Effects Of Short-Distance Transport Stress On Blood Physiology And Biochemistry Of Qinchuan Cattle And Analysis Of Transcriptome Characteristics

Transportation is a key link in beef cattle production.Transport causes transport stress.Transportation stress will lead to disorders of beef cattle nervous system,immune system and metabolic system.Transportation stress causes huge economic losses to the beef cattle industry every year.Most studies have focused on the effects of long-distance transport stress on animal health,but little known about the stress molecular mechanisms underlying short-distance transport.This study aimed to investigate the effect of short-distance transportation stress on beef cattle by measuring hematological indicators and blood transcriptomic analysis and to explore the molecular mechanism of its occurrence.In this study,10 adult Qinchuan cattles in good health were used as the research objects,and jugular vein blood was collected before and after transportation for subsequent experiments.（1）Detection of blood physiological and biochemical indicators before and after transportation:The results showed that the number of leukocytes,the stress-related marker,increased significantly after transportation（P<0.05）;the number of hemoglobin,red blood cells and hematocrit decreased significantly（P<0.05）.It showed that the metabolic level of Qinchuan cattle decreased after transportation;the contents of triglyceride,cholesterol,high-density lipoprotein and low-density lipoprotein decreased significantly（P<0.05）.The glucose level showed a downward trend after transportation,indicating that transportation caused increasing energy consumption of Qinchuan cattle,but dosage was not enough to activate glycogenolysis.（2）Analysis of the results of transcriptome sequencing for blood samples:The collected blood samples were removed from red blood cells and RNA was extracted,and then send to Shengyi Biotechnology Co.,Ltd.for transcriptome sequencing,including mRNA-seq and miRNA-seq.After sequencing,the sequencing data were processed for quality control.According to the criteria of log2|fold change|>1 and FDR<0.05,1092 differentially expressed genes（329 up-regulated,763 down-regulated）and 114 differentially expressed miRNAs（40 up-regulated,74 down-regulated）were screened.Twelve differentially expressed genes and differentially expressed miRNAs were randomly selected for real-time quantitative PCR verification.The results showed that the consistency of the verification of differentially expressed genes was 75%,and the consistency of verification of differentially expressed miRNAs was 100%.GO and KEGG enrichment analysis of differentially expressed genes showed that differentially expressed genes were enriched in pathways related to metabolism and B cell function.（3）Construction of mRNA and miRNA co-expression network:Since the functional enrichment analysis of differential expressed genes failed to identify key genes related to short-distance transportation stress in beef cattle,we further screened all genes with an absolute median difference of top 5000.Gene co-expression networks were constructed and 11 independent modules were identified by weighted co-expression network analysis.Thirty key genes were obtained through protein-protein interaction analysis.Combined with the verification of qRT-PCR and their correlation with B cell function,three genes,SMC3,JUN and CXCL10 were used as potential molecular markers for identifying short-distance transport stress.After data preprocessing,co-expression network construction was performed on miRNAs,and four independent modules were identified by weighted co-expression network analysis.Based on their association with 30 key genes,cyan modules in miRNAs were identified as the key module in this study.The regulatory network between miRNAs and targeted genes in the cyan module was constructed through miRNA targeting relationship prediction,and miRNAs targeting hub genes were defined as key miRNAs.Furthermore,CD40 and ITPKB share the same targeted miRNA,miR-339a/b.In conclusion,the disorder of B cell differentiation,proliferation,survival and apoptosis is an important manifestation of beef cattle subjected to short-distance transport stress.SMC3,JUN and CXCL10 can be used as potential molecular markers,the targeting relationship between hub miRNAs and hub genes can provide practical value for prediction and treatment of short-distance transportation stress in beef cattle for beef cattle production.