The aim of this study was to investigate the complete genomic sequence,genomic characteristics and genetic variation of bovine papilloma virus genotype 1 (BPV-1) GX01 strain from Guangxi,and the histopathological changes of host was caused by it as well.Firstly,histopathological examination was conducted using the cutaneous papilloma samples of cattle collected from Hezhou city of Guangxi.DNA were extracted and employed as the template of PCR for BPV detection and genotyping using FAP59/FAP64 primers.Based on the complete genomes of the BPV-1 reference strains,specific primer pairs were designed,and the complete genome of the GX01 strain was further amplified and sequenced.Histopathological analysis of cutaneous papilloma showed hyperkeratosis,acanthosis and koilocytosis,which were the typical histopathological characteristics of BPV infection.Genotyping showed GX01 belonged to BPV-1.The full-length genome of GX01 was 7 945 bp,and contained 8 open reading frames (E1,E2,E4,E5,E6,E7,L1 and L2),which matched the structural characteristics of BPV-1.GX01 shared 98.6% to 99.6% homology with the complete genome sequences of BPV-1 reference strains,while 86.9% and 87.2% homology with those of the BPV-2 (M20219.1) and BPV-13 (JQ798171.1) reference strains,respectively.The GX01 strain was the first BPV-1 strain from Guangxi being confirmed and fully sequenced.This study provided theoretical basis for pathogen identification,epidemic regularity,genetic variation,epidemic source tracking and scientific prevention and control of bovine papilloma in Guangxi and China.

In order to understand the variation of bovine coronavirus (BCoV) S gene and establish an ELISA detection method,total RNA was extracted from neonatal calf diarrhea (CD) and adult cattle winter dysentery (WD) diarrhea samples from different farms.cDNA was synthesized,and S and S1 genes were supplemented by PCR.The target fragment S1 was lighted into the expression vector pET-32a(+) and transformed into E.coli BL21 (DE3).The recombinant plasmid was induced by IPTG after being verified by PCR,double enzyme digestion and sequencing.The results showed that the nucleotide identitie of S gene of CD and WD isolates was 98.4%,and the isolates had the highest nucleotide homology with the reference strain BCoV-ENT,which were 98.4% and 98.5%,respectively.The homology of CD isolate with the reference strain SUN5 was the lowest,which was 97.5%,and the homology of WD isolate with strain FRA/EPI/Caen/2004/13 was the lowest,which was 97.3%.The comparison showed that there were a big difference between the isolated strains and the known strains,and provided a basis for screening the vaccine candidate strains.At the same time,pET-32a-S1 expression vector was constructed.The recombinant bacteria could produce a large amount of fusion S1 protein in E.coli BL21 and obtain 58 ku expression product,induced by 0.2 mmol/L IPTG for 5 h.In this study,S1 protein was successfully expressed,and nucleotide evolution analysis of BCoV were carried out,which laid the foundation for the establishment of the vaccine immune effect evaluation method.

In order to clarify the structure and function of paraoxonase 1 (PON1) gene,all exon sequences of PON1 gene in Longdong cattle were amplified by PCR method,the complete sequence was assembled by Seqman software,and the base composition,open reading frame,physical and chemical properties of coding protein,atomic composition,number of amino acid residues,hydrophilicity and hydrophobicity,molecular weight,isoelectric point,secondary structure,higher structure,etc were predicted and analyzed by bioinformatics method.The results showed that the CDS sequence of PON1 gene in Longdong cattle was 1 068 bp,encoding 355 amino acids,Leu was the most abundant,Cys and Trp were the least.The content of A+T (56.55%) was higher than that of G+C (43.26%) in base composition.Compared with the published sequence (GenBank accession No.:EU289337) of cattle,we found that there were mutations which were a synonymous mutation in exons 4,6,8 and 9,but none of them changed amino acids.PON1 protein was a stable water-soluble protein,the atomic composition was C₁₈₁₀H₂₇₉₆N₄₅₄O₅₃₃S₁₂,the molecular mass was 39.83 ku,and the theoretical isoelectric point was 5.24.The number of amino acids of random coil,extended strand,α-helix and β-turn in the secondary structure of PON1 protein was 159,116,55 and 25,respectively,eventually formed a hybrid model with random coil and extended strand.Hydrophobic results of PON1 protein showed that the 8th Thr had the strongest hydrophobicity (the highest score:2.878) and the 299th Pro at the highest hydrophilicity (the lowest score:-2.222).The results of this study would lay a foundation for further study on the function of PON1 protein in Longdong cattle and explore the effect of PON1 gene mutation on the carcass and meat quality traits of local cattle in Gansu province.

This study was aimed to clone and sequence the buffalo staphylococcal nuclease and tudor domain containing 1 (SND1) gene using the electronic cloning method,and analyze its genetic struction with bioinformatics,which laid a foundation for investigating the effect of SND1 gene on the milk performance in buffaloes.In this experiment,three pairs of specific primers were designed by Primer Premier 5.0 according the sequence of Bos taurus SND1 gene in GenBank (accession No.NM_205784.1),the buffalo DNA was used as the template,the sequence of buffalo SND1 gene mRNA was ampified by PCR,the target fragment linked into the cloning vector pMD18-T was sequenced and systemically analyzed by bioinformatics.The results showed that the length of SND1 gene mRNA was 3 503 bp,containing a CDS of 2 733 bp,which encoded 910 amino acids.The protein molecular formula was C₄₅₂₃H₇₁₈₃N₁₂₈₁O₁₃₅₄S₂₆, the molecular weight was 102.01 ku,the theory isoelectric point was 6.74,the instability coefficient was 42.07,grand average of hydropathy was -0.419,SND1 was the soluble acid protein.The secondary structures prediction of buffalo SND1 protein showed that alpha helices and random coils accounted for 37.80% and 34.18%,which were the main structure of the protein.Gene function prediction showed that the possibility of the gene function in the purine and pyrimidine,central intermediary metabolism,energy metabolism,biosynthesis of amino acids were 0.449,0.401,0.303 and 0.262,respectively.Sequence homology analysis indicated that the buffalo SND1 showed 98.7%,97.8%,97.8%,93.8%,93.1% and 91.7% identity with those of Bos taurus,Ovis aries,Capra hircus,Sus scrofa,Equus caballus and Homo sapiens,respectively.Sequence analysis revealed that buffalo SND1 gene was conserved,and showed high sequence similarity with other species.Online predicting tool SMART was used to predict protein structure domain,it showed that buffalo SND1 protein contains four SN area.This results suggested that the SND1 gene CDS was more conservative in the evolution process.

