【Objective】 The purpose of this experiment was to conduct bioinformatics analysis of plasminogen activator inhibitor 1 (PAI1) gene in Qinchuan beef cattle,detect its expression in different tissues and confirm its function by experiments,which provided the theoretical basis for molecular breeding to promote intramuscular fat deposition in Qinchuan beef cattle.【Method】 Intramuscular adipocytes were isolated from longissimus dorsi muscle of Qinchuan beef cattle as the research object.The CDS region of PAI1 gene was amplified by PCR.The similarity was analyzed and the phylogenetic tree was constructed,the structure and function of the encoding protein of PAI1 gene was predicted by bioinformatics software.The expression of PAI1 gene in different tissues of Qinchuan beef cattle was detected by Real-time quantitative PCR.The intramuscular adipocytes was transfected with siRNA of PAI1 gene,the experiment was divided into two groups:Control group (NC) and interference group (si-PAI1).Flow cytometry,EdU staining,CCK-8 assay,and Oil red O staining were used to detect the cell phenotypic alterations.Real-time quantitative PCR and Western blotting were used to identify the expression of marker genes (PCNA,CDK1,CCND2,PPARG,PLIN2 and FABP4) at the transcriptional and translational levels.【Result】 The CDS region (1 054 bp) of PAI1 gene was successfully amplified.The similarity of amino acid sequence of PAI1 protein between Bos taurus and Bubalus bubalis,Capra hircus,Homo sapiens,Mus musculus,Ovis aries and Pan troglodytes were 96.8%,95.8%,85.3%,76.9%,95.8% and 85.3%,respectively.The phylogenetic tree results showed that Bos taurus had the closest genetic relationship with Bubalus bubalis,and the farthest genetic relationship with Mus musculus.Bioinformatics prediction showed that cell cycle-related kinase was involved in the phosphorylation of PAI1 gene.The secondary structure of PAI1 protein were mainly alpha helix and random coil.The binding sites of adipogenic transcription factors were also identified in the upstream 2 000 bp of PAI1 gene promoter.Real-time quantitative PCR results showed that PAI1 gene was expressed in all tissues,with the highest expression in perirenal fat and the lowest expression in longissimus dorsi muscle.After interfering with PAI1 gene,compared with control group,the cell number of the proliferative phase in interference group was significantly increased,the mRNA expression of CDK1,PCNA and CCND2 genes were extremely significantly or significantly increased (P＜0.01 or P＜0.05),and the protein expression of CDK1 and PCNA were also significantly increased.With the differentiation of intramuscular adipocytes,compared with control group,the number of lipid droplets in interference group was significantly increased,and the mRNA expression of PPARγ,PLIN2 and FABP4 genes were extremely significantly increased (P＜0.01).【Conclusion】 PAI1 gene was highly conserved among species.PAI1 might participate in the proliferation and differentiation of intramuscular adipocytes through phosphorylation and transcription factors.The interfering with PAI1 gene could promote the proliferation and differentiation of intramuscular adipocytes,which suggested that PAI1 gene might play an important role in regulating the deposition of intramuscular fat.

【Objective】 This study was aimed to understand the structural characteristics of CRISPR-Cas system and the distribution of cas gene in Pasteurella multocida,and provide a basis for the application of different CRISPR subtypes and the antiviral evolution of Pasteurella multocida.【Method】 The CRISPR cluster and cas gene were screened in the genome of Pasteurella multocida via CRISPRCasfinder online website.The conservation of repeat and spacer sequence,the secondary structure of repeat,the conservation of nucleotide sequence of cas gene,and the similarity of CRISPR cluster in various subtype system were investigated using bioinformatics analysis.【Result】 There were three subtypes(Ⅰ-F,Ⅱ-C and Ⅲ-A) of CRISPR-Cas system identified in 19 selected Pasteurella multocida strains.The repeat sequences in the same CRISPR subtypes had a relatively conservative palindrome repeat sequence,whereas the palindromic repeats varied in the different CRISPR subtypes.The tracrRNA could bind with crRNA in subtype Ⅱ-C system and form conservative structures.In CRISPR system of each subtype,the newly acquired spacer was integrated both at the 5'-end,3'-end or internal locations of CRISPR cluster.Through the analysis of the spacer sequence of each strain,it was found that the same spacer sequence existed in the CRISPR sequence within or between strains,which might relate to the sequence recombination of repeat-spacer in bacteria or convergent evolution of gene level shifts during genetic evolution.【Conclusion】 The 19 strains of Pasteurella multocida contained 3 CRISPR-Cas systems of Ⅰ-F,Ⅱ-C and Ⅲ-A.The repeat sequence of different CRISPR subtypes was significantly different.The distribution of spacer characteristic repeats might be related to the sequence recombination of repeat spacer in Pasteurella multocida during genetic evolution.

【Objective】 This study was amied to obtain the full-length transcriptome database of granulosa cells of small yellow follicle (SYFG),and provide references for further exploring the internal molecular regulation of chicken follicular development.【Method】 The SYFG of Hy-Line variety Brown was sequenced using PacBio high-throughput sequencing platform,and the full-length transcriptome sequence was finally obtained by filtering, clustering and correction.Bioinformatics analysis software was used to compare the full-length transcriptome data for function annotation,transcription factor annotation,coding sequence (CDS) prediction,long non-coding RNA (lncRNA) prediction and screening,simple sequence repeats (SSR) detecting and alternative splicing (AS),and alternative polyadenylation (APA) analysis.【Result】 A total of 52 311 full-length transcripts of SYFG of Hy-Line variety Brown were obtained,and the length was mainly distributed in 1-3 500 bp.By comparing with GO,KEGG,COG,NR,Swiss-Prot,and Pfam databases,the full-length transcriptomes were annotated,of which 26 525 full-length transcriptomes were annotated in GO database.The KEGG database annotated 25 233 full-length transcriptomes.The COG database annotated 31 303 full-length transcriptomes.The NR database annotated 34 284 full-length transcriptomes.The Swiss-Prot database annotated 30 701 full-length transcriptomes.The Pfam database annotated 24 622 full-length transcriptomes.In addition,559 transcription factor,40 944 CDS,14 699 lncRNAs,7 types of SSR,28 423 AS,and 2 747 APA events were identified or predicted.【Conclusion】 This study obtained the full-length transcriptome sequencing data of SYFG of chicken,which improved the number and length of transcripts,perfected the functional annotation of transcripts,analyzed the type of AS and APA,and revealed the complexity of transcriptome,which could provide data support to further analyze the molecular regulatory mechanism of chicken follicular development from the transcriptome level.

【Objective】 The aim of this study was to analyze the sequence characteristics of 2-deoxyriboaldolase (DERA) CDS region and its expression levels in different tissues and different growth stages,so as to lay a foundation for further study of the regulatory mechanism of DERA gene in the muscle of Jingyuan chickens.【Method】 The complete CDS region of DERA gene was cloned,sequenced and bioinformatics analyzed using the breast muscle cDNA of 42 days of age chickens as template.Real-time quantitative PCR was used to detect the expression of DERA gene in chest muscle,leg muscle and different tissues (heart,spleen,liver,kidney,lung,chest muscle and leg muscle) of Jingyuan chickens at different growth and development stages(7,42,126 and 180 days of age).【Result】 The CDS region of DERA gene in Jingyuan chickens was 699 bp in length,encoding 232 amino acids,and had the highest similarity (99.1%) with Phasianus colch.The results showed that the amino acid sequence of DERA protein was highly conserved during the evolutionary process.DERA was a stable protein,the average hydrophilic coefficient was 0.175,which was a hydrophobic protein.DERA protein had a transmembrane structure without signal peptide,which was a transmembrane protein.The protein domain was a deoC/LacD family aldolase domain.There were alpha helix,extended chain and random coil in secondary structure of DERA protein. Protein interaction network analysis showed that DERA protein interacted with PGM2,TPI1,TKT and other proteins.DERA gene was expressed in kidney,chest muscle,leg muscle,heart,liver,lung and spleen,among which the expression of kidney and chest muscle were higher,which were significantly higher than that of other tissues (P＜0.05),and the expression of lung and spleen were lower,which significantly lower than that of other tissues (P＜0.05).The expression of DERA gene in chest muscle and leg muscle at 7 days of age were significantly higher than that at 42,126 and 180 days of age (P＜0.05).【Conclusion】 The complete CDS sequence of DERA gene in Jingyuan chickens was successfully obtained in this experiment.The expression of DERA gene in chest muscle and leg muscle of Jingyuan chickens at different stages of growth and development was the highest at 7 days of age,and the expression of DERA gene in kidney and chast muscle of Jingyuan chickens were higher at 42 days of age.The results provided a reference for further study on the mechanism of DERA gene in muscle development of Jingyuan chickens.

