To obtain the biological information of differentially expressed genes of Eimeria tenella oocysts treated with Eupatorium adenophorum,0.5% Eupatorium adenophorum were adopted to treat oocyst sporulation process of coccidiosis,suppression subtractive hybridization (SSH) was used to screen differential expression genes of chicken coccidian oocysts after treatment, GO and COG function prediction were used to analyze those genes,and Real-time PCR was used to verify the results of SSH.The results showed that a subtractive cDNA library of chicken Eimeria tenella before and after treatment of Eupatorium adenophorum was successfully constructed,86 ESTs sequences and 31 Unigenes were obtained after splicing and clustering,of which 23 Unigenes were with function annotation,8 Unigenes with no functional annotation,and 4 Unigenes had no homology matching.3 differentially expressed genes were randomly selected to further verify the specificity of the cDNA library by Real-time PCR,and the results showed that the expression of surface antigen 13, 3-hydroxyacyl coenzyme A dehydrogenase and cytochrome P450 genes in Eimeria tenella sporulated oocysts in Eupatorium adenophorum treatment group was obviously lower than that in untreated group,indicating that Eupatorium adenophorum had an inhibitory effect on the activity of oocysts.The main regulation effect of Eupatorium adenophorum on genes were obtained in this study,which laid a theoretical foundation for the development of Eupatorium adenophorum environmental pesticides,and could provide references for the screening of coccidiosis attenuated vaccine or gene deletion vaccine targets.

The study was aimed to clone sterol carrier protein 2 (SCP2) of buffalo,analyze the sequences by bioinformatics software,and measure the expression of SCP2 gene in different tissues of buffalo.The complete CDS of buffalo SCP2 gene was successfully cloned which was 1 632 bp in length and encoded 543 amino acids using the SCP2 gene (GenBank accession No.:NM_001033990.3) of Bos taurus as the seed sequence.The CDS of buffalo SCP2 gene showed 95.9%,93.4%,92.4%,89.4%,88.3%,86.3% and 86.9% identity with that of Bos taurus,Ovis aries,Capra hircus,Delphinapterus leucas,Homo sapiens,Felis catus familiaris and Canis lupus,and the results of phylogenetic tree showed that the nearest relationship existed between buffalo and Bos taurus.The results of amino acid sequence analysis showed that the formula of protein encoded by SCP2 gene in buffalo was C₂₆₀₂H₄₁₃₁N₇₀₉O₇₇₄S₂₉₈, and the molecular weight,theory isoelectric point,instability index and average of hydropathicity was 58.66 ku,8.59,27.94 and -0.215 respectively,showing it was an alkaline,hydrophilic and stable protein.The secondary structure of SCP2 protein mainly consisted of α-helices (35.54%),random coil (48.99%) and extended strand (15.47%),which was consistent with the results of tertiary structure prediction.The results of subcellular localization showed that SCP2 protein was located in the cytoplasm (43.5%),peroxisome (21.7%),mitochondria (17.4%),nuclear (13.0%),and cytoskeleton (4.4%).There was no transmembrane domain and signal peptide,indicating it was an non secretory protein.There were 13 Ser,3 Thr and 2 Tyr which might be a protein kinase phosphorylation site.Prediction of protein binding sites showed that buffalo SCP2 protein contained 12 protein binding sites and 1 polynucleotide binding site.The Real-time PCR results showed that the expression of buffalo SCP2 gene in the liver was obviously higher than other tissues,which followed by mammary gland,lymph,kidney,large intestine,stomach,lung,spleen,ovary,pituitary,brain and heart ranged from high to low.This study provided the basis for further studies on the function of SCP2 gene.

To analyze the different characteristics between VIL-10 gene from the vaccine and wild strains of orf virus (ORFV),a pair of specific primers was designed according to ORFV NZ2 strain VIL-10 gene sequence reported in GenBank.The genomic DNA was taken as templates extracted from the vaccine and wild strains of ORFV.The full lengths of VIL-10 gene were obtained by PCR and then sequenced.The variation of nucleic acids,amino acids and protein structures were predicted and analyzed using bioinformatics software.The results showed that VIL-10 gene from ORFV vaccine strain and wild strain shared 94.4% identity at nucleotide level and the main difference was a single base mutation,deletion in the nucleotide sequence which occurred at 132 to 134 bp.92.5% identity at amino acid level and a mutation of 15 amino acid sites occurred,of which the asparagine was missing at 42 amino acid position.There were some differences in primary structure and physical and chemical properties of protein,secondary structure,tertiary structure,physicochemical properties,epitope parameters and the presence or absence of signal peptide.However,no transmembrane domains were identified both in the vaccine and virulent strains of ORFV.Phylogenetic analysis revealed that the wild strain and the vaccine strain belonged to different branches and the genetic relationship was far away.The results suggested that the VIL-10 gene of wild virus strains and vaccine strains had obvious mutations,and these mutations might be related to the virulence of the vaccine strains.

To study the sequences characteristics of pdhc gene in M.bovis Wuwei strain and its location in M.bovis cells,the primers of pdhc gene were designed according to M.bovis HB0801 strain (accession No.:CP002058.1) in GenBank,and the pdhc gene of M.bovis Wuwei strain was amplified by PCR.Based on sequencing and gene analysis,the prokaryotic expression vector pET-pdhc was constructed,and then transformed into Escherichia coli Rosetta (DE3).After induced by IPTG,the M.bovis fusion protein PDHc-E2 was purified and New Zealand rabbits (Oryctolagus cuniculus) was immunized to prepare anti-serum against M.bovis rocombinant protein PDHc-E2.Subsequently,the distribution of PDHc-E2 in M.bovis cells was analyzed using iELISA and Western blotting.The results showed that the full-length sequence of pdhc gene CDS of M.bovis Wuwei strain was 735 bp and encoded 244 amino acids.It had 100.0% homology with the domestic M.bovis isolates,such as HB0801,Hubei-1,CQ-W70 and NM2012,and had 99.2% homology with the international standard strain PG45,the homology with Mycoplasma agalactiae (M.agalactiae) were 90.9% to 91.2%,the homology with the ST6 strain of Mycoplasma californicum (M.californicum) was only 78.4%,and the gene sequence was very conservative.By Overlap PCR,four TGA codons encoding tryptophan in this gene mutated to TGG;After point mutation the pdhc gene successfully expressed in Escherichia coli.The molecular weight of recombinant protein was approximately 29 ku,and it mainly existed in the form of soluble protein.iELISA results showed that recombinant protein PDHc-E2 had a high immunogenicity,and could stimulate New Zealand rabbit to produce high levels of antibodies,the iELISA titer of antiserum was approximately up to 1:100 000.The results of subcellular localization showed that the preparation of polyclonal antibody could bind specifically to recombinant protein PDHc-E2,total protein,membrane protein and cytoplasm of M.bovis,which indicated that the PDHc-E2 of M.bovis was distributed in both the membrane and the cytoplasm,and was a membrane-related protein,but the distribution in the cytoplasm was more than in the cell membrane.The results of this study laid a theoretical basis for further investigation on biological functions of M.bovis.

