In order to obtain bovine pregnancy-associated glycoprotein 2 (BoPAG2) with high expression and purification and good antigenicity,the structure and function of the protein were analyzed by bioinformatics prediction.The BoPAG2 gene of Holstein dairy cow was amplified by PCR,and the gene was ligated into the expression vector pET-28a,transformed into E.coli (BL21) to induce expression and purification.SDS-PAGE and Western blotting were used to verify and detect the size and immunological characteristics of the protein.The glycosylation modification,B-cell epitope,hydrophilicity,signal peptide,secondary structure and 3D model,conservative domain and protein interaction of BoPAG2 protein were predicted and analyzed by bioinformatics.The results showed that the BoPAG2 gene was successfully cloned,and the corresponding protein was expressed and purified by induction.The size of the BoPAG2 protein was consistent with the expected results by SDS-PAGE and Western blotting.The results of bioinformatics prediction analysis showed that five sites of BoPAG2 protein occurred glycosylation modification,Asp⁵¹,Asp⁷¹,Asp¹¹⁴,Asp²⁵² and Asp³⁴³,respectively.The protein contained 15 B-cell epitopes and a signal peptide sequence consisting of 15 amino acids in the N terminal.The 3D model of the BoPAG2 protein was successfully established.The protein contained 4 superfamily domains.The results of protein interaction analysis showed that there were 7 proteins that interact with BoPAG2.The protein might be involved in digestion and absorption.Other processes in the maternal regulatory function during pregnancy.In summary,the BoPAG2 protein was successfully expressed and purified in this experiment.The bioinformatics prediction analysis of the structure and function of BoPAG2 protein provided a theoretical basis for the preparation and function research of BoPAG2 protein antibody.

In order to study the regulatory effect of microRNA-124-3p(miR-124-3p) on lung injury caused by H1N1 subtype swine influenza virus (SIV) in mice,the expression vector of miR-124-3p adenovirus was constructed,and the miR-124-3p differentially expressed mouse models were induced by intravenous injection in the tail of mice which were divided it into three groups (overexpression,inhibition and control groups).48 h later,mice in each group were inoculated with H1N1 subtype SIV in the nasal cavity,with 10⁵ EID₅₀ (50 μL) per mouse.The average body weight change rate,pathological section observation and relative mRNA expression level of related inflammatory factors IL-1β,TNF-α and IL-6 in mice were observed for 14 consecutive days.The results showed that the pre-miR sequence and its sponge sequence had been successfully inserted into the shuttle plasmid of adenovirus,and co-transfected into 293A cells.Real-time PCR detection confirmed that compared with control group,the expression level of miR-124-3p in overexpression and inhibition groups were extremely significantly increased (P<0.01) and significantly decreased (P<0.05),respectively,indicating that the adenovirus expression vectors were successfully constructed.The rates of weight change of overexpression,inhibition and control groups were －5.5%,－12.4% and －8.6%,respectively.The alveolar wall of inhibition and control groups were thickened,in which there were a lot of lymphocyte infiltration,some of the alveoli showed fibrin exudation,and the pathological changes were more serious in the inhibition group,there were a lot of RBC infiltration in alveoli.There were only a little lymphocyte infiltration in overexpression group,and the lung tissue was normal.Compared with control group,the mRNA expression levels of IL-1β,TNF-α and IL-6 in overexpression group were significantly decreased (P<0.05);The mRNA expression levels of inflammatory factors in inhibition group were significantly increased (P<0.05).The results showed that miR-124-3p inhibited the expression of pulmonary inflammatory factors induced by H1N1 subtype SIV in mice,and also alleviated pathological injury of lung.

It aimed to identify the differential methylation region (DMR) and differential methylation gene (DMG) through the analysis of the genome-wide methylation difference of the longest muscle of the sheep's back of different breeds of sheep,and laid the foundation for the analysis of the differences in sheep skeletal muscle development.In this study,one-year-old sheep(Tan sheep and Hu sheep and Tan-Hu F2) were sanned by the whole genome bisulfite sequencing method(WGBS).The degree of methylation and differentially methylated region in the whole genome DNA of longissimus dorsi(LD) muscle were studied to explore the difference of DNA methylation in level in sheep.The results showed that the methylation (mC) rates of cytosine (C) were 3.55%,3.18% and 3.56% in Tan sheep,Hu sheep and Tan-Hu F2,respectively.A total of 97 731 DMRs and 10 784 related DMGs were detected.In CG,CHH and CHG sequences,the methylation levels of the three groups were not significantly different.419 GO terms and 20 related signal pathways were detected by GO and KEGG analysis,respectively,which were showed to be significantly enriched in cellular process,cell part,binding,long-term depression,and so on.Five candidate genes,ACTA2、ROCK1、CALD1、MYH3 and MYH10 were sreened out in muscle tissues.This study provided genome-wide methylation of pattern of three groups (Tan sheep and Hu sheep and Tan-Hu F2).The results provided reference information for the epigenetic study of Tan sheep and screened candidate genes related to muscle development and meat quality.

The aim of this study was to investigate the prevalence of canine circovirus (CCV) in Chongqing in recent years,and to explore the characteristics of the field strains in Chongqing.In this study,100 dog serum samples collected in Chongqing in 2017 were tested for CCV nucleic acid by PCR,and the obtained CCV positive samples were amplified and sequenced with the full length genome,and the Chongqing CCV strain isolated was analyzed by MegAlign,Mega 6.0 and RDP4 software.Four positive sample were found,with a positive rate of 4%,and three full length CCV genomes (CQ76,CQ79,and CQ82) were obtained,with a total length of 2 062 nt.Compared with the previously reported genome of CCV with a length of 2 063 nt,one base was missing,which was located downstream of the stem ring structure in the 5'-intergenic region.The length of the 5'-intergenic region and 3'-intergenic region between the two ORFs of the three strains were 134 (1 929-2 062 nt) and 203 nt (913-1 115 nt),respectively.The homology of CQ76,CQ79,and CQ82 ranged from 99.8% to 100%,among which,CQ76 and CQ82 only had synonymous mutations at the 2 019 locus.The homology of genome sequences of the three Chongqing strains and other strains was 82.7% to 97.1%,among which,the degree of variation of ORF2 was greater than that of ORF1.Based on virus ORF2 sequences and genome-wide respectively build Neighbor-Joining in the evolutionary tree,CQ76,CQ79 and CQ82 all belonged to the same subgroup,which was close to the Chinese strain 204,which belonged to genotype Ⅱ,and the homology was 96.5% to 96.7%.RDP4 recombinant analysis indicated that the genomes of CQ76,CQ79 and CQ82 strains were all the recombinant sequences of 204 and 388 strains from Guangxi,and the recombinant region was located in ORF2.Above results enriched the epidemiological and genetic evolution information of CCV and provided further basic data for prevention and control research.

This study was aimed to further investigate the structure and function of Brucella outer membrane protein 19 (OMP19),obtain the recombinant protein of OMP19 with reactivity,and establish the new method for diagnosing brucellosis based on indirect ELISA.The amino acid sequence of OMP19 protein was analyzed by bioinformatics software,Omp19 gene was amplified by PCR,and the recombinant expression vector pET-32a-Omp19 was constructed by seamless cloning method,it was transformed into E.coli BL21 (DE3) competent cells,induced and purified OMP19 protein.The immunogenicity of OMP19 protein was detected by Western blotting and ELISA,respectively,to establish an indirect ELISA method based on the protein.The results showed that there were 19 signal peptide sequences in the N terminal of OMP19 protein,and the secondary structure was random coil,and OMP19 protein contained dominant antigenic epitopes.The prokaryotic expression vector pET-32a-Omp19 was successfully constructed by PCR and seamless cloning method,the OMP19 fusion protein was successfully induced,expressed and purified,the size of protein was about 35 ku,which was consistent with the expect size.The reactivity of OMP19 was identified by Western blotting and ELISA,the results showed that the fusion protein had good reactivity,and the coating concentration was 10 μg/mL.The dilution ratio of the first antibody was 1：50 and the second antibody dilution ratio was 1：8 000,the P/N value reach the highest at 2.80.The results laid a foundation for the establishment of rapid diagnosis of brucellosis and the development of new vaccine.