【Objective】 This study was aimed to screen and identify the members of matrix metalloproteinases (MMPs) family and their functional related genes specifically expressed in skeletal muscle of yak,in order to provide a basis for studying the formation mechanism of skeletal muscle structure in yak.【Method】 Glutei and pectoral muscle of adult male yaks were collected.The characteristics of muscle fibers and the number of collagen fibers were analyzed by histochemical staining (HE and Masson).The mRNA expression of MMPs and matrix metalloproteinase inhibitors (TIMPs) were detected by Real-time quantitative PCR.Western blotting and immunohistochemistry were used to analyze the protein expression and tissue distribution of MMPs,types Ⅰ and Ⅲ collagen.Protein-protein interaction networks analysis of MMPs and their functionally related genes was constructed by STRING website.EnzChek^TM Gelatinase/Collagenase kit was used to detect the differences in enzyme activities of gelatinase/collagenase between gluteus and pectoral muscles.【Result】 Masson staining and Real-time quantitative PCR analysis results showed that the area of collagen fiber,the expression of MMP-2 and MMP-14 genes in gluteus of yak was significantly lower than that in pectoral muscle (P＜0.05),whereas the expression of TIMP-3 gene was the opposite (P＜0.05).Western blotting results showed that the expression of MMP-3,MMP-14,types Ⅰ and Ⅲ collagen in gluteus were extremely significantly or significantly lower than those in pectoral muscle (P＜0.01 or P＜0.05).Immunohistochemical analysis results showed that the expression of MMP-2,MMP-9,MMP-14 and type Ⅰ collagen in gluteus were significantly or extremely significantly lower than that in pectoral muscle (P＜0.05 or P＜0.01),but the expression of MMP-16 was the opposite (P＜0.05).Protein function correlation clustering analysis showed that MMP-2,MMP-14,MMP-16,TIMP-2,TIMP-3,COL1A2 and COL3A1 could cluster together,and MMP-3,MMP-9 and TIMP-1 could cluster together.The enzyme activities of gelatinase/collagenase in gluteus and pectoral muscle showed a similar tendency,and the difference was not significant (P＞0.05).【Conclusion】 Members of the MMPs family and their functionally related genes were widely expressed in skeletal muscle of yak,and the expression were different between gluteus and pectoral muscle,which provided reference basis for further study on the mechanism of MMPs regulating the structure formation of skeletal muscle in yak.

【Objective】 The purpose of this study was to characterize the envelope protein (env) of an Avian leukosis virus (ALV) strain by bioinformatics method.【Method】 The serum samples from suspected avian leukosis ckickens were cultured in chicken fibroblast (DF-1),and the subgroups of ALV was identified by ELISA and Real-time quantitative PCR.The PCR products of env gene were sequenced and spliced by SeqMan.The env gene sequence was compared with several ALV subpopulations both domestically and abroad,and the genetic evolution analysis was performed.The env proteins were analyzed with a variety of bioinformatics software,including physicochemical properties,hydrophilicity and hydrophobicity,transmembrane domain,signal peptide,glycosylation site,phosphorylation site,secondary structure,tertiary structure,acetylation site,epitope and B cell epitopes prediction.【Result】 The full length of env gene of ALV-J strain was 1 719 bp,and GenBank accession No.:OP837418.It was located in the same evolutionary branch with ALV-J subgroup,and the similarity was 89.3%-94.8%.The env protein had two transmembrane domains and was a hydrophilic and unstable protein composed of 572 amino acids with a predicted instability coefficient of 43.49 and a total average hydrophilicity of -0.201.The env protein was a non-secreted protein,without signal peptide,with 17 N-glycosylation modification sites,77 O-glycosylation modification sites,42 phosphorylation modification sites,21 potential epitope sites and 9 linear B cell epitopes with more than 10 consecutive bases.In the secondary structure of the env protein,the proportion of randorn coil was the largest (38.29%).【Conclusion】 The env gene was a key gene that caused genetic variation in ALV, and its encoded envelope protein env was unstable, with multiple protein modification sites and antigenic epitopes.

【Objective】 The pourse of this study was to investigate the structure and function of CDS region sequence of myosin light chain 2 (MYL2) gene and its encoded protein of Luchuan pigs,and to explore the effects of MYL2 gene on muscle growth and development,so as to provide molecular basis for the development and utilization of Landchuan pigs.【Method】 The CDS region of MYL2 gene in Luchuan pigs was amplified by RT-PCR.MegAlign software was used to compare MYL2 gene in Luchuan pigs with different species and construct phylogenetic tree.The physical and chemical properties,hydrophobicity and transmembrane structure of MYL2 protein were analyzed by bioinformatics software.The eukaryotic expression vector of MYL2 gene was constructure,the recombinant plasmid was transfected into C2C12 cells by the liposome method, and then the fluorescence was observed. Real-time quantitative PCR was used to detect the expression of MYL2 gene in different tissues of Luchuan pigs.【Result】 The results showed that the CDS region of MYL2 gene in Luchuan pigs was 501 bp length,encoded 166 amino acids,and the similarity was 99.6% with the wild boar MYL2 gene published in NCBI.There were two mutations,C mutates to T at 156 bp,which was synonymous mutation.Mutation from T to C at 404 bp caused amino acid mutation from isoleucine (I) to threonine (T).Bioinformatics analysis showed that the total number of MYL2 protein atoms was 2 633,the molecular mass was 18.879 ku,the theoretical isoelectric point (pI) was 4.83.There was no transmembrane structure,no signal peptide,and it was a non-secreted protein.The MYL2 protein had 16 sites that might be phosphorylated and 1 N-glycosylation site at the 78-position amino acid.The subcellular localization results showed that MYL2 protein was mainly found in cytoplasm (56.5%),cytoskeleton (21.7%), mitochondria (8.7%),nucleus (8.7%) and vacuoles (4.3%).The secondary structure of MYL2 protein was mainly composed of alpha helix,random coil,beta turn and extended chain,accounting for 57.83%,29.52%,9.04% and 3.61%,respectively.The results of cell tests showed that fluorescence was observed in both the experimental group and control group after 48 h.Compared with control group,the expression of MYL2 gene in experiment group was extremely significantly increased (P＜0.01).The expression of MYL2 gene in different tissues from high to low were heart,longissimus dorsi muscle,liver,subcutaneous fat,lung,spleen and kidney,and the expression in longissimus dorsi muscle was significantly higher than that in other tissues except heart (P＜0.05).【Conclusion】 In this study,the CDS region of MYL2 gene in Luchuan pigs was cloned successfully,the eukaryotic expression vector and tissue expression profile were constructed.The structure and function of the protein encoded by MYL2 gene were analyzed,which provided molecular basis for exploring the role of MYL2 gene in the growth and development of muscle and the development and utilization of Luchuan pigs.

【Objective】 This study was aimed to clone,express and bioinformatics analyze the CDS region of chicken embryonic lethal abnormal vision like 1 (ELAVL1) gene.【Method】 The CDS region of ELAVL1 gene was amplified by PCR using the cDNA of chicken embryonic fibroblast (CEF) as a template to construct pMD19-T-ELAVL1 clone plasmid.The pET-32a-ELAVL1 prokaryotic recombinant expression plasmid was constructed using pMD19-T-ELAVL1 template.IPTG was used to induce the expression of the recombinant strain,and the recombinant protein expression were detected by SDS-PAGE and Western blotting. Bioinformatics analysis of ELAVL1 protein was performed by online software. 【Results】 The length of ELAVL1 gene CDS in chicken was 981 bp and encoded 326 amino acids, which had the closet relationship with duck and the furthest relationship with zebrafish.SDS-PAGE and Western blotting results showed that the recombinant protein was mainly expressed in soluble form,and its molecular weight was about 55 ku.Bioinformatics analysis showed that the molecular weight of ELAVL1 protein was 36.10 ku,which was a non-fat soluble,weakly alkaline hydrophilic stable protein.The ELAVL1 protein contained 1 N-glycosylation site,3 O-glycosylation sites,32 phosphorylation sites and 10 linear B cell epitope binding site.Subcellular localization revealed that the protein was located in the nucleus.The secondary and tertiary structures were mainly composed of alpha helix and random coil. The ELAVL1 protein contained 3 RNA recognition motif (RRM), which were predicted to interact with various RNA-binding proteins and protein kinases.【Conclusion】 This study successfully cloned the CDS region of ELAVL1 gene in chicken,and ELAVL1 was a non-fat soluble and weakly basic hydrophilic stable protein.After induced expression,soluble expression of ELAVL1 recombinant protein was successfully obtained.The results provided scientific basis for further function of chicken ELAVL1 gene.