This study was aimed to construct membrane occupation and recognition nexus protein 2 (MORN2) gene knockout strain of Toxoplasma gondii type Ⅰ RH strain,and evaluate the functions of MORN2 gene.The sgRNA target of MORN2 gene was screened to design primers of sgRNA using CRISPR/Cas9 technology.MORN2 CRISPR-targeted plasmid (sgMORN2) was constructed using pSAG1∷CAS9-U6∷sgUPRT as a template under the effect of Q5 enzyme.One pair of homologous primers was designed according to the sgMORN2 target sequence to amplify a DHFR fragment with pUPRT∷DHFR-D plasmid as a template.The MORN2-CRISPR plasmid and the homologous DHFR fragment were cotransfected into Toxoplasma gondii tachyzoites to perform pyrimethamine screening,monoclonal selection in 96 well plates and PCR identification.The results showed that the MORN2 knockout strain (△MORN2) was successfully created.The plaque assays showed that the knockout strain had the same growth rate as the wild strain in vitro plaque assay.Mice were injected intraperitoneally with tachyzoites of 100 and 1 000 knockout strains in vivo infection test,and the death time of mice was basically the same as that of control group.These results indicated MORN2 gene knockout had no effect on the growth rate of Toxoplasma gondii RH strain in vitro and the virulence of mouse infection.This study demonstrated that MORN2 gene had no phenotypic function in RH strain,which laid the foundation for further exploration of the functional genes of Toxoplasma gondii.

To construct red fluorescent protein expression vector of Brucella host cells and individuals,RBS-Red and BDNA (Brucella DNA) fragments were obtained by PCR,and the RBS-Red-BDNA overlapping fragments were obtained by overlapping extension PCR method in this experiment.The overlapped fragments were inserted into pLB vector,the recombinant pLB vector and pMC-221 plasmid were digested by Sma Ⅰ and Sac Ⅱ double enzyme,the ligation fragments were transformed into E.coli DH5α,the extract plasmid was electroporated into Brucella 16M competent cell,16M-pMC-Red strain was coated with Brucella culture medium containing chloramphenicol resistance,and observed the strain color under inverted fluorescence microscope.The 16M-pMC-Red strain was used to infect the mouse macrophages and make the cell crawling slices,observation of mouse macrophages under laser confocal fluorescence microscopy and identification of red fluorescent expression.The results showed that the double promoter expression vector of red fluorescent protein in Brucella was successfully transformed by overlapping extension PCR method,the transformed plasmid was electroporated into Brucella strain 16M,and the strain could stably express the red fluorescent protein.The 16M-pMC-Red strain in mouse macrophages could stably express the red fluorescent protein.The red light-producing plasmid pMC-Red could be used together with the green light-emitting plasmid pMC-221,which laid a foundation for the research of different strains of Brucella,and also provided a new strategy using fluorescence detection as an indicator for the detection of brucellosis.

This study was aimed to clone and express BPSL1467 gene of Burkholderia pseudomallei (B.pseudomallea),and performe bioinformatics analysis of its protein.A pair of primers was designed according to the BPSL1467 gene sequence of B.pseudomallea K96243 strain in GenBank.BPSL1467 gene fragment of B.pseudomallea BPHN1 strain was amplified by PCR amplification.The BPSL1467 gene fragment was ligated into the pET-28a(+) vector to construct the pET-28a(+)-BPSL1467 recombinant plasmid.Then,the pET-28a(+)-BPSL1467 was transformed into E.coli BL21 (DE3) competent cells.The expressed product induced by IPTG was analyzed by SDS-PAGE and Western blotting.Bioinformatics analysis of BPSL1467 gene sequence was carried out using DNAMAN,ProtParam,SOPMA and Protscale softwares.The results showed that BPSL1467 gene was cloned with the length of 462 bp.The expressed recombinant protein was about 22 ku and was mainly in the form of inclusion body;The molecular weight of the BPSL1467 recombinant protein was 16 890.58 u (C₇₆₃H₁₂₀₉N₂₀₃O₂₁₇S₆);Its instability coefficient,theoretical isoelectric point (pI) and total average hydrophobicity (GRAVY) were 33.95,8.85 and -0.190 respectively,which was a stable hydrophilic basic protein;The secondary structure of the protein was mainly random coil (42.48%) and alpha helix (32.68%).This results provided a reference for the further study of molecular mechanism of Burkholderia pseudomallei BPSL1467 gene.

The purpose of this study was to clone the TACR1 gene of Funiu White goat and analyze its structure and encoded protein by bioinformatics methods to lay a foundation for further study on the function of TACR1 gene and its role in reproductive physiology and immune regulation.5 pairs of primers were designed based on the goat TACR1 gene sequence in GenBank (accession No:NC_030818.1).The Funiu White goat TACR1 gene was amplified by PCR amplification and sequenced,and the complete coding region (CDS) of TACR1 gene was obtained after splicing the fragments.The results showed that the open reading frame(CRF) of TACR1 gene in Funiu White goat was 852 bp in length and encoded a total of 283 amino acids.The content of base composition was A (23.47%),T (25.49%),C (27.76%) and G (23.27%).Compared with sheep TACR1 gene, the TACR1 gene CDS region of the Funiu White goat had a two-base mutation which did not cause amino acid changes.The non-coding region at the 5'and 3' ends was 860 and 271 bp in length, and had 9 and 2 base mutations,respectively.The results of homology analysis showed that the identity of TACR1 gene between Funiu White goat and Capra hircus,Pantholops hodgsonii,Ovis aries,Bos indicus,Odocoileus virginianus,Lipotes vexillifer,Homo sapiens,Sus scrofa,Panthera pardus,Cavia porcellus,Equus asinus were 99.9%,99.3%,99.2%,97.5%,96.7%,92.9%,89.7%,88.6%, 86.3%,86.2% and 85.0%,respectively.There was a signal peptide at the N-terminus of TACR1 and there might be a cleavage site at 21th amino acid predicted by SignalP4.1 online software.Through the prediction of protein structure,it was found that the protein encoded by TACR1 gene of the FuNiu White goat had no transmembrane domain.

The purpose of the research was to clone heat shock protein 70 (HSP70) gene CDS region,construct the HSP70 prokaryotic expression vector,and induce the expression of HSP70 fusion protein to further study the effect of HSP70 on the freezing semen motility of river buffalo.The buffalo sperm genome DNA was extracted as a template, the HSP70 gene CDS region was amplified by PCR,and the PCR products was connected with pET30a plasmid to construct recombinant prokaryotic expression plasmid and induce the expression of HSP70.The obtained HSP70 was added to the buffalo frozen semen and it's motility was evaluated.The results showed that the CDS region of HSP70 gene was amplified successfully with 2 155 bp in length.The SDS-PAGE and Western blotting analysis showed that the fusion protein had the molecular weight of 70 ku and could react with antibodies.The final concentration of 2,4 and 8 mg/mL HSP70 could improve the motilities of frozen semen,and 8 mg/mL HSP70 had the best effect on the motilities,but there were no significant differences among groups (P>0.05).In conclusion,the purified HSP70 obtained in this study had good quality,and could improve the frozen semen motility.The study laid a theoretical foundation for further studying the antifreeze protection mechanism of buffalo HSP70 protein.