This study was conducted to construct zinc finger and BTB domain containing 38 (Zbtb38) gene lentiviral overexpression plasmid vector.Firstly,the CDS sequence of mouse Zbtb38 gene was amplified by overlap extension PCR method using mouse spinal cord tissue,and the Zbtb38 CDS sequence front amplification primer 1 and the latter amplification primer 2 were designed respectively.Using the amplification products of the above two primers as template,primer 3 was designed and subjected to fusion PCR amplification,thereby obtaining the full CDS sequence of Zbtb38.Then,the obtained Zbtb38 gene CDS full sequence was ligated to the plasmid pGM-T,and the recombinant plasmid was transferred into E.coli DH5α competent cells,the positive clones were identified and verified by direct PCR and sequencing.Finally,the Zbtb38 CDS sequence was ligated into the lentiviral vector Plvx-puro by XhoⅠ and BamHⅠ double digestion to obtain the shuttle plasmid Plvx-puro-Zbtb38,which was co-transfected with lentiviral packaging plasmids to produce lentiviral particles in 293T cells.The results showed that the primer 1 amplification product contained the first 1-1 794 bp of the Zbtb38 CDS sequence,and the actual sequencing length of the amplified product was 1 772 bp.The primer 2 amplification product contained the 1 762-3 594 bp of the entire sequence of Zbtb38 CDS,and the actual sequencing length of the amplified product was 1 818 bp,indicating that the fragmented amplification target fragment was successfully obtained.The expected fusion products amplified by primer 3 and the PCR amplification products of the colonies both contained the complete sequence of Zbtb38 CDS (3 594 bp),which was compared with the CDS sequence of Zbtb38 gene in NCBI database,and the coincidence rate was 99.99%,indicating that the Zbtb38 CDS sequence was successfully amplified.The Real-time PCR results showed that the virus titer reached 3.25×10⁸ Tu/mL,which met the experimental animal injection standard requirement,indicating that the lentiviral vector was successfully constructed.In summary,the mouse Zbtb38 gene lentiviral overexpression plasmid vector was successfully constructed in this experiment.

The aim of the experiment was to construct the recombinant rabies virus SRV₉ vaccine strain with EgM123 gene by reverse genetics technology and provide the technical means for effective prevention and control of rabies and hydatidosis in China's agricultural and pastoral areas.In this study,the structural protein N,P and L genes of rabies virus SRV₉ were synthesized using gene synthesis technology,which was based on the complete genome sequence of rabies virus SRV₉ and the fusion fragment of the N-P-M fusion fragment and the rabies G gene,through the carrier of enzyme insertion connection methods,the recombinant rabies virus L gene,N-P-M gene fusion fragment and G+EgM123+eGFP gene fusion fragment were successively recombined on the expression vector pcDNA3.1(-) to construct the full-length cDNA of recombinant rabies virus SRV₉ with EgM123 gene.The synthesized genes were constructed on pcDNA3.1(-) expression vector,and the results of transformation,plasmid digestion and gene sequencing showed that the length of N,P,L,N+P+M and G+EgM123+eGFP gene fragments were 1 365,1 107,6 471,3 160 and 3 256 bp,respectively.The full-length cDNA fragment of EgM123 gene recombinant rabies virus full-length cDNA was 12 465 bp,and the sequencing results of each gene fragment were 100%.In this experiment,the full-length cDNA fragment of recombinant EgM123 rabies and eukaryotic expression vectors of the N,P and L genes of rabies virus were successfully constructed,which could save EgM123 gene recombinant rabies by reverse genetics,it also provided the reference for the development of rabies and hydatid disease combined gene recombinant oral live vaccine.

The purpose of the experiment was to screen psychrotrophic lactic acid bacteria with strong stress-resistant growth ability,acid production ability and good bacteriostatic characteristics in vitro,and analyze its related biological characteristics,so as to provide excellent psychrotrophic lactic acid bacteria resources for low temperature silage in Northeast China.The culture condition of 10 ℃ was used to screen the psychrotrophic lactic acid bacteria.After the determination of the growth characteristics of Lactobacillus under different pH,temperature and salinity conditions,the species identification of the candidate strains was carried out,and the growth curve,acid production curve,acid and bile salt resistance,bacteriostatic performance and feeding test of mice were conducted for the candidate strains.The results showed that 11 strains of psychrotrophic lactic acid bacteria could grow well at 10 ℃.The strains C37,C34 and C4212 were identified as Lactobacillus plantarum by 16S rRNA sequencing analysis.The pH of C37,C34 and C4212 decreased to below 4.25 after 7 days of culture.In addition,C37,C34 and C4212 could grow well at 4 ℃,45 ℃,pH 3.0 and 6.5% NaCl.The analysis of growth curve showed that the three strains of Lactobacillus all had a fast growth rate,entered the logarithmic growth period at 4 h,and entered the stable growth period in 10-12 h after inoculation.In the aspect of acid production rate,C37,C34 and C4212 could reduce pH below 4.04 at 24 h of culture.At pH 3.0,C37 and C4212 had a high survival rate,they all had a certain tolerance to 0.5% bile salt,and the survival rate was more than 80%.C37,C34 and C4212 had inhibitory effect on enterotoxin producing Escherichia coli,Salmonella Typhimurium,Staphylococcus aureus and Salmonella Enteritis,and the animal test of three Lactobacillus plantarum were safe.Based on the results of this experiment,Lactobacillus C37,C34 and C4212 were screened and identified from the forest soil of Changbai Mountain,which could provide excellent lactic acid bacteria resources for low temperature silage in winter in Northeast China.

The study was aimed to investigate the effects of high temperature on serum oxidative stress indexes and jejunal mucosal mechanical barrier function in Tongcheng pigs.Thirty 100-day-old female Tongcheng pigs with similar genetic backgrounds and average initial body weight of 45 kg±5 kg were randomly divided into six groups.All pigs were firstly raised under (28±1) ℃ with ad libitum feeding for 7 d.Then the control group was still raised under (28±1) ℃,the high temperature groups were treated with heat preservation lamps,and the temperature was rapidly raised to (38±1) ℃.Pigs were slaughtered and samples were collected at 2,4,6,8,and 10 days,respectively.Serum oxidative stress indexes and the expression levels of mechanical barrier and inflammatory factor-related mRNA in jejunum mucosa were detected.HE sections and immunohistochemical staining were performed on jejunum tissues.The results showed that ①Compared with the control group,the content of serum MDA and GSH-Px activity decreased significantly on the 2nd day of high temperature (P<0.05),and then stabilized.There was no significant change in serum SOD in high temperature groups (P>0.05).Serum T-AOC increased significantly on the 2nd and 8th day of heat treatment (P<0.05).②Compared with the control group,the expression of tight junction proteins ZO-1,OCLN,CLDN5 and CLDN7 mRNA decreased significantly (P<0.05),while the expression of cytoskeleton proteins IQGAP1,IQGAP2 and IQGAP3 mRNA decreased significantly (P<0.05),and the expression of Rac1 mRNA decreased significantly (P<0.05),the expression of Rac2 and Rac3 mRNA did not change significantly (P>0.05) in the heat stress group (2 days).Compared with the control group,the expression of HSP70 in jejunal mucosa increased,mucosal structure was damaged and inflammation occurred in heat stress group.In conclusion,Tongcheng pigs had good antioxidant stress regulation ability under high temperature environment,but high temperature would weaken jejunal barrier function.

The topic of this research was to investigate the expression pattern of myogenic regulatory factors (MRFs) in the myocardium and skeletal muscle of sheep at different fetal stages,the fetuses of Tan sheep at 60 (E60),70 (E70),80 (E80) and 90 days (E90) of gestation were selected as the research objects,and the myocardium and skeletal muscle were collected.The histogenesis of myocardium and skeletal muscle during fetal development were observed by HE and oil red staining.The expression patterns of MRFs in myocardium and skeletal muscle during fetal development and the expression differences of MRFs between myocardium and skeletal muscle at the same stage were tested by Real-time quantitative PCR.The results of HE staining showed that the microstructure of myocardium and skeletal muscle was very different,myocardial fibers crisscross into a network with high density,which were more mature;the density of skeletal muscle fibers were low with large gaps,the number of fibers from E60 to E90 increased gradually,and the structure fiber bundles was very clear.The results of oil red staining showed that there were no fat droplets stained red in myocardium and skeletal muscle tissues,which means that these two tissues form E60 to E90 did not have fat differentiation.The results of Real-time quantitative PCR showed that the expression of MYOG,Myf5 and Myf6 genes in myocardium decreased continuously from E60 to E90 day,but there was no MYOD gene expression.The expression of MYOD,MYOG and Myf6 genes in skeletal muscle from E60 to E90 increased gradually,among which the level of MYOG gene was the highest,and the expression of Myf5 gene fluctuated periodically.The above results indicated that the skeletal muscle was at the stage of rapid development from E60 to E90,however,the myocardial tissue had been roughly formed,which led to the expression of MRFs in skeletal muscle tissue was much higher than that in myocardial tissue,and MYOG gene played an important role in maintaining the rapid development of skeletal muscle.