This study were aimed to construct the eukaryotic expression vectors for porcine orexin 2 receptor (pOX2R) mutants and explore the difference in constitutive activity between wild type and mutants.We used the eukaryotic expression vector pcDNA3.1(+)/pOX2R-myc WT as the template to construct four mutants,named pcDNA3.1(+)-myc/pOX2R-P10S,pcDNA3.1(+)-myc/pOX2R-P11T,pcDNA3.1(+)-myc/pOX2R-V308I and pcDNA3.1(+)-myc/pOX2R-T401I,and then transient transfected the plasmids into HEK293T cell.The basal activities of pOX2R wild-type and mutants were determined using dual luciferase reporter assays,the intracellular cAMP levels were measured at different concentrations of agonists,the wild-type and mutants were stimulated with the endogenous agonists orexin A (OXA) and orexin B (OXB),respectively.The results showed that the recombinant plasmid pcDNA3.1(+)/pOX2R-myc-P10S,pcDNA3.1(+)-myc/pOX2R-P11T,pcDNA3.1(+)-myc/pOX2R-V308I and pcDNA3.1(+)-myc/pOX2R-T401I were constructed successfully.The 10,11,308 and 401 amino acids of the pOX2R were successfully mutated to serine,threonine,isoleucine and valine,respectively.There were no significant difference between reconstruction plasmid of pcDNA3.1(+)-myc/pOX2R WT and four mutants in basal activity (P>0.05).It showed that there was no significant effect of amino acid mutations at 4 sites on the basal expression signal of their receptors.Compared with the wild-type receptor,there was no significant difference in the response of the mutant receptor to OXB (P>0.05),but the EC₅₀ of the mutant P10S,P11T and T401I to agonist OXA was significantly lower (P<0.05),there was no significant difference in R_max (P>0.05).The mutants (P10S,P11T and T401I) might be affect the binding between agonist OXA ligand and receptor,and reduce the agonistic effect of the agonist.The study provided foundation for the further exploration of the function of pOX2R.The results laid the foundation for further study of pOX2R function in vitro.

The aim of the experiment was to construct the expression vector of V5-labeled porcine circovirus type 2 (PCV2) Cap protein using eukaryotic expression system.The V5 tag was introduced into the end of the PCV2 ORF2 gene using genetic engineering,and the tagged gene was cloned into the pFastBacHTA vector.Recombinant transfer vector pFastBacHTA-ORF2-V5 was transformed into E.coli DH10Bac.The identified recombinant bacmid was transfected into Sf9 cells using liposome-mediated method;Indirect immunofluorescence assay (IFA),SDS-PAGE and Western blotting were used to test the expression of V5-labeled Cap protein in Sf9 cells.The results showed that this experiment successfully introduced the V5 tag to the end of the PCV2 ORF2 gene.After the recombinant transfer vector pFastBacHTA-ORF2-V5 transformed into E.coli DH10Bac,Bacmid-ORF2-V5 recombinant bacmid was obtained.The result of IFA,SDS-PAGE and Western blotting showed that it could be expressed in Sf9 cells.The recombinant baculovirus V5-labeled PCV2 Cap protein was detected to have good reactogenicity,indicating that this experiment successfully obtained a rAcMNPV-Cap-V5 recombinant baculovirus.The test results provided a theoretical basis for the development of PCV2 marker subunit vaccine.

In order to explore the effects of different biotin levels on lipolysis and relevant gene expression of 3T3-L1 adipocytes,3T3-L1 cells were differentiate into 3T3-L1 adipocytes by the triplet induction method.3T3-L1 adipocytes were treated with 0 (control group),0.2, 0.5 and 1.0 μmol/L biotin upon being concentrated,and relative expression levels of ATGL,HSL,perilipin A and ADRP were measured at 12,24 and 48 h,respectively.The results showed that:At 12 h,the glycerol emission and ATGL gene mRNA relative expression of 1.0 μmol/L group were extremely significantly lower than that of control group (P<0.01).The HSL gene mRNA relative expression of 0.5 and 1.0 μmol/L groups were significantly lower than that of control group (P<0.05),while the mRNA relative expression of perilipin A and ADRP genes in 1.0 μmol/L group were extremely significantly higher than that of control group and 0.2 μmol/L group (P<0.01).At 24 h,the glycerol emission of three test groups and ATGL gene mRNA relative expression of 1.0 μmol/L group were extremely significantly lower than that of control group (P<0.01).The perilipin A gene mRNA relative expression of 0.5 and 1.0 μmol/L groups were extremely significantly higher than that of control group and 0.2 μmol/L group (P<0.01),and the mRNA relative expression of ADRP gene of 1.0 μmol/L group were extremely significantly higher than that of other groups (P<0.01).At 48 h,the glycerol emission and ATGL gene mRNA relative expression of 1.0 μmol/L group were extremely significantly or significatly lower than that of control group (P<0.01;P<0.05).The perilipin A gene mRNA relative expression of 1.0 μmol/L group was significantly higher than that of control group and 0.2 μmol/L group (P<0.05),and the mRNA relative expression of ADRP gene of 0.5 and 1.0 μmol/L groups were extremely significantly higher than that of control group (P<0.01).In conclusion,the biotin could inhibit the lipolysis through promoting the relative expression of perilipin A and ADRP and reducing the relative expression of ATGL and HSL.Moreover,when the concentration was 1.0 μmol/L,the effect was optimal.

In order to construct an eukaryotic expression vector of chicken retinoid acid receptor related orphan receptor alpha (RORα) and detect its expression efficiency in MSB1 cell,the specific primers of RORα gene were designed according to the gene sequence of chicken RORα gene from GenBank (accession No.:NM_001289887.1).Total RNA was extracted from chicken liver tissue,the first strand of cDNA was synthesized by reverse transcription,and the chicken RORα gene fragment was amplified from cDNA,then cloning vector pMD18-T-RORα was constructed.The eukaryotic expression vector pEGFP-N1 and cloning vector pMD18-T-RORα were digested by the restriction enzyme Sal Ⅰ and BamH Ⅰ,the RORα and pEGFP-N1 fragments were recycled,then the RORα gene fragment were connected to pEGFP-N1 to construct the recombinant vector pEGFP-N1-RORα,and the recombinant vector was identified by PCR,double enzyme digestion and squencing.The results showed that the 1 600 bp fragment was amplified from the recombinant eukaryotic expression vector,and two segments length about 4 700 and 1 600 bp were cut using restriction endonuclease Sal Ⅰ and BamH Ⅰ from the recombinant vector pEGFP-N1-RORα.Then the recombinant vector pEGFP-N1-RORα was transfected into the MSB1 cells,the EGFP-positive cells was observed under the fluorescence microscope,and the expression level of RORα in MSB1 cell was evaluated which measured by Real-time quantitative PCR at 48 h after transfection.Compared with control group,the expression level of RORα was significantly increased in pEGFP-N1-RORα transfected MSB1 cell (P<0.05).The result suggested that the eukaryotic expression vector of RORα was successfully constructed,and the recombinant vector could express RORα in MSB1 cell,which laid a foundation to further study the effect of RORα on the biological behavior of MSB1 cell.

