Effect Of Heat Stress On Protein Synthesis In Mammary Gland Of Dairy Cow And The Relative Mechanism

In dairy cows,mammary gland is an important place for milk protein synthesis.Amino acids are important precursors for the synthesis of milk protein.The bovine mammary gland cells uptake amino acids from blood to synthesize milk protein.The level of blood amino acid and the blood flow through the mammary gland determine the amount of amino acids taken by the mammary gland.It is well known that heat stress negatively influences dairy cow's health and lead to sharp decline in milk production,milk protein synthesis and ultimately,milk protein yield.Traditionally,the main reason for lower dry matter intake(DMI)was considered for lower milk production,milk protein synthesis and ultimately milk protein yield in heat stressed dairy cows.However,the utilization of pair-fed thermal neutral(PFTN)founded that the decrease in DMI during heat stress could only partially explain(35-50%)the reduction in milk production,milk protein synthesis and milk protein yield.Furthermore,it has been established that in addition to the decrease of DMI,blood hormone levels during heat stress can decrease milk protein yield.It has also been founded in different studies that the specific downregulation of milk synthesis activity in mammary gland cells could be the reason for the decrease of milk production,milk protein synthesis and milk protein yield.However,previous findings did not reveal the direct effect of heat stress on the decline in milk protein synthesis.Therefore,it was necessary to explore the direct mechanism of heat stress on milk protein synthesis.In the current study,heat stressed-animal model was established to study the effect of heat stress on the uptake of amino acids by the mammary gland,and RNA-seq technology was used to find the differential expressed genes in mammary gland tissues of non-heat-stressed and heat-stressed cows.At the cellular level,the effect of heat stress on lactation related genes or pathways were studied to reveal the direct impact of heat stress on milk protein synthesis in bovine mammary epithelial cells.On the basis of our study findings,it is concluded that the increased level of Methionine could alleviate the effect of heat stress,and promot milk protein synthesis.Experiment 1 The effect of heat stress on the milk production,milk quality and blood biochemical index of lactating dairy cowsIn spring(March to April 2018)and summer(July to August 2018)period,20 Holstein dairy cows were selected respectively to explore the effect of heat stress on milk production,milk quality and blood biochemical index.All cows in the experiment were similar in parity,days in milk,milk yield,and body weight.The temperature and humidity index(THI)was recorded in the cowshed to establish the heat stressed animal model.The results showed that the average THI(79.5)of cowshed in the summer period was higher(P < 0.05)than that in the spring period(59.8).Heat stress reduced DMI,milk protein yield and milk protein contents of dairy cows(P < 0.05).Heat stress also reduced the level of plasma glucose(P <0.05),but heat stress increased the level of serum Glutamic oxalacetic transaminase,Alkaline phosphatase,Glutamic-pyruvic transaminase and non-esterified fatty acids(P <0.05).However,the content of Thyroxine and the activity of plasma Superoxide dismutase,Glutathione peroxidase and Catalase were decreased(P < 0.05)by heat stress.The concentration of Malondialdehyde,Triiodothyronine,Lipopolysaccharide,Cortisol and Heat shock protein 70 were elevated(P < 0.05)by heat stress.These experiment results indicated that the THI in summer period exceeded the minimum value(THI>68)that caused the heat stress in experimental dairy cows-combined with the significant increase in respiration rate and rectal temperature.Results of THI,respiration rate and rectal temperature indicated that the dairy cows in the summer period were exposed to moderate heat stress.Furthermore,the decline of milk production,milk quality and the alteration in antioxidant,endocrine and immune functions of dairy cows indicated that the heat stressed-animal model was successfully established.Experiment 2 The effect of heat stress on the supply of nutrient and amino acid uptake in mammary glandBased on the successful