This study was aimed to explore the role of methionine adenosyltransferase 2A (MAT2A) in porcine intramuscular adipocyte differentiation by constructing the adenovirus mediated overexpression vector in vitro.The primers was designed according to the MAT2A gene mRNA sequence in GenBank (accession No.:NM_001167650.1),the total RNA sequence of porcine adipose tissue cell was extracted and reversed to get cDNA,which was as the template to proceed PCR amplification and connected to the pAdTrack-CMV adenovirus shuttle vector for sequencing identification,the recombinant plasmid was named pAd-MAT2A.pAd-MAT2A was digested by Pac Ⅰ and transfected into 293A cell.The expression level of MAT2A gene was detected by Real-time PCR,and the MAT2A protein was analyzed by Western blotting.The cells differentiated on the 8th day were used for Oil red O staining.The results showed that the shuttle vector (pAdTrack-CMV-MAT2A) was successfully constructed and homologous recombinated with backbone vector (pAdEasy-1).The virus titer was 1E+6 PFU/mL after transfecting the 293A cell with adenovirus vector pAd-MAT2A.Real-time PCR and Western blotting showed that the mRNA and protein levels of MAT2A gene were significantly upregulated by transfecting pAd-MAT2A in porcine intramuscular preadipocytes.Oil red O staining results showed that the overexpression of MAT2A gene promoted lipid accumulation in porcine intramuscular adipocytes.In conclusion,MAT2A gene was overexpressed in expression vector mediated by adenovirus and promoted the lipid increasement in porcine intramuscular adipocytes.

The production and quality of breast milk is crucial for the survival rate,growth rate and health condition of piglets.Hyperthermia is one of the important environmental factors that affect the health of animal lactation.Especially in summer,animals are easily subjected to heat stress due to the long-time high temperature.In addition,the process of lactation is strictly regulated by the body's endocrine system,involving a variety of hormones,such as prolactin (PRL),growth hormone (GH) and insulin-like growth factor 1 (IGF-1).Many production data and researches indicated that hyperthermia induced the decline of food intake and lactation performance and causes changes in the endocrine state of the animal,which seriously affected sows and piglets.Therefore,in order to guarantee the health status of sows and obtain a better growth performance of piglets,it is necessary to find out how hyperthermia influences the sows and related hormones during the process of lactation.In this review,the authors summarized the effects of hyperthermia on milk production,milk composition and hormones related to lactation,founding that hyperthermia will reduce milk production and change the contents of lactose,milk fat,and lactoprotein,as well as the level of hormones related to lactation.It is expected to provide some references for researches on endocrine regulation mechanisms of environmental factors in animal lactation,as well as explorations on appropriate mitigation measures.

This study was conducted to study the effect of tributyrin (TB) on small intestine mucosal growth and gut barrier function of acetic acid (ACA)-treated piglets.Eighteen crossbred healthy piglets (Ducoc×Landrace×Yorkshire,28 days old) were randomly allocated into three treatment groups (control,ACA and TB).The pigs in control and ACA groups were fed basal diet,and TB group was fed the basal diet supplemented with 0.1% TB.On day 15 of the trial,ACA and TB groups were intracolonically administrated with 10 mL of 10% ACA solution,control group were given sterile saline at the same volume.On days 18 and 21,blood samples were collected from jugular vein for the determination of DAO activity.On day 21 of the trial,piglets were killed and the samples of small intestinal mucosa were collected for the measuring of intestinal morphological structure and mucosal injury correlated genes mRNA levels.The results showed that:①Before ACA administration,comparing to control group,dietary supplementation with TB could increase ADG,but the difference was not significant (P=0.089),while reduced F/G significantly (P<0.05);②Compared with control group,ACA administration significantly decreased the villus height and surface area of villus in the jejunum,and the ratio of villus height to crypt depth in the ileum (P<0.05);③Compared with ACA group,TB supplementation significantly decreased the plasma DAO activity (P<0.05),and decreased the expression of AREG gene in the ileum mucosa extreme significantly (P<0.01).Collectively,dietary supplementation with 0.1% TB could alleviate ACA-induced damages on mucosal growth and barrier function in the small intestine of piglets.

The experimtent was conducted to investigate the effects of water extraction of Moringa oleifera and Morinda citrifolia (LNS) on performance and egg quality of Hyline Brown laying hens.A total of 540 Hyline Brown laying hens at peak period of laying eggs (22 weeks) were randomly allocated into 6 groups with 6 replicates per group and 15 laying hens per replicate.The hens in negative control group(NC)were fed a corn-soybean meal basal diet,in positive control group (PC) were fed the basal diet supplemented with 50 mg/kg zinc bacitracin,and the others in the experimental groups were fed the basal diet supplemented with 0.25,0.50,0.75 and 1.00 g/kg LNS,respectively.The experiment lasted for 24 weeks.The results showed as follows:①Different diet treatments had no significant effects on the production performance in the earlier and later stages(P>0.05),while the diets supplemented with 0.50 and 0.75 g/kg LNS had the tendency to improve the ratio of feed to egg in the later period of the experiment(P=0.087);Diet treatments had no significant effects on shape index,egg strength,shell thickness,yolk weight and haugh unit(P>0.05).However,diet supplemented with zinc bacitracin and LNS had the tendency to improve the egg strength(P=0.062).②Compared to NC and PC groups,diets supplemented with LNS significantly improved eggshell color of raw and boiled egg yolk (P<0.05),and significantly decreased egg yolk cholesterol content (except 0.25 LNS group)(P<0.05).③Compared to NC group,diet supplemented with 0.50 g/kg LNS had significantly increased egg contents of total flavonoids,docose hexaenoie acid (DHA),lecithin,linolenic acid and linoleic acid (P<0.05).In conclusion,as feed additive,LNS could improve egg's nutritional quality,and the dietary suitable supplementation of LNS for laying hens was 0.25 to 0.50 g/kg.

