In order to improve the production efficiency of hirudin to meet the demand of medical application, the prokaryotic expression system of recombinant hirudin was constructed by optimizing codon of hirudin gene in this study. The four induction factors of D_{600 nm} value, induction time, inducer IPTG concentration and induction temperature were optimized, and the DOE experimental scheme was designed to study the interaction of four factors. The recombinant protein was purified by His-Trap affinity chromatography and the recombinant hirudin activity was determined by thrombin titration. The results showed that the recombinant hirudin prokaryotic expression vector was successfully constructed and the expressed hirudin protein was soluble. The best induction condition acquired through single factor optimization was addition 1 mmol/L IPTG at D_{600 nm}=0.4 and 37℃ for 7 h, which collected the recombinant protein accounting for 66.5% of the total protein. However, the DOE experiment result suggested that the best induction condition was addition 0.82 mmol/L IPTG at D_{600 nm}=0.6 and 31.9℃ for 7.6 h, the recombinant hirudin protein accounted for 72.7% of the total protein, which was significantly higher than that of the single factor method (P<0.05). Further analysis showed that there were obivous interaction among all four influence factors, which might affect the accuracy of the single factor experiment. The recombinant hirudin protein purified by affinity chromatography could achieve 99% purity, and its anticoagulant activity was 114 ATU/mg. Finally, the recombinant hirudin prokaryotic expression vector was successfully constructed, its expression condition was optimized, and the recombinant hirudin protein was obtained, which laid the foundation for the application of hirudin in medical application.

This study was aimed to establish a Real-time and easy-to-diagnose method for the detection of goose Newcastle disease virus (GNDV) at the grassroots level. Based on the highly homologous conserved sequence of GNDV F gene published in GenBank, specific primers were designed and the calcein/manganese chloride indicator was added to the reaction system to establish a visual RT-LAMP detection method. The results showed that the detection method could specifically detect GNDV and NDV Lasota vaccine strains, and other viruses such as GPV, AIV and DTMUV were negative. The minimum detection limit of RNA was 10 pg. The reaction procedure did not require complex instruments such as PCR instrument, and the reaction was completed within 50 min. The results were observed by the naked eye. This method was specificity, high sensitivity, and safe operation, simple and fast, could meet the primary screening of GNDV.

In order to develop a multiplex RT-PCR method for detecting three genotypes of bovine parainfluenza virus type 3 (BPIV3), according to the hemagglutinin-neuraminidase protein genomic sequences of three BPIV3 genotypes obtained from GenBank, three pairs of specific PCR primers were designed. A multiplex RT-PCR detecting three genotypes of BPIV3 was established and optimized. The results showed no cross-reactivity with IBRV, BRSV, BVDV, M.bovis, PPRV, B.melitensis, B.abortus, bovine P.multocida serotype A and bovine P.multocida serotype B. In this study, the amplification produced a series of specific fragments with lengths of 150 bp (BPIV3a), 253 bp (BPIV3b) and 342 bp(BPIV3c), respectively. The limit detection of three recombinant plasmids were 0.89×10⁴, 0.92×10⁴ and 1.53×10⁴ copies/μL. This multiplex RT-PCR method was high specificity and simplicity of operator. It was applied to detect clinical samples and could rapidly detect three genotypes of BPIV3.

This study was aimed to clone the cDNA sequence of Equus asinus Zfy gene and analyzed its genetic structure with bioinformatics. Specific primers were designed according to the mRNA sequence (accession No.:NM_177491.1) of Bos taurus Zfy gene published in GenBank, and Zfy gene were amplified and cloned by RT-PCR. The nucleotide sequence and protein structure of CDS region were analyzed through bioinformatic method. The results showed that the length of CDS region of Equus asinus Zfy gene was 2 325 bp, encoding 774 amino acids. Compared with Bos taurus, Ovis aries, Homo sapiens, Felis catus, Canis lupus, Cervus elaphus, Pan troglodytes and Pan tholops hodgosonii, the homology of the CDS region of Equus asinus Zfy gene were 92.9%, 92.6%, 91.8%, 93.8%, 92.5%, 92.3%, 91.6% and 92.6%, respectively. The phylogenetic tree, constructed from the CDS region of Equus asinus and eight other mammalian Zfy gene cDNA, displays that Equus asinus and Ovis aries, Pantholops hodgosonii, Bos taurus, Cervus elaphus had a closer genetic relationship, however the relationship was far away among Equus asinus, Felis catus and Canis lupus, and the relationship with Homo sapiens and Pan troglodytes was the farthest. In this study, we cloned the CDS region of Equus asinus Zfy gene, which laid the foundation for the further study of the function of Zfy gene.

In this study,to investigate the core promoter activity region, transcription factor binding site,the structure and function of encoding protein and the expression and regulation mechanism of Blue fox MITF-M gene,the skin tissue samples (1 cm×1 cm) was collected from adult Blue fox,the partial sequence of MITF-M gene was obtained by RT-PCR and sequencing technique for the first time. The length of the Blue fox MITF-M gene was 3 880 bp (including 2 262 bp 5' flanking region,1 260 bp CDS region and 358 bp 3' flanking region), and encoding 419 amino acid residues. The bioinformatics analysis showed that there were potential promoter regions at -86 to -336 bp in the 5' flanking region of MITF-M gene, and some regulatory elements (TATA box,CAAT box,GC box and CRE) and transcription factors (CREB,LSF and Sp1) were found. The protein encoded by MITF-M gene was unstable and hydrophilic which mainly located in nucleus and cytoplasm. Subcellular localization results showed that the protein had a higher probability in nucleus (65.2%),cytoplasm (17.4%)and mitochondrion (8.7%),the results of secondary structure prediction by DNAMAN software found that the protein was consist of α helix (33.66%),β sheet (16.66%) and random coil (49.68%),and there was no transmembrane region and signal peptide. Multiple sequence alignment and phylogenetic tree analysis showed that Blue fox had the closet relationship with dog and a high homology with mammal and poultry (>89.1%) indicated that the MITF-M gene coding region was conservative in the course of evolution. This study laid the theoretical foundation for the in-depth study of the molecular genetic mechanism of the MITF-M gene regulating the hair color of the fox.

