Metabolism Of Milk Protein Precursors In The Rumen And Mammary Tissues Of Dairy Cows Under Heat Stress

Milk protein is an important economic variable for the dairy industry and its content is decreased during the warm summer months.Besides the decrease of DMI,the declined free amino acids in blood were also the important factor inducing the decrease of milk protein content under heat stress condition.However,the reasons for the decrease of blood free amino acid content during heat stress,the physiological metabolic changes in mammary tissue during heat stress,and whether physical cooling can alleviate the decrease in milk protein content induced by heat stress and its mechanism are still unclear.Therefore,the following research were conducted in this study:1.For the reasons of the decrease in blood free amino acid content during heat stress.Eight Holstein lactating cows fitted with rumen fistulas were randomly divided into two groups.The experiment was divided into two periods,and 1 of the 2 groups was selected for each period.Each period consisted of two trial phases(control phase and trial phase).During the control phase,all cows were raised in a thermalneutral environment.During the trial phase,4 cattle were randomly assigned to HS or PFTN groups.Dairy cows in HS group(n=4)were kept in heat stress environment and were ad libitum feed.Cows in the PFTN group(n=4)were kept in a thermal-neutral environment,and their feed intake was consistent with the average feed intake of HS group in the last day.Rumen fluid and blood samples were collected on the 2nd,4th and 8th day of each trial phase.The rumen fluid and blood were analyzed by metabonomics.The results showed that heat stress decreased the contents of amino acids in blood(Ile: 31%,Met: 18%,Phe: 19%,Arg: 20%,Lys: 30%,VIP > 1.15),increased the contents of creatinine in blood(12%,VIP =1.2),and decreased the contents of amino acids in rumen fluid(Ile: 32%,Met: 47%,Phe: 31%,Tyr: 27%,Lys: 31%,VIP > 1.2)of dairy cows.Correlation analysis of the significantly different metabolites of HS and PFTN in blood and rumen fluid showed that there was a significantly negative correlation between creatinine in blood and the contents of maultipule amino acids in rumen fluid.Taken together,these results suggested that the decrease of nutrient content in rumen fluid during heat stress may be one of the reasons for the decreased amino acid in blood.2.For the effect of heat stress on the metabolic changes in mammary tissue.Four dairy cows were used in a crossover design where HS was induced using environmental chambers.There was a 30 d washout between periods.Mammary tissue was collected via biopsy at the end of each environmental period [HS or pair-fed and thermal neutral(PFTN)] for transcriptomic analysis.RNAseq analysis revealed HS affected >2,777 genes(FDR < 0.05)in mammary tissue.Expression of main milk protein encoding genes(CSN1S2,CSN1S1,CSN3,and LALBA)and several key genes related to regulation of protein synthesis(LAMTOR2,JAK1,STAT5 A,STAT5B,and ELF5)and amino acid and glucose transport(SLC38A10,SLC38A3,SLC2A1,and SLC2A8)were downregulated by HS.Bioinformatics analysis revealed an overall decrease of mammary tissue metabolic activity by HS(especially carbohydrate and lipid metabolism)and an increase in immune activation and inflammation.Network analysis revealed a major role of TNF,IFNG,S100A8,S100A9,and IGF-1 in inducing/controlling the inflammatory response,with a central role of NF-?B in the process of immunoactivation.The same analysis indicated an overall inhibition of PPAR.In addition,16 differently expressed miRNAs were dected in the mammary gland of HS cows compared with PFTN cows.Thereinto,bta-miR-451 may involve in the change of energy metabolism,bta-miR-184,bta-miR-218,and bta-miR-493 may involve in the cell proliferation,bta-miR-132 may involve in the inflammation response,while bta-miR-382 might have synergistic effect with the regulation in m RNA expression in mammary gland under HS.Collectively,these data suggest HS directly controls milk protein synthesis via reducing the transcription of metabolic-related genes and increasing inflammation-related genes,as well as miRNA expression.3.For the question that whether physical cooling can alleviate the decrease in milk protein content induced by heat stress and its mechanism(s).Thirty lactating multiparous Holstein cows(DIM 175±25 d,milk yield 27.5±2.5 kg/d)were assigned to 1 of 3 treatments: heat stress(HS,n=10),cooling(CL,n=10),and cooling with pair-feeding(PFCL,n=10).The barns for PFCL and CL cows were equipped with sprinklers and fans,whereas the barn for HS cows were not.The average THI during the experiment ranged from 74 to 83.The spraying was activated automatically two times per day(1130 to 1330 h and1500 to 1600 h)with 3 min on and 6 min off during the first 2 weeks,and 1.5 min on and 3 min off during the last 2 weeks,while the fans operated 24 h/d.The experiment lasted for 4 weeks in total.Milk,urine,feces,TMR,blood,and rumen fluid samples were collected weekly.Compared with HS,feed efficiency(1.24 and 1.49,P < 0.05),milk protein yield(0.82 and 0.94 kg/d,P < 0.05),and milk fat yield(0.98 and1.26 kg/d,P < 0.05)were increased in PFCL compared with HS,while the differences between CL and HS were not significant.Compared with HS cows,PFCL and CL cows had lower respiratory rate(70.6,59.1,and 60.3 bpm,respectively,P < 0.05),rectal temperature(38.95,38.61,and 38.51?,P < 0.01),and sholder skin temperature(33.95,33.25,33.40?,P < 0.01),and had higher milk protein content(3.41,3.72,and 3.69 %,P < 0.05)and milk fat percent(4.08,4.97,4.65%,P < 0.01).Both the blood activity of CAT(increased by 12.8 and 41.0%,P < 0.01)and GSH-Px(12.6 and 40.4%,P < 0.01)of PFCL and CL cows were higher than the HS cows.Compared with HS,cooling increased the blood content of glucose,methionine,threonine,and cystathionine by 10.7% and 10.3%(P < 0.05),19.0% and 9.5%(P < 0.05),15.8% and 12.0%(P < 0.05),and 9.5% and 23.8%(P < 0.05)in PFCL and CL respectively.In conclusion,the results indicate that cooling partially rescues milk protein synthesis induced by heat stress,and the potential mechanism may due to decreased oxidative stress,and increased blood glucose and key amino acids.In conclusion,the present study showed that: 1)The decrease of nutrient supply in rumen fluid during heat stress may be one of the reasons for the decrease amino acid in the blood of heat-stressed cows;2)Heat stress can directly control milk protein synthesis by decreasing transcription of metabolism-related genes and increasing transcription of inflammation-related genes in mammary gland as well as miRNA expression;3)Cooling can alleviate the decrease of milk protein synthesis induced by heat stress to a certain extent,and the mechanism may be related to the decrease of oxidative stress and the increase of blood glucose and key amino acid levels.