In order to investigate the expression changes of type Ⅲ fibronectin domain contains protein 5 (FNDC5) in adipose tissue of mice during aging process,healthy mice at different developmental stages were used as research objects.In this experiment,HE staining,immunohistochemical staining,Real-time PCR and Western blotting techniques were used to study the expression and localization of FNDC5 in adipose tissue during aging process.The results showed that FNDC5 was expressed to varying degrees in the fats of all developmental stages in the aging process of mice and showed significant differences.HE staining showed that with age,the size of fat cells gradually increased and then shrank.Immunohistochemical staining showed that the expression level of FNDC5 in adipose tissue of young mice was the highest,followed by that of sexual mature adipose tissue,and that of aged adipose tissue was lower than that of sexual maturation,and the expression level of FNDC5 was the lowest in mature adipose tissue of young mice.Subsequent Real-time PCR and Western blotting results were also consistent with immunohistochemical staining trends.The above results indicated that the FNDC5 gene might play a certain role in adipose tissue,which would provide new ideas for the study of fat metabolism and obesity-related diseases.

The purpose of this study was to construct the adenovirus pDC316-LPIN1-eGFP vector and package the Ad-LPIN1 adenovirus particles,in order to explore the effect of LPIN1 gene on triglyceride and fatty acid synthesis in buffalo mammary epithelial cells.The expression of LPIN1 gene and lipin1 protein in buffalo mammary epithelial cell were detected by Real-time quantitative PCR and immunofluorescence (IF) after Ad-LPIN1 infected.The triglyceride was detected by oil red O staining and triglyceride measurement and the genes expression of triglyceride synthesis and fatty acid synthesis after overexpressing LPIN1 were detected by Real-time quantitative PCR.The results showed that high-quality Ad-LPIN1 adenovirus particles were packaged with the pDC316-LPIN1-eGFP vector.Ad-LPIN1 adenovirus particles infected the buffalo mammary epithelial cell (MOI=50),LPIN1 mRNA was relatively up-regulated more than 5 000-fold,which was extremely significant difference (P<0.01).lipin1 protein expression was up-regulated and mainly expressed in the nucleus.Oil red O staining results showed that lipid droplets in cells were significantly reduced after overexpressing LPIN1 gene.The results of triglyceride measurement showed that the triglyceride content in cells decreased after overexpressing LPIN1 gene,and the difference was extremely significant (P<0.01).The mRNA of peroxisome proliferator activated receptor gama (PPARγ),peroxisome proliferator activated receptor alpha (PPARα) and sterol regulatory element binding transcription factor 1 (SREBP1) after overexpressing LPIN1 gene were significantly or extremely significant increased (P<0.05;P<0.01).And the mRNA of stearoyl-CoA desaturase (SCD),fatty acid synthase (FASN),acyl-CoA synthetase long chain family member 1 (ACSL1) and acetyl-CoA carboxylase alpha (ACACA) were decreased to 83.17%,51.30%,44.24%,41.18% (P<0.01),respectively.The above-mentioned results showed that adenovirus efficiently mediated the expression of LPIN1 gene in buffalo mammary epithelial cell,and increased the expression of lipin1 protein in the nucleus.lipin1 protein,as a common transcription activation factor,inhibited the synthesis of triglycerides by down-regulating mRNA of ACACA,FASN,ACSL1 and SCD genes.

The aim of this study was to investigate the effect of P48 protein on proliferation and apoptosis of embryonic bovine lung (EBL) cells.In this study,samples which co-incubated with P48 protein and EBL cells in different concentrations at different time points were collected,the proliferation rate of the cells was detected by MTT method,and the changes in the nuclear morphology of EBL cells were observed by DAPI staining method.Meanwhile,flow cytometry was used to detect the apoptosis rate of EBL cells induced by P48 protein.Real-time quantitative PCR was used to detect the changes in mRNA level of apoptotic marker genes,and Western blotting tested the Bax and Beclin-1 protein expressions.The results showed that under the condition of 72 h and 10 μg/mL of protein concentration treatment,P48 extremely significantly inhibited EBL cells proliferation (P<0.01),while 0.1 and 0.5 μg/mL protein concentration had no inhibitory effect (P>0.05).The nuclear morphology showed no significant change after protein induction for 12 h,but wrinkled and condensed at 24 h.The nucleus was fragmented,and a sprouted apoptotic body was appeared at 48 and 72 h.Apoptosis related genes expression showed no obvious increase at 2 and 12 h at mRNA level,but gradually increased at 24,48 and 72 h,and it showed a time-dependent manner.Accordingly,the expression of apoptosis marker proteins Bax and Beclin-1 significantly increased.Flow cytometry analysis showed that the apoptosis rate of EBL cells induced by P48 protein was 48.44%.In conclusion,P48 recombinant protein of Mycoplasmas bovis inhibited the proliferation of EBL cells and promoted their apoptosis,which provided reference for revealing the pathogenic mechanism of Mycoplasmas bovis.

The aim of this study was to explore the optimal technological conditions for the synergistic enzymatic hydrolysis of feather processing by-products by recombinant keratinase and disulfide bond reductase from Bacillus licheniformis CP-16.Firstly,the appropriate proportion of keratinase and disulfide bond reductase was explored in this experiment.Secondly,the factors affecting the treatment of combined enzymes were studied,including steam pressure treatment time,enzymatic hydrolysis time,combined enzyme addition level and water content.Finally,L9(3⁴) orthogonal test was used to further optimize the processing conditions.The results were as follows:When the enzyme activity ratio of keratinase and disulfide bond reductase was 80：1 and the final concentration of keratinase was 90 U/mL,the combined enzyme had the best efficiency in feather hydrolysis.Pressure treatment of feather keratin for 2 h was more conducive to hydrolysis of feathers processing by-products by combined enzymes.Feather hydrolysis rate was the highest at 60 h hydrolysis,but it was not significantly different from that at 48 h (P>0.05).The efficiency of feather enzymatic hydrolysis in the total reaction system with 5.5 mL buffer was significantly higher than control group(P<0.05),but the difference was not significant compare to the group with 4.5 mL buffer (P>0.05).The results of orthogonal test showed that,the factors affecting the hydrolysis efficiency were pressure treatment time,enzymolysis time and enzyme dosage from large to small.The optimal conditions for feather enzymatic hydrolysis were:The ratio of keratinase to disulphide bond reductase was 80：1,121 ℃ for 2 h,the final concentration of keratinase was 90 U/mL,and the enzymatic hydrolysis was conducted at 50 ℃ for 48 h.At this condition,the soluble protein content of supernatant produced of each gram feather was 352.72 mg,which was 640.26% higher than that of the control group.In conclusion,the combination of multiple treatment processes was more conducive to improving the efficiency of hydrolysis of feathers processing by-products by combined enzymes.The study could provide guidance for the development and utilization of feather resources.