In order to study the effect of puerarin on differentiation of 3T3-L1 pre-adipocytes, explore the potential mechanism of puerarin in adipogenic differentiation. Puerarin was added into the medium to induce differentiation with final concentration of 0,10 and 50 μmol/L in the process of adipogenesis. The effects of puerarin on accumulation of lipid droplets and the content of triglyceride were measured by oil red O staining and triglyceride assay kit in 3T3-L1 adipocytes, respectively. The mRNA expression of CCAAT-enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) in adipocytes were detected by quantitative Real-time PCR, and the protein expression levels of adipogenic transcription factor and Akt signaling pathway were qualified by Western blotting. The results showed that 10 μmol/L puerarin extremely significantly increased the accumulation of lipid droplets and triacylglycerol (TG) in mature adipocytes, which extremely significantly promoted the mRNA and protein expression levels of adipogenic transcription factor C/EBPα and PPARγ (P<0.01). Further studies showed that stimulation of puerarin enhanced the phosphorylation level of the Akt signaling pathway Ser473 protein compared to control group, suggested that the phosphorylation of Akt signaling pathway played a major role in promoting adipogenic differentiation. In conclusion, puerarin could promote the differentiation of 3T3-L1 pre-adipocytes and improve the sensitivity of insulin, and its mechanism was related to the phosphorylation level of Ser473, which activated Akt signaling pathway. This research would provide a new insights into the mechanism of insulin and a new ideas for treatment of insulin resistance related diseases.

To study the genotypes of EHV-1-XJ2015 strain isolated from Ili area of Xinjiang, this study investigated the potential risk factors for EHV-1-XJ2015.In this experiment, specific primers was designed and using PCR technique to amplify corresponding region of EHV-1-XJ2015 isolate ORF30 gene, connected to vector pMD19-T,and the recombinant plasmid pMD19-T-ORF30 was successfully constructed. Sequencing results showed that EHV-1-XJ2015 strain ORF30 gene 2 254 bp site was A, and encoding asparagine (N).It was proved that EHV-1-XJ2015 was a non-neurotoxic strain, virulent strains tend to behave as a miscarriage. The phylogenetic analysis showed that the EHV-1-XJ2015 strain was the same branch as the 90c16, 00c19, HH1, NY03 isolated from Japan, amino acid homology was the highest and reached 100%. The results provided a theoretical basis for the study of EHV-1 molecular epidemiology and the assessment of existing diagnostic methods and prevention and control measures in Xinjiang, and ultimately achieved the purpose of prevention and control of the disease.

This study was aimed to construct and express the prokaryotic expression plasmid of EfaA gene. One pair of PCR primers was designed according to the EfaA gene sequence from GenBank (accession No.:U03756). EfaA was used as target gene to clone and analyze by biological information. EfaA gene was sub-cloned into pGEX-4T-1, PGEX-4T-EfaA prokaryotic expression plasmid was constructed, and the plasmid was transformed into E.coli BL21(DE3), which was induced by IPTG. The best induction time was screened and biological characteristics of the protein was analyzed. The results showed that 873 bp fragment of Enterococcus faecalis EfaA gene was obtained successfully. Induced expression was operated by IPTG and 59 ku EfaA recombinant protein was obtained. The best way of this react was 6 h in 37℃ by IPTG (0.5 mmol/L). The Western blotting result showed good reactogenicity. The prokaryotic expression plasmid pGEX-4T-EfaA was successfully constructed and expressed in E.coli BL21 (DE3).

Misuse of antibiotics imperils the development of animal husbandry in China, it is important to find reliable alternatives to antibiotics to reduce the use of antibiotics. This review summarizes the most common antibiotic feed additives already being used in ruminant feed, and gives an overview of the mechanism of alternative feed additives, such as probiotics, plant extracts, exogenous enzymes, organic acid, antibiotic peptides and oligosaccharides. The influences of alternative feed additives on rumen fermentation, immunity, microbial flora and production performance are presented. Their utility and limitations in modern ruminant production are also discussed. It can provide basis and references for further research and application of alternatives to antibiotics.

The experiment was carried out to investigate the effects of tea tree oil on growth performance,serum biochemical indexes and viscera indexes of weaned piglets,and discuss the possibility of tea tree oil replace the antibiotics. One hundred and twenty Duroc pig×Landrace pig×Yorkshire pig crossbreed weaned piglets (21-day-old,6.73 kg±0.12 kg) were randomly divided into five groups:CON group (basal diet),ANT group (basal diet+200 mg/kg colistin (10%)+75 mg/kg chlortetracycline (15%)),LTO group (basal diet+50 mg/kg tea tree oil),MTO group (basal diet+100 mg/kg tea tree oil) and HTO group (basal diet+150 mg/kg tea tree oil),with six replicates per group and four pigs per replicate. The experiment lasted for 21 d. During the experiment,the growth performance was measured (ADG,ADFI and F/G),and at the end of the experiment,one piglet was chosen from each replicate for blood sampling and then were slaughtered,the heart,liver,spleen,lung,kidney,thymus and pancreas were collected to determine the organ indexes.The results showed as follow:① Compared with CON group,ADG of MTO and HTO groups increased by 8.33% and 14.84% (P>0.05),respectively, F/G were decreased 9.58% and 7.78%,and the two indexes were not significant different with ANT group (P>0.05).②Diarrhea rate and diarrhea index of ANT,MTO and HTO groups were significantly lower than that of CON (P<0.05),in which MTO was the lowest.③Compared with ANT group,the activities of AST and GGT in MTO and HTO groups were significantly reduced (P<0.05),GLU in LTO and HTO groups were significantly decreased (P<0.05),there were no significant difference in ALT,TP,ALB,GLOB and A/G among groups (P>0.05).④The liver index in MTO and MTO groups and thymus index in LTO group were significantly higher than ANT group (P<0.05).In summary,adding a certain percentage of tea tree oil (100 mg/kg) to diet of weaned piglets could promote the growth performance,reduce diarrhea, promote the development of liver and thymus with a certain degree of regulate metabolism.

