Analysis Of Blood Physiology And Biochemistry And Genetic Characteristics Of Four Types Of SPF Pigs

In recent years,China’s pig industry has entered a stage of rapid development,followed by the deterioration of pig health and the increase of disease,which has seriously affecting the development of pig industry.In order to carry out research on diseases and preventive drugs,animal disease models should be established.Specific Pathogen Free(SPF)pigs do not carry major potential infections and pathogens that interfere with scientific experiments,and are more suitable for the establishment of animal disease models.Biological characteristics mainly include physiology,biochemistry and genetics.Blood physiology and blood biochemistry are not only the most intuitive reflection of the physiological health status of pigs,but also an indispensable basis for veterinary clinical diagnosis of diseases and prevention and treatment of diseases.Mitochondrial DNA can clearly reflect the kinship,and has a wide range of applications in genetics and breeding.The Swine Leukocyte Antigen(SLA)gene is highly polymorphic and analysing of the genetic background is great important to breeding.This study carried out biological characteristics research on four types of SPF pigs,laying the foundation for disease diagnosis,drug treatment,drug and vaccine development.First,we studied the blood physiology and blood biochemical characteristics of SPF pigs.The blood of 200 SPF pigs was collected,including 50 SPF Yorkshire pigs,50 Landrace pigs,50 Yorkshire × Landrace pigs and 50 Landrace × Yorkshire pigs.23 blood physiological indexes were measured by five-class blood cell analyzer and 19 blood biochemical indicators were measured by automatic biochemical analyzer.The measured data were analyzed by SPSS software.We have also carried out Mitochondrial DNA and SLA-1 gene characteristics research.The ear tissue of 40 SPF pigs was randomly taken for genomic DNA extraction,including 10 SPF Yorkshire pigs,10 Landrace pigs,10 Yorkshire × Landrace pigs and 10 Landrace × Yorkshire pigs.Primer design was performed on the D-loop hypervariable region of mitochondrial DNA and the second exon and third exon of SLA-1 gene using primer 5.0 software.The target fragment was amplified by PCR amplification technique,and the amplified product was subjected to 1% polyacrylamide gel electrophoresis to observe the band size.PCR product was sent to Shanghai Bioengineering Co.for sequencing.The sequencing results were analyzed using Chromas 2.3 software,MEGA 5.0 software,Clustal X software and Dnasp 4.10 software for data editing and statistic analysis of mitochondrial DNA D-loop hypervariable region genetic diversity and polymorphism of SLA-1 gene alleles.The research results are as follows:(1)Through the statistics and analysis of the blood physiology and blood biochemical indexes of the four types of SPF pigs,we established the blood physiology and blood biochemical reference intervals of four types of SPF pigs.The results show that the number of red blood cells(RBC)in Landrace has a wider reference interval than the other three types of pigs.For hemoglobin(HGB),Landrace has a higher reference interval than Yorkshire pigs.In terms of blood biochemistry,the reference interval of alanine aminotransferase(ALT)in Yorkshire pigs is higher than that of Landrace × Yorkshire pigs.For glucose(GLU),Landrace × Yorkshire pigs have a higher reference interval than the other three types of pigs.Although there are differences in the reference interval for the four types of SPF pigs,but the differences are not obvious.(2)The genetic diversity analysis of the D-loop hypervariable region of mitochondrial DNA showed that 13 difference sites were detected in the 435 bp nucleotide sequence,and 10 haplotypes were defined and the shared haplotypes exist between hybrid pigs and their female parents.Analysis of the difference sites and nucleotide diversity of the four types of pigs showed that new mutations occurred in the hybrid offspring,which improved the diversity of the population.Nucleotide polymorphism analysis of all D-loop sequences showed that the peaks of base variation(S)and nucleotide diversity(Pi)appeared near the 220 th base,which is more susceptible to mutation.However,the 180 th bp to the 200 th bp and the 310 th bp to the 340 th bp are very conservative and there is no variation.Finally,by establishing a phylogenetic tree,the analysis shows that it is divided into two categories,which reflects the genetic stability of D-loop and provides a more detailed reference for genetic resources.(3)The analysis of SLA-1 allele polymorphism showed that 9 alleles were obtained in the second exon and the third exon of SLA-1 gene,and the base variation distribution was mainly concentrated in 151-175 bp and 451-575 bp two base regions,the number of mutations are more than 20 times,the highest peak reached 32 times.The highest frequency of Yorkshire pigs is SLA-1*08:11,the frequency is 55%;the highest frequency of Landrace pigs is SLA-1*14:01,the frequency is 35%;the highest frequency of Yorkshire × Landrace pigs is SLA-1*08:11,the frequency is 40%;the highest frequency of Landrace × Yorkshire pigs is SLA-1*12:01 with a frequency of 35%.The amino acids encoded by the nine SLA-1 alleles of the four types of pigs were aligned with the amino acid sequence of the human HLA-A gene,the results were highly similar,and only the 87-91 of the second exon was continuous multiple amino acid differences,other sites are similar.Finally,through phylogenetic tree analysis,in order to obtain more genetic advantages,female pigs with more allele distribution can be used as the female parent to cross with the male.In summary: In this study,the blood physiological reference interval and blood biochemical reference interval of four types of one-month-old SPF pigs were established.The genetic diversity analysis of the D-loop hypervariable region of mitochondrial DNA showed that 13 difference sites,and 10 haplotypes were defined and the shared haplotypes exist between hybrid pigs and their female parents,which reflects the genetic stability of D-loop.It was found that the trend of nucleotide diversity and the distribution trend of base variation were basically the same and the amino acids encoded by the nine SLA-1 alleles were aligned with the amino acid sequence of the human HLA-A gene following the analysis of the polymorphism of leukocyte antigen alleles,the results were highly similar.