This experiment was conducted to study the effects of cotton straw fermentated by Candida utilis CU-3,in order to obtain a compound preparation with high efficiency to improve the quality of cotton straw feed.The gossypol tolerance of the strain was tested through the plate tolerance with gossypol acetate as the sole carbon source,and the optimal detoxification conditions of CU-3 were optimized by the solid fermentation.CU-3 mixed fermentation with other preparations were applied to the realistic production,the chemical substances such as crude protein,crude fiber and crude fat in the cotton straw were detected to verify the fermentation effect.The results showed that CU-3 could be grown in 1 000 mg/kg gossypol acetate medium,which was the sole carbon source.The increase of fermentation time and inoculation amount,the detoxification rate of CU-3 on cotton straw increased significantly (P<0.05),and the detoxification rate was the best when the water content was 60% in single-factor cotton straw solid fermentation.The variance analysis of response surface model showed that the equation regression was significant (P<0.01),the maximum degradation rate was 70%,when the conditions were 60% water content of cotton straw,10% inoculation amount and 30 days fermentation time.CU-3 mixed with other preparations,the sensory evaluation scores were all excellent,cotton straw had golden color,clear structure and mellow smell.The free gossypol content of cotton straw was significantly reduced (P<0.05),the minimum content of free gossypol was 62.00 mg/kg.The crude protein content was significantly increased (P<0.05),the highest reached 9.41%.The crude fat content was up to 14.50 g/kg.The crude fiber content was significantly reduced (P<0.05).The cadmium,lead and chromium contents of each treatment group reached the standard.The best compound preparation was CU-3 with 2% corn flour.The application effect tests were carried out in the three farms of 142 Regiment,Manas and Beiwucha.The pH of the cotton straw were between 5.05 and 5.20 after fermentation,the detoxification rates were all above 70% and the highest reached 78.5%,the crude protein contents were 7.97%,8.24% and 9.89%,the crude fiber content were 35.4%,34.5% and 31.2%,respectively,the crude fat content was up to 10%.In this experiment,CU-3 and its compound preparation could effectively improve the detoxification efficiency and feed quality of cotton straw under optimized fermentation conditions,and provide experimental basis for the application of cotton straw feed in Xinjiang.

The present study was conducted to evaluate the threonine requirement for Pekin ducks in low protein diets by determining the effects of threonine on growth performance,carcass traits,and serum parameters.In total,240 1-day-old were randomly allocated to one of five dietary treatments with six replicate cages with eight duck for each treatment according to average body weight.They were fed low protein diets (17.65%) with 0.41%,0.48%,0.55%,0.62% or 0.69% threonine from 1 to 21 days of age,respectively.The results showed that threonine supplementation in low protein diets improved body weight,weight gain,feed intake,and breast muscle percentage,and reduced the ratio of feed to gain (P<0.01).Threonine supplementation in low protein diets had no influence on thigh muscle percentage,and the activity of ALT and AST,and the contents of TP,ALB,GLB and GLU in serum (P>0.05),but reduced the contents of CHO,TG,HDLC and LDLC in serum (P<0.05).The evaluated threonine requirement in low protein diets based on linear broken-line regression with weight,weight gain,feed intake,ratio of feed to gain,and breast muscle percentage were 0.594%,0.594%,0.595%,0.513% and 0.607%,respectively.In summary,the optimal dietary threonine levels would be 0.607% for Pekin ducks fed low protein diets from 1 to 21 days of age.Although which failed to support equal growth performance to that of high protein diets,it was possible to reduce nitrogen emissions.

This study was conducted to investigate the effects of amino acid by-products(ABP) on fermentation quality and digestibility of red sorghum and sweet corn,and explore the mechanism of action by scanning electron microscopy(SEM).There was a control group without additives,an experimental group Ⅰ with 2.0% ABP,and an experimental group Ⅱ with 2.0% ABP mixed with forage fungus for red sorghum and sweet corn,respectively.The results showed that:①2.0% ABP reduced the pH of red sorghum and sweet corn silage to 3.90 and 3.28 respectively,and they were belonged to the high-quality silage range by sensory evaluations.②For the red sorghum silage,compared with the control group,the dry matter (DM) contents of the experimental groups were slightly increased,but the difference were not significant (P>0.05),while the crude protein (CP) contents were significantly increased (P<0.05).The fiber content of the experimental group Ⅱ was slightly lower than the control group,but the difference was not significant (P>0.05).The lactic acid and acetic acid contents of experimental groups were significantly higher than that of control groups (P<0.05).The content of butyric acid in experimental group Ⅱ was significantly lower than that of the other groups (P<0.05).The dry matter and neutral washing fiber (NDF) digestibilities of the experimental groups were higher than the control group,but the difference were not significantly (P>0.05),and acidic washing fiber(ADF) digestibility was significantly higher than control group (P<0.05).③ For sweet corn silage,the dry matter and fiber contents of experimental groups were lower than those in the control group,but the differences were not significant (P>0.05),the crude protein content was significantly higher than the control group (P<0.05).The lactic acid content of the experimental group Ⅰ was significantly higher than the other groups (P<0.05),the acetic acid content of experimental group Ⅱ was significantly higher than that of the other groups (P<0.05),and the butyric acid content of each experimental group was significantly higher than the control group (P<0.05).The digestibilities of the experimental groups were lower than the control group but higher than the raw materials,and the differences of dry matter and acid washing fiber digestibilities were significant (P<0.05).④SEM results showed that ABP promoted the adhesion of forage bacteria by destroying the waxy layer on the surface of red sorghum silage,and degraded the cell wall fiber components to improve the quality of the silage fermentation and improve the digestibility during the silage process.But the destructive effect of ABP on sweet corn silage was not obvious.In conclusion,the addition of ABP could improve the fermentation quality and digestibility of red sorghum,but the effect on sweet corn silage was not obvious.

With the development of intensive cultivation in dairy cows,the production efficiency of dairy farms has been improved.However,issues such as animal welfare,environmental protection and product quality of dairy farm have received more attention.As judging criteria in the pattern,outdoor grazing and grass intake necessary in grass-fed farming,and its products are gradually being favored by consumers in developed countries,such as Europe and the United States.The effect of grass-fed on milk and dairy products mainly depend on the quality of forage grass.The high fiber content in forage grass maintains rumen health,high unsaturated fatty acids increase the proportion of unsaturated fatty acids in milk fat,and rich degradable proteins promote milk protein synthesis.The metabolism of fatty acids,amino acids,carbohydrates and other nutrients from forage in dairy cows may affect the sensory quality of milk and dairy products.This review focused on the actual situation and standard requirements of grass-fed in some countries,and their differences compared with organic farming,and analyzed the effects of feed composition differences on rumen metabolism of dairy cows under grazing farming and total mixed diet feeding conditions,as well as resulting differences in milk fat,protein and sensory indicators.

Finding antibiotic alternatives is a major issue in the breeding industry.Pediococcus, a kind of Gram-positive lactic acid bacteria,is one of the probiotics allowed in the feed addition catalogue.A large number of studies have shown that Pediococcus has great application value in the food,medical and breeding industries.Their good colonization ability on the surface of the host intestinal mucosal cells reduces the adhesion of other bacteria.Meanwhile,Pediococcus is able to produce many antibacterial substances including pediocin,organic acids and hydrogen peroxide.Therefore,it's showing their good probiotic performance in inhibiting the growth of pathogenic bacteria,improving the intestinal microbial community structure,stimulating the host's immunity,promoting the health of farmed animals,and improving animal growth performance,However,compared with other probiotics,its degree of attention is not high.This article summarizes the types of bacteriostatic substances produced by Pediococcus and their bacteriostatic mechanisms,introduces Pediococcus's adhesion and immune regulation,and the application of Pediococcus in the production of animals such as pigs and chickens.The aim was to provide a theoretical reference for Pediococcus's reasonable application in animal production.The results discussed in this article indicated that Pediococcus had broad-spectrum,stable bacteriostatic effects and rich probiotic properties.It's beneficial to the healthy growth of livestock and poultry,prevents pathogenic bacterial infections,and reduces the incidence of diseases.Therefore,Pediococcus is worth increasing its promotion and application.

This study was aimed to screen the selection signatures on autosome of Tibetan goats and discover genes with important germplasm characteristics.Based on the Illumina 50K chip genotyping data of Tibetan goats,Xinjiang goats and Taihang goats,high quality SNP markers were obtained after filtering out SNPs with low allele frequency and not located.The genetic structure was analyzed by genetic differentiation coefficients (Fst).Meanwhile,the principal component analysis (PCA) and phylogenetic tree construction were conducted to determine the population structure.The selected genes in Tibetan goats were also identified through genome selective signals testing,which contained Di and XP-EHH with top 5% valued as a significant threshold.To identify the genes that were under selection,bioinformatics databases were examined that contained relevant data on goats.The results showed that 48 358 SNPs were identified in these three populations.Population genetic analysis showed that the three groups had similar genetic distance (Fst<0.05),but the degree of genetic differentiation of Tibetan goats (Fst=0.0376) was significantly higher than that of Xinjiang goats (Fst=0.0256) and Taihang goats (Fst=0.0257),indicating that Tibetan goats breed had already generated a certain degree of genetic differentiation.Based on these SNPs,36 SNPs and 211 genes were identified in Tibetan goats by Fst and XP-EHH.Among them,EGFR,AKT1,PDHB and PFKP genes were related to high altitude adaptation.These genes were found to be mainly enriched in purine metabolism pathway,metabolic pathway and HIF-1 signaling pathway.In conclusion,the genomic SNPs had more advantage in revealing the selection of Tibetan goats in high-altitude adaptability,and provided new theoretical references for the protection and utilization of germplasm resources.