Milk fat is a kind of natural fat and a main nutritional component of milk. Milk fat percentage is an important index to evaluate milk quality. Lower milk fat percentage could limit dairy industry development.Recently,the milk yield increased,but not milk fat percentage. The milk fat percentage is 3% to 4% in usual,however,milk yield restricts the increase of milk fat percentage and how to increase it become a hot area of lactating research. To further study and solve this problem,we reviewed the research progress on milk fat synthesis and the factors influencing milk fat including diet,rumen microorganism and genetic factor. We also reviewed the genes and signaling pathways related to milk fat synthesis,which includes SREBPs,PPARs,CIDEC,as well as mTOR signaling pathway. Milk fat synthesis is a dynamic process and complex network regulated to a lot of genes,and the researches of the interaction between the various signaling pathways can provide theoretical basis for artificial regulation milk fat synthesis. These information will provide the idea to improve milk fat content,as well as lactation biology.

The study was conducted to study the protective effect of Ginko biloba extract (GBE) on oxidative damage in cardiomyocytes of chicken induced by heat stress. The primary cardiomyocytes of 12-day-old AA broiler were prepared,0,5,10,50,100,150 mg/mL GBE were added and the cell vability was tested by CKK-8 method.And then the cardiomyocytes were inoculated and cultured for 48 h in 96-well plates,0,5,10,50,100,200,500 and 1 000 μg/mL GBE were added and the cell apoptosis were studied by Hoechst 33258 method to identify the proper concentration of GBE.The chicken cardiomyocyte heat stress model was established at 42℃ for 0,0.5,1,2 and 4 h,respectively. The LDH,GSH-Px,MDA and SOD were measured by ELISA method.Western blotting was used to detect the expression of Hsp72 protein.The results showed that high concentration of GBE was cytotoxic and 50,100,150 mg/mL GBE could extremely significantly inhibit cell growth (P<0.01),and cell proliferation rate was extremely significantly increased by 50 μg/mL GBE (P<0.01).Heat stress could induce cell apoptosis and oxidative damage through increasing the levels of MDA and LDH and reducing the GSH-Px level and SOD activity. GBE could reduce the levels of LDH and MDA and increase the SOD activity and GSH-Px level after heat stress,and the effect was more obvious when heat stress for 2 and 4 h. Furthermore,Western blotting results showed that GBE enhanced the expression of Hsp72.In conclusion,GBE could protect cardiomyocytes from oxidative damage induced by heat stress by increasing activities of antioxidative enzymes and Hsp72 expression.

To study the effects of bamboo vinegar powder on the antioxidant and immune indexes of blood and colostrums in late-pregnant Hu ewe, ten healthy late-pregnant primiparous Hu ewes at about 120 days were chosen and randomly divided into 2 groups with 5 ewes per group. The basic diet and the basic diet added with 2% of bamboo vinegar powder were fed to the ewes in control and experimental groups respectively, and the trial period were 3 weeks including 1 week of preparatory feeding and 2 weeks of formal trial period. The blood samples were collected and detected for the biochemical, immune and antioxidant indexes after finishing experiment. The colostrum was collected within a week after parturition and measured for immune and antioxidant indexes also. The results showed that:①Compared with the ewes in control group, the total protein (TP) content and glutamic-pyruvic transaminase (GPT) activity of blood in experimental ewes were significantly increased (P<0.05), while the glutamic oxalacetic transaminase (GOT)/GPT and platelet counts were significantly decreased (P<0.05), and the red blood cell counts and the hemoglobin concentration were increased with no significant difference (P>0.05);②Compared with the control group, the serum interleukin-6(IL-6) and immune globulin A(IgA) contents of ewes in experimental group were increased significantly (P<0.05), the total antioxidant capacity (T-AOC), immune globulin G(IgG) and immune globulin M(IgM) contents were extremely significantly increased (P<0.01), the tumour necrosis factor-α(TNF-α) content was decreased with no significant difference (P>0.05);③Bamboo vinegar powder significantly decreased the TNF-α content (P<0.05), increased the superoxide dismutase(SOD) activity (P<0.05) of colostrum, but the immunoglobulin content had no significant changes(P>0.05). In conclusion, diet supplemented with bamboo vinegar powder could improve the healthy and enhance the antioxidant and immune performance of late-pregnant Hu ewe.

