| Mastitis is a common disease in dairy farms and causes huge losses to dairy production every year.The rumen and gut bacteria play an important role in the homeostasis and health of the host.In this study,microbiome analysis and metabolomics were applied to explore the cows' physiology and gastrointestinal microbiological changes under different mammary gland health conditions,and to screen bacterial indicators related to mastitis.Firstly,based on milk somatic cell count(SCC),a common indicator of udder health,the differences were compared in rumen microbiomes in lactating cows with different levels of SCC.On this basis,the differences were further explored in physiological status and gastrointestinal bacteria among healthy,subclinical mastitis and clinical mastitis cows;then the changes of gastrointestinal bacteria in dairy cows after recovered from mastitis were investigated.Metabolomics were applied to explore the metabolic changes in rumen,hindgut,blood and milk of cows before and after mastitis was cured.Finally,the potential rumen and hindgut bacterial indicators for cow mastitis were selected through random forest models.A scientific basis was provided to judge the health of dairy mammary glands.Part 1:Physiology and rumen bacterial statuses of cows with different levels of SCCThe main purpose of this part was to investigate the physiology and rumen bacterial statuses of cows with different levels of SCC in milk.The DHI data of 334 dairy cows were selected from a dairy farm.According to the SCC of these cows,they were divided into four groups according to SCC:SCC1(?200,000 cells/mL),SCC2(200,001-500,000 cells/mL),and SCC3(500,001-1,000,000 cells/mL)and SCC4(>1,000,000 cells/mL).Milk yield of dairy cows was recorded.Samples of milk,blood,and rumen fluid were collected to analyze milk compositions,blood biochemical parameters and rumen fermentation characterics;rumen samples were also analyzed for the microbiota.At the same time,20 cows with highest SCC(H_SCC)and lowest SCC(L_SCC)were selected respectively,to compare the differences in these indicators.Milk yield of cows was significantly lower(P<0.01),but percentage of milk protein and milk fat was higher(P<0.01)in SCC4 than in other three groups.Similar differences were found between H_SCC and L_SCC.Blood total antioxidant capacity,total cholesterol,total protein and glucose content were significantly different among the four groups(P<0.05),with no differences in other indicators(P>0.05).Significant difference was found in the acetate to propionate ratio in the rumen among four groups(P<0.05),with no differences in rumen pH,volatile fatty acid,and ammonia nitrogen among groups(P>0.05).Content of propionate and valerate was significantly lower in H_SCC cows than in the L_SCC cows(P<0.05),while the acetate and propionate ratio was significantly higher than in the L_SCC group(P<0.05).In terms of rumen bacteria,there was no significant difference in the ? diversity and predominant rumen bacteria in cows with different SCC levels(P>0.05),but the relative abundances of SR1 and Actinobacteria at the phylum level and unclassified_f_Clostridiales and Butyrivibrio at the genus level showed significant differences.The results showed that there were differences in rumen bacteria among cows with different levels of SCC.Part 2:Physiological and gastrointestinal microbial changes in dairy cows with mastitisIn this part,cows with mastitis and healthy cows were selected as below:19 healthy cows(HE,SCC?500 × 103 cells/mL,no clinical signs of mastitis),20 subclinical cows(SCM,SCC>500 × 103 cells/mL,no visible symptoms)and 10 cows with clinical mastitis(CM,visible symptoms of mastitis).Milk yield was recorded.Samples of milk,blood,rumen fluid and feces(hindgut)were collected to analyze milk compositions,blood biochemical variables and rumen fermentation characterics.Rumen and hindgut samples were analyzed for the microbiotal compositionsto explore the relationship between mastitis and gastrointestinal bacteria.Significant differences(P<0.01)existed in SCC,milk yield and milk composition among the three groups;Significant differences were(P<0.05)also observed in total antioxidant capacity,and blood urea nitrogen among the groups.Cows with mastitis(SCM and CM)were significantly lower in total VFA concentrations than healthy cows(P<0.05),while concentration of acetate,propionate,butyrate,and isovalerate were significantly different among the three groups(P<0.05).Results of 16S rRNA gene amplicon sequencing showed that the rumen and hindgut bacterial richness and evenness were higher in cows with mastitis than in healthy cows.In cows with mastitis,the relative abundance of 6 phyla in the rumen and 4 phyla in the hindgut were higher than in healthy cows,while the relative abundance of 4 phyla in the hindgut of healthy cows was higher than in mastitis cows.Compared with cows suffering from mastitis,the relative abundance of 42 rumen bacteria was significantly lower(P<0.05),while the relative abundance of 22 bacteria was significantly higher(P<0.05)in healthy cows.In the hindgut,cows suffering from mastitis had