The purpose of this experiment was to study the effect of gender on the slaughter performance and meat quality of Sunite sheep.Twelve 6-month-old Sunite rams and ewes at the same body condition were selected from the grazing herds of the Sunite Zuoqi Improvement Station Breeding Farm to determine their slaughter performance and meat quality.The slaughter test results showed that the bone quality and bone-meat ratio of the rams were significantly higher than that of the ewes (P<0.05),and the net meat quality and net meat rate of the ewes were significantly higher than that of the rams (P<0.05).The results of meat quality traits showed that the drip loss of ewes were significantly lower than that of rams (P<0.05),and the other indicators were not significantly different (P>0.05).The results of conventional nutrient composition of muscles showed that the intramuscular moisture content of the rams were significantly higher than that of the ewes (P<0.05),and the intramuscular fat content of the ewes were significantly higher than that of the rams (P<0.05),while the other indicators were not significantly different (P>0.05).The results of amino acid determination showed that the essential amino acid ratio in muscle of sunite ewes were significantly higher than that of rams (P<0.05),but there was no significant difference between the rams and ewes that formed the precursor amino acid content of meat flavor (P>0.05).Fatty acid measurement results showed that the saturated fatty acid ratio in the subcutaneous fat of rams was significantly lower than that of ewes (P<0.05).There was no significant difference in the ratio of saturated fatty acids in muscle fat and tail fat (P>0.05);The ratio of monounsaturated fatty acids in ewes tail fat was significantly higher than that of rams (P<0.05).The ratio of monounsaturated fatty acids in subcutaneous fat and intermuscular fat was not significantly different between rams and ewes (P>0.05).The ratio of polyunsaturated fatty acids in intermuscular fat of rams was significantly higher than that of ewes (P<0.05),and there was no significant difference in the content of polyunsaturated fatty acids in subcutaneous fat and tail fat (P>0.05).In summary,it showed that gender had a certain influence on the slaughter performance and meat quality of 6-month-old Sunite sheep.

This study was aimed to evaluate the information of their genetic background of Frizzle chicken (FM),Naked-neck chicken (CB) and YN chicken (YN) that were three newly discovered native chicken genetic resources with excellent characteristics,which had been found in Nujiang prefecture and mountainous area of Yunnan province.The variation in a total of 168 individuals sampled from the three chicken populations was assessed using the mitochondrial DNA (mtDNA) D-loop region sequences as genetic marker.The results showed that there were a total of 27 haplotypes were defined in the three native chicken as well as the haplotype diversity of these three chicken were 0.947,0.938 and 0.596,respectively.The nucleotide diversity of these three chicken were 0.01268,0.01434 and 0.00239,respectively.Phylogenetic tree displayed that all 168 individuals distributed in maternal lineage A,B,C,E,F and G.Frizzle chicken contained lineages E,F and G,and Naked-neck chicken contained all 6 lineages,of which the main lineages were E,F and G.YN chicken included lineages E,F and G,and lineage E was the highest percentage in this population.Also,it found that YN chicken was closely related to Gallus gallus murghi,White Plymouth Rock chicken,White Leghorn chicken and New Hampshires chicken,while Naked-neck chicken was closely related to Gallus gallus spadiceus,Gallus gallus jabouillei,Gallus gallus gallus Linnaeus,Indonesian cockfight and Laos chicken.The Frizzle chickens shared more haplotypes with Naked-neck chicken,and the evolutionary relationship between the two species was closer.This study provided a basis for the origin and genetic assessment of three newly discovered Yunnan local chicken breeds.

Jingxing-Yellow 103 is the unique commercial breed (synthetic line) that contains Beijing-You consanguinity by far.In this study,carcass performance,meat qualities and main nutrient content of breast muscle were compared between the synthetic line and pure line.A total of 120 Jingxing-Yellow 103 and 120 Beijing-You pure lines (half male and half female) were incubated and hatched contemporarily,and housed under the same management with the same feed.Each 20 birds with average body weight of the synthetic line and pure line were selected and slaughtered at 90 and 120 days of age,respectively,for the measurement of related traits.As the results showed,Jingxing-Yellow 103 grew faster than Beijing-You pure line.The average body weight of Jingxing-Yellow 103 was 1.48 and 1.88 kg at 90 and 120 days of age,respectively,which was 24.4% and 24.5% higher than those of pure line (P<0.05).The breast muscle and thigh muscle mass was 24.5% and 19.2% increased than the pure line at 90 days of age,and 30.7% and 28.9% at 120 days of age.The breast muscle and thigh muscle yield did not shown significant difference between breeds (P>0.05).The intramuscular fat content and crude protein content of breast muscle in Jingxing-Yellow 103 were higher than those of Beijing-You pure line (P<0.05).The main fatty acid in the breast muscle included saturated fatty acid like stearic acid and palmitic acid,and unsaturated fatty acid like arachidonic acid,linoleic acid and oleic acid.The fatty acid composition was not different between breeds (P>0.05).In all,Jingxing-Yellow 103 showed faster growth and reached the market weight of typical medium type of Yellow-feathered broilers by 90 days,and they had higher muscle mass and basically retained favourable meat quality like high intramuscular fat content as compared to Beijing-You pure line.

In order to study the muscle growth and development process of Simmental cattle in vitro and establish a primary cell model of bovine skeletal muscle satellite cells.Bovine skeletal satellite cells (BSSC) were obtained by digestion with 0.2% type Ⅱ collagenase and then digested with 0.25% trypsin-EDTA,and using the differential adhesion method to separate and obtain purified BSSC,reverse transcription PCR (RT-PCR),immunofluorescence staining and Western blotting were used to identify BSSC.BSSC was induced differentiation into adipocytes by adipogenic inducers.Oil red O staining was used to identify the adipogenic capacity of differentiated cells.It was also induced differentiation into myocytes by 2% horse serum,and RT-PCR was used to identify the expression of PAX7 and MyoG marker genes in resting phase before and after induction.The results showed that the isolated and purified BSSC was fusiform or spindle-shaped,with full cell morphology and strong refraction.With the extension of the culture time,the cells changed from disordered growth to ordered growth.It was found that BSSC surface marker genes Desmin,c-Met,Myf5 and specific marker Paired box 7 (PAX7) were all positively expressed by RT-PCR detection.Immunofluorescence staining and Western blotting showed that PAX7 and MyoD gene were both positively expressed.Adipocyte induction after differentiation,it was heavily stained with oil red O and a large number of lipid droplets were observed.The expression of PAX7 gene in the resting period after induction of myoblasts was higher than that before induction,but the expression of myogenin marker gene MyoG was opposite.The above results indicated that this study successfully isolated BSSC and proved that BSSC had the ability to differentiate into adipocytes and myocytes,which provided a primary cell model for the in vitro study of the regulation mechanism of Simmental beef products.

With the rapid development of dairy breeding and the scale feeding level,the average individual milk yield of dairy herds in China has been greatly improved over the last few decades.However,the reproductive performance of dairy cows,especially in high-producing dairy cows,has declined continuously and affected severely dairy farm profitability.Reproductive performance of dairy cows was influenced by many factors including environment,herd management,genetics and various diseases.It has become more evident that genital tract diseases(also known as reproductive diseases) decreased reproductive performance of dairy cows during the last 10 years.The effect of reproductive diseases on reproduction of dairy cows has received more attention.This paper briefly described the incidence of reproductive diseases such as metritis,various types of endometritis and retained fetal membrane and ovarian diseases such as ovarian cyst,persistent corpus luteum in China and abroad.This paper highlighted the effects of reproductive diseases on reproductive performance such as days open,calving interval,pregnancy rate,estrus detection,days to first breeding and breeding index.Moreover,we analyzed in more detail the possible mechanisms by disrupting endocrine signaling,damaging intrauterine environment and causing ovarian dysregulation.Lastly,we looked forward to the future research direction of reproductive diseases of dairy cows that provided data reference and theoretical support for improving the management measures of scale feeding,reducing the incidence of reproductive diseases,improving the production and reproductive capacity of dairy herds in China.