This experiment was aimed to study the genetic polymorphism of heart fatty acid binding protein (H-FABP) gene in sheep, and find the molecular marker-assisted selection. Tan sheep (250) and Tan×Hu hybrid F1 generation (174) were used as the experimental animals to detect single nucleotide polymorphism (SNP) of H-FABP gene (GenBank accession No.:AY157617) using SNaPshot technology. Gene frequency and genotype frequency statistics, Hardy-Weinberg equilibrium (HWE), expected heterozygosity (He), polymorphism information content (PIC) and effective number of alleles (Ne) were calculated. The correlation among the gene in different genotypes and body weight, body length, body height, chest circumference, chest depth, chest width and cannon circumference traits was analyzed. The results showed that 9 polymorphic loci were detected:939[A/G], 980[G/A], 1018[T/C], 2878[C/T], 2956[C/T], 3017[G/A], 3341[G/C], 3394[T/A] and 1056[-/G], including 6 transitions, 2 transversions and 1 single-base insertion. The He at 939[A/G], 980[G/A], 2956[C/T], 3341[G/C], 3394[T/A] and 1018[T/C] was 0.3200 to 0.4666, PIC ranged from 0.2688 to 0.3577; The He at 2878[C/T] and 3017[G/A] was 0.0283 to 0.1272, PIC ranged from 0.0279 to 0.1191 for low polymorphism; The He at 1056[-/G] in Tan sheep was 0.0120,whereas that of Tan×Hu hybrid F1 generation was 0, PIC in Tan sheep was 0.0119, whereas that of Tan×Hu hybrid F1 generation was 0.9 polymorphic loci in Tan sheep and Tan×Hu hybrid F1 generation followed Hardy-Weinberg equilibrium. 5 polymorphic loci of 939[A/G], 980[G/A], 2956[C/T], 3341[G/C] and 3394[T/A] were tightly linked (D'>0.99), and H-FABP gene was divided into 3 haplotypes:AA,AB and BB. BB haplotype had the maximum value in each production traits in 41 Tan×Hu hybrid F1 generation, but the difference was not significant (P>0.05). H-FABP gene in sheep had abundant genetic polymorphism,5 polymorphic loci of 939[A/G], 980[G/A], 2956[C/T], 3341[G/C] and 3394[T/A] tightly linked,BB haplotype might be the dominant haplotype associated with production traits in sheep.

In order to obtain the genes related to meat quality traits, the transcriptome of longissimus dorsi of 3 Small-tail Han sheep and 3 Bamei mutton sheep were analyzed using RNA-Seq technology. The sequencing data was analyzed by bioinformatics methods then obtained the interrelated candidate genes which affect the meat quality traits. After quality control, 120 Gb clean data were obtained from 6 libraries. The differential expression gene were 333, which the expression of 82 genes was higher and 251 genes was lower. The results showed that 6 candidate genes (PDK4, MYF6, PPARGC1A, SLCO4A1, FABP4 and LEP) related to meat quality traits were screened by GO functional enrichment and KEGG Pathway analysis. Through comparison of the longissimus dorsi transcriptome data of Small-tail Han sheep and Bamei mutton sheep, we obtained the genes of meat quality traits, and enriched the information of sheep genome. This results laid a foundation for further elaboration of the molecular mechanism of meat quality traits in different sheep breeds.

This study was aimed to constract the recombiant plasmid of DKK1 gene in Aohan Fine Wool sheep and detect its expression in fibroblasts. The genomic DNA of biceps brachii in Aohan Fine Wool sheep was extracted, a pair of primers was designed according to DKK1 gene sequence in GenBank, and the DKK1 gene fragment was amplified by PCR method. DKK1 gene was ligated into pEASYTM-T1 vector to construct pEASYTM-T1-DKK1 recombinant plasmid and transformed into E.coli DH5α competent cell, the plasmid was identified by restriction enzyme digestion. The recombinant plasimd PmaxGFP-DKK1 was constructed and transformed into E.coli DH5α competent cell. Then PmaxGFP-DKK1 plasmid was transfected into fibroblasts, and Real-time quantitative PCR was used to detect the expression level of DKK1 gene. The results showed that the PmaxGFP-DKK1 plasmid was constructed successfully (3 956 bp), and was identified by enzyme and sequencing. The expression level of DKK1 gene in fibroblasts was extremely significantly higher than control group (P<0.01). The plasmid was constructed and transfected into fibroblasts successfully, the results would lay a foundation for the further research.

This study was aimed to investigate the association between single nucleotide polymorphisms (SNPs) of prostaglandin-endoperoxide synthase 2 (PTGS2) gene exon 2 and the reproductive traits in Large White pigs, which provided a method for the marker-assisted selection. DNA of American and France Large White sows were used to detect the single nucleotide mutation of PTGS2 gene by sequencing alignment, the genotype of polymorphic loci was identified by PCR-RFLP method, the association with reproductive traits in Large White pigs was analyzed. The results showed that a mutation site (g.86A>G) was found of PTGS2 gene exon 2, which changed MspⅠ restriction enzyme cutting site. Three genotypes of AA, AG and GG were detected in Large White pigs. The polymorphism distribution of PTGS2 gene g.86A>G fitted for the Hardy-Weinberg equilibrium in France Large White pigs (P>0.05). In American Large White pigs group, the number of weak piglets in primiparous sow with AA genotype was significantly lower than that of AG genotype (P<0.05), while the litter weight of multiparous sows with AA genotype was significantly higher than that of AG genotype (P<0.05). In France Large White pigs group, the total number born, number of strong piglets and litter weight of GG genotype were higher than those of AA and AG genotypes (P>0.05). The mutation site g.86A>G of PTGS2 gene exon 2 was significant association with the litter weight of multiparous sows and the number of weak piglets,so the polymorphic loci might be one of the candiate molecular markers used in pig breeding.