The method of grazing+feeding with two kinds of complete pellet feed of corn straw and wheat straw was adopted to supplement the Gansu Alpine Fine-wool sheep in the late pregnancy in cold season in the study.The supplementary effect was studied by the measurement of body weight,serum biochemical parameters and rumen fluid parameters of ewes during late pregnancy,and the birth weight of lambs.60 healthy ewes in late pregnancy with similar body weight were chosen and randomly divided into 3 groups with 20 ewes per group.The nutritional requirement of groups Ⅰ and Ⅱ was determined according to the feeding standards of the Chinese Merino sheep at the late stage of gestation (deducting grazing feed intake).The ewes in groups Ⅰ and Ⅱ were supplemented with 1.19 kg/d wheat straw,corn stalk complete pellet feed,respectively.The ewes in control group were fed with traditional method of the farmers (corn 0.05 kg and oat grass 0.20 kg per ewe),and the formal test lasted for 60 d.The results showed that the average daily gain of pregnant ewes and birth weight of lambs in two treatment groups were significantly higher than that in control group (P<0.05),while there was no significant difference between two groups (P>0.05).The concentrations of total protein, albumin,glucose,total cholesterol,alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase in two treatment groups were significantly higher than that in control group (P<0.05);The mole ratio of propionic acid and the concentration of total nitrogen,protein nitrogen,volatile fatty acids in the rumen fluid of pregnant ewes in two treatment groups were significantly higher than that in control group (P<0.05),however the mole ratio of acetic acid were significantly lower than that in control group (P<0.05).It could be seen that supplementing wheat stalk or corn stalk complete pellet feed could improve the nutritional status of Gansu Alpine Fine-wool sheep in late pregnancy,and promote the growth and development of lambs.

The experiments of six kinds of probiotics screened from the rumen were conducted to provide references for their further development and utilization in livestock and poultry husbandry.56 male 5-week-old Kunming mice were chosen and randomly divided into 7 groups.The mice in control group was fed with 0.2 mL of normal saline,while that of the other experimental groups were fed with 0.2 mL/(10 g·BW) of Bacillus amyloliquefaciens Lw-J21,Bacillus subtilis Lw-K16,Lactobacillus Lw-R35,Bifidobacterium Lw-D48,cellulose-producing bacteria Lw-X21,Candida dattila Lw-JM70 (1×10⁷ CFU/mL) for 42 d,respectively.The effects of different probiotics on body weight,intestinal flora,fat accumulation,immune indexes and hypoglycemia of mice were compared and analyzed.The results showed that compared with the control group,the body weight of mice in Lw-J21 and Lw-X21 groups were higher (P>0.05), and the fat accumulation in mice were significantly reduced at the same time (P<0.05).The number of Lactobacillus and total anaerobic bacteria in the intestinal tract of mice in Lw-R35 and Lw-X21 groups were significantly or extremely significantly increased (P<0.05;P<0.01).The spleen index and thymus index of mice in probiotics groups significantly or extremely significantly enhanced (P<0.05;P<0.01),meanwhile the hypoglycemia of mice was improved.In conclusion,the screed probiotics had different degrees of influence on intestinal microenvironment, immune indexes and blood glucose regulation ability of mice.

Meat and meat products are easy to oxidize during processing and storage,leading to quality degradation.Dietary supplementation with plant polyphenols in animal feeding will help to inhibit lipid and protein oxidation, and further enhance the oxidative stability of the meat and meat products during the processing and storage.Plant polyphenols has the ability of scavenging free radical anti-oxidant properties due to their phenolic hydroxyl structure.It can improve the content of non-enzymatic antioxidants,or enhance the activity of antioxidant enzymes in the organism or raw meat,or activate the Nrf2/ARE signal pathway to improve the antioxidant capacity of meat or/and meat products during processing and storage.Dietary of plant polyphenols can promote digestion and absorption of nutrients in feed and improve production performance in livestock and poultry.Furthermore,it can increase the antioxidant enzyme activity and total antioxidant capacity of the animal, decrease the content of MDA,and enhance the quality and extended the shelf life of the products.In the paper,the types and representative structures of plant phenolic compounds were introduced, including the structure of flavonoids,phenolic acids,non-flavonoids,and the antioxidant mechanism of plant polyphenols and its application in animal production aiming to provide theoretical references for the application of plant polyphenols to improve the oxidation stability and safety of meat and meat products.

In order to clarify the expression levels of gonadotropin-inhibitory hormone (GnIH) and gonadotropin-releasing hormone (GnRH) genes in hypothalamus of pigeons at different reproductive stages,and explore the relationship between GnIH and GnRH genes and reproductive performance,the CDS sequence of GnIH and GnRH genes were amplified,cloned and analyzed,and the expression level of GnIH and GnRH genes was detected in hypothalamus of pigeons in pre-laying,post-laying and feeding periods,respectively.Sequence analysis results showed that 522 bp of pigeon GnIH gene complete CDS (GenBank accession No.:MG589638) was obtained which encoded 173 amino acids,and 276 bp of pigeon GnRH gene complete CDS (GenBank accession No.:MG589639) was obtained which encoded 91 amino acids.The homology between GnIH and GnRH genes of pigeon and those of other birds were more than 85% and 80%,respectively.GnIH precursor included a GnIH peptide and two GnIH-related peptides (GnIH-RP-1,GnIH-RP-2) which had the typical C-terminal "LPXRF-amide" sequence.GnRH precursor included a signal peptide,a GnRH peptide and a GnRH-associated peptide (GAP).Real-time PCR results indicated that the expression level of GnIH gene in hypothalamus of pigeons in pre-laying period was extremely significantly higher than that in post-laying and feeding periods (P<0.01).The expression level of GnRH gene in hypothalamus of pigeons in post-laying period was extremely significantly higher than that in pre-laying and feeding periods (P<0.01).The expression level of GnIH gene in hypothalamus of pigeons in pre-laying period was extremely significantly higher than GnRH gene (P<0.01).The expression levels of GnRH gene in hypothalamus of pigeons in post-laying and feeding periods were extremely significantly and significantly higher than GnIH gene (P<0.01;P<0.05).The results revealed that the expression of GnIH and GnRH genes was related to the transformation of female pigeons in different breeding stages,which laid a foundation for discussing the molecular mechanism of pigeon production.

Capacitation is an important physiological prerequisite before sperm cells undergo acrosome reaction and fertilize oocytes.Capacitation is a more sophisticated phenomenon that occurs in the female reproductive tract,it gives sperm the ability to enhance activity,interact with zona pellucida (ZP),perform acrosome reaction and initiate the fusion with the oocyte plasma membrane,and complete the fertilization process.However,the molecular mechanism of sperm capacitation is very complex,although it is not yet fully defined,but the sperm has many structural and biochemical changes after capacitation,such as protein tyrosine phosphorylation,the cholesterol exodus of the sperm membrane,the production of active oxygen and the hyperpolarization of the sperm membrane.Proteins regulate some important phenomena such as capacitation and acrosome reaction by phosphorylation or dephosphorylation,this is the process that sperm must undergo to reach,bind,penetrate and fuse the oocyte.Therefore,protein phosphorylation is a very important process of capacitation,especially phosphorylation at tyrosine residues,which is one of the most important events in the process of capacitation,and tyrosine phosphorylation may be the main or even the only indicator of signal transduction pathways in cells.This text focuses on protein phosphorylation,the discovery,role and localization of tyrosine phosphorylation in sperm,and several factors affecting tyrosine phosphorylation in the process of capacitation.

