Mechanism And Regulation Of Functional Amino Acids (Leucine And Arginine) On Milk Protein Synthesis In Dairy Cows

Functional amino acids are not only building blocks for the protein but also bioactivators involving in the signal transduction in cell.Many studies have demonstrated that functional amino acid plays an important role in the regulation of milk protein synthesis.This experiment was conducted to study the effect of leucine and arginine on milk protein synthesis via assessing the change of nutrients metabolism and endocrine of dairy cows,and analyzing the expression of amino acid transporters and key signals related casein synthesis of bovine mammary epithelial cell(BMEC)using the nutrients infusion technique in vivo and BMEC culture technique in vitro.Experiment 1.A study of comparing the amino acid supplies of jugular and mammary gland in lactating dairy cows.Three lactating Holstein cows(BW 523±27 kg)averaging 88±13 DIM were used.The study was conducted to compare the plasma flow of jugular and mammary gland in dairy cows using the downstream dilution method and Fick principle,respectively,and to analyze the relationship of amino acid supplies in two sites.All experimental cows were surgically equipped with a permanent catheter inserted into left carotid artery in one week before the experiment.Temporary indwelling catheters were placed in the left V.jugularis externa for infusion of the blood flow marker para-aminohippuric acid(pAH)on the day before the experiment.The results show that the mean of mammary plasma flow(MPF)tended to be higher than the one of jugular(P=0.085).The supplies of threonine(Thr)(r=0.768),valine(Val)(r=0.483),isoleucine(Ile)(r=0.573),leucine(Leu)(r=0.7),histidine(His)(r=0.844)and arginine(Arg)(r=0.569)existed strong correlation between mammary gland and jugular.Although the correlation of supplied methionine(Met)(r=0.161),phenylalanine(Phe)(r=0.339)and lysine(Lys)(r=0.383)between the two sites was weak,because the concentrations of these three AA between the coccygeal artery and the jugular(C/J are 97.2%,108.1%and 97.2%respectively)were similar,additionally,the plasma flow of mammary gland was higher than that of jugular vein,the absolute supplies of them were still enough for mammary gland,hence,it is feasible that jugular-infused method is used to monitor the metabolism regulation of amino acids on mammary gland in dairy cows.Experiment 2.Milk protein responses to jugular-infused amino acid mixtures of the two profiles in lactating dairy cows.Eight lactating Holstein cows averaging 82 ± 11 DIM were used for this study.The cows were randomly assigned to 1 to 2 treatments in a block design with a 5-d infusion period.The treatments were randomly distributed as jugular infusions of(1)EAA mixture imitating the composition of casein(160 g EAA,Casein),and(2)EAA mixture of recommended model(160 g EAA,R),and taken themselves before infusion as control respectively(C1 and C2).The cows were given a 14-d adaptation period before exposure to the experimental treatment.Total mixed ration(TMR)was adopted and formulated by corn grain,soybeen meal,cottonseed meal,corn silage,alfalfa hay and Chinese wildrye according to NRC(2001).The results demonstrate that milk protein yield and content had upward trends with Casein treatment after infusion(7.14%,P=0.078;3.27%,P=0.072),in addition,the arterious plasma concentrations of Ile,Leu,Lys and His were increased in different extent(31.5%,P=0.097;65.9%,P=0.041;36.9%,P=0.088;40.1%,P=0.01),the arterious plasma concentrations of Thr,Val,Met,Phe and Arg were higher numerically in Casein treatment than the C1,but no significant difference.Infusion of ideal amino acid profile tended to increase the milk protein content(5.78%,.P=0.064)and decrease markedly the milk fat yield(8.57%,P=0.015)than C2 treatment.There were an upward trend of Arg concentration(18%,P=0.093)of arterious plasma and a downward trend of tyrosine(Tyr)concentration(47.8%,P=0.074)in R treatment after infusion.However,the increment of milk fat yield(P=0.012)of Casein treatment was higher than that of R treatment,and the increments of milk yield(P=0.095)and milk lactose yield(P=0.091)tended to be higher for cows receiving EAA infusion of casein profile than for those receiving EAA infusion of ideal profile,but the increments of other parameters were not different between Casein treatment and R treatment(P>0.05).Under this experiment condition,the milk protein content