【Objective】 This study was aimed to investigate the effects of rosiglitazone (RSG) on the oocyte maturation in vitro and subsequent development potential of embryos in mice,in order to provide reference for the optimization of in vitro maturation (IVM) culture system.【Method】 The optimum concentration of RSG was determined by adding 0,10,20,50 and 100 μmol/L RSG to IVM medium,and calculating the first polar body (PBⅠ) rate. According to the screening results of the RSG optimal concentration,they were divided into control and rosiglitazone groups.Reactive oxygen species (ROS),glutathione (GSH),cathepsin B (CB),mitochondrial membrane potential (MMP),total cell number (Hoechst 33342) and apoptosis ratio (TUNNEL) in oocytes or blastocysts were detected by immunofluorescence staining.The transcription levels of antioxidant genes (Sod-2,Gpx-3 and Cat) and apoptosis-related genes (Caspase-3,Bcl-2 and Bcl-xl) in blastocysts were detected by Real-time quantitative PCR.【Result】 Compared with control group,20 μmol/L RSG supplementation could significantly increase the PBⅠ rate,cleavage rate and blastocyst rate of oocytes in mice (P＜0.05);The intracellular ROS and CB levels were significantly decreased,the intracellular GSH and mitochondrial membrane potential levels were significantly increased (P＜0.05);The total cell number in blastocysts was significantly increased,and the apoptosis rate in blastocysts was significantly decreased (P＜0.05).The transcription levels of antioxidant genes (Sod-2, Gpx-3 and Cat) and anti-apoptotic genes (Bcl-2 and Bcl-xl) were significantly increased, and the transcription level of apoptotic gene Caspase-3 was significantly decreased (P＜0.05).【Conclusion】 The addition of RSG to IVM medium was beneficial to intracellular redox homeostasis of oocytes,which could improve the mitochondrial function,increase the developmental ability of oocytes after IVF,and decrease the intracellular CB level and apoptosis of cells in blastocysts.

【Objective】 This study was aimed to explore the effect of Agrocybe aegerita polysaccharid (AAP) on immunomodulatory of splenic lymphocytes in mice,in order to provide a basis for elucidating the action mechanism and developing the clinical application of AAP.【Method】 The splenic lymphocytes in mice were taken as the subjects,the MTT method was used to determine the safe concentration of AAP on lymphocytes,and the safe concentrations were selected for subsequent experiments.MTT assay was also used to detect the effect of different concentrations of AAP on the proliferation of splenic lymphocytes in mice.Three control groups of cell,lipopolysaccharide (LPS) and plant hemagglutinin (PHA) were also set at the same time to screen out AAP with the stronger proliferation ability on splenic lymphocytes in mice,flow cytometry was used to detect the effect of AAP on the values of CD4⁺/CD3⁺ and CD8⁺/CD3⁺ of T lymphocyte subtypes.ELISA method was used to determine the effect of AAP on the contents of interleukin-2 (IL-2),IL-4,tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) in the supernatant of lymphocytes.【Result】 AAP concentration lower than 1 000 μg/mL had not any toxic effect on splenic lymphocytes in mice.Compared with control group,different doses of AAP could extremely significantly cooperate with LPS and PHA to stimulate lymphocyte proliferation (P＜0.01),125 and 250 μg/mL AAP could extremely significantly increase the values of CD4⁺/CD3⁺ and CD8⁺/CD3⁺ (P＜0.01),and significantly or extremely significantly increase the contents of IL-2,IL-4,TNF-α and IFN-γ secreted by lymphocytes (P＜0.05 or P＜0.01).【Conclusion】 AAP could cooperate with LPS and PHA to stimulate lymphocyte proliferation in spleen of mice,increase the values of CD4⁺/CD3⁺ and CD8⁺/CD3⁺ on the surface of lymphocytes and the ability of lymphocytes to release cytokines,thus improving the immune function of the body.

【Objective】 Taking the antioxidant peptide of fly maggot (FTP) as the research object,the protective effects of FTP on DPPH and ABTS⁺· free radicals scavenging ability and oxidative stress injury of IPEC-J2 cells were investigated using in vitro chemical and cell tests.【Method】 By measuring different concentrations of FTP (500,1 000,2 000,3 000 and 4 000 μg/mL) scavenging rate of DPPH,ABTS⁺· free radicals to detect the antioxidant capacity of FTP in vitro. IPEC-J2 cells were cultured with different concentrations of FTP (0,100,250,500,1 000 and 2 000 μg/mL),and the cell survival rate of each group was measured by MTT method after 24 h of incubation.The appropriate hydrogen peroxide H₂O₂ concentration was selected by MTT method to construct oxidative stress model.According to the constructed model of oxidative stress and the appropriate concentration gradient for FTP,IPEC-J2 cells were randomly divided into blank,model,and test groups (100,250,500 and 1 000 μg/mL),and the levels of cell viability,reactive oxygen species (ROS),and intracellular SOD,CAT and MDA were measured in each group.【Result】 When the concentration of FTP was 4 000 μg/mL,the scavenging rates of DPPH and ABTS⁺· radicals reached 54.82% and 81.90%,respectively.Different concentrations of FTP had no inhibitory effect on the growth of IPEC-J2 cells,and the cell survival rate was higher than 100%,showing a concentration gradient growth,and the cell survival rate of the FTP concentration of 2 000 μg/mL was 1.22 times higher than that of blank group.The suitable concentration for H₂O₂ to construct the cellular oxidative stress model was 1 000 μmol/L.Compared with blank group,the cell survival rate of model group was extremely significantly decreased,the levels of ROS and MDA were extremely significantly increased,and the activities of SOD and CAT were extremely significantly decreased (P＜0.01).Compared with model group,the cell survival rate of 4 test groups was extremely significantly increased (P＜0.01),and the cell survival rate of 500 μg/mL FTP group was 2.37 times that of model group.The levels of ROS and MDA in 500 and 1 000 μg/mL FTP groups were significantly decreased (P＜0.05),and the activities of SOD and CAT were extremely significantly or significantly increased(P＜0.01 or P＜0.05),both in a dose-effect relationship.【Conclusion】 FTP had a significant protective effect on oxidative stress injury of IPEC-J2 cells in a dose-dependent manner.

【Objective】 This experiment was aimed to study the effects of Eucommia ulmoides leaves fermented by different probiotics on the performance,antioxidant performance,organ and intestinal immune related gene expression of broilers,so as to provide scientific basis for the research and development of new additives to replace antibiotics.【Method】 In this study,broilers were fed with basal diet (group A), and diets supplied with Eucommia ulmoides leaves fermented by Enterococcus faecium (group B), Eucommia ulmoides leaves fermented by Lactobacillus plantarum (group C) and Eucommia ulmoides leaves mixed fermented by Enterococcus faecium and Lactobacillus plantarum (group D).The average daily feed intake (ADFI),average daily gain (ADG) and feed/gain (F/G) were weighed and calculated on the 28th and 56th days,respectively.The indexes of glutathione peroxidase (GSH-Px),malondialdehyde (MDA) and total antioxidant capacity (T-AOC) in serum were detected by cardiac blood sampling.The spleen,liver and ileal tissues of broilers were collected to detect the expression of gamma interferon (IFN-γ),nitrick oxide synthase (iNOS),interleukin-6 (IL-6) and IL-10 by Real-time quantitative PCR.【Result】 ① Compared with group A,ADG were significantly increased and F/G were significantly decreased in groups B,C and D from 1 to 28 and 1 to 56 d (P＜0.05);ADG in groups B and C were significantly increased,and F/G in groups B,C and D were significantly reduced from 29 to 56 d (P＜0.05).② The activity of GSX-Px in groups B and C were significantly increased at 28 and 56 d (P＜0.05).③ Compared with group A,the mRNA expression of IFN-γ gene in spleen of groups B, C and D and ileum of group D were significantly increased after feeding for 28 d (P＜0.05);The mRNA expression of IFN-γ gene in liver of groups B and D,spleen and cecum of groups B,C and D were significantly increased after feeding for 56 d (P＜0.05).④ Compared with group A,the mRNA expression of IL-6 gene in ileum of groups B and D were significantly decreased after feeding for 28 d (P＜0.05);The mRNA expression of IL-6 gene in liver of group D,spleen of groups C and D,and ileum of groups B and D were significantly decreased after feeding for 56 d (P＜0.05).⑤ Compared with group A,the mRNA expression of IL-10 gene in ileum of groups B and D were increased after feeding for 28 d (P＜0.05);The mRNA expression of IL-10 gene in liver and ileum of groups B,C and D were significantly increased after feeding for 56 d(P＜0.05).⑥ Compared with group A,the mRNA expression of iNOS gene in spleen of group D and ileum of groups B,C and D were significantly increased after feeding for 28 d (P＜0.05);The mRNA expression of iNOS gene in liver and spleen of groups C and D,and ileum of groups B,C and D were significantly increased after feeding for 56 d (P＜0.05).【Conclusion】 Adding different probiotics to ferment Eucommia ulmoides leaves in diet could promote the performance of broilers,and affect the serum antioxidant indexes and intestinal immune related genes.

