Metabolomics And Ionomics Analysis Of Serum Of Cows With Deformed Hoof Based On LC-MS/MS And ICP-OES/MS Techniques

Lameness caused by hoof disease seriously affects pasture performance,second only to mastitis and reproductive disorders.Hoof deformation is caused by various adverse factors,resulting in abnormal growth of hoof horn,hoof shape changes from normal hoof,also known as deformed hoof.Hoof deformation is a common productive disease of dairy cows,which can lead to lameness in mild cases,and reduce performance and service life in severe cases,which seriously affect breeding benefits.At present,the research on hoof deformation in dairy cows mainly focuses on diagnosis and treatment,but there are few reports on the pathogenesis of hoof deformation,and there are no reports on the metabolism and ionomics of hoof deformation in dairy cows.This study explored the changes of metabolism and ions in dairy cows when hoof deformation occurred from the perspective of metabolomics and ionomics.The experiment mainly includes four parts:1.Investigation of foot disease in large-scale cattle farms.The incidence of hoof disease was investigated by filling out a form.A total of 1459 cows were registered for the incidence of hoof disease,and the data were collected from the veterinary diagnosis records of the ranch,and the cycle was one year.Hoof diseases were classified into seven types: hoof dermatitis,hoof ulcer,white line disease,laminitis,foot rot,hoof deformation and other hoof diseases(hoof crack,interphalangeal hyperplasia,hoof erosion,etc.).The results showed that 433 dairy cows had hoof disease,with an incidence of 28.96%.The incidence of hoof dermatitis was the highest(16.19%).The incidence of hoof deformity was 5.82%.The incidence of hoof ulcer was 3.41%.The incidence of other hoof diseases,such as white line disease,laminitis,and foot rot,is low.The incidence of hind hoof disease was significantly higher than that of front hoof.2.LC-MS/MS non-targeted metabolomics detection and MRM targeted metabolomics verification.Serum samples from 10 healthy cows(control group,group C)and 10 deformed hoof cows(experimental group,group T)were selected for LC-MS/MS non-targeted metabolomics.Principal component analysis(PCA),orthogonal partial least squares discriminant analysis(OPLS-DA)and t-test were used to screen the metabonomics results.Hierarchical clustering analysis was performed on the differential metabolites between the screened groups,and then KEGG and Metabo Analyst4.0 were used to map the metabolic pathways of the differential metabolites and perform pathway enrichment analysis.Finally,the target differential metabolites were quantitatively detected by targeted metabolomics technology based on multiple reaction monitoring(MRM).The results of LC-MS/MS metabolomics showed that a total of 127 metabolites were screened,of which 81 differential metabolites were significantly up-regulated(P < 0.05)and 46 differential metabolites were significantly decreased(P < 0.05).A total of 28 metabolic pathways were enriched by KEGG metabolic pathway enrichment analysis,of which 9metabolic pathways were significantly enriched,and the enrichment degree of phenylalanine tyrosine and tryptophan biosynthetic metabolic pathways was extremely significant.According to the results of LC-MS/MS metabolomics,three target metabolites(L-proline,L-phenylalanine,L-tryptophan)were selected for MRM target metabolomics verification,and the verification results were consistent with the results of LC-MS/MS non-targeted metabolomics.3.ICP-OES/MS ionomics.Serum samples from 10 healthy dairy cows(group C,control group)and 10 dairy cows(group T,experimental group)with abnormal hoof were selected for ICP-OES/MS analysis,which included 31 common ions(elements).PCA analysis,OPLS-DA analysis and t-test were used to screen the results of ICP-OES/MS.The screening value of ICP-OES/MS ionomics data was VIP greater than 0 and P less than 1.The selected data were analyzed by matchstick plot analysis,principal component analysis(PCA)and hierarchical cluster analysis(HCA).The results showed that the contents of 13 ions were up-regulated and 18 ions were down-regulated in serum of dairy cows.The content of K ion(P < 0.01)and Li ion(P< 0.05)were significantly increased.The contents of Al ions(P < 0.01)and Cu ions(P < 0.01)were extremely significantly decreased,and the contents of Sb,Na,Sn,and B ions(P < 0.05)were significantly decreased.4.Combined analysis of metabolomics and ionomics.The results of metabolomics and ionomics were integrated and analyzed by ggplot2 software in R language.It was found that potassium was positively correlated with L-tyrosine,L-proline,thiamine and negatively correlated with L-valine.Sodium ion was positively correlated with L-valine and negatively correlated with α-D-glucose.Moreover,a variety of ions were significantly correlated with differential metabolites.In summary,there are significant differences in serum metabolites and ions between hoof deforming dairy cows and healthy dairy cows.It is speculated that these differential metabolites and ions may be closely related to the occurrence and development of hoof deforming.This experiment provides a theoretical basis for further research on the pathogenesis of hoof deforming in dairy cows,and for solving prevention and control strategies for hoof deforming in dairy cows.