Long non-coding RNAs（lncRNAs）are a novel class of RNAs，which are longer than 200 nucleotides and have no protein-coding potential.In comparison with short noncoding RNAs which was studied extensively，a variety of long noncoding RNAs account for the majority of the transcripts in mammalian genome and the functions of lncRNAs are little understood．The recent studies have indicated that lncRNAs’ function involved in epigenetic modification，transcriptional regulation，cell differentiation，embryonic development and disease occurrence.In this review，we summarized the classification and molecular mechanisms of lncRNA，and research advances in the regulation of skeletal muscle development and domestic animal.

The evaluation of semen quality was the primary method of predicting livestock fertility ability and the fertility related protein markers in semen were the basic materials to determine the potential of fertilization.With the development of proteomic technology，an increasing number of biological markers related to sperm fertility were found in livestock，which affected both the pregnancy rate and the litter size directly or indirectly.The discovery and identification of these protein biomarkers made it possible to improve reproductive performance through directive breeding，and provided theoretical basis for the prediction of sperm fertility difference between subspecies of sire.The research progress in semen protein biomarkers related to reproduction in male livestock was summarized in this article.

The Myozenin2 (MYOZ2) gene promoter of bovine was cloned and sequenced to analyze the active region，which will provide theoretical basis for function determination and expression regulation of MYOZ2 gene.Applying 5′-rapid amplification of cDNA end analysis (RACE)，we determined the transcription initiation site of MYOZ2 gene.Using genome DNA as the template，the promoter of MYOZ2 gene was obtained by PCR.Online software was used to analyze putative transcription factor binding sites.Primer were redesigned according to the results of online software analysis，seven promoter fragments in different length were obtained by unidirectional deletion and cloned into luciferase reporter gene expression vectors.Then，the vectors were transfected into C2C12 cells，their expression activity were measured using the dual reporter assay.The promoter sequence of 2 065 bp of MYOZ2 gene in bovine was obtained and the transcription initiation site of MYOZ2 gene was determined.Luciferase assays indicated that the transcriptional activity of pMYOZ2-84/+125 was significantly higher than pGL3-Basic (P＜0.01)，and the transcriptional activity of pMYOZ2-683/+125 also was significantly higher than pMYOZ2-263/+125 (P＜0.01).The core functional promoter of bovine MYOZ2 was located in the region -84/+125 relative to the TSS，and the transcriptional factors such as MEF2，SRF，MyoD and YY1 might be involved in the transcriptional regulation of MYOZ2 gene.

The aim of this study was to provide evidence about the role of ADIG gene in bovine adipose tissue during growth and development，and to lay a theoretical basis for further study of the molecular mechanisms of bovine fat metabolism and meat quality improvement through inducing bovine myoblasts trans-differentiating into fat with overexpressing ADIG gene in bovine myoblasts.We induced bovine myoblasts trans-differentiating with AD-ADIG recombinant adenovirus，observed the lipid droplets after Oil Red O staining，collected cells to extract RNA at the 0^th，2^nd，4^th，6^th，8^th，10^th，12^th，14^th day，respectively，and detected relative expressions of fat-related genes ADIG，PPARγ，C/EBPα，SREBPF1，FABP4，FAS as well as the muscle-related gene MyoD by real-time quantitative PCR.The real-time quantitative PCR results showed that during the induced trans-differentiation process，ADIG gene was expressed at high levels at the 2^nd，4^th，6^th，8^th，10^th，12^th，14^th day，and lipid droplets increased significantly.The relative expression of the fat-related genes PPARγ，SREBPF1 and FAS at late stages were obviously higher in AD-ADIG group than that in other groups，and the relative expression of muscle-related gene MyoD decreased at early stages，and was obviously lower in AD-ADIG group than that in other groups.The result indicate that ADIG gene play important roles in bovine myoblasts induced into fat and bovine myoblast has the potential of differentiating to adipocyte according to the results of Oil Red O staining and related gene relative expression.