Individual identification technology was the primary requirement of automatic production techniques,including intelligence weighting,body condition scoring,body shape identification,and behavior monitoring.Individual cattle identification methods were categorized and elaborated in current review,that the research progress of different individual cattle identification methods including traditional,biometric,and deep learning methods were introduced,especially the difficulties of applying deep learning method in practice were discussed.Moreover,the advantages and disadvantages of different identification methods were analyzed.Traditional identification methods such as ear notching,ear tattooing,and hot branding identified cattle relies on manual labor,which had low identification accuracy and efficiency,and ignored animal welfare and mark persistence.Radio frequency identification techniques provided an automatic identifying protocol that elevated the efficiency but the data security could not be guaranteed.With the development of image recognition technology and deep learning methods,non-contact,safe and efficient cattle intelligent recognition had been realized with the technology based on biometric recognition and deep learning methods.However,the biometric identification method based on nose print,retinal blood vessels and iris had poor practicability,because the ideal image was difficult to obtain.Based on deep neural networks learning of image features,the deep learning method was more practical and promising in complex dairy farm circumstances.What’s more,different individual identification methods were compared,and the research on individual cattle identification technology was prospected in current review.

【Objective】 The aim of this experiment was to investigate the effects of different concentrations of curcumin supplementation on production performance,serum biochemical indexes and apparent feed digestibility of growing Hainan Tunchang pigs.【Method】 Eighty Hainan Tunchang pigs weighted 35.88 kg±0.25 kg were randomly divided into five groups,each group had 4 replicates,and 4 pigs for each replicate.The pigs in control group were fed with basal diet,and the pigs in antibiotic group were supplemented with 300 mg/kg mucocitractin sulfate in basal diet.The pigs in other 3 treatment groups were supplemented with 200 (Cur group 1),400 (Cur group 2) and 600 mg/kg(Cur group 3) curcumin in the basal diet, respectively.The pre-feeding period was 7 days,and the trial period was lasted 50 days.The experimental pigs were weighted on the 51 st day morning for the calculation of growth performance.Six pigs’serum in each group were randomly collected for the measurement of serum biochemical indicators.The fresh feces of 3 pigs were randomly collected for each repeat to determine the apparent digestibility of feed nutrients.【Result】 Compared with control group,average daily weight gain (ADG) was improved by 23.82% (P＜0.05),the ratio of feed and gain(F/G) was reduced by 20.06% (P＜0.05),the activity of serum AST and DAO content was reduced by 22.23% and 29.31%,respectively (P＜0.05),and serum IgA concentration was increased by 21.14% (P＜0.05) in Cur group 3. Compared with antibiotic group,the ADG of pigs was improved by 23.09% (P＜0.05),the apparent digestibility of crude fiber was increased by 42.14% (P＜0.05), and the activity of serum AST was reduced by 31.75% (P＜0.05) in Cur group 3.Compared with control and antibiotic groups,ADG of Cur groups 1 and 2 showed no significant difference(P＞0.05).Compared with control group,both of serum’D-lactic acid and DAO concentrations in Cur groups 1 and 2 were significantly reduced (P＜0.05),and the apparent digestibility of crude fiber of Cur group 2 was significantly increased than control and antibiotic groups(P＜0.05).【Conclusion】 Adding with 600 mg/kg curcumin could increase the growth performance and apparent digestibility of crude fiber in growing Hainan Tuncahng pigs,and improve the immune state of pigs,which could play an potential role for replacing antibiotics.

【Objective】 The effects of fermented hybrid Broussonetia papyrifera on growth performance,meat quality,digestive enzyme activity and intestinal flora of Hu sheep were studied to provide scientific data for the application of hybrid Broussonetia papyrifera in mutton sheep breeding.【Method】 Seventy-two Hu sheep with good health and similar weight were randomly divided into 4 groups,with 6 replicates in each group and 3 sheep in each replicate.The sheep in control group (CG) was fed with basic diet,while the sheep in experimental groups (EG1,EG2 and EG3) was fed with 16%,24% and 32% fermented hybrid Broussonetia papyrifera respectively.The pre-feeding period was 14 days and the experimental period was 90 days.After the experiment,the weight of Hu sheep was measured,and one sheep was slaughtered randomly from each repetition.Femoral muscles of hindlegs and longissimus dorsi,contents of the middle jejunum and cecum were taken to determine the meat quality,digestive enzyme activity and intestinal flora changes.【Result】 Compared with the control group,①There was no significant difference in average daily feed intake,average daily gain and feed-to-weight ratio of the sheep fed with different proportions of fermented hybrid Broussonetia papyrifera(P>0.05).②In femoral muscles of hindlegs,the shear force of EG3 group was significantly reduced (P＜0.05), the cholesterol content in EG2 and EG3 groups decreased significantly (P＜0.05), the crude fat content of the three experimental groups decreased significantly (P＜0.05),while the hydraulic capacity increased significantly (P＜0.05),and there was no significant difference in pH,crude ash,crude protein and amino acid content among three groups (P>0.05). The contents of palmitic acid, oleic acid, linoleic acid and α-linolenic acid in EG1, EG2 and EG3 groups were significantly increased (P＜0.05), and the contents of Ca, Fe, Zn and Se in EG2 and EG3 groups were also increased (P＜0.05). In longissimus dorsi, the shear force,pH,crude fat and cholesterol contents in EG2 and EG3 groups were significantly decreased (P＜0.05),while the crude ash content in EG2 group was significantly decreased (P＜0.05).There was no significant difference in hydraulic power, crude protein and amino acid content among three experimental groups (P>0.05). The contents of palmitic acid, oleic acid, linoleic acid, α-linolenic acid and arachidonic acid, and Ca and Zn contents in EG1, EG2 and EG3 groups increased significantly (P＜0.05). Se content increased significantly in EG2 and EG3 groups, and Fe content increased significantly in EG3 group (P＜0.05).③Intestinal trypsin and lipase activities increased significantly (P＜0.05), while the concentration of total protein in intestine decreased significantly (P＜0.05) in EG2 and EG3 groups.④Adding fermented hybrid Broussonetia papyrifera could significantly improve the diversity and richness of intestinal flora of experimental sheep,increase the relative abundance of probiotics such as Firmicutes,and decrease the relative abundance of harmful bacteria such as Proteobacteria. 【Conclusion】 Adding 24%-32% fermented hybrid Broussonetia papyrifera in the diet could improve the meat quality and digestive enzyme activity of Hu sheep,and improve the richness and diversity of intestinal flora which was conducive to promoting the absorption of nutrients by animals.

【Objective】 The purpose of this survey was to investigate the distribution of magnesium contents in various feed materials from different regions in China,so as to provide scientific basis for reasonable supplement of magnesium in the diet.【Method】 A total of 4 054 samples of 37 kinds of feed materials for livestock and poultry were collected from 31 provinces,municipalities and autonomous regions in China.After pretreatment,microwave digestion was performed and then the magnesium contents were determined by an ion chromatography-inductively coupled plasma-mass spectrometry (IC-ICP-MS).The One-Way ANOVA was used to analyze the magnesium content in different feedstuffs from the same category and some feedstuffs from different provincial (district) with SAS software,respectively.The differences in magnesium contents among different feedstuffs and different regions were compared.【Result】 The distribution regulation of magnesium contents in various categories of feed materials was as follows:Mineral feeds (average magnesium contents were 12 740 mg/kg) ＞ vegetable protein feeds (average magnesium contents were 4 335 mg/kg) ＞ cereal by-products (average magnesium contents were 3 275 mg/kg) ＞ forage feeds (average magnesium contents were 2 482 mg/kg) ＞ straw feeds (average magnesium contents were 2 346 mg/kg) ＞ animal protein feeds (average magnesium contents were 1 436 mg/kg) ＞ cereal seeds (average magnesium contents were 1 220 mg/kg).There were significant differences in magnesium contents among different feed materials from the same categories (P＜0.05).In the cereals,the magnesium content in wheat or barley (1 358 mg/kg) was the highest,while that in rice (1 115 mg/kg) was the lowest.In the cereal by-products,the highest (5 791 mg/kg) and lowest (387 mg/kg) magnesium contents were observed in rice bran and broken rice,respectively.In the vegetable protein feeds,the highest (6 664 mg/kg) and lowest (2 605 mg/kg) magnesium contents were detected in cottonseed meal and expanded soybean,respectively.As for animal protein feeds,the highest (3 479 mg/kg) and lowest (269 mg/kg) magnesium contents were detected in fish meal and dried blood cells,respectively.As for straw feeds,the highest (4 024 mg/kg) and lowest (1 136 mg/kg) magnesium contents were found in sweet potato vine and wheat straw,respectively.As for pasture feeds,the highest (2 996 mg/kg) and lowest (1 617 mg/kg) magnesium contents were found in alfalfa and Leymus chinensis,respectively.As for mineral feeds,the highest (18 290 mg/kg) and lowest (3 118 mg/kg) magnesium contents were found in calcium hydrogen phosphate and shell powder,respectively.A comparison was made among the magnesium contents of corn,wheat or soybean meal in different provinces (regions),and the results showed that there were significant differences in the magnesium contents of the same feed materials from different provinces (regions) (P＜0.05).According to 152 feed formulas commonly used in swine and chickens all over the country,the calculated magnesium contents in the diets were ranged from 1 848 to 2 100 mg/kg.In accordance with the magnesium requirements suggested by feeding standards of swine and chicken (2004) in China or the NRC (1994) of the United States for swine and chickens,about 1/3 of the magnesium contents in the diets could meet all the nutritional requirements of magnesium for swine and chickens,but the above estimations had not considered the availabilities of magnesium in different feed materials.【Conclusion】 The content of the mineral element magnesium varies greatly among regions and various types of feed ingredients in China.The magnesium contents in the commonly used diet formulas in the country could provide all the magnesium nutritional requirements of swine and chickens.Therefore,the actual production should fully consider the total magnesium content of the ration and its utilization rate in different regions,and reduce the addition of magnesium and the waste of resources under the premise of ensuring the health of livestock and poultry and efficient production.

