreased(P < 0.05)the Km of above three enzymes.Trial 3 Comparison of anhydrous betaine and hydrochloride betaine on meat quality of partridge shank broiler chickensThis trial was to compare the effects of anhydrous betaine and hydrochloride betaine on meat quality,texture,nutrition composition,relaxometry,glycolysis,and antioxidant capacity of partridge shank broiler chickens.The experimental design was the same to the trial 2.Compared with the control group,diet supplemented with low and high doses of anhydrous betaine significantly decreased(P < 0.05)drip loss24 h in breast and thigh muscles of broilers.The high-dose anhydrous betaine group further increased(P < 0.05)p H24 h,immobile water proportion(P21),the contents of crude protein and glutathione(GSH),the activities of creatine kinase(CK)and glutathione peroxidase(GPx),the m RNA expressions of glucose transporter 4(GLUT4),protein kinase AMP-activated non-catalytic subunit gamma 3(PRKAG3),nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase 1(HO-1)in breast muscle,and a*45 min,GLUT4 m RNA expression in thigh muscle,and decreased(P < 0.05)drip loss48 h,free water proportion(P22),the contents of lactate and malondialdehyde(MDA)in breast muscle.Moreover,the high-dose hydrochloride betaine significantly increased(P < 0.05)p H24 h,P21 proportion,the activities of CK,total antioxidant capacity(TAOC),total superoxide dismutase(T-SOD)and GPx,the content of GSH,the m RNA levels of Nrf2,HO-1,GPx and γ-glutamate-cysteine ligase catalytic subunit(γ-GCLc)in breast muscle,and the activity and m RNA expression of GPx in thigh muscle,and decreased(P < 0.05)drip loss24 h,P22 proportion in breast muscle,and MDA content in breast and thigh muscles.In addition,in terms of main effects,betaine variety had no significant effects on muscle color,p H,water-holding capacity,texture,nutrient composition,glycolysis and antioxidant enzyme activities(P > 0.05).Trial 4 Effects of anhydrous betaine on methyl and amino acids transport and metabolism of partridge shank broiler chickensThis trial was aimed to investigate the effects of betaine on methyl metabolism,serum hormones and metabolites,tissue amino acids contents and amino acids transporters genes expressions in partridge shank broiler chickens.A total of 160 one-day-old partridge shank broiler chickens were randomly divided into 2 groups with 8 replicates of 10 chickens each.Broilers were fed a basal diet(Control group)and a basal diet supplemented with 1 000 mg/kg anhydrous betaine(Betaine group)for a 52-d feeding trial.Compared with the control group,the betaine group significantly increased(P < 0.05)the ADG,and decreased(P < 0.05)the F/G of broilers.Diets supplemented with anhydrous betaine could significantly increase(P < 0.05)the contents of betaine,S-adenosylmethionine(SAM),and SAM/SAH,and decrease(P < 0.05)homocysteine(Hcy)and cystathionine concentration in serum of broilers.Dietary betaine could increase(P < 0.05)the contents of betaine,SAM,and free carnitine in breast muscle,and increase(P < 0.05)the contents of SAM and free carnitine,the activities of betaine homocysteine methyltransferase(BHMT),methionine adenosyltransferase(MAT),glycine-N-methyltransferase(GNMT)and DNA methyltransferase(DNMT),the m RNA levels of BHMT,MAT2 A,S-adenosine homocysteine water-like protein 1(AHCYL1)and DNMT1 in liver of broilers.Moreover,compared with the control group,the betaine group significantly increased(P < 0.05)the concentrations of serum growth hormone(GH),IGF-1,TP,Gly,Ala,Met and Lys,decreased(P < 0.05)serum BUN content,increased(P < 0.05)the activity of aspartate aminotransferase(AST),the content of Ser,Gly,Ala and Met,the m RNA level of ASC amino acid transporter 1(ASCT1)in the liver,increased(P < 0.05)the content of Ser,Glu,Met and Phe,the m RNA levels of ASCT1,y+L amino acid transporter 1(y+LAT1),and proton-assisted amino acid transporter 1(PAT1)in breast muscle,and increased(P < 0.05)the m RNA expressions of peptide transporter 1(Pep T1),bo,+ amino acid transporter(bo,+AT),cationic amino acid transporter 2(CAT2),and y+LAT1 in ileal mucosa.Trial 5 Effects of anhydrous betaine on muscle protein deposition,nucleic acid content,and proteomes of partridge shank broiler chickensThis trial was conducted to investigate the effects of betaine on muscle protein deposition,nucleic acid content,and proteomes of partridge shank broiler chickens.The experimental design was the same to the trial 4.Compared with the control group,the contents of absolute protein deposition,RNA,and the ratio of RNA/DNA in breast muscle were significantly increased(P < 0.05)by dietary anhydrous betaine supplementation,but there was no significant different in DNA content(P > 0.05).Moreover,i TRAQ-base proteomic analysis revealed 35 differentially expression proteins(DEPs)in the betaine group compared with the control group,including 27 up-regulated proteins and 8 down-regulated proteins(P < 0.05).Gene Ontology(GO)enrichment analysis showed that the enriched biological process categories of these DEPs were related to cell differentiation,small molecule metabolism process,tissue development,regulation of hormone levels,and inorganic ion homeostasis.Cytoplasm,intracellular organelle part and macromolecular complex were involved in the cell component categories.And protein binding,RNA binding,protein heterodimerization activity,actin binding and transferring sulfur-containing group activity were the most abundant subcategories in the molecular function categories.In addition,the up-regulated DEPs were mainly involved in cellular processes,metabolic processes,and single-organism process,while the down-regulated DEPs were mainly participated in single-organism biosynthetic process,nucleobase-containing small molecule metabolic process,and single-organism biosynthetic process.In summary,the following conclusions were drawn:1.Diets supplemented with 500 or 1000 mg/kg anhydrous betaine improved the growth performance of partridge shank broiler chickens,and 1000 mg/kg betaine further increased the breast muscle yield.Betaine could improve muscle growth via altering myogenic genes expressions and IGF-1/m TOR signaling pathway.2.Anhydrous betaine was better than hydrochloride betaine in improving broilers growth performance,earlier stage apparent nutrient utilization and digestive enzyme activity.In vitro experiment showed that hydrochloride betaine affected the activity of digestive enzymes mainly by reducing the affinity between enzymes and substrates.3.Anhydrous betaine and hydrochloride betaine supplementation could improve the meat quality of partridge shank broiler chickens by reducing lactate content to increase muscle p H,increasing immobile water proportion and decreasing free water proportion to decrease drip loss,and enhancing muscle antioxidant capacity.4.Anhydrous betaine could promote liver methionine cycle and enhance the methyl metabolism of broilers.In addition,betaine could regulate the amino acids transport metabolism by affecting serum hormone levels,tissue amino acids contents and amino acids transporter genes expressions.5.Anhydrous betaine could increase protein deposition and RNA content in breast muscle of broilers.i TRAQ-base proteomic analysis revealed 35 DEPs in the betaine group compared with the control group,including 27 up-regulated proteins and 8 downregulated proteins.GO enrichment analysis showed that betaine regulated muscle growth mainly by cell differentiation,small molecule metabolism process,tissue development,and regulation of hormone levels.