The Application Of Milk Cell Types And Differential Expression Of Milk Protein In The Assessment Of Cow's Early Breast Inflammation

Breast health of dairy cows is a key issue in milk production.Therefor,to monitor the health of the breast is very important for the dairy farm economy.Although the mastitis in the dairy cows is the most common production disease,it can affect dairy farm incomes by reducing milk yield and quality,harming the dairy cow welfare,fertility and even life expectancy.However,the immune response in the breast at the time of inflammation is a crucial indicator of the inflammatory stage.As a reasult,understanding the immune response process of inflammation in the mammary gland,and then grasping the status of breast health in the dairy cow,can improve the quality of milk production and reduce the cost of treatment of breast inflammation.Trail 1.The milk somatic cell type detection and the change in lactation performance under the different breast health status of dairy cows.The aim of this study was to evaluate the breast health status and lactation performance of dairy cowss by studying the characteristics of somatic cell classification in different milk SCC levels.(Method)A total of 102 Holstein dairy cowss were selected for milk sample collection and tested for SCC,milk production and milk composition.According to the results of SCC test,102 samples were divided into three groups,low SCC group(SCC <100,000 cells / mL),medium SCC group(SCC: 100,000-400,000 cells / mL)and high SCC group(SCC> 400,000 cells / mL).The percentage of lymphocytes,macrophages and polymorphonuclear neutrophils(PMNs)were detected by flow cytometry.The total number of colonies in milk samples was counted by standard plate count method,and the bacterial species in milk samples were classified and identified by bacterial routine identification method.The results showed that: 1)The average percentage of lymphocytes in the low SCC group was 89.43% when the SCC was in the range of 13,000 ~ 76,000 cells / mL,which was the main cell population in the healthy mammary gland.While the SCC increased to 76,000 ~100,000 cells / mL,the proportion of PMN increased significantly(P <0.01),reaching 79.68% with the increase of SCC,and gradually became the major cell population in the mammary gland,indicating that early inflammation had begun to occur.When the SCC> 200,000 cells / mL,the proportion of PMN was positively correlated with SCC(r = 0.3604);lymphocyte ratio was negatively correlated with SCC(r =-0.3207);macrophages had no significant correlation with SCC(r = 0.1672).2)identified the main pathogens are Staphylococcus aureus,coagulase-negative staphylococcus(CNS),Streptococcus agalactiae;secondary pathogens Pseudomonas aeruginosa,Bacillus,Lactococcus lactis and so on.Compared with the positive samples(24.91-37.63%),the average lymphocyte ratio(58.48%)was significantly higher in the milk samples(P <0.01).(18.93%)(P <0.01),among which the average percentage of PMN was between the major pathogens and the secondary pathogens,and the proportion of PMN was significantly higher than that of the negative pathogens(38.90 ~ 55.96%)There was significant difference(P <0.05).3)Compared with the healthy group,the milk production of dairy cowss infected with infectious pathogens decreased significantly(P <0.01),the number of casein and the content of lactose were significantly decreased(P <0.05),the total protein,casein,?s1-casein and ?-casein were significantly decreased(P <0.05).In case of bacterial culture negative milk samples,the ratio of casein to protein and the content of lactose were significantly different in medium to high SCC milk samples(P <0.05).(Conclusion)It can be seen that the detection of milk somatic cells classification can play an important role in the inflammatory response and immune process of the three important immune cells(lymphocytes,macrophages and PMN)in the mammary gland.They can judge the real situation of breast health status and milk compositions more detailedly in the dairy cowss,especially in the case of extremely low SCC milk samples,It can detect the occurrence of early inflammation.Thus,the somatic cells classification can be used as a new parameter to assess the health status of dairy cows in detail.Trail 2.Differentially expressed proteins in different breast health status of dairy cows based on proteomicsThe aim of this experiment was to analyze the differences in expression of whey protein in different breast health status of dairy cows based on the Label free non-labeled quantitative proteomics techniques.According to the results of trail 1,the milk samples were divided into seven groups,which were healthy(milk,SCC <100,000 cells / mL,no bacterial culture)group,positive bacteria culture(positive cultures with contagious,environmental,and opportunistic breast infections)group and negative bacterial culture group(culture negative-low SCC group,culture negative-medium SCC group,and culture negative-high SCC group).Differences in protein expression in different groups of whey samples were identified.The results showed that: 1)The health status of mammary gland was not related to the presence or absence of specific protein,but mainly related to the protein expression level.2)A total of 692 expressed proteins were identified,of which 58 showed significant regulatory changes.Compared with the bacterial culture-negative whey samples,20 kinds of expressed proteins were significantly up-regulated in the bacterial culture-positive whey samples,whereas the functions of most up-regulated protein were related to specific immune responses.Some of these significantly up-regulated proteins are acute phase protein(APP),which are useful diagnostic markers of intra-breast infection(IMI).The significantly down-regulated expressed proteins were 38,most of the proteins were unrelated to immune protection but associated with other biological functions such as lipid metabolism,lactose synthesis,milk secretion,and immune cells(T-lymphocytes,Neutrophils,etc.)induced chemotaxis.3)There were 21 bacterial proteins identified only in culture-positive whey samples.The presence of these bacterial proteins verified the results of bacterial culture on the one hand,on the other hand,the molecular mechanism of pathogenic microorganism-induced inflammation were elucidated to some extent,from the perspective of bacterial protein characteristics and functions.In summary,the detection of milk somatic cell type and the identification of differential expression milk protein under different breast status have significant meaning.On the one hand,it provides a method of occurring the early and subclinical mastitis timely in dairy cows,on the other hand,indicates some potential biomarkers of the diagnosis in mastitis.