The aim of the experiment was to clone the ORF129 gene of orf virus,express and purify its encoded protein.According to the published ORF129 gene sequence of OV-IA82 strain in GenBank (Accession No.:AY386263.1),a pair of specific primers was designed and synthesized using Primer Premier 5.0 software,and the ORF129 gene was amplified by PCR.The whole gene sequence was ligated with the plasmid pET-28a(+) by BamH Ⅰ and Hind Ⅲ,and the recombinant plasmid pET-28a(+)-ORF129 was constructed.After identified by double digestion and sequencing,the recombinant plasmids were transformed into E.coli Rosttta,and the expression of ORF129 gene was induced by IPTG.After SDS-PAGE analysis,the expression products were disrupted,washed and dissolved by ultrasound,and then urea gradient dialysis method was used.After renaturation,the target protein was purified by affinity chromatography and identified by Western blotting.Double restriction enzyme digestion and sequencing results showed that the recombinant plasmid was successfully constructed with the correct reading frame.The ORF129 inclusion body protein was obtained when the initial D_{600 nm} value was 0.4 to 0.8,37℃,220 r/min,the final concentration of IPTG was 1 mmol/L,and the induction time was 3 h.And the protein size was 58 ku.In the arginine and glycerol refolding solution,the protein refolding rate was higher,and the protein was refolded after binding to the Ni column.When the concentration of imidazole was 500 mmol/L,the protein could be eluted.The purified protein was identified by Western blotting and proved to be successfully expressed.In this study,the prokaryotic expression recombinant plasmid of ORF129 gene was successfully constructed,and the ORF129 protein was successfully expressed and purified,which laid a foundation for the establishment of the detection method for orf virus.

In order to establish a TaqMan Real-time PCR for rapid,sensitive and specific distinguishing the wild strain and gE-deleted vaccine strain of pseudorabies virus (PRV),two sets of primers and TaqMan probes were designed for gD and gE genes of PRV,respectively,then a set of 2 novel Real-time PCR were developed for quantitative detection and differentiation of wild-type strain from gE-deleted vaccine strain of PRV.The concentration of primers and probes,the annealing temperature in TaqMan Real-time PCR were optimized,the sensitivity,specificity and reproducibility of the assays were determined,and were applied to the detection of clinical samples.The R² value of the TaqMan Real-time PCR standard curve were 0.996 and 0.980,respectively,both showed good linear response.The detection limit of the assays were 39.4 and 12.1 copies/μL,respectively.The assays were highly specific for PRV,without cross-reaction with other common swine viral pathogens,such as porcine circovirus type 2 (PCV2),classical swine fever virus (CSFV),porcine reproductive and respiratory syndrome virus (PRRSV).Intra-batch and inter-batch reproducibility tests showed that the coefficient of variation (CV) were 1.43% to 1.86%,1.10% to 2.07% (gD gene) and 0.98% to 1.41%,1.12% to 1.86% (gE gene),respectively.Applying the TaqMan Real-time PCR and the conventional PCR to detect 11 PR suspected clinical samples,they got 36.4% and 27.3% positivity,respectively.All these results indicated that the TaqMan Real-time PCR were both sensitive,specific and reproducible,and could be used as a useful tool for quantitative detection and differentiation of wild-type strain from gE-deleted vaccine strain of PRV.

The study was aimed to analyze the status of batching fidelity of protein feedstuffs by statistical process control (SPC) and the causes of batching error,in order to offer references for the quality management of batching process in feed mill.Batching preset values and actual bacthing values of three protein feedstuffs including soybean meal, fish meal and extruded soybean of piglet feed from two feed mills (A and B) in 2015 were used as the research data,and the status of batching fidelity was analyzed by individual and moving range control charts (X-MR) and process capability method.The results showed that for feed mill A,process capability index (C_Pk) of soybean meal,fish meal and extruded soybean were 0.20,0.11 and 0.01,respectively,and the feed batching fidelity of extruded soybean was the worst;Batching values of three protein feedstuffs were much higher than preset values.For feed mill B,the C_Pk of soybean meal,fish meal and extruded soybean were 0.24,0.12 and 0.24,respectively.The feed batching fidelity of fish meal was the worst,and the batching process of extruded soybean had out of control situation.The batching errors of protein feedstuffs exceeded the demands of ±0.3% FS,indicating that the batching fidelity of protein feedstuffs of two feed mills was low.The reasons of low batching fidelity were the technology parameters of batching scale in two feed mills were unreasonable.The parameters of air dropping values and inlet amount should be improved, and there were operational mistakes in feed mill B.In conclusion,using SPC could visually monitor the feed batching process,improve batching accuracy of protein feedstuffs, reduce waste of protein feedstuffs and feed processing cost,so improve quality management level of feed mills.

The study was aimed to investigate the effect of Lactobacillus plantarum and Lactobacillus buchneri on the quality and aerobic stability of sugarcane tops silage.There were 10 groups in this experiment:Group A served as control group and no additives;The sugarcane tops silage in groups B,C,D were treated with Lactobacillus plantarum with 10,20,30 mL/kg, respectively;The sugarcane tops silage in groups E,F,G were treated with Lactobacillus buchneri with 10,20,30 mL/kg, respectively;The sugarcane tops silage in group H,I,J were treated with Lactobacillus plantarum+Lactobacillus buchneri (1:1) with 10,20,30 mL/kg, respectively.There were 3 replicates in each group,and the sugarcane tops was stored at room temperature for 40 d,and the quality and aerobic stability of silage were determined.The results showed that:Compare to control group,the pH was significantly reduced and lactic acid concentration was increased of sugarcane tops silage in Lactobacillus plantarum adding groups (P<0.05),and with the increase of Lactobacillus plantarum,the concentration of lactic acid increased.Compare to Lactobacillus plantarum adding groups,the concentration of lactic acid of Lactobacillus buchneri adding groups was significantly decreased (P<0.05),while the acetic acid content was significantly increased (P<0.05),and the effect was more obvious with the increase of Lactobacillus buchneri adding dose,and the aerobic stability was increased by 48 h compared with the control group.The concentration of lactic acid of Lactobacillus plantarum and Lactobacillus buchneri adding groups were significantly increased (P<0.05),and the DM losses was reduced and WSC utilization efficiency was improved compare to Lactobacillus buchneri adding groups.In conclusion,Lactobacillus plantarum and Lactobacillus buchneri adding together (2×10⁶ CFU/g FW) could improve the quality and aerobic stability of sugarcane tops silage.