The cashmere-goat intramuscular adipocytes were used as experimental materials，and aimed to establish the stable intramuscular adipocyte line for the interference through target interrupting the peroxisome proliferation-activated receptor(PPARγ) gene，so as to explore the function of cashmere-goat PPARγ gene during the proliferation and differentiation processes of the intramuscular adipocytes.Lentivirural plasmids pack-aging system was adopted to establish a lentiviral vector for RNA interference of specific targeting White Cashmere-goat PPARγ gene，which was used to establish the intramuscular adipocyte line to stabilize and silence the PPARγ gene.Real-time quantitative and Western blot methods were used to detect the expression of PPARγ gene at different points in time in the interference group and control group，as well as the MTT and Oil Red O Staining methods were used to research the influence of cashmere-goat PPARγ gene on the proliferation and differentiation of intramuscular adipocytes.It was confirmed by sequencing that oligonucleotide chains containing PPARγ-shRNA lentivirus vector were inserted properly，its infection efficiency to intramuscular adipocytes was above 80%，lentivirus-mediated shRNA detected by fluorescence quantitative and Western blot could effectively reduce the expression of PPARγ gene，the reaction time interval between mRNA and protein level was 24 h.After silencing of PPARγ gene，the concentration of triglyceride within adipocytes was significantly lower than that in the control group；Contrarily，the cell proliferation ability in MTT test interference group was higher than that in control group.This study established cashmere-goat shRNA-PPARγ lentivirus interference vector，after successfully transfecting into the intramuscular adipocytes，it obviously inhibited the differentiation of adipocytes，and promoted the proliferation of intramuscular adipocytes，which lay foundation for further research the role of PPARγ gene in cashmere-goat metabolic pathways and in the fat deposition mechanism.

To investigate the function difference of the different Melanocortin-4 receptor (MC4R) gene mutants，the complete coding sequence( CDS) of MC4R gene were cloned from pig muscle tissues.The CDS sequence was linked with pcDNA3.1 vector by digested with restriction endonuclease of Kpn Ⅰ and EcoR Ⅰ and the recombinant eukaryotic expression vector MC4R1 was constructed.After recombinant MC4R1 plasmid transfected into BHK cells，the protein expression of MC4R1 was confirmed by Western blotting.Simultaneously，3 site-directed MC4R cDNA mutant (707/892 site) vectors were generated.The protein expression of MC4R mutations vector was observed in transiently transfected BHK cell by Laser Scanning Confocal Microscopy and Flow Cytometry ananlysis.The results indicated that the CDS sequence of MC4R1 gene was about 1 000 bp and the sequence of 707/892 site was G/G.The protein expression of MC4R1 was confirmed in BHK cells using Western blotting.Three site-directed mutant eukaryotic vector MC4R2，MC4R3，MC4R4 were constructed and the sequence of 707/892 site was G/A，A/G，A/A，respectively.Confocal microscopy showed that every mutant of MC4R receptor were expressed well on the cell surface by the Immunofluorescence.There were no significant difference of the fluorescence intensity between mutants of MC4R receptors（P＞0.05）by FCM.The construction and functional research of MC4R vector suggest that the mutants of 707/892 site has no significant impact on the expression of MC4R receptor on cell surface.This will provide technological platform for further research in the role of MC4R gene.

To study the effects of gender and breed on blood biochemical parameters and the correlations between blood glucose，blood lipids and fat deposition，we measured the blood physiological and biochemical parameters and fat deposition traits in Laiwu and Sutai pigs.Total 6 blood physiological and biochemical parameters were measured in both 251 Laiwu and 248 Sutai pigs by using automatic biochemical analyzer.The results showed that，compared to the gilts，the castrated male pigs had significantly higher LDL-C level (P<0.01)，but no significant difference was identified in other blood biochemical parameters between genders (P>0.05) in Laiwu pig.We further compared the phenotypic values of blood glucose and lipids between Laiwu and Sutai pigs，and found that GLU，GSP，LDL-C were significantly higher in Laiwu pigs than that in Sutai pigs (P<0.01).However，Laiwu pigs had significantly lower HDL-C than Sutai pigs (P<0.01).The other 2 blood biochemical parameters had no significant differences between Laiwu and Sutai pigs.Blood glucose and some blood lipid traits were significantly associated with fat deposition traits in Laiwu and Sutai pigs.Correlation analysis found that T-CHOL and LDL-C had extremely significant positive correlation with chest (6-7 ribs) backfat thickness (P<0.01)，LDL-C showed significantly positive correlation with hip backfat thickness in Laiwu pigs (P<0.05).In Sutai pigs，GLU，T-CHOL and HDL-C exhibited significantly (P<0.05) or extremely significant (P<0.01) positive correlations with shoulder backfat thickness，chest backfat thickness，waist backfat thickness and hip backfat thickness，GSP had significantly positive correlation with hip backfat thickness (P<0.05)，further，TG showed significantly positive correlation with shoulder backfat thickness and waist backfat thickness (P<0.05).In conclusion，we suggest that gender may have effect on porcine blood glucose and lipid levels，therefore，breed has significant effect on blood physiological and biochemical parameters.Blood glucose and lipids are significantly correlated with fat deposition traits in Laiwu and Sutai pigs.