In order to investigate the expression of the relate genes in the differentiation of intramuscular preadipocyte cells in Congjiang Xiang pig,the longissimus dorsi was collected from the 3-day-old Congjiang Xiang pig,type Ⅱ collagenase was adopted to digest the cells,and the intramuscular preadipocyte cells were separated and cultured,and then the morphology were observed.After induction,the intramuscular preadipocyte cells were identified by Oil red O staining method.Real-time PCR method was used to detect the expression levels of pyruvate dehydrogenase kinase 4(PDK4),fibroblast growth factor 10(FGF10),adiponectin (ADIPOQ),lipoprotein lipase(LPL),CCAAT/enhancer-binding protein α (C/EBPα),fatty acid synthase (FAS),adipocyte fatty acid binding protein 4 (FABP4) and protein kinase B (AKT2) at 0,24,48,72 and 144 h of cell differentiation,0 h was selected as control group.The results showed that the primary culture of intramuscular preadipocyte cells were started adherent in about 5 h,the shapes of adherent cells were round and transparent.After sub-culturing,the cell morphology was uniform,the cells showed red by Oil red O staining after induced culture.Real-time PCR results showed that the mRNA expression levels of PDK4,ADIPOQ,C/EBPα,FAS,FABP4 and AKT2 genes were all highly expressed at 48 h,which were extremely significantly higher than that in other stages (P<0.01);The mRNA expression level of FGF10 gene was higher in the induction of 24 and 48 h;The mRNA expression level of LPL gene at 72 h was extremely significantly higher than control group (P<0.01);The mRNA expression levels of PDK4,ADIPOQ and FGF10 genes at 144 h were extremely significantly lower than control group (P<0.01);The mRNA expression levels of C/EBPα gene at 144 h was significantly higher than control group (P<0.05);The mRNA expression levels of FAS gene at 144 h was significantly lower than control group (P<0.05);The mRNA expression levels of AKT2 and LPL genes at 144 h were not significantly different from that in control group (P>0.05).In this study,we successfully cultured the intramuscular preadipocyte cells,and the expression of adipose-related genes in different induction stages were detected,which provided the foundation for further study on lipid metabolism and deposition of Congjiang Xiang pig.

The sperm maturation in the epididymis is the key to mammalian male gametes gaining fertility capacity.Glutathione peroxidase-5 (GPx5) has a strong antioxidant effect as an antioxidant enzyme specifically expressed in the epididymis,which can regulate the concentration of reactive oxygen species in the epididymis microenvironment and protect spermatozoa from lipid peroxidation to maintain DNA integrity of sperm.GPx5 may also have a certain effect on sperm motility and acrosome reaction.The chromosomal location and the number of exons of GPx5 gene have certain interspecific differences.The expression of GPx5 gene is specific in different species,parts and developmental stages.The gene expression is regulated by factors such as androgen,PEA3 factor and ETV4 family.The authors reviewed the research advance on the structure and location,expression characteristics,function and regulation mechanism of specific GPx5 gene of mammalian epididymis in order to provide theoretical basis for further study on sperm antioxidant mechanism and improving male animal fertility.

In order to study the effects of cold and heat stress at different time on the development of porcine parthenogenetic embryos in vitro,the porcine parthenogenetic embryos were used as the material in this study,immunofluorescence staining and Real-time PCR were adopted to detect the rate of blastocyst,cell count,cell apoptosis,and expression of autophagy related gene and transcription level of apoptosis related gene mRNA.The results showed that compared with control group,the rate of blastocyst decreased significantly after 12 h heat stress or cold stress for 18 h (P<0.05),while the rate of blastocyst in cold stress group was higher than that in heat stress group.After 12 h heat stress or cold stress for 18 h,the number of cells in blastocyst decreased significantly (P<0.05),the apoptotic rate increased significantly (P<0.05),and the apoptotic rate of cold stress group was lower than that in heat stress group.The expression of autophagy related genes Atg6 and Atg8 in cold and heat stress groups was extremely significantly higher than that in control group (P<0.01),Lamp2 expression was significantly higher than that in control group (P<0.05),and Atg6 and Atg8 genes expression in heat stress group were higher than that of cold stress group.By detecting the transcription level of apoptosis-related gene mRNA,the expression of apoptosis-related genes Bak,Casp-3 and FAs in cold and heat stress group was extremely significantly higher than that in control group (P<0.01),and Bcl-xl expression was significantly lower than that in control group (P<0.05).In summary,the tolerance of porcine parthenogenetic embryos to cold stress (31℃) was stronger than that of heat stress (41℃),and heat stress could induce the expression of autophagy and apoptosis-related genes in porcine parthenogenetic embryos cultured in vitro,that could reduce the development ability of parthenogenetic embryos.

Somatic cell nuclear transfer (SCNT),also named as somatic cell cloning technology,has vast importance and implications for biomedicine,genetically modified animals research and livestock breeding or expansion of excellent individuals.Currently,mammalian cloning technology remains inefficient and cloned animals usually accompanies various phenotypic defects.In recent years,researchers have been exploring many ideas,techniques and methods to improve cloning efficiency,which include addition of small molecules in culture media,seeking for superior donor cells like iPS cells,optimization of conditioning parameters for nuclear transfer manipulations and genetic regulation of developmentally important genes such as XIST and H3K9me3 demethylase genes.This paper was aimed to summary above-mentioned progress on cloning efficiency promotion.

Leptin is a protein hormone secreted by white adipose tissue.In mammals,leptin is a 16-ku peptide hormone that plays an important role in the energy balance of neuroendocrine and peripheral regulation,which is an important signal factor for reacting body fat content and regulating body weight and feeding.In human diseases,the expression of leptin gene plays an important regulatory role in many diseases.In particular,the mutation of leptin gene may lead to diseases such as obesity,diabetes and breast cancer.In animal husbandry,the expression of leptin gene has an significant impact on the intake and growth traits of cattle,sheep and pigs.In order to deepen the understanding of leptin gene,the distribution,structure and function of leptin are summarized,and the research advances on disease and animal husbandry in recent years are reviewed in this paper.

DNA methylation is one of the most studied epigenetic,which plays a significant role in mediating biological progress such as gene regulation and expression,the stability of chromosome,genomic imprinting,X-chromosome inactivation.Although DNA methylation is stable,it is dynamic in the process of individual development.The function of DNA methylation modification in early embryonic developmental is not comprehensive.As the research into DNA methylation deepens,its function in pre-implantation embryos is also gradually revealed.This article mainly discusses the discovery and regulation of DNA methyltransferases (DNMTs) and the role of DNA methylation in pre-implantation embryos.