The experiment was conducted to study the effects of plant additives (mulberry leaf powder,garlic powder and green tea powder) on content and composition of amino acids and fatty acids in muscle in Huiyang Bearded chicken.120 one hundred and twenty-day-old female chickens were randomly divided into 4 treatments,3 replicates per treatment,10 chickens per replicate.The different treatments were fed the following diets:Group Ⅰ,fedding basal diet + 2% green tea powder;Group Ⅱ,feeding basal diet + 4% mulberry leaf powder;Group Ⅲ,feeding basal diet + 1.5% garlic powder;Control group,feeding basal diet.After preliminary experiment for 7 days,the feeding trial lasted for 30 days.After the trial,10 chickens per treatment were selected to be slaughtered.100 g of meat from breast muscle of every individual from the slaughtered chickens was used to analyze the content and composition of fatty acids and amino acids by gas chromatography and automatic amino acid analyzer,respectively.The results showed that:①The plant additives had no effect on the composition of amino acids in muscle,but significantly improved the content of essential amino acid in muscle compared with the control group (P<0.05);②The plant additives had no effect on the composition of fatty acids in muscle,but significantly decreased content of the saturated fatty acids (P<0.05),while significantly improving percentage of polyunsaturated fatty acids in muscle compared with the control group.Overall,the plant additives could improve the meat quality by significantly improving the content of essential amino acid,percentage of polyunsaturated fatty acids in muscle,and significantly decreasing content of the saturated fatty acids in muscle.

The experiment was aimed to study the effects of adding different levels of sodium butyrate on the growth performance,diarrhea rate and blood biochemical indexes of weaned piglets.240 healthy weaned piglets (Duroc×Landrace×Yorkshire) at age of (21±2) days were selected and randomly divided into five groups according to the principles of nest and body weight,namely,groups Ⅰ to Ⅴ,with 3 replicates per group and 16 piglets per replicate.Group Ⅰ was the control group,the diet of group Ⅱ was supplemented with 0.01% antibiotics while that of groups Ⅱ,Ⅳ and Ⅴ were added 0.1%, 0.2% and 0.3% sodium butyrate, respectively.The experiment lasted for 28 days.The body weight of weaned piglets were measured at 0,7,14,24 and 28 d,and ADG,ADFI, F/G,diarrhea rate and diarrhea indexes were measured during the experiment.The blood samples were collected from 6 piglets per group to determine the blood biochemical indexes.The results showed that compared with group Ⅰ,the addition of sodium butyrate in weaned piglets diets significantly increased the ADG in groups Ⅳ and Ⅴ at first,second,third weeks and the whole period (P<0.05).Supplementation of sodium butyrate significantly increased the ADFI in groups Ⅲ,Ⅳ and Ⅴ at the second,third weeks and whole period (P<0.05).The F/G of groups Ⅱ,Ⅲ,Ⅳ and Ⅴ were decreased at second,third,four weeks and the whole period (P>0.05),while that in groups Ⅳ and Ⅴ were decreased significantly at first week (P<0.05).The diarrhea rate and diarrhea index in groups Ⅱ,Ⅲ and Ⅳ did not change significantly (P>0.05).The diarrhea rate (P<0.05) and the diarrhea index (P>0.05) in group Ⅴ was increased.Compared with group Ⅰ,the levels of serum phosphorus in groups Ⅲ,Ⅳ and Ⅴ were significantly increased (P<0.05), and the levels of serum calcium and total protein in group Ⅴ were significantly increased (P<0.05),while the triglyceride content of groups Ⅳ and Ⅴ were significantly increased than that of groups Ⅰ,Ⅱ and Ⅲ (P<0.05).There was no significant change in serum total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, urea nitrogen and albumin among all groups (P>0.05).In conclusion, adding sodium butyrate to the diet of weaned piglets had the effect of promoting growth performance and improving health,and the optimum amount of sodium butyrate was 0.2%.

This experiment was aimed to study the effects of different adding amounts of yeast-germanium culture on intestinal flora,immunity and antioxidant properties of weaned piglets.One hundred healthy piglets((6.0±0.5) kg) were randomly divided into 5 groups,with 5 replicates per group and 4 piglets per replicate.The control group was fed basal diet,and the content of yeast-germanium culture in basal diet of test Ⅰ,Ⅱ,Ⅲ and Ⅳ groups were 15,25,35 and 45 mg/kg,respectively.The prefeeding period was 7 days.The test period was 42 days,which was divided into 3 stages,14 days per stage.The number of Lactobacilli and Escherichia coli in intestinal tract of piglets at all stages was measured.The content of blood immunoglobulin and the related indexes of antioxidant activity were measured at the end of the test.The result shows that:Compared with the control group,in the first stage of the test,the number of Escherichia coli in all experimental groups were extremely significantly or significantly decreased (P<0.01; P<0.05),and the number of Lactobacilli in all experimental groups were extremely significantly or significantly increased (P<0.01; P<0.05).The serum IgG levels in groups Ⅲ and Ⅳ were extremely significantly increased (P<0.01).For serum MDA content,group Ⅱ was significantly decreased (P<0.05),groups Ⅲ and Ⅳ were extremely significantly decreased (P<0.01).For serum GSH-Px,SOD and T-AOC,group Ⅱ was significantly increased (P<0.05),groups Ⅲ and Ⅳ were extremely significantly increased (P<0.01).In summary,the yeast-germanium culture could effectively improve the intestinal flora environment and improve the immunity and antioxidant properties of piglets.Based on the consideration of economic cost,it was suggested that the concentration of yeast-germanium culture in weaned piglets diet was 35 mg/kg.

The experiment was aimed to study the effect of compound preparations of negative ion and Chinese medicine on indexes of growth performance,nutrient apparent digestibility and blood biochemical of Yanbian Yellow cattle.Twelve thirty-month Yanbian Yellow cattle with body weights of (351±20) kg were selected and divided into control and test groups randomly.The cattler in control group were fed the basal diet,and in test group were fed the basal diet added with 20 mg/kg compound negative ion and Chinese medicine.The experimental period was 100 d,including preliminary trial period for 10 d and trial period for 90 d.During the experimental period,feed consumption and residue were recorded accurately.The cattle were weighed at the end of the preliminary trial and before slaughter.At the end of the test,the blood samples were colleted without feeding to measure the routine biochemical indexes.The nutrients apparent digestibility of cattle were measured using total collection method at day 90 of the test.The results showed that,compared with the control group,the ADG,FCR and the apparent digestibilities of CP,EE and CF of the cattle in test group increased significantly (P<0.05),while the apparent digestibilities of DM,crude ash,Ca and P had no significant change (P>0.05).The blood WBC and LYM count,LYM% increased signicantly (P<0.05),but other indexes in blood had no significant difference(P>0.05).In conclusion,adding compound preparations of negative ion and Chinese medicine could improve the growth performance,nutrient absorption and immunity of Yanbian Yellow cattle in the latter finishing period.