 In order to study the effects of Lipopolysaccharide（LPS） on proliferation，apoptosis and estradiol (E₂) secretion of porcine granulosa cells.Primary porcine granulosa cells were cultured in vitro and treated by LPS at different concentrations (0，500，1 000 and 2 000 ng•mL^-1).Then cell proliferation，apoptosis，secretion of E₂ and related gene expressions，FN1，IGF2，IGFBP2，Cyclin D1，Cyclin D2，P27^kip and P450arom were determined individually.The results showed that cell proliferation and survival ratio were significantly promoted at LPS concentrations of 1 000 or 2 000 ng•mL^-1(P<0.05)，on the contrary，cell apoptosis was inhibited.However，gene expression levels of FN1 (P<0.01)，IGF2，IGFBP2 (P<0.05)，Cyclin D1 and Cyclin D2 (P<0.01) were up-regulated at 500 ng•mL^-1 of LPS，P27^kip (P<0.01) which hindered cell cycle was inhibited.P450arom gene expression was decreased，resulting the inhibition of E₂ secretion when treated by LPS.These results suggested that granulosa cell proliferation could be promoted by LPS，while cell apoptosis and E₂ secretion were inhibited.

To explore the relationship between ACAA2 gene and liver fatty metabolism in goose，35 Landes geese were divided into overfeeding group(15) and control group(20)，the overfeeding group included 3 periods：overfeeding 7，14 and 19 days.The full coding sequence (CDS) of ACAA2 was cloned by RT-PCR and analyzed，its expression levels at different stages of overfeeding were determined in both normal and fatty liver by qRT-PCR in Landes geese.In addition，goose primary hepatocytes were treated with glucose，fatty acids or insulin to determine how its expression was regulated during fatty liver formation.Data indicated that the complete CDS of goose ACAA2 was 1 194 bp and encoded 397 amino acids.The ACAA2 expression level in the livers of the overfed geese was significantly higher (P<0.05) than the control at every stage，and presented a declining trend with the extension of overfeeding time.Moreover，compared to the control，the expression of ACAA2 gene was significantly increased in the goose primary hepatocytes treated with 0.5 mmol•L^-1 oleate，but was decreased in the primary hepatocytes treated with 0.25 mmol•L^-1 palmitate and insulin with different concentrations (P<0.05).The results suggeste that ACAA2 expression is closely related to the development of goose fatty liver，which will provide a basis for further study the role of ACAA2 gene in the formation of goose fatty liver.

This experiment was conducted to investigate the role of c-Myc/LIN28B/let-7a pathway in chicken puberty onset.The Wenchang chicken breed was used as the material.The comb size，ovary weight，oviduct length and follicle size for hens were measured from 5 to 17 weeks.qRT-PCR was performed to analyze the hypothalalmic expression changes of c-Myc/LIN28B/let-7a pathways.The results showed that：1) The ovary and oviduct developed slowly and the follicles kept primitive in early period of development in Wenchang chicken.This period would last about 12 weeks.Then，the gonad tissues developed explosively，the ovary weight and oviduct length of 13 weeks increased 125% and 120% respectively compared to that of 12 weeks，and accompanied by emergence of pre-hierarchical follicles.Based on these，the crucial “timing” for transition from juvenile to puberty onset for Wenchang hens was determined as age of 13 weeks.2) qRT-PCR test revealed the relative expression levels of c-Myc and LIN28B genes in hypothalamus decreased significantly (P<0.05，P<0.01) at the transition timing of puberty onset in Wenchang chicken.The lower level of c-Myc could last to the age of first laying，but that of LIN28B recovered to higher level before the age of first laying.Accompanied with these changes was the significant increase of let-7a (P<0.01)，which could kept higher until the age of first laying.These results indicated that c-Myc→LIN28B┤let-7a regulating pathway was involved in chicken puberty onset.

This study was designed to apply ERIC-PCR genetic fingerprinting technology to analyze the microbes in rabbit manure，and establish a rapid analysis and detection method of rabbit manure bacterium changes.The ERIC-PCR DNA fingerprint of bacteria in rabbit manure was established by optimizing PCR reaction conditions.The differences between DNA fingerprints of microbiome and dominant strains isolated from rabbit manure was compared，and then the characteristics of the ERIC fingerprint of bacteria in rabbit manure was analyzed.The ERIC-PCR reaction system for bacteria in rabbit manure was established.The characteristic bands for Bifidobacteria were located at 450 bp，the characteristic bands for E.coli were located at 1 300 and 4 000 bp.Anaerobic fermentation experiment proved that the trends of the bands’ brightness were consistent with the results of the colony count (the correlation coefficient between the relative brightness and lg cfu was 0.967 6).A rapid analysis and detection method of rabbit manure bacterium changes by using ERIC-PCR genetic fingerprinting technology was established.This method could reflect the changes of beneficial bacteria in rabbit manure，thus providing a basis for prevention，diagnosis and treatment of rabbit intestinal diseases.