of lactating dairy cows can be increased by the jugular infusions of amino acids mixtures of casein and ideal profiles,and the milk protein yield has an upward trend when lactating dairy cows are received jugular infusion of amino acid mixture of casein profile.So,the application effect of jugular-infused amino acid mixture of casein profile is superior to the jugular-infused amino acid mixture of ideal profile.Experiment 3.Milk protein responses to jugular-infused amino acid mixture of casein profile with or without exclussion leucine or arginine in lactating dairy cows.Eight healthy Holstein cows averaging 108±11 DIM and 509±56 kg of body weight were used for this study.The cows were randomly assigned to 5-d continuous jugular infusions of saline(CTL),EAA mixture prepared on the profile of casein(Casein),EAA mixture excluding Leu(-Leu)or EAA mixture excluding Arg(-Arg)in a duplicated 4×4 Latin square design with 4 infusion periods separated by 7-d interval period.Total mixed ration(TMR)was adopted and formulated by corn grain,soybeen meal,cottonseed meal,com silage,alfalfa hay and Chinese wildrye according to NRC(2001).The results reveal that dry matter intake was not affected by treatments.The Casein treatment increased milk yield(P<0.001),milk protein yield(P<0.001)and milk protein efficiency(P=0.099)than CTL treatment.However,the-Leu treatment decreased the responses of above measured parameters(P<0.06)(compared with Casein).These effects of Leu were related to decreased Leu(48%,P<0.001)concentration and improved concentration of Ile(34%,P=0.006)and Val(20%,P=0.008)in plasma.The-Arg treatment decreased the plasma Arg(16%,P=0.073)concentration than the Casein treatment,whereby resulted in the decrease of milk yield(P=0.073),milk protein yield(P=0.011)and milk protein efficiency(P=0.037).Apparent digestibilities of nutrients and haematological parameters of dairy cows was not affected by infusing amino acid mixtures(P>0.05).In conclusion,supply of balanced EAA profile can improve the lactation production in dairy cows.When the supply of Leu dropped to 8.4%MP,the response of milk protein was partly inhibited by colimiting effect of Leu and other BCAA due to a plasma flow of low-Leu,high-Ile and high-Val into the mammary gland of dairy cows.There was a negative effect on milk protein synthesis when the supply of Arg reduced to 4.6%MP.Arg and Leu are important functional amino acids for producing milk protein,as evidenced by an inhibition of low supply of these two amino acids on milk protein synthesis under this experiment condition.Experiment 4.Leucine modulate mTOR signaling pathway via amino acid transporters(LAT1)in bovine mammary epithelial cellThrough the test of selecting the starvation time of bovine mammary epithelial cell(BMEC),12 hours was determined as the optimum starvation time in the following study when BMEC was cultured in starvation medium 1.BMEC was used to culture in individual prepared mediums of(1)amino acids of casein composition(CTL),(2)removal of Leu from casein profile(-Leu),(3)removal of Gln from casein profile(-Gln),and(4)removal of Leu and Gln from casein profile(-Leu-Gln).The results display that the expressions of L system amino acid transporter(LAT1)(P<0.05)were inhibited when BMEC was cultured in the absence of Leu and Gln solely or both of the medium,meanwhile,the expressions of Leucyl-tRNA synthetase(LARS)(P<0.05)were declined significantly in-Leu treatment and-Leu-Gln treatment,but no significant decrease in-Gln treatment.As expected,the expressions of mTOR and p-mTOR were decreased in different extent,so that the downstream signaling molecules S6K1 activation(P<0.05)were inhibited markedly,accordingly resulted in the expressions of κ-casein(P<0.05)were reduced obviously when the medium was short of Leu or Gln or both.Therefore,Leucine may mediate mTOR signaling pathway to promote the synthesis of κ-casein via stimulating the activation of LAT1 and LARS.Above all,it is feasible that jugular-infused method is used to monitor the regulation of amino acids on mammary gland in dairy cows.Supply of balanced amino acids to dairy cows can improve milk protein efficiency,and milk protein synthesis will be inhibited when the ratio of supply of Leu or Arg and metabolic protein is down to 8.4%or 4.6%respectively in dairy cows.Leucine may mediate mTOR signaling pathway to promote the synthesis of K-casein via stimulating the activation of LAT1 and LARS in vitro study.