To reveal the codon usage pattern of porcine α-(1,2) fucosyltransferase 2 (FUT2) gene and increase its expression level in exogenous host,codon usage bias parameters of porcine FUT2 gene was analyzed using CodonW and EMBOSS Explorer,and then compared it with Drosophila, yeast and Escherichia coli genomes.Finally,according to the most closely codon usage pattern of model organism,the porcine FUT2 gene codon was optimized using JCat and OPTIMIZER.The results showed the expression level of porcine FUT2 gene was low,and it had 24 biased codons,23 of which biased to end with G/C at the third position.The differences in codon usage frequency between porcine FUT2 gene and Drosophila genome were less than those in Escherichia coli and yeast genomes,which proved Drosophila was more suitable for expressing the porcine FUT2 gene than Escherichia coli and yeast.The Drosophila genome was employed to optimize porcine FUT2 gene.The codon adaptation index of optimized porcine FUT2 gene was significantly improved,while the GC content of its CDS sequence was not obviously changed,indicating that the porcine FUT2 gene was successfully optimized in theory.This research revealed the codon usage pattern of porcine FUT2 gene from the translation level,and provided theoretical basis for selecting the best exogenous expression system and improving the expression level of the porcine FUT2 gene in host cells.

To explore the innate immunization level and breed characteristics of Qinghai local and introduced breeds,186 individuals of two local breeds (Euler and White Tibetan sheep) and one introduced breed (Poll Dorset sheep) were chose,and fresh blood samples were collected for routine blood test (WBC,RBC,HGB,HCT,MCV,MCH,MCHC,PLT,RDW-CV and LYM%);Serum was prepared to detect parameters of IL-1,IL-2,IL-6,IL-18,IFN-γ,IgG and MHC using ELISA-Kit,the comparative results were analyzed using different breed and breed origin as dependent variables.When breed was used as the dependent variable,Poll Dorset sheep contained the highest immune level and the disease resistance of White Tibetan sheep was slightly higher than that of Euler sheep.When breed origin was used as the dependent variable,the analysis results showed that the innate immunity level of local breeds was lower than that of introduced breed,the diseases resistance was weak.The blood biochemical level of Poll Dorset sheep was higher than local sheep except for MCH,MCHC and RDW-CV.This results provided reference data for the study of innate immunity of different sheep breeds,and had guiding for breeding and breed improvement of Tibetan sheep in Qinghai province.

This study was aimed to analyze the genetic diversity of Alashan Gobi Bactrian camel using microsatellite markers,find the microsatellite markers which associated with body size trait,and provide reference for marker-assisted selection of Alashan Gobi Bactrian camel.17 microsatellite markers were selected to detect the polymorphism in Alashan Gobi Bactrian camel population by non-denaturing polyacrylamide gel electrophoresis technique,and the correlation analysis was performed between microsatellite markers and body size traits.The results showed that 60 alleles were detected in 17 microsatellite markers,the average effective allele number was 2.7302,the average heterozygosity was 0.6205,the average polymorphic information content was 0.5308,which indicated that the genetic diversity of the Alashan Gobi Bactrian camel population was rich.The correlation analysis results indicated that 8 microsatellite markers (LCA82,LCA90,CMS15,CMS36,CMS104,CVRL01,YWLL36 and YWLL44) were associated with different body size traits,especially CMS36 and YWLL44,which were related to all body size traits.For CMS36 marker,there was significant difference between different genotypes and body length (P<0.05),the order was BB > AA > AB,the body height,chest circumference and cannon circumference of AA and BB genotypes were significantly higher than that of AB genotype (P<0.05).For YWLL44 marker,the body height,chest circumference and body weight of AC genotype were significantly higher than that of BD genotype (P<0.05),the body length of AC genotype was significantly higher than that of AB and BD genotypes (P<0.05),and the chest circumference of AB and AC genotypes was significantly higher than that of BD genotype (P<0.05).Summarily,8 microsatellite markers including LCA82,LCA90,CMS15,CMS36,CMS104,CVRL01,YWLL36 and YWLL44 had certain effects on the body length of Alashan Gobi Bactrian camel,CMS36 and YWLL44 had influence on the body height;LCA82,LCA90,CMS36,CVRL01,YWLL36 and YWLL44 had influence on the chest circumference;LCA82,CMS15,CMS36,CMS104,CRVL01 and YWLL44 had influence on the cannon circumference;LCA82,LCA90,CMS15,CMS36,CVRL01 and YWLL44 had influence on the body weight.

This study was aimed to explore characteristics of fatty acid oxidation in the testes of sterile yak hybrids (cattle-yaks) and its association with male sterility,and elucidate the molecular mechanism of male sterility of cattle-yaks.A Real-time quantative PCR method was established to assay the mRNA levels of liver type-carnitine palmitoyl transferase 1a (CPT1a) and sulfide dioxygenase 1 (ETHE1) genes in yak and cattle-yak testes using GAPDH and 18S rRNA as reference genes.This Real-time quantative PCR showed high amplification specificity,with amplification rate ranging from 98.5% to 107.8% and qualified standard curves.The testes and epididymides of adult yaks (n=9) and sterile male cattle-yaks (n=7) were collected and detected with conventional histological sections and HE staining.Sperms were found in the testes and epididymides of yaks but not cattle-yaks.Total RNA were extracted from the testes of yaks and cattle-yaks for the analysis of CPT1a and ETHE1 genes mRNA.The results showed that the CPT1a and ETHE1 genes mRNA level of testicular in cattle-yaks were significantly and extremely significantly higher than those of yaks (P<0.05;P<0.01).The increased CPT1a gene mRNA level indicated the higher β-oxidation capacity in cattle-yak testes;While the elevated ETHE1 gene mRNA level could facilitate the electron transfer and fatty acid oxidation in mitochondria,or influence other energy metabolism pathways.The results preliminarily demonstrated that the testes of sterile cattle-yaks had increased oxidation of fatty acid for energy supply compared to yak testes,and might had more dependent on fatty acids as fuel.

This study was aimed to investigate the genetic effect of cluster of differentiation antigen 14 (CD14) gene polymorphism on partial immune indexes and reproductive performance in Meishan pigs.The genetic polymorphism of CD14 gene -61 bp locus in Meishan pigs was detected by PCR-RFLP method,the level of the partial vital cytokines (IL-1β,IL-4,IL-6,IL-8,IL-10,IFN-γ,TNF-α and TGF-β1) and reproductive performances (total litter size,number of weaning piglets,birth litter weight and weaning litter weight) were determined,and the association of CD14 gene polymorphism with these immune indexes and reproductive performance was analyzed.The results showed that the -61 bp locus of CD14 gene in Meishan pigs was digested by BamH Ⅰ,and three genotypes were detected named as AA,AG and GG,respectively.The Chi-square test results showed that the genotype distribution of Meishan pigs were in Hardy-Weinberg equilibrium (P>0.05),the polymorphism information content (PIC) were moderate polymorphism in Meishan pigs.The correlation analysis indicated that the level of IL-10 of AG and GG genotypes individuals was significantly higher than that of GG genotype individuals (P<0.05).There was no significant difference of reproductive performances among three genotypes in primiparous sow (P>0.05),while the weaning litter weight of AA and AG genotypes individuals was significantly higher than that of GG genotype individuals in multiparous sows (P<0.05).The results of this experiment showed that GG genotype might be unfavorable genotype both in general disease resistance and reproductive performance when Meishan pigs were molecularly selected based on the -61 bp locus of CD14 gene,and the -61 bp locus of CD14 gene could be used as a potential genetic marker for further systematic verification and analysis.