The aim of this study was to research single nucleotide polymorphisms of agouti signal protein(Agouti)gene and analyze the association of SNPs with coat color phenotype in American mink(Neovison vison).The DNA of blood from 5 mink breeds(Jinzhou black，Jilin white，Silverblue，Coffee and Pearl mink)that possessed significant difference in coat color phenotype were selected as templates，SNP sites were screened by PCR and Sanger double chain termination sequencing technology，and then the relationship between the mutation sites of Agouti gene and coat color phenotype were analyzed in 430 minks.The results showed that the obtained mink Agouti gene was 2 510 bp in length.Total 10 SNPs were screened from 430 individuals of 5 coat color breeds，of which 4 SNPs (g.18G>A，g.159A>G，g.235G>T and g.1189C>T)were located in intron 2 and 6 SNPs(g.252C>T，g.290A>C，g.298G>C，g.340A>G，g.343T>C and g.379T>C)were detected from partial intron 3 of Agouti gene，while no SNPs was discovered in exon 2 and 3.Association analysis of 7 SNPs in Agouti gene with coat color phenotype indicated that all sites were very significantly(P<0.000 1)correlated with coat color phenotype of American mink.In addition，the 5 loci including g.290A>C，g.298G>C，g.340A>G，g.343T>C and g.379T>C might show closely linkaged phenomenon.Results of the present study indicated that Agouti gene might be the candidate gene or linked with the major gene affecting the coat color phenotype of American mink.

This experiment was conducted to study the effect of different rearing systems on hepatic gene expression in twin lambs.Twenty-four pairs of Hu male twin lambs were equally divided into 2 groups.Twenty-four subjects were artificially reared(AR) with milk replacer after 7 days colostrum while 24 others were ewe reared(ER) with mother milk as control.All lambs were weaned at day 60 and then fed with starter till day 90.When the lambs reached 60 and 90 days old，three pairs of twin lambs at each time point were slaughter to collect liver samples.Hepatic gene expression were analyzed via microarray and bioinformatics method.We found many differentially expressed genes(DEGs).They were OR1E2，S1PR3，KMO，APOA4，ORM2，HTR4，FADS1 in both 60AR vs 60ER and 90AR vs 90ER，and ACO1，HBA1，MFSD2A，CYSJ，LOC785954，FCN，GRB2，LOC100037663，ORM2，RXRG，LOC100602447，SQLE，HSD17B2，CYP4F2，INHBE，EAAT2 in both 60AR vs 90AR and 60ER vs 90ER，among which 4 unknown genes were identified with the probe ID 14780690，14815720，14845504，14854080.ORM2 was differentially expressed among all the comparisons.Those DEGs involved in the amino acid metabolism，small molecular metabolism，enzyme activities and immune response of GO process and amino acid metabolism，lipid metabolism，hormone biosynthesis and signaling transductions of KEGG pathways.In summary，different rearing systems altered hepatic gene expression.The pathways and functions those DEGs involved in could shed lights on the mechanisms of early cultivation of lambs.

This study was designed to measure cows’ rectal temperature (RT)，and to explore the pattern of dairy cows’ RT and effect of RT on milk yield under the heat stress during summer in Beijing area.Dairy cows’ morning & afternoon RT，body condition score (BCS) and ambient temperature in the barn were measured from 3 scaled dairy farms in Beijing area from July to August，2014，and DHI data of researched dairy farms was collected.The effects of farm，parity，stage of lactation and BCS on RT and test-day milk yield were analyzed using a fixed model.The result showed that：1) The simple statistics of 803 cows’ morning，afternoon RT and their difference were (38.69±0.45)℃，(38.98±0.50)℃ and (0.29±0.53)℃，respectively；2) RT among different farms was significantly different，the RT level at the first stage of lactation (day 1 to 50) was significantly higher than that at other stages (P＜0.05)，BCS had significant effect on afternoon RT(P＜0.05)；3) Both afternoon RT and squared BCS had significant effect on test-day milk yield (P＜0.05)，the average daily milk yield decreased 1.26 kg with per unit increase of afternoon RT.These results indicated that afternoon RT was relatively higher and might cause loss of test-day milk yield under heat stress during summer in Beijing area.Therefore，herd managers should strengthen cooling measures in summer in Beijing，monitor cows’ BCS，and pay more attention to early lactating dairy cows.RT can be used as an indicator in the study related to heat stress in dairy cows，and RT could provide indirect information for breeding plan for selecting heat tolerance ability in dairy cow.