This study was aimed to investigate the effect of carboxyethylgermanium sesquioxide (Ge-132) on the development rate of embryos,the number of embryonic cells,the early embryonic reactive oxygen species (ROS) level and the related apoptotic genes in the embryo after activation of bovine parthenogenesis.The effects of different concentrations of Ge-132 (0,10,100 and 200 μg/mL) on the embryo development of parthenogenetically activated bovine embryos were studied,Hoechst staining was performed on the 8th day embryos,the number of cells was counted under the microscope.The ROS level was detected in the early embryos by DCFH-DA staining,and the data were analyzed statistically after the measurement of fluorescence intensity by Image J.In addition,the apoptosis related genes (Caspase-3,Bax,Bcl-xl and Survivin) in the embryo were analyzed by RT-PCR method.The results showed that there was no significant difference in the embryo rate between 10 μg/mL Ge-132 treatment and control groups (P>0.05),but 10 μg/mL Ge-132 could reduce the ROS level in the one cell stage.The number of total cells increased significantly in 10 μg/mL Ge-132 treatment group than that of control group (P<0.05).Furthermore,by detecting the mRNA transcriptional level of apoptosis related genes,it was found that compared with control group,the expression level of the apoptotic gene Caspase-3 was significantly decreased in 10 μg/mL Ge-132 treatment group (P<0.05),whereas the expression level of anti-apoptotic gene Survivin was significantly increased in 10 μg/mL Ge-132 treatment group (P<0.05).In conclusion,the supplementation of 10 μg/mL Ge-132 to the early embryo culture medium could reduce the intracellular ROS level and the apoptosis induced by oxidative stress in the embryo,thereby improving the developmental potential of the bovine embryos after parthenogenetic activation.

In order to study the expression pattern of melatonin receptor 1 (MT1) in epididymis of sheep at different age,epididymis were collected from lamb (2 to 3 months old),young ram (6 to 8 months old) and adult sheep (2 to 3 years old),and the expression of MT1 mRNA and the distribution of MT1 in different parts of the epididymis were detected by the Real-time quantitative PCR and immunohistochemistry.The results showed that the MT1 transcription level in the lambs cauda was extremely significantly higher than that in the caput and corpus (P<0.01).The MT1 transcription level in corpus and cauda of young ram were significantly higher than that in the caput (P<0.05).The transcription level of MT1 in adult ram cauda was significantly higher than that in the caput and corpus (P<0.05).The transcription level of MT1 in epididymal decreased with age.The immunohistochemistry results showed that MT1 could be detected in all parts of sheep epididymis and mainly distributed in the epididymal epithelial cells.It was concluded that MT1 were expressed and distributed in different parts of the epididymis of sheep at different age,and the MT1 expression in epididymis decreased with age.The MT1 expression level was higher in cauda than other parts of epididymis for sheep at the same age.

In order to understand the drug resistance,virulence and genetic characteristics of chicken source Proteus mirabilis,2 strains of bacteria (YLF1 and WS strains) were prepared by isolation from the Liangshan area,and they were identified by culture characteristics,microscopic characteristics and 16S rRNA sequence determination,and pathogenicity test.Then the sensitivity of the bacteria to 14 commonly used antibiotics (including β-lactams,ceftenes,aminoglycosides,tetracyclines,fulro quinolones and macrolides) was determined using the (K-B) method;The phylogenetic analysis of the 16S rRNA gene sequences of Proteus from 21 different sources published in GenBank was carried out.The results showed that the YLF1 and WS strains all had migratory and β-hemolytic growth characteristics.The microscopic examination showed gram-negative bacilli or cocci varied in length,the homology of them with Proteus mirabilis in GenBank through the 16S rRNA gene identification was above 99%,YLF1 and WS strains were identified as Proteus mirabilis;YLF1 and WS strains showed multiple drug resistance,the drug resistance rates were 78.6%(11/14) and 71.4%(10/14),respectively,the mortality rate of chicks was 80%,the YLF1 and WS strains had the highest homology with Japanese isolate from human,Xinjiang isolate from cattle and Guangdong isolate from soil (99.9%),and they were highly homologous to chicken source Proteus mirabilis from Henan,Xinjiang and India (98.9% to 99.3%),and the genetic relationship was close.These results indicated that Proteus mirabilis infection existed in the chicken populations of Liangshan area.The isolates had high virulence and strong resistance,it also suggested that the isolates might be from the environment,infected chickens or other animals,and there was a potential risk of zoonotic diseases.

The aim of this experiment was to construct the plasmid DNA expressing ENO gene of Mycoplasma suis and determine its immune effect.The Mycoplasma suis ENO gene was cloned into PVAX1 eukaryotic expression vector,then transfected into Vero cells to express and determine its immune effect.18 BALB/c mice (half male and female) were randomly divided into 3 groups (PVAX1-ENO plasmid DNA immunization group,PVAX1 empty vector control group and PBS control group),6 mice in each group.Mice in each group were immunized with PVAX1-ENO plasmid DNA,PVAX1 empty plasmid and PBS,respectively.Serum was separated and serum ENO protein antibody titers,IgG₁ and IgG_2a antibody levels and IFN-γ and IL-4 cytokine levels were determined by ELISA.Two weeks after the third immunization,3 mice from each group were selected to measure the levels of CD4⁺ and CD8⁺.The results showed that the PVAX1-ENO plasmid DNA was successfully constructed and identified by PCR and restriction endonuclease digestion,and it could be successfully expressed in Vero cells.The level of ENO protein antibody,IgG₁ and IgG_2a antibody,IFN-γ and IL-4 cytokines and CD4⁺ and CD8⁺ in PVAX1-ENO plasmid DNA immunized group were significantly higher than those in PVAX1 empty vector control group and PBS control group (P<0.05).The results showed that PVAX1-ENO plasmid DNA could significantly improve the humoral immunity of BALB/c mice.The cellular immune of BALB/c mice was stimulated to some extent.

This study was aimed to establish an indirect competition ELISA (ci-ELISA) method for determination of chloramphenicol (CAP) residue in animal-derived foods.An active carboxyl group (-COOH) was introduced to the hydroxy (-OH) locus of phenyl of CAP to form CAP-HS.Via the method of mixed anhydride,CAP-HS was conjugated with BSA and OVA to obtain artificial immunogen (CAP-HS-BSA) and the coating antigen (CAP-HS-OVA) was obtained in the same way.CAP-HS-BSA was used to immunize BALB/c mice.Cell fusion spare mice were screened by indirect ELISA and ci-ELISA.CAP mAb were established using hybridoma technology.ci-ELISA based on CAP mAb and CAP-HS-OVA for CAP was developed.The result showed that one hybridoma cell line (2C4) was isolated,which produced mAb that could bind CAP,the titer of mAb was 4.8×10^-5.The optimal conditions for ci-ELISA based on 2C4 for CAP were as follows:0.4 μg/mL CAP-HS-OVA coating at 37℃ for 2 h;5% pig serum blocking at 37℃ for 1 h; 1:6.4×10⁴ CAP mAb incubating at 37℃ for 15 min;1:1 000 goat anti-mouse enzyme-conjugated secondary antibody (GaMIgG-HRP) incubating at 37℃ for 30 min;TMB was used for color development at room temperature for 9 min.The calibration curve of ci-ELISA CAP showed typical sigmoid and fitted to the four parameters logistic equation.The IC₅₀ in ci-ELISA was 0.53 ng/mL.The recoveries of CAP in negative carp meat and pork were 93.3% to 96.6% and 93.7% to 96.8%,respectively;The coefficient variation of the variability of intra-assay were 2.3% to 5.0% and 2.2% to 4.6%,respectively;The coefficient variation of the variability of inter-assay were 2.7% to 4.1% and 2.3% to 3.6%, respectively.HPLC comparative test results showed no significant difference between ci-ELISA and HPLC.The ELISA method for residue detection of CAP was successfully established in this experiment.This method had high sensitivity,accuracy and precision,and could meet the requirements for CAP residue detection in animal-derived foods.