Gaoyou ducks were used as the experiment objects in this study,12 Gaoyou ducks were chosen from high and low double-yolk egg laying groups (6 ducks per group),and hypothalamus,pituitary gland,liver,fallopian tube,ovary tissues were collected immediately after bleeding to death by carotid artery cut, HE staining was made to observe the ovary morphological structure and compare the development levels of follicle in two groups.FSHR immunohistochemical staining was conducted to observe its expression in positive cells and the distribution of ovarian granulosa cells.The total RNA was extracted from the hypothalamus, pituitary gland,liver,fallopian tube and ovary tissues, Real-time PCR method was used to detect the expression of FSHR gene in these tissues.The results showed that there were significant differences in ovary structure between high and low laying Gaoyou ducks.The follicles were dense and well developed,numerous small yellow follicles, small and big white follicles were distributed, and the sizes of the follicles at all levels were significantly different in high laying Gaoyou ducks'ovary;While low laying Gaoyou ducks' ovary had less and undeveloped follicles.The results of Real-time PCR showed that the expression of FSHR gene in hypothalamus,hypophysis gland, liver and fallopian tube of high laying ducks were extremely significantly higher than those in the low laying ducks (P<0.01), but there were no significant difference in ovary between two groups (P>0.05).In conlusion,the ovary structure and follicle development of high and low laying Gaoyou ducks had significant differences,and the microenvironment in the ovary might affect double-yolk egg laying performance of Gaoyou duck.

In this paper,the research progress of immune-related partial resistance genes (MHC,Nramp1,MBL,TLR and IFN) in cattle at home and abroad are briefly reviewed.Because of the relationship between the disease resistance genes of cattle and their own disease resistance and immunity,it is very meaningful to study the related resistance genes to improve the autoimmunity of cattle and to breed the fine offspring.This article focuses on the domestic and foreign study progress on the candidate resistance genes (Nramp 1,MHC,MBL,TLR and IFN) to certain diseases in cattle and whether it can be used for future generations of breeding molecular markers,such as MHC is closely related to dairy cow's mastitis;The CC genotype in the 1760 C>T mutation of TLR4 gene in cattle can be used as a molecular marker in the screening of resistance to mastitis in dairy cows.We believe that the study of these disease-resistance genes can give us a deeper understanding of the disease-resistant mechanism.At the same time, advanced techniques such as molecular markers and genetic engineering can be used to breed disease-resistant offspring,and the application of anti-disease genes in breeding will be the main direction of studying disease resistance and immunity in the future.

Nowadays many women suffer from many reproductive-related diseases such as infertility,spontaneous abortion and birth defects.The main internal factors are the germ cell abnormal meiosis and the decline of oocyte quality.It is well known that estrogen not only regulates the estrus and sexual behavior of mammals, but also involves in the regulation of the growth and development of oocytes.Estrogen receptor, as a kind of steroid receptors, plays a vital role in mediating the genomics and non-genome effects of estrogen.At present,the reports about estrogen receptors regulating oocyte maturation are mostly focused on the teleosts, especially the novel membrane receptor GPR30, but few reports on mammalian.So the structure and distribution of estrogen nuclear receptor ER α,ER β and new membrane receptor GPR30 are described in this review.Moreover,the research progress of different types of ligands for membrane receptor GPR30 and the interaction between estrogen receptors and oocyte development are introduced.The analysis have showed that estrogen's nuclear receptors and membrane receptor may play a different regulatory role in the process of oocyte maturation.Besides, this article further reveals the potential mechanism of estrogen and its receptors in the regulation of maturation of oocyte.

In order to explore the regulatory mechanism of cadherin 13 (CDH13) gene methylation in mastitis caused by Staphylococcus aureus in dairy cows,the CDH13 gene methylation levels of healthy and mastitis infected mammary tissue were tested by bisulfite sequencing PCR (BSP),and the relationship between the level of CDH13 gene methylation and expression was analyzed.The results showed that,all of the CpG islands except the twelfth CpG of experimental group showed different levels of methylation.The methylation level was the highest in -259 (50% to 60%),and those at -144,-135 and -126 were low,which were all about 5%.The overall methylation level of experimen and control groups ware 10.13%±1.81% and 14.43%±0.55%,respectively,and there was no significant difference between different loci and overall methylation rate of this two groups (P>0.05).Compared with the healthy tissue,the expression of CDH13 gene in mammary tissue which were inflected by mastitis was up-regulation,the methylation level of -162 and -93 CpG islands were significantly negatively correlated with gene expression (P<0.05).The methylation of CDH13 gene affected its expression,thus affecting the occurrence of mastitis.These provided a reference for further exploring the pathogenesis of Staphylococcus aureus pathogenic mastitis.

In order to study the genetic variation of current domestic canine parvovirus (CPV),the intestinal tissue were collected from a suspected CPV infected canine in Beijing.After sample tissues were processed by asepsis work,virus was inoculated in F81 cells and identified by electron microscopy,serology,molecular biology and challenge test.The results indicated that the virus produced typical CPE on F81 cells and the HA blood coagulation rate was 1:256.The diameter of virus particle was about 20 nm observed by electron microscopy.The specific band was at 570 bp in PCR identification which proved that the virus isolated from the samples was CPV,and named CPV-BJ03/17.The results of whole-genome sequencing and analysis indicated that the full length virus genome was 4 620 bp,GenBank accession No.:MF134808,and this virus strain had a closer relationship with virus isolated from Guangdong (SC02/2011),Shenzhen (CPV-s5) and Gansu (CPV-LZ1) whose nucleotide identities were all 99.7%.The analysis of VP2 amino acid sequence suggested that CPV-BJ03/17 belonged to a New CPV-2a subtype.The major amino acid sites of CPV-BJ03/17 showed no significant changes compared with the recent isolated stain BJ15-1.Animal experiments showed that CPV-BJ03/17 was a high CPV virulent strain.In conclusion,a high CPV virulent strain was isolated in this study,according to the comparison of CPV prevalence and genetic variation,this results also provided a reference for CPV disease treatment and vaccine research.

To determine the molecular characteristics of F1L gene of orf virus (ORFV) isolate from Sichuan,and predict the antigenic epitopes,this experiment used bioinformatics software to analyze ORFV F1L gene of Sichuan isolate and predict the secondary and tertiary structure and B cell epitopes of the encoded protein.The F1L gene was cloned,transformed and sequenced.The mutation of ORFV F1L gene from other regions was analyzed by Mega 7.0 software.The secondary structure of F1L protein was predicted by SOPMA server,and the hydrophilicity was predicted by DNAStar software.Surface accessibility,flexibility,and antigenicity were validated using ABCpred protocol, and the B-cell epitopes were exported.After using SWISS-MODEL server to predict the tertiary structure,re-validated the epitopes in the modeling area,final returned to the protein sequence alignment.The results showed that the ORFV F1L gene of Sichuan isolate was 1 029 bp in size and encoded 342 amino acids.F1L gene and its encoded protein had a certain variation compared with other regions.F1L protein predicted 9 potential B cell epitopes.Its 122 to 137 peptide was the optimal epitope after comparison of tertiary structure;The predicted ORFV F1L protein antigen of Sichuan isolates had certain specificity compared with other regions,the 124,131 and 137 amino acids were different in many strains of other regions.The results of this study provided theoretical basis for the research of ORFV genetic engineering vaccines and specific detection methods in Sichuan.