Myosin heavy chain 3 (MYH3) gene encoded an embryonic myosin heavy chain protein that controlled the traction and sliding of muscles.MYH3 gene was an important marker gene for muscle differentiation,could regulated muscle development and energy metabolism,and played an important role in the entire muscle development of animals. MYH3 gene was highly conserved among different species and expressed in many tissues in animals,and it was highly expressed during embryonic muscle tissue and muscle regeneration.It was affected by various factors such as transcription factors,microRNA,lncRNA and environmental nutrition factors,and also regulated the funtion of other genes.MYH3 gene mutation could also change the phosphorylation level of TGF-β signaling pathway and MAPK signaling pathway related proteins.After mutation of MYH3 gene,it would affect the activity of ATPase,prolong the hydrolysis time of ATP and the cycle of transverse bridge,which could affect the energy metabolism of muscle and eventually lead to the disease of muscle energy metabolism.The change of copy number or expression level of MYH3 gene was significantly correlated with the size,carcass weight,slaughter weight and growth performance of animals.MYH3 gene was highly expressed in muscle tissues with high marbling and high intramuscular fat,and also to be considered an important candidate gene affecting muscle tenderness,shear strength and red flesh color of muscle in animals.The high expression of MYH3 gene was related to the content of oxidized type Ⅰ muscle fibers,diameter of muscle fibers and content of slow muscle fibers.This article introduced the basic structural characteristics of MYH3 gene,pointed out its regulatory role with muscle tissue development and related influencing factors,and then explained the relationship between MYH3 gene and animal muscle energy metabolism,growth performance and meat quality,which provided a reference for further research on MYH3 gene and muscle development regulation and meat quality improvement.

microRNA (miRNA) is a class of endogenous single-stranded non-coding small RNA that is widely found in eukaryotes,with a length of about 21-22 nt,which regulate gene expression in a specific sequence complementary binding manner.In the classical miRNA mechanism of action,miRNAs are usually completely or incompletely complementary to the 3'UTR seed region sequence of the target gene,thereby regulating the expression of the target gene at the post-transcriptional level.More and more reports have confirmed that miRNA can also be combined with the target gene 5'UTR region,coding region and promoter region,and then participate in complex gene regulation processes.With the development of high-throughput sequencing technology and molecular biology experimental technology,a large number of miRNA have been identified and their biological mechanisms involved in regulation have been gradually revealed.A large number of studies have shown that miRNA can participate in biological processes such as animal reproduction,growth and development,metabolism and disease regulation,and is of great significance for maintaining normal life activities.In the genetic improvement of pigs,the improvement of the main economic traits (reproduction,growth and development,carcass quality,disease resistance,etc.) and the selection of excellent varieties are inevitable trends in the future development of the breeding industry.Therefore,the author reviews the mechanism of miRNA synthesis and action,as well as recent studies on swine reproductive regulation,muscle development,fat deposition and resistance to disease infections.It provides a reference for systematically understanding the application of miRNA in the regulation of important economic traits in pigs,and provides a basis for further research on related genetic breeding.

The purpose of this study was to investigate the effect of growth differentiation factor 9 (GDF9) on the gene expression of cumulus cells expansion and hormone receptors as well as hormone secretion,in order to provide evidence for the role of GDF9 in the development of sheep cumulus cells.Sheep cumulus cells were used as the research object in this study,and were cultured for 48 h by adding different concentrations (0,50,100,200,400 ng/mL) GDF9 to low serum cell culture medium.Total RNA were extracted from the cells,using β-actin as the reference gene,Real-time quantitative PCR technology were used to detect the cumulus cells expansion related genes hyaluronic acid synthase gene 2 (HAS2),prostaglandin lead oxide synthase 2 (PTGS2),pentraxin 3 (PTX3) and hormone receptor genes follicle-stimulating hormone receptor (FSHR),luteinizing hormone receptor (LHR) and estrogen receptors (E₂R).Using the enzyme-linked immunosorbent assay (ELISA) method to test the content of E₂ and P₄.The results showed that HAS2,PTX3,FSHR,E₂R and LHR mRNA relative expression of 200 ng/mL GDF9 group was extremely significantly higher than the control group and other GDF9 groups (P<0.01),PTGS2 mRNA relative expression was extremely significantly higher than the control group and 50,400 ng/mL GDF9 groups (P<0.01),and significantly higher than 100 ng/mL GDF9 group (P<0.05).When added 400 ng/mL GDF9,the relative mRNA expression of all the mentioned-above genes were all extremely significantly lower than that of the 200 ng/mL GDF9 group.Moreover,the E₂ secretion level was extremely significantly higher than that of the control group and 50 ng/mL GDF9 group (P<0.01),significantly higher than that of the 100 ng/mL GDF9 group(P<0.05),while had no significant difference from the 200 ng/mL GDF9 group (P>0.05).When added 100,200 and 400 ng/mL GDF9,the concentration of P₄ was significantly higher than the control group (P<0.05),and there was no significant difference from the 50 ng/mL group (P>0.05),and there was no significant difference between 100,200 and 400 ng/mL GDF9 groups (P>0.05).To sum up,GDF9 could promote the expansion of sheep cumulus cells and participated in the regulation of hormone secretion of sheep cumulus cells.

Selection and breeding are very important in production of livestock and poultry,and breeding value estimation is the core of selection and breeding.Genomic selection (GS) is a novel molecular breeding method to estimate genomic breeding value using high-density markers across the whole genome.At present,GS has been successfully applied in cattle,pig,chicken and so on,and made significant progress.This method can achieve early selection,decrease the testing costs,shorten generation interval,improve the accuracy of breeding value estimation and accelerate genomic progress.GS estimates the effect of SNP by phenotype information and SNP genotype of each individual in the reference population,and measures the SNP genotype to calculate the genomic estimated breeding value in the candidate population,then selects the best individuals according to the genomic estimated breeding value.With the rapid development of genotyping technology and the decrease of detection cost,and the continuous optimization and high efficiency of genomic selection methods,genomic selection has become an important research method in the selection and breeding of livestock and poultry.The authors reviewed some of the widely used genomic selection methods,compared the differences between different methods,analyzed the problems and challenges of genomic selection,and looked forward to its application prospects in breeding.

In order to explore the polymorphism site and difference of expression level of creatine kinase,mitochondrial 2 (CKMT2) gene in muscle segments of Tibetan and Yorkshire pigs,in this experiment,Sanger sequencing method was used to screen and genotype the single nucleotide polymorphism (SNPs) in the 1 000 bp region upstream of the initiation codon of CKMT2 gene in 50 Tibetan and 60 Yorkshire pigs.The expression of CKMT2 gene was detected by Real-time quantitative PCR in heart,back fat and longissimus dorsi muscle of 180-day-old of 10 Tibetan and 10 Yorkshire pigs.The results showed that 5 SNPs (G-357A,T-469C,G-649T,A-784G and A-953G) were found in the upstream 1 000 bp region of the initiation codon of CKMT2 gene,the allele frequency of Tibetan pigs at all sites were significantly different from those of Yorkshire pigs.These SNPs were related to skeletal muscle growth and development,muscle energy metabolism and anti-hypoxia.The mRNA expression of CKMT2 gene in longissimus dorsi muscle and heart of Tibetan pigs were extremely significantly higher than Yorkshire pigs (P<0.01),and the mRNA expression in back fat of Tibetan pigs was extremely significantly lower than Yorkshire pig (P<0.01),which indicated that CKMT2 gene could positively regulate anti-hypoxia and muscle tissue production,and negatively regulate fat production,the analysis results were consistent with the breed characteristics of Tibetan and Yorkshire pigs.This experiment results provided some new ideas for further exploration of pig growth and development,fat deposition and hypoxia adaptability.