【Objective】 This experiment was aimed to study the effects of earthworm hydrolysate on laying performance,serum biochemical indexes,antioxidant indexes and immune-related indexes of laying hens aged 105 to 175 days,provide data reference for the rational application of earthworm in animal husbandry.【Method】 A total of 720 healthy 105-day-old Hy-line laying hens with similar body weight were randomly divided into 4 groups with 6 replicates per group and 30 hens per replicate.Hens in control group were fed a basal diet,and hens in experimental groups were fed a basal diet supplemented with 1%,2% and 3% earthworm hydrolysate,respectively.The pre-test lasted for 15 days and the positive test lasted for 55 days.During the trial period,daily feed intake,egg production and egg weight were recorded in each replicate.On the 55th day of the trial period,6 eggs were randomly selected from each replicate for egg quality determination,and 6 laying hens were randomly selected from each replicate to collect blood for the determination of serum biochemical indices,serum antioxidant indices and serum immune indices.【Result】 Compared with control group,the differences in the egg laying performance and egg quality index of each test group were not significant (P＞0.05).The content of uric acid was significantly lower, the total antioxidant capacity (T-AOC) were significantly higher of 1% and 3% earthworm hydrolysate groups than that in control group (P＜0.05). The activities of catalase (CAT) and superoxide dismutase (SOD) were significantly higher, the contents of malondialdehyde (MDA), immunoglobulin G (IgG) and interleukin-2 (IL-2) in 1%,2% and 3% earthworm hydrolysate groups were significantly lower than those in control group (P＜0.05).The levels of tumor necrosis factor α(TNF-α) and IL-17 in 1% and 3% earthworm hydrolysate groups were extremely significantly lower than those in control group (P＜0.01).【Conclusion】 Earthworm hydrolysate could promote the excretion of uric acid in 105-175 days old laying hens,enhance the antioxidant capacity and immunity of laying hens,and it was advisable to add 1%.

【Objective】 The purpose of this study was to investigate the effects of ten-eleven translocation (TET) demethylase on the development of porcine oocytes.【Method】 The collected porcine cumulus-oocyte complexes (COCs) were cultured in vitro maturation medium with different concentrations (0 (control group),100,200 and 400 μmol/L) Bobcat339 inhibitors for 42 h.Cumulus expansion diameter was measured,maturity rate was calculated,and the minimum effective concentration was determined.After 27 h culture in in vitro mature medium,the signal strength of 5mC and 5hmC and the spindle assembly were detected by immunofluorescence (IF).Reactive oxygen species (ROS) were detected by DCFH-DA dye.Mitochondrial membrane potential (MMP) was detected by JC-1 staining.ATP level of oocyte was detected by ATP detection reagent.The expression levels of cumulus expansion related genes at 42 h and antioxidation-related genes in oocytes at 27 h were detected by Real-time quantitative PCR.【Result】 Compared with control group,the cumulus expansion level of 100,200 and 400 μmol/L groups were extremely significantly decreased (P＜0.01),the maturity rate of 100,200 and 400 μmol/L groups were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).The minimum effective concentration was determined to be 100 μmol/L,which was used in subsequent tests.IF test results showed that compared with the control group,5mC signal intensity in 100 μmol/L group was significantly increased at 27 h (P＜0.05),while 5hmC signal intensity was not significantly decreased (P＞0.05).Compared with control group,ROS levels were extremely significantly increased (P＜0.01),while MMP and ATP levels were extremely significantly decreased (P＜0.01).Compared with control group,the expression level of mound expansion related gene hyaluronic acid synthetase 2 (Has2) in the COCs of 100 μmol/L group was significantly decreased at 42 h (P＜0.05),and the expression level of penetrin 3 (Ptx3) gene was extremely significantly decreased (P＜0.01).The expression of antioxidation-related genes superoxide dismutase 1 (Sod1) and Sod2 in 100 μmol/L group were significantly decreased at 27 h (P＜0.05).【Conclusion】 TET demethylase could guide the normal assembly and arrangement of spindles,maintain the normal expansion of COCs and ensure the normal maturation of porcine oocytes by clearing 5mC in porcine oocytes,reducing ROS level and increasing MMP level.

【Objective】 The purpose of this study was to explore the genetic polymorphism of swine leukocyte antigen-1(SLA-1) and analyze its disease resistance potential,in order to provide a theoretical basis for molecular breeding of Suzi pig.【Method】 The SLA-1 gene was amplified by PCR using blood cell cDNA in Suzi pig as a template and sequenced. Similarity analysis and phylogenetic tree construction were conducted using DNAStar and Mega 5.0 software, respectively.NetMHCpan 4.1 server was used to analyze the ability and characteristics of SLA-1 molecule binding to African swine fever virus (ASFV) antigenic epitopes.The physical and chemical properties,hydrophilic and hydrophobic properties,transmembrane region,structure and function of the encoded proteins were predicted by biological software.【Result】 Six SLA-1 allele sequences of Suzi pig were successfully amplified,and namely SLA-1*sz01,SLA-1*sz02,SLA-1*sz03,SLA-1*sz04,SLA-1*sz05 and SLA-1*sz06.The highly variable sites were mainly located in the α1 and α2 regions of the peptide binding groove (PBG).The similarity comparison results showed that the similarity of the six SLA-1 alleles nucleotide and amino acid sequences of Suzi pig were between 92.9% to 96.9% and 87.0% to 94.8%,respectively.The similarity of the six SLA-1 alleles nucleotide and amino acid sequences of Suzi pig with different pig breeds were between 92.8% to 98.8% and 84.3% to 98.0%,respectively.The prediction results of the binding ability of ASFV derived polypeptides showed that the SLA-1*sz01 protein had a strong ASFV antigen presentation ability,and had highly affinity with B354L protein polypeptide fragment FIDKTTVLY in ASFV.Prediction of functional domain and tertiary structure showed that SLA-1*sz01 was related to the immune system and had a classic SLA-1 tertiary structure.【Conclusion】 In this study,six SLA-1 gene sequences were obtained,among which SLA-1*sz01 could restrict the binding of more ASFV-derived peptides,and had the highest affinity with FIDKTTVLY,which could be used as a candidate gene for disease resistance breeding of Suzi pig.The results provided a reference basis for the research and development of ASFV vaccine.

【Objective】 The aim of this study was to investigate the function and mechanism of miR-137-3p in the differentiation of preadipocytes 3T3-L1.【Method】 3T3-L1 cells at the 0,2,4,6 and 8 days of induced differentiation were collected and the expression of miR-137-3p was detected by Real-time quantitative PCR.miR-137-3p mimics,miR-137-3p inhibitor and negative control (NC) were transfected into 3T3-L1 cells and induced lipid differentiation.Oil red O staining was performed to observe the formation of lipid droplets.The relative expressions of lipogenic marker genes CAAT enhancer binding protein α (C/EBPα),C/EBPβ,peroxisome proliferator-activated receptor γ (PPARγ) and fatty acid binding protein 4 (FABP4) were detected by Real-time quantitative PCR.The protein expression of C/EBPβ,PPARγ and FABP4 were detected by Western blotting.The target genes of miR-137-3p were predicted using TargetScan online website,and the binding sites with miR-137-3p in the 3'-UTR region of candidate target genes were compared for sequence conservation between different species.miR-137-3p mimics,NC and 3'-UTR-WT and 3'-UTR-Mut vectors were co-transfected into HEK293T cells,respectively.Dual luciferase reporting assay was used to detect the targeting relationship between miR-137-3p and MYC-related factor X (MAX) gene.3T3-L1 cells were transfected with siMAX and siNC,and the relative expression of C/EBPα,C/EBPβ,PPARγ and FABP4 genes were detected by Real-time quantitative PCR,and the expression of C/EBPβ,PPARγ and FABP4 proteins were detected by Western blotting.【Result】 In the process of lipogenic differentiation of 3T3-L1 cells,compared with day 0,the relative expression of miR-137-3p on days 2,4,6 and 8 was extremely significantly decreased (P＜0.01).Compared with NC group,the number and size of lipid droplets in miR-137-3p mimics group were significantly decreased,the relative expression of C/EBPα,C/EBPβ,PPARγ and FABP genes in miR-137-3p mimics group were extremely significantly or significantly decreased (P＜0.01 or P＜0.05),the expression of C/EBPβ,PPARγ and FABP4 proteins were down-regulated.The results of oil red O staining,mRNA and protein expression of adipogenic differentiation marker genes in miR-137-3p inhibitor group were opposite.The results of target gene prediction indicated that the 3'-UTR region sequence of MAX had a predicted binding site with miR-137-3p,which was highly conserved among different species.Compared with NC group,the expression of MAX gene in mimics group was extremely significantly decreased (P＜0.01),while that of inhibitor group was significantly increased (P＜0.05).Compared with siNC group,knockdown of MAX gene significantly reduced the number and size of lipid droplets,the mRNA expression of C/EBPα,C/EBPβ,PPARγ and FABP4 genes were extremely significantly down-regulated (P＜0.01),and the protein expressions of C/EBPβ,PPARγ and FABP4 were decreased.【Conclusion】 The expression of endogenous miR-137-3p was decreased during the differentiation of 3T3-L1 preadipocytes,and miR-137-3p might inhibit the adipogenic differentiation of 3T3-L1 preadipocytes by down-regulating the expression of MAX gene.