establishment of heat stressed cows' model in experiment 1,A total of 16 cows(with approximately similar milk yield)were selected to study the effect of heat stress on the supply of nutrient and the uptake of amino acid in the mammary glands.The experiment 2 was started ten days before finishing of experiment I.The analysis of arteriovenous amino acid difference was carried out through the external pudic artery and mammary vein intubation operation.The result of the experiment showed that the mammary blood flow was reduced by heat stress in experimental dairy cows.Result also showed that heat stress significantly increased the blood concentrations of total amino acid,Alanine,Glycine,Isoleucine,Leucine,Threonine and Serine of the external pudic artery(P < 0.05).However,the blood level of Methionine,Lysine and Tryptophan of the external pudic artery were decreased(P < 0.05)due to heat stress in experimental dairy cows.Furthermore,heat stress significantly reduced the uptake of almost all of the essential amino acids by mammary gland(total amino acid,Alanine,Methionine,Glycine,Arginine,Lysine,Leucine,Leucine,Tryptophan,Threonine and Valine)in experimental dairy cows(P < 0.05).Moreover,statistical results explored that the uptake of the first(Metionine)and second(Lysine)limiting amino acids of dairy cows were reduced 53.1% and 24.2% respectively.The results of experiment indicated that heat stress reduced the supply of amino acid and the uptake of amino acid in mammary gland of dairy cows which revealed that the synthesis of milk protein was affected by heat stress.Experiment 3 The effect of heat stress on the transcription level of dairy cow's mammary gland tissueTo reveal the direct mechanism of heat stress on the synthesis of milk protein at the transcriptional level,the third experiment was carried out.Based on the results of experiment II,3 Holstein dairy cows(with approximately similar milk yield,good body condition and similar body weight)were selected for mammary gland tissue sampling in the last week of the experiment.The RNA-seq technology was employed to study the effect of heat stress on the whole gene transcriptional in dairy cow's mammary gland tissue.The objective of the study was to find out the differential expressed genes affected by heat stress.The results of experiement revealed that a total of 213 differentially expressed genes were affected by heat stress,among which 89 genes were up-regulated and 124 genes were down-regulated.Heat stress significantly inhibited the expression of casein genes(CSN1S1,CSN2 and CSN3)(P<0.01)and the genes which related to Bovine leucocyte antigen(BOLA)(BOLA-DYA,BOLA-DRB3).While heat stress significantly increased the gene expression of heat shock protein 70(HSP70)and heat shock Protein 90B1(HSP90B1)in mammary gland tissue(P< 0.05).The gene expression of amino acid transporter(AAT),Late endosomal/lysosomal adaptor,MAPK and MTOR activator 2(LAMTOR2),Late endosomal/lysosomal adaptor,MAPK and MTOR activator 3(LAMTOR3)and E74-like transcription factor 5(ELF5)were significantly inhibited by heat stress(P < 0.05).These results showed that heat stress inhibited antigen processing and presentation signalling pathways.Inhibition of antigen processing and presentation signalling pathways due to heat stress,was not favourable forthe timely clearance of the antigens of mammary gland tissue that may affect the health of heat stressed cow's mammary gland,and ultimately reduced the gene expression of the related genes of lactation.Experiment 4 The effect of heat stress on the apoptosis of bovine mammary epithelial cells and the regulation of mTOR pathwayTo further explore the mechanism by which heat stress may affect the synthesis of protein in mammary gland cells,foruth experiment was carried out.The aim of the experiment was to explore the effect of heat stress on apoptosis of bovine mammary epithelial cells(BMEC),and the gene and protein expression of mTOR signaling pathway by heat treatment of bovine mammary epithelial cell's model.In the present study,there were 2 experimental treatments with 6 replicates per treatment and 1 blank for each replicate was also carried out.The results of current experiment showed that heat stress promoted the apoptosis of BMEC and reduced viability of BMEC(P < 0.05).Heat stress