Skeletal muscle is the most abundant tissue and the main component of muscle in animals.The process of skeletal muscle cell's proliferation and differentiation is the basis of muscle development and it's directly affects the meat production performance of domestic animals.It has been found that epigenetic modification plays an important role in regulating the proliferation and differentiation of skeletal muscle cells.In this study,the effects of epigenetics on skeletal muscle in terms of the effects of DNA methylation on the proliferation and differentiation of skeletal muscle cells,the selection and expression of factors regulated by histone acetylation,the regulation of non-coding RNA,and the effects of chromosome remodeling.Research progress in the process of muscle cell proliferation and differentiation,briefly describes the effects of different modification methods and factors on the two processes of skeletal muscle proliferation and differentiation.At the same time,the methods and means used by predecessor in the study of skeletal muscle proliferation and differentiation were reviewed,and then the role of apparent regulatory factors in skeletal muscle growth was analyzed.The purpose was to further explain the important role of epigenetic modification in the proliferation and differentiation of skeletal muscle,enhanced the understanding of the regulation of skeletal muscle proliferation and differentiation,provided a reference path for integration with animal production,and also provided skeletal muscle.Molecular regulation of such as growth and development provided more reference materials.

This experiment was aimed to analyze the changes in the duodenal mucosal transcriptome level of duck plague virus (DPV) infected Cherry Valley ducks,and screen out genes and regulatory pathways involved in inflammation after DPV infection.DEV-GZ strain was used to inoculate 35 days old ducks with leg muscles,and the samples of duck duodenal mucosa tissues were collected at 24,48 and 72 h after inoculation,total RNA was extracted,and transcriptome sequencing was performed.Quality control and annotation of data were performed to screen differentially expressed genes and regulatory pathways related to inflammation,and some of the differentially expressed genes were verified by Real-time quantitative PCR.The results showed that a total of 21.39 Gb Clean Bases was obtained in control and experiment groups,the effective sequence of sequencing samples accounted for more than 99% of the original sequence,and the mapping rate was higher than 83%.There were 221 differentially expressed genes in duck duodenal mucosa after exposure 24 h with DEV-GZ,and 3 genes involved in inflammation-related biological processes,all of which were down-regulated genes.499 differentially expressed genes in duck duodenal mucosa after exposure 48 h with DEV,and 17 genes involved in inflammation-related biological processes,all of which were all down-regulated genes.798 differentially expressed genes in duck duodenal mucosa after exposure 72 h with DEV-GZ,and 20 were involved in inflammation-related biological processes,including 13 up-regulated genes and 7 down-regulated genes.GO database analysis showed that the differentially expressed genes involved in inflammation were mainly CCR8,CCL19,CCL4,IL-17,IRF1,IFN-γ,SLC11A1,etc.,involving acute inflammation,chemokine receptor activity,acute phase reaction and inflammation biological processes such as the positive regulation of reactions and inflammation.KEGG database analysis showed that differentially expressed inflammation-related genes were mainly enriched in Toll-like receptor signaling pathway,NF-κB signaling pathway,IL-17 signaling pathway,TNF signaling pathway and inflammatory bowel disease.Real-time quantitative PCR results showed IFN-γ and MALT genes were basically consistent with the transcriptome results.This experiment completed the transcriptome sequencing analysis of the duodenum of Cherry Valley duck infected by DPV,obtained the functional annotation information of differentially expressed genes,and initially revealed the inflammation-related pathways involved in DPV infection,provided a theoretical reference for further explore the pathogenic mechanism of DPV.

In order to improve the efficiency of virus isolation,Vero cells were designed and constructed to express the epithelial cell receptor of canine distemper virus (CDV).In order to improve the accuracy of protein localization and easy identification,Ig κ signal peptide was used to replace the original signal peptide sequence and HA tag was added.IRES-Puro sequence was concatenated with Nectin4 ORF and inserted into pCI-Neo eukaryotic expression vector to obtain the complete transfection vector pCI-N4.The minimum dose of puromycin to kill all monolayer Vero cell was 6 μg/mL.Vero cell transfected with pCI-N4 plasmid would form significant cell clusters in presense of 6 μg/mL puromycin after 5-7 d.The stable cell line expressing Nectin4 receptor was obtained by 3 times limiting dilution method.The F15 cell could still detect Nectin4 mRNA transcription and almost 60 ku interest protein by Western blotting.By indirect immunofluorescence,the Nectin4 protein expressed abundantly and equably.Cellular localization of Nectin4 protein was on the cytomembrane in the laser confocal microscope field.The Nectin4 protein correctly expressing on membrane met the structural requirement as viral receptor.The stable cell line incubated with a canine distemper positive asepsis simple generated obvious syncytium CPE.And ordinary Vero had no CPE after 3 times blind passage.TCID₅₀ of isolated wild-type strain was 10^-5.9/0.1 mL.In conclusion,the Vero cell which expressed the modified Nectin4 receptor stably was available for CDV isolation.

This study was aimed to reveal the trend of drug resistance about Haemophilus parasuis (Hps) in Guizhou province and guide clinical use of Glasser's disease then slow the emergence of drug-resistant Hps strains.K-B slip-agar diffusion method was used to test the 15 common antibiotics resistance of 517 Hps strains isolated from 9 districts in Guizhou province from 2013 to 2019.The drug resistance differences of strains isolated from different time and districts were analyzed.The results showed that 14.36% to 70.58% isolates indicated drug resistance to 15 antibiotics,the proportion of EFT resistant strains was the lowest and SXT resistant strains was the highest.Compared with 2013,the resistance rate of Hps strains isolated in 2019 to 13 antibiotics increased by 4.52% to 324.68%.Among them,the proportion of drug-resistant strains of 13 drugs increased which EFT resistant strains increased at the fastest rate to 324.68% while STR resistant strains slowest rate to only 4.52%.The rate of TET resistant strains showed negative growth which decreased by 2.21% and 8.63%,respectively.Compared with Kaili,Bijie and Xingyi of Guizhou,the strains isolated from Anshun,Zunyi and Guiyang districts showed higher drug resistance rate of EFT,MC and DO which with low resistance in Guizhou province,but lower of SXT,AML and STR which with high resistance in Guizhou province.The results indicated that the resistance of Hps strains to 15 commonly antibiotics in Guizhou province was serious with rising tendency and the resistance rate of certain strains showed obvious differences between different districts.

The aim of this study was to investigate the effect of immunosuppressant cyclosporine A (CsA) on the expression of proinflammatory cytokines interleukin 1 beta (IL-1β) and interleukin 6 (IL-6) in LPS-induced uterine endometrial stromas cells (ESCs).After cultured and identified in vitro,the inflammation model of the ESCs of female rabbits were successfully constructed by 1 μg/mL lipopolysaccharide (LPS),and 0 μg/mL LPS was used as the control.Then the expression levels of IL-1β and IL-6 were detected after treatment with 0,50,100,200 and 500 ng/mL CsA.The results showed that LPS significantly induced the expression of IL-1β and IL-6 in ESCs (P<0.05).Although CsA had no effect on the expression of IL-1β and IL-6,but 50,100,200 and 500 ng/mL CsA extremely significantly inhibited the upregulation of IL-1β and IL-6 induced by 1 μg/mL LPS (P<0.01).It suggested that a certain dose of CsA could effectively inhibit the LPS-induced immune stress in ESCs.This study provided a basis for the feasibility of CsA treatment after the genital tract of female infected by gram-negative bacteria,but its clinical application and mechanism still need to be further studied.

In order to get more information about the distribution of virulence genes and drug resistance genes of Escherichia coli(E.coli) isolated from calf diarrhea in some large-scale cattle farms in Heilongjiang,sensitive drugs were screened to analyze the preventive and therapeutic effect of sodium humate on E.coli.In this study,40 samples were collected and identified by separation and purification,staining microscopy,biochemical identification and molecular biology,and the virulence and drug resistance genes were detected by PCR and the drug sensitivity was detected by K-B test.The inhibitory effect of different concentrations of sodium humate on E.coli was measured by serial dilution and plate counts.Moreover,the protective effect of sodium humate on mice were studied through gastric administration.The results showed that 30 strains of E.coli were isolated,of which 25 strains had virulence genes and 8 virulence genes were detected out of 10,with detection rate of 83.3%.There were many virulence gene combinations.The highest detection rates for bla_TEM and bla_SHV genes were 100% and 13.3% respectively,and the detection rate of other resistance genes was also high.The results of drug sensitive test showed that 30 isolated strains were most sensitive to meropenem,followed by amikacin.The results of serial dilution and plate counts showed that 5% sodium humate was the most bacteriostatically effective,and the results of toxicity test showed that sodium humate could improve the survival rate of mice.This study provided a basis for the rational use of antibiotics and the clinical application of sodium humate in this area.