Two samples labeling methods of microarray detection for diarrheal viruses are compared respectively in this study.Primers were designed based on the sequences of S and M gene of Porcine Epidemic Diarrhea Virus(PEDV)，S and N gene of Transmissible Gastroenteritis Virus(TGEV)，VP7 and NSP4 gene of Group A Porcine Rotavirus(GAR)，and then were used to amplify the target genes by PCR.The target genes were purified using ethanol precipitation to manufacture simultaneous detection microarray.Fluorescence molecules were imported to PCR products by direct-labeling method and indirect-labeling method respectively after extraction of virus RNA from samples.The specificity and sensitivity of two labeling methods were compared by scanning and analyzing the results after PCR products hybridized with microarray.The results showed that microarray based on two different labeling methods could both hybridize with target genes with high specificity.However，the median signal volumes of direct-labeling were higher than those of indirect-labeling and Signal to Noise Ratio(SNR) of direct-labeling was more than five times higher compared with that of indirect-labeling.Specific test results showed that two labeling methods were both specific and CSFV，PRRSV，PCV-2，JEV detection showed negative result.Sensitivity test results suggested that the sensitivity of direct-labeling were 100 times higher than that of indirect-labeling.The minimum concentration of direct-labeling method and indirect-labeling method can be detected reliably were 10⁵ copies•μL^-1 and 10⁷ copies•μL^-1，respectively.A total of 56 clinical samples were treated with direct-labeling and hybridized with the microarray，the results were consistent with those of RT-PCR.This study indicate that direct-labeling technology could effectively improve the detection effect of diarrhea microarray.

CRISPR/Cas 9 system is a novel gene editing technology.This study was aimed to construct CRISPR/Cas 9-based knockout system for E.coli aroA gene，and analyze knockout efficiencies in different E.colis. In the present study，aroA gene-targeting short guide RNA (sgRNA) was designed and constructed together with Donor sequence for homologous repair，then this resulting vector and pEwt-Cas 9 vector co-constituted the CRISPR/Cas 9 system.Preliminary application was performed on E.coli DH10B，DH5α and JM109，respectively，and then knockout efficiencies of aroA gene were analyzed by PCR amplification，TA cloning and subsequent DNA sequencing.The restriction enzyme digestion analysis and sequencing results showed that homologous repair vector was successfully constructed.PCR results indicated that the established CRISPR/Cas 9 system could be used for aroA gene knockout in multiple E.coli with efficiency ranging from 46% to 50%.Sequencing results further confirmed the successful and accurate knockout of aroA gene.In conclusion，CRISPR/Cas 9-based knockout system for E.coli aroA gene was successfully established，which would provide a novel and effective tool for functional studies on aroA genes of other pathogenic microorganisms and further development of attenuated vaccine.

The aim of this study was to understand the role of rpoN gene on biofilm formation of S.Typhimurium.Two S.Typhimurium strains with strong biofilm-forming ability were selected for construction of deletion mutants with deficiency of gene rpoN using the Red recombination system，and complemented strains were constructed by using prokaryotic expression vector.The biofilm-forming ability，and resistance to environmental stress of the wild-type，rpoN mutant and rpoN complemented strains were compared.A quantitative real-time PCR method based on csgA and bcsA genes was established to compare the expression of their biofilm components.The results showed that the biofilm formation，mainly contributed by increased expression of curli，was significantly enhanced in the rpoN gene deletion mutants when compared with that of the wild-type strains.The biofilm formation of revertants was similar to that of the wild-type strains.Furthermore，deletion of rpoN gene of S.Typhimurium strains resulted in their increased resistance to acid and alkali environment.Therefore， RpoN was identified as one of the associated sigma factors involved in biofilm formation of S.Typhimurium.These data may be helpful for elucidating the regulatory mechanism of Salmonella biofilm formation.