This study was aimed to evaluate the effect of vitrification freezing on the development of oocytes in Equus asinus, and seek the best freezing conditions of oocytes in Equus asinus. The oocytes of Equus asinus in different development stages were vitrified and frozen, after the resuscitation, the mature culture and parthenogenetic activation were performed, respectively. The oocytes were stained with microfilaments and mitochondrial ultrastructure, and the oocytes were divided into GV stage without freezing group (control group), GV stage freezing group, IVM-M Ⅱ freezing group. The normal rate of oocyte morphology, maturation rate, the cleavage rate of partial activation, the normal rate of ultrastructural were counted. The results showed that there was no significant difference in the normal rate of morphology between GV stage freezing group and GV stage without freezing (P>0.05), and the maturation rate and cleavage rate were significantly lower than those in control group (P<0.05), the cleavage rate of IVM-M Ⅱ freezing group was significantly lower than that of control group (P<0.05), and the cell development was blocked after cleavage. The distribution of microfilaments in the cortex of most oocytes in freezing group was significantly reduced, and the number of mitochondria in freezing group was significantly lower than that in control group, so it could be explained that freezing caused damage to the oocyte's ultrastructure, resulting in decreased resuscitation after the maturity rate, affecting the oocyte fertilization and in vitro development, and the GV stage freezing group had less damage to microfilaments and mitochondrial structure than IVM-M Ⅱ freezing group, and the developmental status was better.

The tail of an animal has the functions of direction changing,movement and speed controlling,body supporting,defense,attack,heat preservation,warning, escape and predation.The tail of birds plays a key role in maintaining balance during flight,but in evolution,the tail appears to shorten and fuse,such as Piao chicken in China and Araucana chicken in Chile. The authors reviewed the anatomy research about rumpless characters of chicken in recent years,the results showed that there had been shown rumples phenomenon in the period of Hamburger and Humilton 19 stage,and rumpless chicken were lack of uropygial gland,rectrix,sickle feather, caudal vertebra and pygostyle. At the same time,the author analyzed the molecular genetic mechanism of caudal vertebrae development arrest,including the transcriptional regulation of the periodic expression genes that influenced the tail extension process, the back-end factors gradient induces tail formation, the Hox gene influenced the process of segmental specialization,and gene mutation that might lead to rumpless phenomenon. The author proposed the key molecular and signaling pathways in embryonic development,including Irx1 and Irx2 genes,Notch signaling pathway,Wnt signaling pathway,Fgf signaling pathway and RA signaling pathway. The study of the molecular mechanism of rumpless chicken not only could understand the mechanism of tail development of vertebrate in embryo development, but also helped to reveal the evolution process of tail shortening and fusion in birds.

This study was conducted to evaluate the microsatellite polymorphism in the selected 20 microsatellite loci among four beef cattle including Simmental cattle, Limousin cattle, Luxi cattle and Lilu cattle. A total of 144 blood or semen samples from Simmental cattle, Limousin cattle, Luxi cattle and Lilu cattle were collected from different bull stations and the sample numbers were 56, 27, 31 and 30, respectively. The DNA from the blood or semen samples were extracted by Lab-Aid DNA separation kit or salt-extraction method, respectively. The DNA polymorphism of the four beef cattle was analyzed by fluorescence capillary electrophoresis method using the selected 20 microsatellite markers. The genetic variation indices (the number of alleles, polymorphism information content and heterozygosity) and genetic relationship (F-statistic and gene flow) among four beef cattle breeds were analyzed. The results showed that the DNA bands of the beef cattle was neat,and was suitable for the research. A total of 211 alleles were detected in the selected 20 microsatellite loci and the average number of allele in each locus was 10.6. The average observed heterozygosity (Ho) and average expected heterozygosity (He) among the beef cattle breeds were 0.6147 to 0.7176 and 0.6735 to 0.7862, respectively. The polymorphism information content (PIC) of four beef cattle breeds was 0.4853 to 0.8714 in the selected 20 microsatellite loci and the average value of PIC was 0.7284. The average inbreeding coefficient of the four beef cattle was 0.085 and the gene flow in each microsatellite locus was less than 1. In total, the selected 20 microsatellite loci had high polymorphic information, and were effective in the analysis of genetic diversity among different beef cattle breeds.

The broodiness is essential to breeding in domestic fowl, which can lead to the decrease of egg production. As the heritability of broodiness is low, it is impacted by three factors,environment, endocrine and genetics. The photoperiod, temperature and feeding management had significant effect on broodiness. Reproductive endocrine hormones such as GnRH, PRL, FSH, LH, P₄ and E₂ play a very important role in broodiness. The genetic mechanism is a major determinant for broodiness in poultry. The researches of candidate gene of broodiness is based on broodiness-related endocrine hormones. With the development of genome sequencing technology, a lot of new broodiness-related genes were identified by DNA sequencing. The discovery of new genes may provide an idea for the researches of molecular genetic mechanism. Until now, genetic mechanism of broodiness is not completely clear. The study on the genetic mechanism of broodiness will greatly improve the laying performance of poultry, and benefit for the protection, exploitation and utilization of the local domestic fowl breeds. The researches of environmental, endocrine and genetic in the regulation of broodiness were summarized in this paper to discuss the occurrence and regulation mechanism of broodiness in domestic fowl.

In order to obtain a porcine epidemic diarrhea virus (PEDV) isolate that could grow and propagate efficiently in cell culture, and to know the genome sequence of the isolate, virus isolation was conducted on Vero cells through inoculating the intestinal content of piglets with watery diarrhea from swine farms in Guangxi. The strain was identified by cytopathic effects (CPE) and RT-PCR. The complete genome sequence of the isolate was determined by the next generation sequencing technology. One PEDV strain named as CH/GX/2015/750A was successfully isolated from the intestinal content of a piglet with watery diarrhea. The isolate could grow efficiently in cell culture, it had been serially propagated for over 25 passages and typical CPE was observed. The infectious titer of the virus gradually increased and kept at 10^7.50 TCID₅₀/mL. The complete genome sequence of CH/GX/2015/750A was 28 038 bp in length. The nucleotide homologies with 22 reference strains were 96.8% to 99.8%, and with 99.8% nucleotide homology to YC2014 PEDV strain. The whole genome and S gene phylogenetic analysis revealed that PEDV CH/GX/2015/750A isolate belonged to Ⅱa subgroup, and closely related with YC2014 and PEDV-WS mutant strains. The results showed that the PEDV strain obtained in the present study was a variant strain.