The study was aimed to separate a Salmonella Pullorum lytic phage with wide host range using the double-layer agar culture method.The feces,sewage and padding were collected from the modern broiler farms.And the biological parameters of the lytic phage were assayed,including the transmission electron microscopy observation,determination of host range,thermal stability,pH stability,optimal multiplicity of infection (MOI) and one-step growth curve drawing.The results showed that a lytic phage against Salmonella Pullorum was successfully isolated and purified which was named as STP98-a.Its plaques were round and transparent and the sizes were about 4 to 5 mm in diameter.It had an elliptical head with approximately 61 nm in diameter,67 nm in transverse diameter and had a long tail with 112 nm in length,10 nm in diameter,indicating it belonged to Siphoviridae.Phage STP98-a could lyse Salmonella Pullorum and Salmonella enterica subsp.enterica with a wide host range.The phage was stable with a wide range of temperature (30 to 60℃) and pH (3.0 to 12.0).The optimal MOI was 0.001.The one-step growth curve showed that the latent time and burst time of STP98-a were 5 and 135 min,respectively,and the burst size was 93.In conclusion,STP98-a was a Salmonella Pullorum lytic phage with wide host range that could serve as a promising phage preparations against Salmonella Pullorum.

The aim of this experiment was to evaluate the safety of Zixiekang oral liquid by acute toxicity test in mice and sub-chronic toxicity test in rats,so as to provide theoretical basis for clinical use of drugs.Acute toxicity test was conducted in 36 mice by oral administration with the maximum dose method.In sub-chronic toxicity test,80 rats were randomly divided into high,medium,low dose and control groups,the respective dose of the experimental groups were 24,12 and 6 g/(kg·BW) by oral administration,and the control group was given saline at the same volume for 30 days.The weight of the rats was measured after withdrawal.The blood routine index,blood biochemical index,viscera index were calculated.The results showed that no mice died in acute toxicity test,LD₅₀ could not be calculated,and there was no death in maximum tolerance test.In the sub-chronic toxicity test,weight gain of Wistar rats in the treatment group had no significant effect;There were no distinct abnormal changes in parenchymal organs other than the hepatocytes at the distal end of the vein showed swelling of varying degrees while no necrosis or inflammatory reaction in high dose treatment group,and there was no significant difference in hematological,biochemical and visceral indexes compared with control group (P>0.05).According to the acute toxicity of xenobiotics classification standard (WHO),the formulations were no toxic substances,high safety.Thus,the Zixiekang oral liquid was safe in clinical treatment.

Campylobacter jejuni is a major pathogen cause of foodborne diarrhea that affects both humans and animals,and the infection rate is increased year by year.Flagella is an unique structure of the bacterial,it is associated with the motility of bacterial,and it also plays an important role in the pathogenicity.Many studies have shown that the pathogenicity of Campylobacter jejuni has a great relationship with the colonization and invasion of flagella in host epithelical cells and the production of toxins.This paper reviewed the research progress on the structure,function,regulatory mechanism and relates genes expression of flagella in Campylobacter jejuni,summed up the influence of known gene deletion mutation of Campylobacter jejuni on flagella to explore the pathogenic mechanism of Campylobacter jejuni from molecular level in order to offer theoretical basis to reduce the infection rate.

The aim of this study was to investigate the acute toxicity,sub-chronic toxicity and target animal safety of Sarcandra glabra Sanqing granules,and provide theoretical basis for clinical safety medication.In the acute toxicity test,the mice were administrated once by gavage and the LD₅₀ was not detected.Therefore,the method of multiple doses administration within 24 h was conducted to determine the maximum tolerance dose.In the sub-chronic toxicity test,the rats were intragastrically administrated Sarcandra glabra Sanqing granules in three different doses of low,medium and high (5,10 and 20 g/(kg·BW)) once a day for 35 days and control group was set up.The clinical signs and weight changes of the rats were observed during the administration,on the 35th body weigh gained,blood examination,blood biochemical indicators,histopathological changes were observed respectively.In the safety test of target animal,the clinical recommended dose of 1,3 and 5 times were administered for continuous 5 d,the clinical signs of experimental chickens were observed and the blood and biochemical indicators were determined.As a result,in the acute toxicity test,mice were not dead in each dose group,the value of LD₅₀ was not measured,the maximum tolerance was 75 g/(kg·BW) (particle gauge).In the sub-chronic toxicity test,there was no significant difference in clinical signs,body weight,blood routine and blood biochemical indexes between the drug-administered rats and blank control rats (P>0.05).Histopathological observations revealed no abnormal lesions in the parenchymal organs.In the safety test of target animals,the weight gained,feed conversion rate,blood routine and biochemical indexes of chickens in each treatment group were not significantly different from those of blank control group (P>0.05).It indicated that the Sarcandra glabra Sanqing granules were actually non-acute toxicity and non-sub-chronic toxicity,and clinical use of Sarcandra glabra Sanqing granules was safe under the 5 times dose of the recommended dose for target animal.

In order to study the biological properties between the bovine type Ⅰ interferon receptor alpha (IFNAR1) chain and its binding ligand, the bovine IFNAR1 (BoIFNAR1) was amplified by RT-PCR from primary kidney cells infected with vesicular stomatitis virus (VSV),and the recombinant plasmid pET30a-BoIFNAR1-EC was constructed to express the recombinant protein of BoIFNAR1 extracellular region (rIFNAR1-EC) in Rosetta^TM (DE3) pLysS.Besides,the polyclonal antibody (PAb) against BoIFNAR1 was prepared in rabbits with purified rBoIFNAR1-EC.Then the binding ability of bovine IFNAR1 to its ligands was identified.The results showed that the cloned CDS length of BoIFNAR1 gene was 1 685 bp.Moreover,the open reading frame of BoIFNAR1 encoded 560 amino acids,which consisted of one singal peptide encoded by 24 amino acids,one extracelluar region encoded by 414 amino acids,one transmembrane region encoded by 23 amino acids and one intracelluar region encoded by 99 amino acids.What's more,the homology of amino acid among BoIFNAR1 and other species ranges from 63% to 91%,indicating it was highly conserved in evolution,and especially close to sheep.And then a recombinant expression vector containing the extracellular region of BoIFNAR1 was constructed and the recombinant protein rBoIFNAR1-EC was induced.The results showed that rBoIFNAR1-EC was expressed in E.coli as a soluble form,which could be used as the immnuogen in preparing specific antibody against BoIFNAR1 after purification and dialysis.The titer of PAb obtained here was 1:204 800.Ligand binding experiment showed that rBoIFNAR1-EC could bind with bovine IFN-αA and IFN-ε.The PAb against BoIFNAR1 could block the specific binding area of bovine IFN-αA and IFN-ε to the cell surface IFNAR1, resulted in blocking the antiviral signal transduction of bovine IFN-αA and IFN-ε.