relatively lower relative abundance of 41 bacteria genera,while healthy cows had higher relative abundance of 24 bacteria(P<0.05).The above results indicated that there were differences in rumen and hindgut bacteria among dairy cows under different udder health conditions.The main differences were the reduced relative abundance of the dominant bacteria,and increased abundance of the bacteria with low relative abundance.Part 3:Gastrointestinal bacteria and metabolic changes after cows recovered from mastitisCows with and recoved from mastitis were selected in this part to analyze the changes in gastrointestinal bacteria and further explore the relationship between cows' udder health and gastrointestinal bacteria.A total of 9 cows,5 from the subclinical mastitis(SCM)and 4 from the clinical mastitis(CM)group,were followed,and rumen and blood samples were collected for analysis before and after recovery(RE).Compared with their status of mastitis,the SCC of RE cows decreased(P<0.05),and the lactose content increased(P<0.05),with no significant changes in other parameters(P>0.05).After recovery,the content of aspartate aminotransferase,?-hydroxybutyric acid and total cholesterol in the blood of cows was reduced(P<0.05),while total bilirubin and creatinine content increased significantly(P<0.05).No significant changes(P>0.05)were observed in the rumen fermentation parameters with the recovery from mastitis.Before and after recovery from mastitis,the relative abundance of 4 rumen genera belonging to Firmicutes changed significantly(P<0.05).In the hindgut,the relative abundance of bacteria in two phyla(Firmicutes and Actinobacteria)and 1 1 genera(of which 10 genera were from Firmicutes)changed in comparison with status of mastitis.Correlation analysis of hindgut metabolomics and microbiome results revealed their potential association with mastitis of dairy cows.Compared with the rumen,bacteria and metabolites in the cow's hindgut may be more closely related to the recovery process of cow mastitis.Metabolomics analysis of rumen fluid,feces(hindgut),blood,and milk yielded 165,256,234 and 143 metabolites,respectively,among which,there were 6,27,11,and 7 differential metabolites(P<0.05,VIP>1).PC A analysis showed no separation in the metabolic pattern of rumen fluid,serum,and milk before and after recovery,while the hindgut showed a clear separation.The above results indicated that the bacteria and metabolism in the hindgut of dairy cows are more closely related to the mastitis recovery process than in the rumen.Part 4:Prediction of udder health statuses in high-SCC with gastrointestinal bacteriaThe purpose of this part was to explore potential gastrointestinal bacteria that may indicate health statuses of mammary gland.Based on the analysis of the results in Part 2,it is found that the rumen and hindgut bacteria of 20 cows with subclinical mastitis showed different biases:partly to health(SCM_H),and partly to clinical mastitis(SCM_C),suggesting that these cows with high SCC statuses were different in the gastrointestinal bacteria,and that certain bacteria may be able to indicate the health statuses of the udder.Therefore,20 cows were divided into groups of SCM_H(N=9)and SCM_C(N=11),and random forest models were constructed to screen potential rumen and hindgut bacteria that could indicate the udder health of high SCC cows.No significant differences existed in parity,lactation days and SCC between the SCM_H and SCM_C cows(P>0.05).Milk yield,lactose,and milk urea nitrogen content were significantly higher(P<0.05)in SCM_H cows than in SCM_C cows.The SCM_H cows had significantly lower acetate content(P<0.01),but significantly higher contents of butyrate,isovalerate and valerate(P<0.05)than SCM_C cows.Plasma contents of aspartate aminotransferase,glucose,and total cholesterol were significantly different between SCM_H and SCM_C cows(P<0.05).The Shannon index of the rumen and hindgut bacteria was extremely significantly lower(P<0.01)in SCM_H than in SCM_C cows.Significant differences were found in the rumen and hindgut microbial diversity between two groups.Based on random forest models,some potential bacteria in the rumen and hindgut were screened out to indicate udder health in high SCC cows.Among the rumen bacteria,Erysipelotrichaceae UCG 004 and[Eubacterium]xylanophilum group were the most important to the model,with MDA values greater than 3.Among the genera from hindgut,Family XIII AD3011 group was the most important(MDA>3);Bacteroides and uncultured_f_F082 were the second and third important genera(MDA>2.5).These genera may be able to be used as potential indicators of the mammary gland health status of high SCC cows.In summary,a corrrelation existed between the gastrointestinal bacteria and the health of the mammary gland in dairy cows,and a relationship was closer of recovery of mastitis to the hindgut bacteria than the rumen ones.Together with milk SCC,specific genera in the gastrointestinal tracts may be used as potential biomarkers of mastitis to further indicate the mammary gland health cows. |
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