In order to determine the immunogenicity of Mycoplasma bovis P48 gene and lay the foundation for further screening of the immunoprotective gene of Mycoplasma bovis.In this study,Mycoplasma bovis Xinjiang isolate as the research object,Overlap PCR method was used to amplify P48 gene point mutation of Mycoplasma bovis Xinjiang isolate and construct the prokaryotic expression vector pET-32a(+)-P48,which was transformed into Escherichia coli BL21 (DE3),the recombinant protein P48 was obtained under the induction of ITPG.BALB/c mice was immunized by purified recombinant P48 protein to produce polyclonal antibody,the reactivity and immunogenicity of the recombinant protein were tested using Western blotting and ELISA methods.The results showed that the prokaryotic expression vector pET-32a(+)-P48 was successfully constructed,and the recombinant protein P48 was 66 ku.The purified recombinant P48 protein of Mycoplasma bovis immunized in mice could produce good immune response,and serum antibody titers reached a higher level (D_{450 nm}=1.126).Western blotting results showed that there was obvious antibody response of anti-Mycoplasma bovis P48 recombinant protein of mouse serum,Mycoplasma bovis P48 recombinant protein and Mycoplasma bovis total protein antigen,P48 recombinant protein had good immunogenicity and reactogenicity,which could be used as a candidate gene of Mycoplasma bovis vaccine.The homology of P48 gene was high between Mycoplasma bovis Xinjiang isolate and 5 domestic Mycoplasma bovis,and the genetic relationship was closer.

Classical swine fever is one of the most important infectious diseases that endanger the health of pigs.It has such characteristics as acuteness,heat and high contact,causing great economic losses to the world pig industry.At present,the traditional vaccination is still the main means to prevent classical swine fever.Although traditional vaccines (such as C-strain) play a huge role in the prevention and control of classical swine fever,some shortcomings exist,such as the inability to differentiate between wild-type and vaccinated animals and the immune failure of classical swine fever.Therefore,the development of a new type of vaccine against classical swine fever is of great significance.With the continuous development of molecular biology technology and recombinant DNA technology,the research on genetic engineering vaccines has been continuously deepened.Six kinds of new classical swine fever vaccines such as subunit vaccines,nucleic acid vaccines,recombinant live vaccine vectors,gene deletion vaccines,full-length infectious cDNA marker vaccines and synthetic peptide vaccines have been developed.Compared with the traditional vaccine,the new vaccine is cheap,safe,efficient,easy to transport and save and could distinguish between wild-type virus infection and vaccine immunization.The authors review the research progress of six kinds of new classical swine fever vaccines so as to provide reference for prevention and control of classical swine fever.

The aim of this study was to construct a recombinant virus carrying green fluorescent protein (GFP) protein,and detect vaccine titer more rapidly and accurately.Recombinant plasmids containing GFP gene were constructed by gene recombination technology and rescued by reverse genetics.The viral infection in Vero cells and protein samples were collected for Western blotting assay to detect the expression of foreign protein GFP.One-step growth curve was compared with the biological characteristics of the original strain.The results showed that we successfully rescued the recombinant GFP-expressing recombinant rabies virus SAD-GFP using the constructed recombinant plasmids.Western blotting result showed that the recombinant virus could express GFP,and the expression of GFP increased with the time of virus infection.The expression of structural protein G was not affected by the expression of foreign proteins.There was no significant difference between the recombinant strains and the original strains.After passage,GFP could stably express in the virus.In this experiment,the recombinant SAD-GFP strain was successfully rescued.In the continuous passage,the GFP gene was not lost,and the protein expression level did not decrease,which proved that the foreign protein expressed at this site could sustainedly and stablly express in the course of virus passage,and did not affect the growth characteristics of the virus.

The technology of increasing cashmere by light control in which special housing was used to control light intensity and the length of time,was to stimulate the pineal gland to increase the secretion of melatonin,which make goat producing cashmere in the non-cashmere producing seasons.The influence of light control on the production performance of cashmere goats was studied in Inner Mongolia Arbas cashmere goats in the non-producing seasons.66 ewes aged 2-year old were randomly divided into the artificial lighting control group (experimental group) and the nature light group (control group),with 33 goats per group.All the goats were weighted and the cashmere samples were collected every month,and the straighted length,fineness,yield of cashmere and body weight changes were analysized.The results showed that the average of fineness of the cashmere in two groups had no significant difference (P>0.05);The length of cashmere in control and experimental groups were 3.20 and 7.85 cm respectively during the experiment period,which had significant difference (P<0.05);The average yield of cashmere in the experimental group was increased by 56.5%~68.2% comparing with control group (P<0.01).The body weights of control and the experimental groups had no significant difference (P>0.05).In conclusion,the cashmere production was promoted by light control during the non-cashmere producing seasons,the individual yield of cashmere increased,the length of cashmere increased,but the fineness of cashmere had no significant change.

Cephalosporins are known for their broad spectrum and strong antibacterial activity in clinic.Cephalosporins are widely used in the medical field, but their application is partly restricted in veterinary medicine because of fewer classes,and cross resistance derived from abusing and misusing.Therefore,the review concluded the development process of cephalosporin, antibacterial mechanism, pharmacokinetics and safety, summarized the main classes of veterinary cephalosporin, antibacterial activity and pharmacokinetic parameters in animals, compared the antibacterial activity of the main veterinary cephalosporin and the therapeutic effect on sensitive bacterial infection diseases.Meanwhile,the resistance mechanism and the situation of resistance of cephalosporin were summarized, and the measures taken by various countries on the resistance control were compared and analyzed.Through the review of the development and clinical application of veterinary cephalosporin,it was made for promoting the scientific and standardized use of cephalosporin in benefit clinic.