In order to determine the pathogenicity of the pseudorabies virus (PRV) variant R13139 strain in pigs,PRV R13139 strain and PRV classic virulent SC strain were inoculated intranasally with 10⁶ TCID₅₀ to inoculate each of 3 healthy piglets at 6 and 9 weeks of age,and blank control group pigs were intranasally with saline.The pathogenicity difference of the two strains were evaluated by clinical manifestations,gross pathology,histopathology,antibody production and virus distribution in organs of infected pigs.The results showed that the lethality rates of SC strain to 6 and 9 weeks old pigs were 66.7%(2/3) and 100%(3/3) respectively,while the lethality rates of R13139 strain to 6 and 9 weeks old pigs were 33.3%(1/3) and 66.7%(2/3) respectively,indicating that R13139 strain was lower mortality than SC strain to 6 and 9 weeks old pigs;However,compared to SC strain infection,R13139 strain caused an earlier onset of clinical signs,higher temperature rise in the same time period and more serious neurologic symptoms in 6 and 9 weeks old pigs;The gross pathology showed that the liver lesions caused by R13139 strain had more obvious necrosis than those of SC strain;The pathological changes of heart,lung,spleen,bladder and brain were not very different;Histopathological examination revealed that the pathological damage caused by R13139 strain of tonsil,lung,liver and trigeminal ganglia of infected pigs was remarkably obvious than those of SC strain;Meanwhile,the PCR results showed the R13139 strain was more widely distributed in tonsil,spleen,lung,trigeminal ganglion and lymph node (inguinal,mesenteric and submandibular) of dead pig's organ samples;The results of ELISA detection showed the R13139-infected pig's PRV-gB antibodies produced earlier for 1 d than those of the SC-infected pigs.The results could provide data for revealing the causes of the reepidemic of pseudorabies in China and further research on the prevention and control technology.

This study was aimed to discuss the effect of drug resistance of Escherichia coli(E.coli) by combining traditional Chinese medicine and antibiotics drugs.50 samples which collected from Henan province were isolated for identification,biochemical experiments and pathogenicity test.12 kinds of traditional Chinese medicine were selected for isolation and identification of pathogenic porcine E.coli,and the K-B method was used to detect the drug resistance.The screened traditional Chinese medicines and antibiotics were used to conduct in vitro antibacterial tests against pathogenic E.coli,and analyze the inhibitory effect of Chinese and Western drugs on E.coli.The results showed that 23 strains of 50 samples were consistent with the isolation and culture characteristics and biochemical characteristics of E.coli.23 strains of bacteria were pathogenic Escherichia coli.Through the drug resistance test,23 test strains had different degrees of resistance to the tested traditional Chinese medicines.The resistance rate of the tested strains to licorice was the highest (82.6%),followed by the birthplace (78.2%),and the resistance to cork(17.3%) and pulsatilla (21.7%) were lower,with certain sensitivity.In vitro antibacterial result showed that the combination of traditional Chinese medicine and antibiotics had different degrees of antibacterial effect,and the antibacterial effect of cork,pulsatilla chinensis combined with antibiotics was the most obvious.It showed that the combination of traditional Chinese medicine and antibiotics could not only inhibit the drug resistance of E.coli,but also delay the emergence of drug resistance of E.coli.

The study was aimed to investigate the pathogens of a suspected Salmonella infection from a large-scale layer farm in Xinxiang city Henan province,and provide a reasonable treatment.The intestinal tube samples of sick chickens were collected under sterile condition,and gram staining,culture characteristics,biochemical test and PCR were carried out,the pathogen regression was performed,also,the drug sensitive of 19 kinds of common antibiotics were analyzed using the disk diffusion (K-B) method.The results showed that a strain of gram-negative bacillus was successfully isolated,and consistent with the culture characteristics and pathologic characteristics of Salmonella Pullorum,the invA gene was amplified successfully by PCR,and the strain was identified as Salmonella Pullorum by comparison with the GenBank database;Using isolated bacterial strain to infect SPF chickens could replicate cases consistent with natural infections.It indicated that the Salmonella Pullorum was the main pathogenic agent of the chickens in this chicken farm.The pathogenic and multiple drug resistance of the Salmonella Pullorum were strong.The isolate strain was resistant to amikacin,oxacillin and penicillin,but it was sensitive to fortum (ceftazidime),ampicillin and ceftriaxone,which could be recommended for clinical treatment positive effect.Above all,this study proposed the specific prevention and treatment measures for the disease and achieved good results.This study initially provided a reference for the diagnosis and treatment of Salmonella Pullorum,with great clinical significance in the early diagnosis and treatment of pathogens of Salmonella Pullorum.

An analytical method was established for the simultaneous determination of tilmicosin,dexamethasone,florfenicol and chloramphenicol in milk by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method.The analytes in milk samples were extracted with acetonitrile,the extract solution was purified by the solid phase extraction cartridge with Oasis PRiME HLB.After concentrated by nitrogen blowing instrument,the analyzed samples were dissolved with sample diluent and excepted fat with n-hexane.The separation was performed on a CAPCELL PAK C18 MG Ⅲ-H column (2 mm×100 mm,3 μm) by gradient elution using methanol(A)-0.1 mmol/L ammonium formate containing 0.01% formic acid (B) as mobile phase:0 to 1.0 min,90% to 60% B;1.0 to 3.0 min,60% to 30% B;3.0 to 4.4 min,30% to 20% B;4.4 to 4.5 min,20% to 90% B;4.5 to 6.0 min,90% B.The analytes were determined by MS/MS under MRM mode,and quantified by matrix-matched external standard method.The results showed that there were good linearities between the peak areas and the concentrations in the range of 1.0 to 100.0 μg/L,with correlation coefficients (r) more than 0.999.The limits of detection (LODs) for the developed method was 0.1 to 0.2 μg/kg,and the limits of quantity (LOQs) was 0.2 to 0.5 μg/kg.Recoveries of 4 compounds ranged from 78.6% to 94.7% when the spiked levels were 0.5,5.0 and 25.0 μg/kg,with the relative standard deviations (RSDs) were 2.4% to 10.1%.Therefore,the developed UPLC-MS/MS method was simple,sensitive and accurate for simultaneous determination of tilmicosin,dexamethasone,florfenicol and chloramphenicol in milk.