In order to determine the cause of calves diarrhea and to provide appropriate treatment and prevention measures,13 anal swab samples and 13 serum samples were taken from calves with diarrhea in a scale dairy farm in Linxia of Gansu province. Identification of antibodies to bovine viral diarrhea virus in serum and pathogenic bacteria in diarrhea feces was performed by means of colloidal gold technology, ELISA, bacterial isolation and identification, and Kirby-Bauer method. The virology test results showed that the antigens of bovine rotavirus (BRV) and bovine coronavirus (BCV) were not detected in 13 fecal samples, however the positive rate of bovine viral diarrhea virus (BVDV) was 23.08%(3/13); No antibodies to BRV and BCV were detected in 13 serum samples, whereas the positive rate of BVDV serological antibody was 38.46%(5/13).Pathogen test results showed that 13 strains of Escherichia coli and 7 strains of Proteus mirabilis were isolated from 13 stool samples. Drug sensitivity test showed that isolated Escherichia coli and Proteus mirabilis had different degrees of resistance to 20 conventional drugs; No drug was found to be effective against both of the bacteria at the same time. The calves diarrhea was caused by mixed infection of BVDV, Escherichia coli and Proteus mirabilis. Escherichia coli and Proteus mirabilis were highly resistant to drugs, which provided an effective treatment basis for calves diarrhea in the cattle farm.

The purpose of this study was to understand the genetic variation and evolution of pseudorabies virus (PRV) in Guangdong province.In this experiment,the PRV strain was primarily identified by PCR from samples (heart,lung,tonsil,liver and spleen) collected from sick pig in Foshan,Guangdong province,and the positive samples were inoculated Vero cells for subculture. And a strain was identifited as PRV by PCR and mice test,and named as PRV FS-2015.The cytopathic effect of the strain was observed and TCID₅₀ was tested,the whole gene of TK and gC were amplified and the sequence were analyzed. The results showed that the titer of TCID₅₀ of isolated virus in Vero cells was 10^-7.5/0.1 mL.The sequence analysis results showed that the nucleotide identities of the gC and TK genes of PRV FS-2015 strain with other PRV strains in GenBank were 95.8% to 100.0% and 99.4% to 100.0%,respectively,the homology of amino acid sequence were 92.3% to 100.0% and 98.7% to 100.0%,respectively. Phylogenetic tree analysis showed that the PRV FS-2015 stain had closer relationship with GY,ZJ01,HB1201,HN1201,JS2012,BJ/YT and BP stains,while had a distant relationship with Kaplan, Becker,NIA3,Kolchis,Bartha,Yangsan,Min-A,SS and SL stains. The experiment showed that PRV FS-2015 strain belonged to the variant popular in recent years,and the results could provide data for the prevention and control of pseudorabies and the selection of vaccine strains in Guangdong province.

Neutrophils,which have become the largest number of immune cells in the peripheral blood,are the important components of the innate immune system in the body and which constitute the first line of defensing against microbial invasion, playing an important role in nonspecific immunity. Besides of the classic phagocytosis of pathogens, degranulation and the release of bactericidal substances, it is found that neutrophils have a new mechanism——Neutrophil extracellular traps (NETs), which could resist the microbial invasion in recent years. Because of the reactive oxygen,peptidylated arginine deaminase 4 and other factors, neutrophils is formed of traps with DNA as the skeleton of the network structure and containing a variety of proteases. NETs can capture and kill pathogens nonspecifically and also play a synergistic role in the production and deterioration of certain diseases. This article is an overview on the formation of NETs,influencing factors and the relationship between NETs and pathogens or diseases to provide the references for studying the function of neutrophils.

To study the impact of season to the distribution of bovine E.coli O157:H7,samples of anus swabs (399),feces (68),water (29) and feed (43) were collected in the spring, summer,autumn and winter from A,B and C farms of Xinjiang. After enrichment by EC broth, SMAC and MUG selective culture were then performed. Finally,PCR was used for identification and virulence gene detection of isolated strains. A total of 5 E.coli O157:H7 strains were isolated from 539 samples from three farms (0.93%,5/539), 2 of them were from spring (1.44%,2/139),1 from autumn(0.56%,1/180),2 from winter (1.38%,2/145) and no strains were isolated from summer samples. One strain were isolated from anus swab samples in farm B (0.69%,1/145) and one were isolated from anus swabs (0.66%,1/152) and three strains were isolated from feed samples in farm C (20.00%,3/15),and no target strains were isolated from water samples. The distribution of bovine E. coli O157:H7 had obvious seasonal characteristics.One E.coli O157:H7 strain of farm B was isolated from autumn and four of farm C were from isolated spring (2 strain) and winter (2 strain),and the isolation rate of E. coli O157:H7 in spring and winter were higher than that in summer and autumn. In conclusion,under the special climate characteristics and feeding mode in Xinjiang,to prevent and control the spreading of E. coli O157:H7 of cattle,we must pay great attention on hygiene management of pens at cold season, specially avoiding the feed contaminated by feces.

