Differential Expression Of Whey Protein And Milk Fat Globule Membrane Protein In Ketosis Cows

Dairy has become a pillar industry in China's husbandry.With the continuous improvement of dairy milk production,as well as some pasture and farmers' unscientific management of dairy cows,the metabolic diseases of dairy cows have also increased.Ketosis is a disorder caused by the imbalance of the supply and demand of energy in postpartum cows,resulting in a disorder of fat and carbohydrate metabolism.It can reduce the production and quality of milk and affect the quality of the offspring,which can cause great economic loss to the husbandry.To explore the influence of the perinatal negative balance energy metabolism ketosis cows on milk proteomics,research the relationship between ketosis cows' negative balance energy metabolism and the changes of milk proteomics,and access to ketosis cows milk proteomics map,looking for early detection markers in ketosis cows milk protein.This experiment analysis ketosis cows milk fat globule membranes protein and whey protein respectively by Label-free quantification,combining with the bioinformatics analysis,obtained the map of whole cow milk protein,and the effect of ketosis on the composition of milk protein was analyzed.Subsequently,the inflammation-related Milk Fat Globule Membrane(MFGM)proteins were selected to checking by enzyme-linked immuno sorbent assay(ELISA).Holstein cows were selected from an intensive cattle farm with 1000 dairy cows in Heilongjiang,China.Collecte blood from the cows for 14 to 21 days before delivery,and collecte the blood and milk every 3 days after the delivery,tracking to the 28 day after delivery.Separate cream and skim milk.Blood alanine aminotransferase(ALT),aspartate aminotransferase(AST),AST/ALT,alkaline phosphatase(ALP),total protein(TP),glucose(GLU),triglyceride(TG),free Fatty acid(NEFA)and ?-hydroxybutyric acid(BHBA)were detected,divided groups according to BHBA concentrations of serum and confirmed using ketone body powder.After mixing the milk fat and skim milk of ketosis and control groups respectively,the MFGM protein and whey protein were extracted for protein quantification,SDS-PAGE electrophoresis and digestion in-gel,followed by LC-MS/MS with three technical repetitions.After searching the library,analysis the screened difference protein by bioinformatics.The effects of ketosis on dairy milk proteomics were determined.Subsequently,ELISA was used to detect the inflammation-related MFGM protein.The cows were also grouped according to the serum BHBA concentration and confirm by theketone body powder.Initial control samples(C1)were collected from the control group on days 7–14 d after delivery.Later,control samples(C2)were collected on days 14–28.Samples(K1)were gathered on days 7–14 after delivery,before the onset of ketosis in the ketosis group,during ketosis on days 14–28(K2),and after recovery(K3),when BHBA levels were<1·40 mol/l more than 28 d postpartum.The regulation of the changes of MFGM protein in normal and ketogenic cows was analyzed.The results of proteomics study showed that 402 MFGM proteins in ketosis group were increased and 308 proteins were down-regulated.22 whey proteins were up-regulated,and 313 whey proteins were down-regulated.Ketosis has caused great influence on biological processes such as transportation,signal transduction,protein transport,vesicle-mediated transport,maturation and immune system response.Antibiotic biosynthesis,ribosome,endocytosis,endoplasmic reticulum protein processing,and cancer pathway changes significance.Comparisons between groups revealed the following: C4 BPA,F2,C3,and ORM1 were significantly higher(p<0·05),and CD14 significantly lower(p<0·05)in C2 than in K2;ITIH4,C9,F2,AHSG,and CD46 were significantly higher(p<0·05),and CD14 and CD46 significantly lower(p<0·05)in K2 than in K1;ITIH4,F2,and ORM1 were significantly higher(p<0·05),and CD14 significantly lower(p<0·05)in K3 than in K1;ITIH4,C4 BPA,C3,and CD46 were significantly higher(p<0·05),and F2,AHSG,and ORM1 significantly lower(p<0·05)in K1 than in C1;CD14 and ORM1 were significantly lower(p<0·05)in K2 than in C2;SAA and ORM1 were significantly lower(p<0·05)in K3 than in K2.It is worth noting that the concentration of NEFA was significantly correlated with BHBA,BC3,BC9,C3,and ORM1 levels.The concentration of BHBA was significantly correlated with BC3,TNF?,BC9,CD14,C3,and ORM1 levels,while GLU concentration was significantly correlated with TNF? and ITIH4 levels.We have found that cows have a reduced production efficiency at the time of ketosis and negative energy balance leads to impaired liver function.Ketosis causes energy metabolism in dairy cows,reduced mammary gland secretion,and reduced milk immune function,which partially elucidated the mechanism by which negative energy balance causes changes in milk protein.The relationship between ketosis in cows and perinatal inflammation-related metabolic pathways was proved.Ketosis caused a change in milk fat globule membrane protein(MFGM)protein in milk and triggered inflammatory responses in the peripartum cow.This experiment hasimportant significance and application value for the prediction,prevention and diagnosis of ketosis in the future.