We attempted to gain insights into the possible mechanism of one Listeria monocytogenes strain M7 with naturally low virulence.Splicing by overlap extension-PCR and homologous recombination were utilized to construct the mutant strains with full deletion of vacuolar lysis related genes hly or (and) plcB.Subsequently，comparisons among mutants and their parent strain M7 together with the reference strain EGD were investigated in terms of hemolytic activity，phospholipase activity，adhesion and intracellular growth in HeLa cells，plaque forming assay in L929 cells，cytotoxicity against Caco-2 cells，as well as virulence to immunocompromised ICR mice.PCR amplification and reverse transcriptional PCR of the target genes indicated the successful construction of the mutants M7-Δhly，M7-ΔplcB and M7-Δhly/plcB.Furthermore，hemolytic and phospholipase activity were devoid in mutants M7-Δhly and M7-ΔplcB，respectively.Neither hemolytic nor phospholipase activity could be seen in the double mutant M7-Δhly/plcB.Besides，mutants M7-Δhly and M7-ΔplcB exhibited similar adhesiveness and intracellular growth in vitro (P＞0.05).Cytotoxicity assay under multiplicity of infection (MOI) ratio 1 000 revealed that the double mutant M7-Δhly/plcB showed the lowest cytotoxic to the host Caco-2 cells with the ratio of 11.10%，followed by mutant M7-Δhly with the ratio of 23.53%.Significant difference could be found between the aforementioned two mutants and other test strains (P<0.05).Plaque forming assay pinpointed that mutant M7-ΔplcB together with its parent strain M7 could form plaques only in the medium without gentamycin.The full deletion of hly or (and) plcB resulted in even less pathogenicity in immunocompromised ICR mice.This study presents some clues as the low virulence of strain M7 probably result from the over-expression of genes hly and plcB.Thereafter，the M7 strain might be exposed to the host immune responses due to its detrimentally stronger cytotoxicity.

The objective of the present study was to explore the effect of MAP0862 in sero-diagnosis of Bovine paratuberculosis，and establish a specific ELISA method based on MAP0862.The specific protein obtained by prokaryotic expression was used as coating antigen after purification and quantification，an indirect ELISA was primary established after a series of optimization.The specificity of this method was verified by MAP antiserum，Brucella antiserum，Bovine tuberculosis (TB) antiserum，BCG antiserum，Escherichia coli antiserum and negative serum.The detection results of 300 serums showed that the coincidence rate of MAP0862 indiret ELISA with commercial kit was 94.3%.The result indicated that the indirect ELISA method primary established based on MAP0862 can detect Bovine paratuberculosis effectively.

The aim of the present study was to analyze the characteristic of genetic variability in Heterakis gallinarum populations from Sichuan province，and provide a foundation for understanding the epidemiology and assist in the control of heterakiasis.The nuclear ribosomal DNA internal transcribed spacer region (ITS1-5.8S-ITS2) of 59 H.gallinarum isolates from chickens belonged to 7 different geographical regions were amplified using the polymerase chain reaction (PCR) and then sequenced.Sequences unit containing ITS1 and ITS2 (excepting 5.8S) were used to determine the genetic variability and genetic structure of H.gallinarum in Sichuan province，China.Meanwhile，the relationships of this nematode with selected members of genus Heterakis were assessed by phylogenetic analysis of ITS1/2 sequence datasets by maximum parsimony (MP) and maximum likelihood (ML).Results showed that 59 H.gallinarum ITS1/2 sequences identity ranged from 98.9% to 100.0%，and ITS1 sequences displayed greater variability than ITS2.Fifty-nine sequences contained 20 variable sites，and were classified into 14 haplotypes (H1-H14).The overall Sichuan population showed high haplotype diversity (Hd = 0.532) and low nucleotide diversity (π=0.000 94).Meanwhile，a frequency of gene flow (Nm=31.72)，a low degree of genetic differentiation (Fst=0.007 82)，and an overwhelming genetic variability (99.22%) were observed within the overall Sichuan population.Phylogenetic trees (MP and ML) and haplotype network were both revealed that 7 different geographical regions did not form significant geographical populations.The different species within genus Heterakis were well distinguished in phylogenetic trees (MP and ML).Our results indicate that there is a high gene flow and low degree of genetic diversity across H.gallinarum population.