The aim of this study was to obtain porcine deltacoronavirus (PDCoV) strain in China and study its biological characteristics. Porcine kidney (PK-15) cell monolayers were inoculated with the samples that were positive for PDCoV only by RT-PCR. PDCoV from the first passage was further serially passed in PK-15 cells. These cell cultures infected with PDCoV were confirmed by cytopathic effect (CPE), sequencing and analysis of PDCoV M gene, acid test; TCID₅₀ was confirmed by Reed-Muench method; The 11-day-old piglets were inoculated with 5×10^4.0 TCID₅₀/mL virus suspension and observed for clinical symptoms. The results indicated that the PDCoV was successfully isolated in cell culture and designated TJ₁, and the infectious titer of the 5th passage of TJ₁ strain was 10^4.5 TCID₅₀/mL; The isolated virus was resistant to chloroform and ether, it was stable at pH 3.0, and the virus was inactivated at 50℃ for 1 h; The TJ₁ M gene shared a homology of 94% to 99% with PDCoV M gene sequences published previously in GenBank; The animal pathogenicity test showed that diarrhea occurred in all piglets by TJ₁. This study successfully isolated a PDCoV TJ₁ strain, and these biological characteristics of the strain were studied preliminarily, which provided a foundation for further research on PDCoV.

The acute toxicity and long-term toxicity tests were carried out in order to explore the safety of Qinhuang qingfei powder (QQP).In the acute toxicity test, mice were given QQP decoction (6.00 mg/(g·BW))for one week, and the LD₅₀ was measured. In long-term toxicity test, 96 SD rats were randomly divided into high, middle and low dose groups (giving QQP decoction 32.00, 16.00 and 8.00 mg/(g·BW), respectively, which was equivalent to 96, 48 and 24 times clinical dose) and control group, each group of 24, half male and half female. QQP was processed and added to rats' feed for 30 d. The rats were weighed every week and the rats' water intake, feed intake, body weight and mental activity were recorded. On the 30th day and the 45th day of feeding, blood samples were taken for measuring the indexes of hematology, blood biochemistry and pathology. The results showed that LD₅₀ of mice was more than 6.00 mg/(g·BW) in acute toxicity test. It was non-toxic actually. In the long-term toxicity test, the differences were not significant in body weight, water intake, feed intake, hematological index or blood biochemical index between dose groups and control group (P>0.05). Most of the major organs of rats were dissected without any lesions and no lesion was observed within histopathological samples (including heart, liver, spleen, lung, kidney and testis/ovary). The test showed that QQP was a safe drug in clinical practice.

This study was aimed to separate a lytic phage against Salmonella Typhimurium from the manure of chicken using double-layer agar culture method. The lytic phage was assessed according to electron microscope observation, thermal stability, pH stability, optimal multiplicity of infection (MOI), One-step growth curve and the inactivation effects on the growth of Salmonella Typhimurium were assayed. As a result, a lytic phage (φBLCC8-0050-3) against Salmonella Typhimurium was successfully isolated from the manure of chicken.φBLCC8-0050-3 had an icosahedral head approximately 65 nm in diameter and had no tail. φBLCC8-0050-3 belonged to family Tectiviridae according to the international classification standard. The plaques of φBLCC8-0050-3 were round and had halo ring around and their sizes which was about 2 to 3 mm in diameter. φBLCC8-0050-3 was stable over a range of temperature (30 to 50℃) and pH (3 to 10). The optimal MOI of φBLCC8-0050-3 was 0.0001. One-step growth experiment showed that the latent time and burst time were 20 and 180 min, respectively, and the burst size was 43 333 PFU/cell. From the control effect of bacteriophage against Salmonella Typhimurium, after 14 days of treatment, the survival rate of mice in the positive control group was 0, was 90% in phage prevention group, and was 70% in treatment group. Compared with the positive control group, the prevention group and the treatment group had a certain effect on mice against Salmonella infection. The results showed that the φ BLCC8-0050-3 was a promising phage, which could be used as a biological antibacterial agent in the prevention and control of Salmonella Typhimurium.

To investigate the effect of lipopolysaccharide (LPS) stimulating rat intestinal mucous membrane microvascular cells (RIMVECs) on lysozyme content of transendothelial migration of neutrophil (PMNs) and the intervention of Pulsatillae (BTW),Coptis chinensis (HL),Cortex phellodendri (HB),Cortex fraxini (QP) and Purslane (MCX),0,0.1,1,10,100 μg/mL LPS were selected to stimulate the RIMVECs in this experiment,and the IL-6 level in cells culture supernatants at 12 and 24 h were determined by ELISA assay to determine the proper concentration of LPS for subsequent trials. To establish a transwell model of PMNs trans RIMVECs adherence and migration,the cell adhesion,migration rate and the release of lysozyme were evaluated by cell counts,staining and ELISA methods after LPS stimulating RIMVECs.The results showed that compared with the control group,the level of IL-6 at 24 h was extremely significantly increased (P<0.001) after RIMVECS was stimulated by 10 μg/mL LPS.Compared with LPS group,the PMNs adhesion rate in HL and BTW groups,the migration rate of QP and BTW groups were significantly decreased (P<0.05),and the amount of lysozyme in HL and BTW groups were extremely significantly increased (P<0.001).The results showed that LPS stimulating endothelial cells could significantly inhibite the release of lysozyme bactericidal enzymes,intervention of Pulsatillae and Coptis chinensis could significantly reduce this inhibition.This study suggested that the release of lysozyme might be related to the functional integrity of endothelial cells,providing a theoretical basis for further screening of water-soluble components in effective Chinese herbal medicine.