increased the gene and protein expression of Bax(apoptosis-promoting factor),increased the ratio of BAX/Bcl-2,and significantly increased the gene expression of apoptosis genes(CASP3 and CASP9)(P < 0.05).Results also showed that heat stress significantly increased the expression of phosphorylated protein of EIF4EBP1(P < 0.05),and significantly increased the ratio of P-EIF4EBP1/T-EIF4EBP1(P < 0.05),reduced the ratio of PRPS6KB1/RPS6KB1,P-mTOR/T-mTOR and P-AKT/T-AKT(P <0.05).Results of the current experiment further revealed that heat stress significantly reduced the gene expression of casein(CSN1S1,CSN2 and CSN3)and amino acid transporter(SLC7A5,SLC38A3,SLC34A2,SLC38A9)(P < 0.05).The results of the current experiment indicate that heat stress weakened cell activity and promoted cell apoptosis.Moreover,the results also explored that inhibition of the mTOR-RPS6KB1-RPS6 signaling pathway and activation of the mTOR-EIF4EBP1-EIF4 E signaling pathway led to inhibiation of the intracellular protein translation and hence,the cell proliferation and milk protein synthesis were affected.Experiment 5 The effect of increased Methionine content on milk protein synthesis of heat stressed-Bovine mammary epithelial cells Under heat stress conditions,the bovine mammary epithelial cell line(BMEC)was used for cell culture through increasing Methionine(Met)supplementation.Methionine(Met)supplementation was ensured to alters the ratio of Lysine(Lys)to Met that was equal to2.5:1,2.2:1 and 1.9:1 based on the ratio of Lys:Met=2.9:1 which was determined in milk.Bovine mammary epithelial cell were incubated(n = 6 replicates/treatment)for 6 h with 4incremental doses of Met to achieve the following ratios: Lys: Met=2.9:1(ideal AA ratio,CON),Lys: Met=2.5:1(LM2.5),Lys: Met=2.2:1(LM2.2)and Lys: Met=1.9:1(LM1.9).The results of the current experiment showed that increasing Met level enhanced the cell viability and depressed the apoptosis of heat stressed BMEC(P < 0.05).Increasing the level of Met significantly increased phosphorylation level of mTOR,RPS6KB1 and RPS6 protein(P <0.05)and LM2.2 treatment had the most obvious effect.The results also expolored that increasing the Met significantly increased the genes expression of CSN1S1 and CSN2(P <0.05),but had no effect on the gene expression of CSN3,SESN2 and CASTOR2.The results also indicated that under heat stress,increasing the level of Met can alleviate cell apoptosis,and is beneficial to activate the proteins expression of mTOR signalling pathway.The LM2.2treatment had the best effect.However,increasing the level of Met had no significant effect on the upstream signaling pathway of mTOR(SESN2-CASTOR2-mTOR).Based on the results it was concluded that Met promoted protein synthesis mainly by activating mTORRPS6KB1-RPS6 and mTOR-EIF4EBP1-EIF4 E signalling pathways.Moreover,Under heat stress,increasing the Met contents affected the gene expression of AAT,thereby controlled the balance of Met concentration inside and outside of BMEC to ensure the activation of the downstream pathway of mTOR.In conclusion,the temperature and humidity index(THI)of cowshed was significantly increased in summer period.The Increased THI,respiration and rectal temperature of dairy cows which raised in summer period and the dairy cows are in the state of moderate heat stress.Heat stress significantly reduced the production performance of Holstein dairy cows,reduced the nutrient supply to the mammary glands and the uptake of amino acid to the mammary glands of dairy cows.Heat stress down-regulated the gene expression of casein,genes related milk protein synthesis,inhibited the gene expression of antigen processing and presentation signaling pathways,activated the immune response of mammary gland cells,and weakened the immune function of the dairy cows.At the cellular level,heat stress promoted apoptosis of BMEC,mainly by affecting the activity of mTOR downstream signalling pathway(mTOR-RPS6KB1-RPS6 and mTOR-EIF4EBP1-EIF4)to inhibit protein synthesis.Under heat stress,increasing Met level could alleviate the effect of heat stress on apoptosis and could enhance milk protein synthesis of heat stressed-BMEC.The supplementation of Methionine at LM2.2(80 ?g/m L)had the best effect to alleviate heat stress on apoptosis and enhance milk protein synthesis of heat stressed-BMEC.