【Objective】 This study was amied to explore the effects of different breeds,seasons and ages of semen collection on semen quality of French boars,and compare the storage effect of semen of different breeds at room temperature.【Method】 278 Duroc,Landrace and Yorkshire boars were selected as the experimental group.17 960 semen records were collected from a boar station in 2021.The general linear model and variance analysis were used to investigate the effects of breeds,seasons and ages of semen collection on semen volume,semen concentration,sperm motility and total sperm count.At the same time,15 samples with sperm motility ≥90% and forward motility ≥80% were selected from each breed respectively in summer, then diluted for 7 days in room temperature preservation test to compare the effects of normal temperature preservation of semen of different breeds of boars.【Result】 The semen volume and total sperm count of Landrace boars were significantly higher than those of Duroc and Yorkshire boars (P＜0.05),the semen concentration of Duroc boars was significantly higher than that of Landrace and Yorkshire boars (P＜0.05),and the sperm motility of Duroc boars was significantly lower than that of Landrace and Yorkshire boars (P＜0.05).The sperm motility and progressive motility decreased gradually with the extension of room temperature storage time.The sperm of Landrace boars had the best effect in room temperature storage in summer,while that of Duroc boars had the worst effect.The semen volume of all breeds in autumn and winter was significantly higher than that in spring and summer (P＜0.05),the total sperm count in spring and winter was significantly higher than that in summer and autumn (P＜0.05),the semen concentration in spring was significantly higher than that in other seasons (P＜0.05),and the sperm motility of all breeds of boars was the highest in winter and the lowest in summer,and the differences were significant (P＜0.05).The semen volume and total sperm count of Landrace boars in different seasons were significantly higher than those of Duroc and Yorkshire boars (P＜0.05).When the semen collection age was ≤30,the semen volume was smaller with the younger boar,but the semen concentration was higher and sperm motility was better.The total sperm count of the boar group,Landrace and Yorkshire boars at 19-24 months and Duroc boars at 13-18 months were significantly higher than those of other months (P＜0.05).【Conclusion】 Breed,season and age of semen collection all affected semen quality of boars,and there were some differences in the storage effect of semen of different breeds at room temperature,which provided reference for making production plan of boar station,and contributed to further fine management of boar station and improving utilization rate of boar.

【Objective】 This study was aimed to further understand the evolutionary dynamics and epidemic characteristics of H9 subtype Avian influenza virus (AIV) in Guangxi,which could provide data support for disease prevention strategy constituted and modified.【Method】 3 600 swabs samples of poultry throat,cloaca and environment were collected in several area markets of Guangxi from 2016 to 2021,then were inoculated in 9-11 days age SPF chicken embryos to isolate virus,the hemagglutinin (HA) and neuraminidase (NA) genes of H9 subtype AIV were amplified,sequenced and genetic evolutionary drynamic analyzed based on hemagglutinin and hemagglutinin-inhibit test,and Real-time quantitative RT-PCR detection assays.【Result】 46 HA and NA genes sequence were acquired,the length of HA gene was 1 683 bp,coding for 560 amino acids and 4 NA gene was 1 410 bp,coding for 469 amino acids except 42 isolates were 466 amino acids with absence of 3 amino acids. BLAST analysis indicated that isolates shared the highest similarity with 97.62%-99.88% and 93.13%-99.79% to HA and NA genes in this study were from Vietnam and several provinces of China,and hosts were not identical,which suggested that the genetic of HA and NA genes was diversity.The nucleotide and amino acid similarity of Guangxi isolates intra were 93.0%-99.1% and 94.6%-99.1%,83.9%-98.2% and 86.1%-98.2%,respectively,which indicated that HA gene was more conserved,the similarity of Guangxi isolates with the reference strains of after 2016 was higher than strains of before,which manifested that the isolates were persisting evolution.The genetic analysis showed that HA and NA genes of Guangxi isolates belonged to G57 branch prevalent in China,the genetic distance of which and vaccine strains was farther.The evolutionary rate estimation of HA and NA genes was 3.99×10^-3and 4.59×10^-3substitution/sites/year,and the most recent common ancestor of which was 66.77 and 58.24 years,which suggested that the rate of HA gene was slower than NA gene.The recombination analysis of HA gene acquired from the chicken,duck and environment was positive signal,respectively.The major and minor parents strains were A/Beijing/1/2017 and A/quail/Hainan/250/2012.【Conclusion】 The HA and NA genes of Guangxi isolates with higher similarity to those of Vietnam and provinces of China,the genetic of them was diversity,and evolution progress was close to the dominant gene type G57,the recombination of HA genes was occurred between AIV and human-origin influenza strain,which could provide outbreak condition for novel subtype influence in future.

【Objective】 The purpose of this study was to analyze the structure and function of ORF2 protein of Swine hepatitis E virus (sHEV) genotype 4,screen the gene fragment with protective antigen and express,so as to provide a candidate protein for the study of its potential protective antigen.【Method】 The bioinformatics analysis of sHEV genotype 4 ORF2 protein was carried out.The gene fragment ORF2 (128/140) with potential protective antigen protein was selected,amplified by PCR and digested,which cloned into insect cell expression vector,and transfected into sf9 cells.The expressed protein p128/p140 were analyzed by Western blotting.【Result】 Bioinformatics analysis showed that the ORF2 protein of sHEV genotype 4 had two stable proteins (p128 and p140),which contained 496 and 476 amino acids,respectively.Both of them were stable proteins,without transmembrane regions,and the secondary structure of the protein was mainly random coil.p128 protein had 11 T cell epitopes and 21 B cell epitopes,and p140 protein had 14 T cell epitopes and 18 B cell epitopes.Insect cell expression vector of ORF2(128) and ORF2(140) genes were successfully constructed,and transfected into sf9 cells,Western blotting results showed that the expressed proteins could be recognized by His-tag monoclonal antibody and positive serum of sHEV antibody.【Conclusion】 p128 and p140 proteins with potential protective antigen of sHEV were found in this study,and the two proteins were expressed in sf9 cells with immune activity,which provide materials for further study of the function of sHEV ORF2 protein and the development of new vaccines.

【Objective】 This study was aimed to investigate the effect of the outer membrane protein OmpA of avian pathogenic Escherichia coli (APEC) on autophagy in DF-1 cells and thus provide a basis for understanding whether OmpA helped APEC evade the autophagy mediated clearance in host cells.【Method】 The genomic DNA of APEC E058 strain was used as a template to recover the ompA gene fragment by PCR amplification,and then cloned into the eukaryotic expression vector pEGFP-N1 after double digestion with EcoR Ⅰ and Xho Ⅰ.The positive recombinant plasmid pEGFP-N1-ompA was screened and confirmed by enzyme digestion and sequencing.After transfected into DF-1 cells,the expression of OmpA was detected by Western blotting and immunofluorescence assay.The effects of OmpA on autophagy in DF-1 cells were detected by transmission electron microscopy,immunofluorescence assay and Western blotting.【Result】 After EcoR Ⅰ/Xho Ⅰ enzyme digestion of the sequencing identified recombinant plasmid,two bands with size of 1 038 and 4 700 bp were obtained,which were consistent with the vector and the target fragment,indicating that the eukaryotic expression plasmid pEGFP-N1-ompA was successfully constructed.After transfection of recombinant plasmid with liposome into DF-1 cells,a large amount of green fluorescence could be observed by immunofluorescence assay and 65 ku protein band was detected by Western blotting,indicating that pEGFP-N1-ompA was successfully transfected into DF-1 cells and ompA-EGFP fusion protein was expressed in large amounts.Meanwhile,Western blotting results showed that overexpression of OmpA caused an increase in the expression of autophagy marker protein LC3 Ⅱ.The autophagsomes in DF-1 cells were observed by transmission electron microscopy.The single fluorescent GFP-LC3 plasmid was transfected into DF-1 cells,and the OmpA protein treated group showed aggregation of green fluorescent spots,indicating that OmpA induced autophagy of DF-1 cells.Furthermore,OmpA affected the degradation of autophagy marker protein p62, and the transfection of double fluorescent mRFP-GFP-LC3 plasmid showed that OmpA could block the oceurrence of autophagic flux.【Conclusion】 The APEC outer membrane protein OmpA could cause autophagy in DF-1 cells,but it blocked the autophagic flux and induced incomplete autophagy in DF-1 cells,which would help to better understand the escape mechanism of APEC from the host cells clearance.