In order to provide new methods and approaches to the treatments of bovine mastitis,this study screened out the best prescription for inhibiting the main pathogens causing mastitis in dairy cows according to the principles of dialectical methods that Chinese veterinarians treat diseases.The Dandelion,Purslane,Honeysuckle,Purple Flower,Radix Paeoniae Rubra,Radix Paeoniae Alba,Chrysanthemi Flos,Scutellariae Radix,Reed and Carthami Flos were used as materials in this experiment.A preliminary screening of antibacterial Chinese herbal medicines was conducted through L₁₂ (2¹¹) orthogonal test,the drug group was further screened by the orthogonal test of L₁₆ (4⁵),and the anti-bacterial effect of Chinese medicine group on the main pathogenic bacteria of bovine mastitis was determined by Oxford cup method in vitro.The minimum antimicrobial concentration (MIC) and minimum bactericidal concentration (MBC) were detected by two-fold dilution method and plate counting.The results showed that the optimal factors of group were Paeoniae Radix Rubra, Scutellariae Radix,Carthami Flos,Paeoniae Radix Alba and Chrysanthemi Flos,and the proportion was 3:3:3:1:2.The bacterial inhibition diameters for Escherichia coli was 17.39 mm,and MIC and MBC were 62.50 and 125.00 mg/mL respectively.The bacterial inhibition diameters for Staphylococcus aureus was 25.44 mm, and MIC and MBC both were 31.25 mg/mL.In conclusion,the study successfully screened out a new traditional Chinese medicine that had good antibacterial and antiseptic effects for the bacteria causing bovine mastitis.

To identify the pathogenesis of duck enteritis virus (DEV),inhibitor and activator of PKC (SP and PMA) were used to research that whether PKC/PKCI could affect the proliferation of DEV,trying to provide some new ideas for understanding the mechanism of DEV infection.Normal duck fibroblasts(DEF) were treated with SP or PMA,and Real-time quantitative PCR method was used to measure the expression levels of PKC and PKCI genes at different time.The DEV was used to infect DEF which treated with SP or PMA,then the cell cultures were collected at different time,and the TCID₅₀ and expression levels of DEV NP gene were measured by Reed-Muench and Real-time quantitative PCR,respectively.The results showed that there was no significant effect of SP treatment on PKC/PKCI genes expression levels in DEF (P>0.05),while the PMA treatment could extremely significantly increased the PKC gene expression (P<0.01),but had no significant impact on PKCI gene (P>0.05);SP/PMA processing could significant or extremely significant affect the ability of DEV replication (P<0.05;P<0.01),and the expression level of DEV NP gene was significantly or extremely significant decreased at the early stage of infection (P<0.05;P<0.01).The above results indicated that the effects of SP and PMA on PKC/PKCI were different,while both of them could effectively inhibit the proliferation of DEV.These results could provide the basis for DEV prevention and its pathogenesis studying.

The study was aimed to investigate the anti-inflammatory and antiasthmatic effects of Zhongjiefeng Sanqing granules.The cell viability that treated with 200,400,800 and 1 600 μg/mL Zhongjiefeng Sanqing granules was measured by MTT method.The inflammation model of RAW264.7 cell was established with LPS in vitro,and then TNF-α and IL-6 levels in the cell supernatant were measured by ELISA after 400,800 and 1 600 μg/mL Zhongjiefeng Sanqing granules treatment.The asthma model established by sensitizing and challenging female BALB/c mice with ovalbumin (OVA) to evaluate the effects of Zhongjiefeng Sanqing granules on asthmatic mice in vivo, the airway hyperresponsiveness (AHR) was measured at 24 h after the final OVA challenge, and the bronchoalveolar lavage fluid (BALF) were harvested to measure the number of inflammatory cells and the concentrations of IL-4,IL-5 and IL-13, and the serum was collected to measure the level of OVA-IgE.The results showed that Zhongjiefeng Sanqing granules in the concentration range of 200 to 1 600 μg/mL could significantly or extremely significantly promote the proliferation of RAW 264.7 cell (P<0.05;P<0.01).Compared with the LPS group,800 and 1 600 μg/mL Zhongjiefeng Sanqing granules could extremely significantly reduce the levels of TNF-α and IL-6 (P<0.01).Compared with the asthma model group, Zhongjiefeng Sanqing granules obviously ameliorated the airway hyperresponsiveness,markedly reduced the number of inflammatory cells in BALF,concentrations of IL-4, IL-5 and IL-13 in BALF and level of OVA-IgE in serum.These results suggested that Zhongjiefeng Sanqing granules had protective effect on inflammatory cell model and asthmatic model of mouse by inhibiting the expression of inflammatory cytokines.

Polyether antibiotics,including monensin,maduramicin,salinomycin,lasalocid,and so on,are produced by saprophytic fungi.They have broad-spectrum anti-parasite and activity against pathogenic Eimeria spp.in poultry and can promote ruminant and pigs growth.But normal using may lead to residue in animal foods,causing human health problems.So we need strength the detection methods of polyether antibiotics residues.In recent years,the polyether antibiotics residues detection methods include spectrophotometry,microbiological detection,chromatography (TLC,HPLC,liquid phase chromatography-tandem mass spectrometry) and immunological methods.In this paper,we summarized the detection methods of polyether antibiotics in recent years,and we also made a forecast of polyether antibiotics detection technology to provide a reference and basis for the future residue detection.

Triazine herbicides and their metabolites may be dangerous for human health because they are suspected to cause interruption of hormone functions.Some of these compounds have been listed as potential environmental endocrine disruptors by some countries.Analysis methods for detecting triazine herbicides and their metabolites residues in environmental and food samples,including sample extraction and instrumental determination,were reviewed here.Liquid-liquid extraction,solid phase extraction and solid phase micro-extraction have been widely used in the pretreatment of triazine herbicide and their metabolites residues due to its easy operation and high recovery over other techniques.In addition,due to the high selectivity and low matrix effect,the technique of molecular imprinting,gel permeation chromatography and accelerated solvent extraction have also been widely applied in the pretreatment of triazine herbicide and their metabolites residues.By far,gas chromatography and liquid chromatography are commonly-used analytical methods for the measure of triazine herbicide and their metabolites residues.Additionally,chromatography coupled mass spectrometry plays more and more important roles in the analysis of triazine herbicides and their metabolites residues because of its lower detection limit and higher selectivity.

In order to provide the basis for the environmental control of sheep houses,the variation of airborne fungal concentration and the aerodynamic characteristics of fungal aerosols were researched in the Southeast Shanxi province.An Andersen six-stage microbial sampler was used to collect air and Rose Bengal Medium (RBM) was used as the medium.Airborne fungi were collected from three sheep houses in Southeastern Shanxi province in four seasons a year.The concentration and particle size characteristics of airborne fungi were analyzed.The results showed that the highest airborne fungal concentration was in autumn,which was significantly higher than the other seasons (P<0.05),and the concentration difference was significant in the 3 periods of morning,noon and afternoon in autumn (P<0.05);The distribution of fungal particle in sampler were same basically in four seasons,and the peak appears at the stage Ⅳ,the fungal particle were mainly distributed from stage Ⅲ to Ⅴ of the sampler,accounting for 72.66%-83.87% of total number;The CMD of airborne fungi ranged from 1.3 to 2.9 μm,The GSD ranged from 1.6 to 2.7 μm;The CMD of summer was significant lower than other seasons (P<0.05).In conclusion,the concentration of airborne fungi in sheep house was closely related to season and had large potential hazards.About 80% of aerosol particles could enter the alveoli of human and animal,the CMD of sheep house was smaller than other animal houses.