In order to investigate the role of mobile plasmids in the horizontal transmission mechanism of drug resistance in Escherichia coli isolated from pigs，the conjugation experiments of fluoroquinolone resistance and PMQR genes positive Escherichia coli were carried out，the minimum inhibitory concentration(MIC) of 8 kinds of common drug in transconjugants were figured by using broth micro dilution method and the plasmid mediated quinolone resistance gene(PMQR) were detected by PCR methods with specific primers designed after the conjugation experiments.The results showed that 16 strains of bacteria were joined successfully within 41 strains of fluoroquinolone resistance and PMQR genes positive donors，successful rate of conjugation experiments was up to 39%，transconjugants showed kinds of resistant phenotypes compared with J53 strain through testing the figure of MIC values，and compared with donor strains，87.5% of the transconjugants reveals changes of drug resistance patterns and the phenomenon of missing a drug resistance with obtaining another drug resistance，the results of PCR showed the decline of gene phenotypes compared with donor strains，qnrS gene revealed the highest successful conjugation rate，the genes of oqxA and oqxB can transferred in the same mobile plasmid，12.5% of the transconjugants displayed that the genes of oqxA，oqxB and qnrS can be transferred by the same mobile plasmid.The results above suggest that different PMQR genes show different success rates in the process of conjugation jointing with mobile plasmid which mediated resistance genes transferred horizontally in strains，which suggest the different PMQR genes may be located in the different mobile plasmid，by comparing the changes of drug resistance and gene phenotypes before and after conjugation experiments，especially the detection rate of PMQR gene，it can be preliminarily confirmed that the mobile plasmids play a very important role in the process that antimicrobial agents resistance genes are transferred horizontally in Escherichia coli strains.

It has been recently reported that PmrA-PmrB are associated with polymyxin B resistance.To determine whether PmrA-PmrB is contributing to colistin resistance in E.coli，we investigated amino acid alterations and transcription level of pmrA-pmrB in colistin resistance strains.All clinical resistance isolated strains were screened for colistin resistance by using MIC determinations and step-wise method was used to induce colistin resistant strains. pmrA-pmrB genes were amplified by PCR from resistant strain and their sequences were characterized by Mega software.Simultaneously，expression levels of PmrA-PmrB were detected by quantitive Real-time PCR.MIC (minimum inhibitory concentration) results showed that 88.5% of 52 clinic resistance strains tested in this study were susceptibility to colistin and 11.5% of the strains were resistant.Five mutations were identified in pmrB in induced colistin-resistant mutants 9R (G55A)，36R (T500C)，53R (T263A)，91R (insertion of 30 bp at position of 229) and 107R (insertion of 189 bp at position of 478).However，six clinical resistance isolates (MIC=4-8 μg•mL^-1) did not show any mutations in pmrA-pmrB.Further study showed that pmrA-B expression levels were significantly activated in three lab-derived colistin resistant strains with point mutations of pmrB.The expression level of pmrA-pmrB genes tended to increase but not significant changed in resistant strains with insertion mutations compared with its correspond parent strains.The transcription level of pmrA-pmrB in 6 clinical resistance isolates remained unchanged compared with susceptible strains.In addition，when MICs were over than 16 μg•mL^-1，mutations in pmrA-pmrB would occur.Mutations in pmrB or high expression levels in pmrA-pmrB contribute to high-level colistin resistance in E.coli strains.

 To explore the distribution of extracellular matrix proteins of epididymis in Plateau Tibetan sheep.The histochemistry of Masson’s，Gomori’s，PAS and AB-PAS（pH 2.5）stainning were used to study the microstructure of epididymis in 7 Plateau Tibetan sheep，also the distribution of the laminin (LN)，type IV collagen (Col IV) and heparan sulfate glycoprotein (HSPG) was identified by SP immunohistochemical method.The observations made with the light microscope showed that the epithelium of epididymal ducts were columnar ciliated epithelium and the cauda was more abundant with collagen and reticular fiber than in the caput and the corpus，and as well as the PAS positive reaction in the cauda，but AB-PAS positive reaction showed that the caput and the cauda were stronger than the corpus.In addition，immunostaining analysis appeared that LN was strongly presented in the epididymis，the relative expression of HSPG was lower than LN，and Col IV was weakly expressed，but the distribution of LN and HSPG without statistical differences in epididymis (P＞0.05).Taken together，the collagen and reticular fiber were more abundant in cauda than caput and corpus in Plateau Tibetan sheep epididymis，and the intensity of the PAS reaction was companied with the changes of the secretion function of epithelium，the Col IV distribution of ECM proteins was participated in basement membrane transport，but HSPG was related to the blood-epididymis barrier，and the distribution of LN may closely related with the membrane construction and secretion the of other extracellular matrix proteins in epididymal ducts.