【Objective】 This experiment was aimed to study the effects of 6-phospho-β-glucosidase encoded by the Lm4b_02324 gene on the biological characteristics of Listeria monocytogenes,and to lay a foundation for the study of the pathogenic mechanism of Listeria monocytogenes.【Method】 In this study,overlapping extended PCR technique was used to construct the Lm4b_02324 gene deletion strain (Lm928ΔLm4b_02324) and Lm4b_02324 gene complement strain (CLm928ΔLm4b_02324) of stable genetic wild type Lm928 through continuous passage.The growth characteristics of Lm928,Lm928ΔLm4b_02324 and CLm928ΔLm4b_02324 were analyzed by detecting D_{600 nm} values of different culture time and drawing growth curves.The mice were injected intraperitoneally with three strains of different concentrations,and the clinical symptoms after infection were observed.The lethal dose 50% (LD₅₀) of the three strains to the mice was calculated by Koch’s calculation method.Lm928,Lm928ΔLm4b_02324 and CLm928ΔLm4b_02324 were infected with multiplicity of infection (MOI) as 10,respectively.The adhesion and invasion ability and intracellular survival ability of each strain were analyzed by detecting the number of bacteria in cells at different time after infection.DNA of wild and deletion strains was extracted.Primers of 9 virulence related factors,such as prfA,mpl and hly,were used to detect their expressions by Real-time quantitative PCR.【Result】 PCR identification results showed that gene deletion strain Lm928ΔLm4b_02324 (wild strain:2 517 bp,deletion strain:1 209 bp) and the complement strain CLm928ΔLm4b_02324 (1 301 bp,consistent with the band of wild strain) were successfully obtained,and the deletion strain did not appear back mutation phenomenon after 15 generations.The results of growth curve showed that there was no significant difference in the growth status of wild strain,deletion strain and complement strain.LD₅₀ determination results showed that the LD₅₀ of the deletion strain was significantly lower than that of the wild strain (P＜0.05).The results of adhesion and invasion test showed that the intracellular survival number of the deletion strain was significantly or extremely significantly higher than that of wild strain and complement strain (P＜0.05 or P＜0.01).Within 12 h after infection with HBMEC cells,the intracellular viable bacteria number of the deletion strain was higher than that of wild strain,and the difference was significant (P＜0.05).Real-time quantitative PCR results showed that the deletion of Lm4b_02324 gene significantly decreased the expression of prfA,inlA and plcA genes(P＜0.05),and the expressions of hly,actA,mpl and inlC were extremely significantly decreased (P＜0.01).【Conclusion】 The results of this study showed that the deletion of Lm4b_02324 gene could increase the mortality of Listeria monocytogenes in mice and enhance its intracellular proliferation ability,which laid a foundation for the study of the role of Lm4b_02324 gene in the pathogenesis of Listeria monocytogenes.

In recent years,with the abuse of antibiotics in aquaculture,bacterial drug resistance has emerged,so green,safe and efficient new strategies are needed to alleviate the drug resistance problem.As a communication mechanism,quorum sensing regulates the behavior and physiological response of bacteria according to the density between cells.Many active substances can affect the pathogenicity of pathogenic bacteria through quorum sensing system without inhibiting the growth of pathogenic bacteria,and at the same time slow down the emergence of drug resistance.Therefore,in recent years,inhibitors that inhibit bacterial quorum sensing system have become a research hotspot.Aeromonas hydrophila,as the main conditional pathogen in aquaculture,seriously harms the economic development of aquaculture.This paper introduced the mechanism of quorum sensing system of Aeromonas hydrophila,the regulation of bacterial biofilm,and the application of quorum sensing system inhibitors of Chinese herbal medicine and probiotics in the prevention and control of Aeromonas hydrophila,aiming at analyzing the development potential of quorum sensing inhibition of Aeromonas hydrophila in the future and providing some reference for the prevention and control of Aeromonas hydrophila in aquaculture.

【Objective】 Bayesian phylodynamics was used to reconstruct the transmission and genetic evolution of Porcine reproductive and respiratory syndrome virus 2 (PRRSV-2) lineage 8 strains among five provinces:Guangdong,Guangxi,Hunan,Jiangxi and Fujian,providing a reference for targeted prevention and control of PRRS.【Method】 In this study,phylogenetic analysis of ORF5 sequences from all available PRRSV-2 strains from the above five provinces was performed,and lineage 8 strains were selected to eliminate recombinant sequences as well as suspicious sequences.The spatial data were combined with the evolution and epidemiological characteristics of pathogens for analysis,using BEAST and related software to calculate the common ancestor of the virus,nucleotide substitution rate,as well as possible transmission direction and migration rate between different geographical locations.【Result】 Phylogenetic analysis showed that PRRSV-2 circulating in pig herds in five southern provinces was divided into four lineages,of which 154 strains were identified as lineage 8 strains.After removing a recombinant strain and three suspected strains,the ORF5 sequences from the above five provinces had a strong temporal signal,and the best-fit model was GTR＋I＋G.Bayesian phylodynamic analysis showed that lineage 8 strains from five provinces had nucleotide evolutionary rates of 2.748×10^-3/site/year (95% HPD:2.053×10^-3-3.428×10^-3),and their most recent common ancestor might have emerged in 2002 (95% HPD:1998-2005).According to SPREAD3 calculations,Guangxi strains spreaded to Guangdong (Rate＝0.876),Hunan (Rate＝0.892),Jiangxi (Rate＝0.883),and Fujian (Rate＝0.889),as well as some Fujian strains spreaded to Guangxi (Rate＝0.897).【Conclusion】 Since 2005,PRRSV-2 circulating in pig herds in Guangdong,Guangxi,Hunan,Jiangxi and Fujian provinces had rich genetic diversity,of which lineage 8 strains had a high nucleotide substitution rate,and the effective population size had increased significantly from 2006 to 2007 and 2009 to 2013.The common ancestor of lineage 8 strains of Guangdong,Guangxi,Hunan,Jiangxi and Fujian provinces might have emerged in 2002,and continued to spread in the region with Guangxi as a potential origin.

【Objective】 This study was aimed to establish an indirect ELISA method for rapid and accurate detection of antibodies to African swine fever virus (ASFV).【Method】 The CP204L gene sequence was extracted from the whole genome of ASFV isolate collected by GenBank,without affecting the amino acid sequence,the codon was optimized,and the pCold TF cold shock expression vector was cloned to construct the recombinant plasmid pCold TF-p30.The prokaryotic expression system of Escherichia coli was used to induce IPTG expression,and the induction time and concentration of IPTG were optimized.The recombinant protein P30 was purified with His labeled nickel column.After identification by SDS-PAGE and Western blotting,the purified recombinant protein P30 was used as antigen to establish an indirect ELISA method for detecting ASFV antibodies and optimize the reaction conditions.The specificity,sensitivity and repeatability of the method were tested and compared with commercial kits.【Result】 The recombinant plasmid was successfully transferred into the competent cells of Escherichia coli BL2 (DE3) and expressed in the form of soluble protein after induced by IPTG.There was a specific band of the target protein at about 82 ku.When the induction condition was 16 ℃ and the induction time was 12 h,the protein expression was the highest.There was no significant difference in the induction effect among different IPTG concentrations.Western blotting showed that P30 recombinant protein could react specifically with ASFV positive serum,which proved that it had good reactivity.The optimal concentration of antigen coating of the established ASFV indirect ELISA antibody detection method was 1 μg/mL,using 1% BSA solution had the best sealing effect,the optimal dilution of serum was 1∶600,and the optimal working concentration of enzyme labeled secondary antibody was 1∶8 000.This method had good specificity,sensitivity and repeatability,and the total coincidence rate was 96.7% when compared with the commercial kit.【Conclusion】 P30 protein of ASFV was successfully expressed and purified,and an indirect ELISA method for detecting ASFV antibody was established,which provided a reference for the monitoring,diagnosis and development of the kit of African swine fever.