In this study,the clinical therapeutic effect of Chinese herbal compound composed of Euphorbia humifusa Willd.and Punica granatum L.on ducklings infected with Escherichia coli (E.coli) was studied in order to provide a new therapeutic method for clinical treatment of E.coli.Fifty healthy one-day-old ducklings were selected and randomly divided into five groups,ten ducklings in each group.The experimental groups were blank control group,model group,amoxicillin group,Chinese medicine compound high-dose group(DS_H) and low-dose group(DS_L).The administration group started administration at 3 days of age,continued administration for 3 days,and continued administration until the end of the experiment,10 days total.Except for control group,when the ducklings were 6 days old,E. coli was intraperitoneally injected for challenge,0.5 mL/head.The mortality of the experimental groups was compared.The dead ducks and the surviving ducks were dissected,the pathological section of liver and small intestine and HE staining sections were observed.The blood routine indexes and serum biochemical analysis were detected to evaluate the therapeutic effect of traditional Chinese medicine compound on E.coli infected ducklings.The results showed that the high and low doses of the traditional Chinese medicine compound and the amoxicillin could effectively reduce the mortality of infected ducklings.Compared with model group,amoxicillin and traditional Chinese medicine high and low dose groups could reduce liver,small intestine and other organs damage.Compared with control group,aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in model group increased significantly (P<0.05),and alkaline phosphatase (ALP),lactate dehydrogenase (LDH),UREA also showed extremely significant upward trend (P<0.01),and the WBC,RBC,HGB,PLT and other indicators changed extremely significantly(P<0.05).Compared with model group,the alkaline phosphatase(ALP)value was significantly or extremely decreased in the administration group(P<0.05;P<0.01),and the blood WBC,RBC,HGB and PLT indexes were extremely significantly increased (P<0.01).The results indicated that the degree of body damage in the administration group was lower than that in the model group.

The purpose of this study was to study the efficacy and effect of compound Banqing soluble powder in preventing and treating chickens infected with infectious bronchitis virus.The chickens were randomly divided into prevention group,treatment group,Shuanghuanglian control group,Maxing Shigan control group,model control group and blank control group.Among them,the prevention group and the treatment group were divided into high,middle and low dose groups respectively.Except for the blank control group,the prevention group was given drugs through drinking water for 3 consecutive days from 12 days old,and then the 15-day-old chicks were exposed to 0.2 mL infectious bronchitis virus by nasal drip,and the treatment group was given drinking water for 6 days.The number of infected and cured chickens were collected during the experiment,the incidence,protection rate and cure rate were calculated,and the index of immune organs,the ability of lymphocyte proliferation and the levels of cytokines IL-2,IL-4,TNF-α and IFN-α in serum were detected to explore the preventive and therapeutic effect of compound Banqing soluble powder on IBV infected chicks.The results of this experiment showed that the protective rate of compound banqing soluble powder against high dose group could reach 66.67%,and the cure rate of the treatment middle dose group was 83.33%.On the other hand,it increased the immune organ index of infected chicks,after exposure 4 and 10 d,the proliferative capacity of T lymphocytes and B lymphocytes in each administration group was significantly higher than that of the model control group (P<0.05).It could also regulate the levels of IL-2,IL-4,TNF-α and IFN-α in serum to maintain dynamic balance,after the challenge (5 and 11 d),the middle-dose group of IL-4 and IFN-α levels were significantly higher than the model control group (P<0.05).Synthesizing the test data,compound Banqing soluble powder had a good effect on the prevention and treatment of infectious bronchitis,and the therapeutic effect was better than the preventive effect.Among them,the preventive high dose group and the middle dose group were the most effective.

The aim of this study was to isolate Enterococcus in clinical dairy cow mastitis,detect its drug resistance and virulence genes,a total of 93 milk samples were collected from 41 dairy cosw with clinical mastitis in eastern,central and western regions of Gansu province,and then construct a prokaryotic expression vector for virulence genes.This experiment used selective medium to isolate and purify bacteria.16S rRNA and biochemical experiments combined method to identify the isolated strains.16 antibiotics were selected for drug sensitivity test,and conventional PCR method was used to detect the carrying of 11 virulence genes.Finally,the detected virulence genes with immunogenicity were selected for the construction of prokaryotic expression vectors.The separation and identification results showed that 18 strains of Enterococcus were isolated and identified from the 93 milk samples,which were divided into 9 species.Drug susceptibility results showed that most of the isolates were multiple resistant to bacteria,accounting for 88.89%.No vancomycin-resistant Enterococcus was found,vancomycin sensitivity rate was 94.12%,and all isolates were resistant to at least one antibiotic.Virulence gene test results demonstrated that 11 virulence genes were detected,the detection rate of cob gene (44.44%) was the highest,the detection rates of efaA,hyl,ccf and esp genes were 33.33%,27.78%,27.78% and 22.22% respectively,the detection rates of Asa1,cylA,EF3314 and gelE genes were all 16.67%,while Ace and cylM genes had the lowest detection rate (11.11%).The virulence genes combination was different due to different strains.Ace and gelE genes with immunogenicity were selected and the prokaryotic expression vector pET32a-Ace and pET32a-gelE were successfully constructed.The results provided basic data and biological materials for subsequent mapping of regional epidemiology of cow mastitis in three regions and preparation of corresponding antibodies and subunit vaccines.

To identify the causative agent of porcine respiratory disease complex (PRDC) occurred at a pig farm in Kaifeng city,Henan province,we identified the potential causative bacteria of the morbid nursing piglets by bacterial test and drug sensitivity test of the clinical samples (lung,liver and spleen),and detected the common potential pathogens causing PRDC diseases by PCR and RT-PCR assays,including porcine reproductive and respiratory syndrome virus (PRRSV),porcine circovirus type 2 (PCV2),swine influenza virus (SIV),pseudorabies virus (PRV),classical swine fever virus (CSFV) and Mycoplasma hyopneumoniae (Mhp),and then sequcing and phylogenetic analysis of the structural gene of positive causative pathogens were carried out.The results showed that a Haemophilus parasuis (Hps) strain were isolated and identified by the observation of bacterial morphology and satellite phenomenon,and the sequence analysis of 16S rRNA gene.The drug sensitivity test showed that the Hps strain was sensitive to ampicillin,amoxicillin-clavulanic acid,ceftiofur and tetracycline.Meanwhile,PCR and RT-PCR assays indicated that all the samples were positive for PRRSV and PCV2,and named the involved strain as PRRSV/HN-2019 and PCV2/HN11-2019,respectively.Sequencing and phylogenetic analysis of the ORF5 gene of the newly identified PRRSV revealed that the PRRSV/HN-2019 strain was closely related to the NADC30-like strains and grouped into NADC30-like genotype clade.And cap gene of the newly identified PCV2 strain the PCV2/HN11-2019 strain was closely related to the PCV-2d strains and grouped into PCV-2d genotype clade.In conclusion,this study demonstrated that the morbid piglets were co-infected with PRRSV,PCV2 and Hps,which provided a basis for the development of effective control strategies in the pig farm.

The abuse of veterinary drugs in production may lead to veterinary drug residues in milk and pose a risk to consumer health.As the kinds of veterinary drugs are too many,the detection method for single target presents low efficiency.Whereas the high-throughput detection technology can detect various veterinary drug residues simultaneously,with time and labor saving,and become a research hot spot.In the aspect of sample pretreatment,dispersed solid phase extraction techniques,such as QuEChERS method,are widely used in sample purification due to its features of simplicity,rapidity and low cost.On the other hand,because of its characteristics of fast separation,high sensitivity and accuracy,ultra-high performance liquid chromatography (UPLC) tandem mass spectrometry (MS) has shown better analytical performance and application prospect than high performance liquid chromatography (HPLC).With the application of new technologies and instruments,veterinary drug residue detection has developed rapidly in terms of pretreatment efficiency,instrument sensitivity and high-throughput detection.In this paper,based on recent literatures and publications,the pretreatment techniques and high-throughput detection technology of veterinary drug residues in milk and dairy products were reviewed,to provide a reference for future research.The pretreatment techniques introduced liquid-liquid extraction (LLE),solid phase extraction (SPE),QuEChERS and salting out support liquid extraction,while the high-throughput detection technology included high-performance liquid chromatography (HPLC),liquid chromatography-mass spectrometry (LC-MS),ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS) and liquid chromatography-time of flight mass spectrometry (LC-TOF-MS).