In order to determine the clinical efficacy of the oral liquid of dried tangerine peel on chicken growth inhibition,three kinds of methods were used to establish the broiler growth inhibition model (sulfamonomethoxine sodium,atropine sulfate and dexamethasone sodium phosphate) for choosing a best modeling method. The results showed that the ADG of dexamethasone group was lowest which was significantly lower than that of control group (P<0.05),and F/G of dexamethasone group was highest which was significantly higher than that of control group (P<0.05),indicating that the dexamethasone had the best efficacy to establish the broiler growth inhibition model. At the experiment of effect evaluation dried tangerine peel of the oral liquid on growth suppression of chicken,50 AA broilers were chosen and divided into 5 groups,the broilers in groups 1 to 4 were intramuscular injection 0.25 mg/(kg·BW) dexamethasone sodium phosphate for 8 d while the blank control group was intramuscular injection normal saline. At the 9th day,the broilers in groups 1,2 and 3 were drenched 0.1,0.2 and 0.3 mL/(kg·BW) for 7 d,the groups 4 and 5 were drenched the normal saline. The results showed that,after treatment,the ADG of groups 2 and 3 were higher and F/G was lower than group 4,while they all had no significant difference with blank control group (P>0.05),the organ and blood biochemical indexes (except AST) of groups 1,2 and 3 had no significant difference with blank control group (P>0.05),indicating that the oral administration of dried tangerine peel could relieve the growth inhibition and repair the damage of the organ,high dose of dried tangerine peel oral liquid (0.3 mL/(kg·BW)) could effectively treat dexamethasone-induced growth inhibition.

To investigate the effect of reduced glutathione (GSH) on the epididymis of mice with fluorosis,50 ICR male mice were randomly divided into five groups:Control group and 4 fluorosis groups,which were given distilled water and distilled water +100 mg/L NaF for 30 d,respectively. Then 3 of 4 fluorosis groups were given 200,400 and 800 mg/kg GSH,respectively. After feeding for 30 d,the epididymis was collected and morphological structure were observed after HE staining,the levels of ROS,MDA and GSH,and the activities of GPx,GSTs and GR in the epididymis were detected. The results showed that compared with the control group,the sperm density,sperm motility,GSH,GPx and GR in 100 mg/L NaF group were all significantly decreased (P<0.05),while that in GSH addition groups were increased than 100 mg/L NaF group,and 800 mg/kg GSH group had the best effect. The rate of teratosperm, ROS and MDA were significantly elevated (P<0.05),while that all decreased in GSH addition groups. Compared with the control group,the epididymis luminal thickness in 100 mg/L NaF group were increased and the number of sperm in the lumen was decreased and the indexes had been restored after GSH supplement,and 800 mg/kg GSH group had the better effect.In summary,after adding GSH,the activity of enzyme in glutathione antioxidant system of mice with fluorosis was increased, epididymis morphology had been restored to a certain extent.

Heat stress syndrome of dairy cows refers to the non-specific defense response of dairy cattle induced by stimulation of high temperature which exceeding the body temperature regulation zone.While suffering the heat stress, dairy cows will activate the mechanism of hypothalamus-pituitary-adrenal axis and change the body's neuroendocrine regulation network,causing cortisol and some other hormones levels change.All of these procession synergized in the body to reduce the influence of heat stress;In addition, after heat stress,the feed intake and digestibility of dairy cows are generally reduced,and result the body is under nutrition and in a negative state of energy balance.So the cow need to increase the body glucose,fat and protein metabolism to provide more energy to relieving heat stress.However,severe heat stress will lead to dairy cows metabolic disorder and immune system damage,then leads to a decrease of digestibility,milk production, reproduction efficiency,and increases the risk of disease susceptibility, finally it takes an negative effect on economic benefits of dairy cow production and huge economic losses in livestock husbandry.At present,most studies in dairy cows heat stress are focused on milk production and reproductive performance,while few reports on physiological and immune function of dairy cows.In current paper,the secretion and regulation of cortisol,the disorder of the three metabolic processes and the expression and secretion of immune cells and cytokines were talked about in dairy cows heat stress. The aim of the paper was to provide theoretical basis for prevention and control,diagnosis and treatment of heat stress syndrome in dairy cow by understanding the effects of heat stress on the physiological status and immune function.

The continuously changes of rectal temperature and blood biochemical indexes were monitored in 10 d in Hu sheep (a southern special sheep breed) exposed to high ambient temperature. Changes of rectal temperature and blood biochemical indexes were analyzed statistically, based on these data, sensitive individuals and resistant individuals were separated in Hu sheep under high temperature. The results showed that the highest rectal temperature of Hu sheep appeared at the highest ambient temperature (11:00-15:00), the lowest rectal temperature generally appeared at about 03:00 and the rectal temperature was circulated in 24 h cycles. The mean difference between the highest temperature and the lowest temperature was significant (P<0.05).After heat stress, the lymphocyte (LYM), red blood cell (RBC), hemoglobin (HGB), and mean hemoglobin (MCHC) were above the normal values for sheep, which were influenced by both sex and age. Basophilic cell (BAS),RBC,HGB, hematocrit (HCT) of ewes were higher than those of rams, but the differences were not significant (P>0.05). The LYM (P>0.05), BAS count (P<0.01) and percentage (P<0.05) of one year old Hu sheep were higher than those of half a year old. Mainly based on changes of rectal temperature and blood biochemical indexes, high temperature stress sensitive sheep and resistance individuals were identified in Hu sheep, and high temperature stress sensitive individuals counted 35.3%, high temperature stress resistant individuals counted 26.5%.Creatine kinase (CK) content was significantly different between sensitive individuals and resistant individuals (P<0.05). In conclusion, the rectal temperature of Hu sheep was significant difference under high environment temperature, and ewes and one year old sheep were sensitive to heat, these results would provide a basis for better to study the principles and molecular mechanism of heat stress in sheep.