In order to study the effects of fluid-increasing decoction for purgation on intestinal structure，concentration of IL-1β，IL-6 and TNF-α in serum of rat in heat accumulation of intestinal type of Qi-fen syndrome，the experiments were carried out to construct the heat accumulation of intestinal type of Qi-fen syndrome，and the fluid-increasing decoction for purgation was given to the rat in preventability or therapeutically，then body temperature，the intestinal organizational structure，and the concentration of inflammatory cytokines in serum of rat were also be studied.The results showed that the intestinal structure in rat in heat accumulation of intestinal type of Qi-fen syndrome were significantly changed，for example，the intestinal mucosa was dropped，inflammatory cells were infiltrated，IELs were increased，goblet cells (GC) were decreased，and the concentration of IL-1β，IL-6 and TNF-α in serum were increased in significantly.The rat were given with the fluid-increasing decoction for purgation in preventability，which will alleviate the damages to intestine of rats，and the concentration of IL-1β，IL-6 and TNF-α decreased significantly；and the rat were given the decoction in therapeutically，which will decrease the body temperature，keep the intestinal structure and mucosal barrier in normal，and decrease the concentration of IL-1β，IL-6 and TNF-α in serum of rats.Therefore，fluid-increasing decoction for purgation which is the classic formula to treat the rats in heat accumulation of intestinal type of Qi-fen syndrome has the preventability and therapeutically effects through decreasing the body temperature，maintaining the intestinal structure in normal，decreasing the IEL’ number and increasing the GC’ number，and then inhibition the secretion of inflammation cytokines.

The aims of this paper was to investigate the protective effect of pulsatillae decoction (PD) on endothelial cells injured by lipopolysaccharide (LPS) and to reveal the pharmacological of PD.The cell proliferation and cytotoxicity of the rat microvascular endothelial cells (RIMVECs) were determined by WST-1 ELISA kit and lactate dehydrogenase (LDH) assay.In addition，the RIMVECs damage stimulated by LPS was observed by hematoxylin-eosin (HE) and Hoechst 33342 staining.The transedothlial neutrophils killing was detected by gentamicin protection assay though the build of Transwell.The results showed that LPS (1 μg•mL^-1) make the nuclei of RIMVECs shriveled and damage the cell membrane of RIMVECs and lead to cell fall off.The rate of cytotoxicity was 53.2%.The rate of separated cells wass 39.6%.PD（20 μg•mL^-1）had the significant proliferative effect for that.LPS induced transendothelial migration of neutrophils can reduce the survival of bacteria in both intracellular and extracellular under the effect of 20 μg•mL^-1 PD (P＜0.01).Results reveal that PD can improve the sterilization function of PMNs by the effective protection of LPS-induced injury of RIMVECs，and might improve the sterilization function of PMNs by protecting the integrity of the RIMVECs to provide a theoretical basis for the dose of clinical treatment of bacterial animal diseases.

In order to improve the accuracy of detecting dairy cow subclinical mastitis by infrared thermography，the effect of smudgy degree on temperature distribution of the udder surface in dairy cow was studied.325 dairy cows with normal udder appearance and SCC<5×10⁵ •mL^-1 were selected.Their rear udders were partitioned into teat cistern，mammary cistern and mammary gland，and the smudgy degree was graded according to a 3-point scoring system.Then different parts of rear udder were divided into 4 groups.The results showed that the difference of skin temperature of left and right sides at the different parts was not significant(P>0.05) in the clean rear udder.It was also found that the mean skin temperature，minimum temperature and maximum temperature from mammary gland，mammary cistern to teat cistern significantly reduced or had a tendency to be lower，and that the decreased amplitude was 1.90，6.90 and 1.62 ℃，respectively.However，with the increase of degree of the smudge，at teat cistern，mammary cistern and mammary gland，the minimum skin temperature，was sharply decreased(P<0.01)，mean temperature had a tendency to reduce，and the variational degree of temperature was significantly increased(P<0.01).These results indicated that smudgy degree of udder surface had an important effect on its temperature distribution，the highest temperature of mammary gland had a smaller effect of smudge，which is optimum to determine subclinical mastitis of dairy cow by IRT method.

In order to explore the epidemiological and genetic character of the H9N2 viruses，a long-term surveillance have been conducted in Shandong province.One thousand two hundred and ninety six strains of H9N2 virus were isolated from clinical samples，which were collected in chicken farms and LBMs，and the HA and NA genes of 40 strains selected were amplified and sequenced.The HA and NA genes of these viruses shared more than 90% identity at the nucleotide level and amino acid level；most viruses carried the amino acid sequence PSRSSR↓GLF at the HA cleavage site and a 3-amino-acid deletion in the NA stalk；new potential N-glycosylation sites were found in the HA and NA in some viruses；the Q226L (H3 numbering) mutation was detected in all viruses at the receptor-binding pocket of HA；phylogenetic analysis showed that the HA and NA belong to the Y280-like sublineage.The H9N2 influenza virus was widely circulating in poultry farms of Shandong province，and reached peak in 2013；all of isolates preferentially bound to the human-like receptor，but still have the characteristic of low-pathogenic AIV.So，keeping monitoring and controlling H9N2 virus is necessary.