Infectious diseases caused by Ehrlichia canis (E.canis) have a serious threat to the health of dogs or human.E.canis can invade the body through wounds on the skin surface or bites of arthropods,triggering a series of reactions in the body and leading to E.canis-related diseases.In addition,the clinical symptoms of diseases caused by E.canis are similar to other diseases,which brings difficulties in ways of rapid diagnosis.E.canis is a strict intracellular parasitic pathogen,and is negative stained by Gram staining and is purple stained by Gimsa staining.It is possible to isolate this pathogen successfully and realize culture in vitro.E.canis is able to induce autophagy of host cells,remaining their own survival and proliferation through capturing nutrients from the host,and can induce the body to have an immune responses.Through producing a large number of inflammatory factors,E.canis causes fever and brings damage to tissues and organs.E.canis has a special mechanism of immune escape to avoid being eliminated by lysosomes or other immune factors of hosts.E.canis can grow and reproduce in macrophages and destroy the anti-inflammatory factors in the host cell in order to destroy the host immune function.However,how E.canis capturing or destroying nutrients and survival of hosts remains unclear,and evading the 'tracking’ of immune system of hosts are still further investigated.In terms of treatment,E.canis related diseases are only sensitive to some antibiotics such as doxycycline.However,the best way to prevent this disease is to remove regularly ectoparasite.Consequently,the aim of the article is to summarize the pathogenesis,epidemiology and pathogenic mechanism of E.canis,including advanced research status between partial pathogens and host cells.

【Objective】 The anti-inhibin α subunit (INHα) nanobody library was constructed by phage display technology,and the specific nanobodies against sheep INHα were screened, so as to prepare for the improvement of peripheral blood follicle-stimulating hormone (FSH) level and ovulation rate in sheep.【Method】 The expression of INHα protein was induced by the pET32a-INHA prokaryotic expression vector preserved in the laboratory,and the induced INHα protein was identified by SDS-PAGE and Western blotting.Then the purified INHα protein was used to immunize Xinjiang bactrian camel.The camel phage display nanobody library was prepared by nested PCR amplification.The specific nanobody against INHα was enriched and screened from the library by three rounds of immunoaffinity screening and phage ELISA.The gene sequence of the nanobody was obtained by sequencing. The affinity of the nanobody to INHα protein was preliminarily identified by protein-protein analog docking.【Result】 The results showed that the recombinant protein of INHα with a size of 36 ku was obtained by prokaryotic induction expression and Ni agarose purification,and a protein band of about 36 ku was detected by Western blotting.Bactrian camel immunized with INHα protein for 6 times and the antibody titer reached 1∶1 024 000.The VHH gene of about 400 bp was obtained by nested PCR,and a nanobody library with a capacity of 1.05×10¹²CFU was constructed by electrotransformation and other experiments.The positive rate of the library was 94%.The anti-INHα nanobody library of 3.0×10⁸ CFU was obtained by three rounds of immunoaffinity screening.Phage ELISA was used to obtain four strains of INHα-specific nanobodies (VHH-4,VHH-29,VHH-89 and VHH-108) and the gene sequence similarity of the four INHα-specific nanobodies was 80.31%.Through protein-protein simulation docking,it was found that all the four nanobodies had strong affinity with INHα.In addition,the total free energy of VHH-4 and INHα was -576.28 kJ/mol,which was the best antibody among the four VHH.【Conclusion】 An anti-INHα nanobody library was successfully constructed,and four INHα-specific nanobodies,VHH-4,VHH-29,VHH-89 and VHH-108,were obtained from the library.The results of this study could provide reference for the application of nanobodies in reproductive immunology.

【Objective】 The aim of this experiment was to express the recombinant Cap-Flagellin protein of Porcine circovirus type 3 (PCV3) by Baculovirus expression system and analyze its reactogenicity and immunological properties,providing important support for the development of PCV3 subunit vaccine.【Method】 The Cap gene was optimized by optimal codon and fused with Flagellin gene of Salmonella Typhimurium then cloned into pFastBac^TMⅠ vector.Recombinant Cap-Flagellin Baculovirus was rescued through the Bac-to-Bac system and identified by SDS-PAGE,indirect immunofluorescence assay (IFA) and Western blotting,and then mice were immunized with recombinant Cap-Flagellin Baculovirus to evaluate its immunogenicity.【Result】 Recombinant Cap-Flagellin Baculovirus could effectively express recombinant proteins in Sf9 cells,the target protein bands of 68 ku were detected by SDS-PAGE.IFA results showed that the specific green fluorescence occurred in the membrane and cytoplasm of Sf9 cells 72 h after recombinant Baculovirus infection.Western blotting showed that the recombinant protein could react specifically with PCV3 positive serum,all indicated it had good reactogenicity.The serum Cap-specific antibody levels of mice immunized with Cap-Flagellin Baculovirus on the 21th and 28th day were higher compared with the Cap Baculovirus group(P＜0.05 or P＜0.01),and IL-8 cytokine level of spleen cells in recombinant Cap-Flagellin group was significantly or extremely significantly higher than that of other groups(P＜0.05 or P＜0.01),which indicated that the recombinant Cap-Flagellin protein had better immunogenicity.【Conclusion】 In this study,PCV3 Cap recombinant flagellin was successfully expressed by Baculovirus expression system and had good biological activity which laid a good foundation for the development of PCV3 subunit vaccine.

【Objective】 The purpose of this study was to express the Hexon protein of Fowl adenovirus serotype 4 (FAdV-4) by prokaryotes expressed system and prepare its polyclonal antibody,so as to provide materials for the study of FAdV-4 Hexon function.【Method】 The method accurate synthesis of long DNA sequences based on PCR (PAS) was used to design the full-length spline primer,and protective bases were designed on both ends of the primer to synthesize FAdV-4 Hexon gene.It was connected to the pCzn1-Hexon expression vector by homologous recombination technology,and the pCzn1-Hexon recombinant plasmid was obtained for prokaryotic expression.The purified recombinant protein was used to immunize New Zealand White rabbits to prepare rabbit polyclonal antibody against Hexon protein.The titer of polyclonal antibody was determined by indirect ELISA,and the specificity of polyclonal antibody was identified by Western blotting and indirect immunofluorescence assay (IFA).【Result】 The recombinant prokaryotic expression plasmid pCzn1-Hexon was confirmed to be constructed correctly by double restriction enzyme digestion and sequencing.The obtained recombinant protein was expressed in the form of inclusion body,and its molecular weight was about 41 ku.After immunizing New Zealand White rabbits with purified recombinant protein,the antibody titer was up to 1∶512 000 by indirect ELISA.Using the prepared polyclonal antibody as the first antibody,the expression of Hexon protein in chicken LMH cells was detected by Western blotting and IFA,indicating that the prepared polyclonal antibody could react specifically with Hexon protein.【Conclusion】 The recombinant Hexon protein had been highly expressed in Escherichia coli,and the prepared and purified anti-Hexon polyclonal antibody showed high reactivity and specificity,which laid a foundation for in-depth analysis of the biological function of Hexon protein and its role in FAdV-4 infection and pathogenesis.

Giardia duodenalis (G.duodenalis) is a common zoonotic protozoan parasite that infects the small intestines of humans,livestock,dogs,cats and other mammals.Giardiasis which caused by G.duodenalis has been noted as one of the neglected diseases endangering human health by the World Health Organization.The intestinal protozoan parasite Giardia has a simple life cycle consisting of disease causing trophozoites and infectious cysts.Giardia trophozoites differentiate into infectious cysts in response to hostile environment (either low cholesterol conditions or alkaline pH and high bile conditions). Encystment is crucial for Giardia’s survival,transmission and pathogenesis.Giardiasis is often caused by the host ingestion of infectious cysts-contaminated food and water.The cyst wall is an important shield for Giardia cysts to resist adverse external environment,maintain their viability and infective activity.About 40% of the encapsulated wall is composed of proteins and the rest is carbohydrates such as N-acetylgalactosamine and lipids.It is shown that the cyst wall of Giardia mainly contains three cyst wall proteins (CWP1,CWP2 and CWP3).Cyst wall proteins not only play an important role in maintaining the viability of the cyst,but also has important value in the diagnosis of giardiosis and the development of oral vaccine.This review mainly introduced the composition and formation of Giardia cyst wall,the induction and regulation of encystation,and the research advances on cyst wall proteins in disease diagnosis and vaccines development,in order to provide reference for the follow-up related research.

【Objective】 This study was aimed to explore the effects of Clostridium perfringens (C.perfringens) infection on intestinal oxidative stress and inflammatory response in Yellow-feathered broilers,so as to lay a foundation for studying the pathogenesis of C.perfringens and developing prevention and control drugs.【Method】 A total of 100 one-day-old healthy Yellow-feathered broilers were randomly divided into 4 groups with 5 replicates per group and 5 broilers per replicate,namely control group and high,medium and low dose infection groups.During the whole period of the experiment,broilers were fed the basal diet.From 14 to 16 days of age,broilers in control group were fed with PBS,and broilers in high,medium and low dose infection groups were fed with 1×10⁹,1×10⁸ and 1×10⁷ CFU C.perfringens for 3 days,respectively.The blood biochemical indexes,oxidative stress and inflammation related factors in jejunum and ileum of broilers on 1 and 3 d post infection were measured,and the histopathological changes of jejunum and ileum were observed.【Result】 Compared with the control group,the inflammation-related factors and oxidative stress indicators in broilers in different dose infection groups showed different degrees of changes,in which the high-dose group significantly increased the activities of serum alkaline phosphatase (AKP),acid phosphatase (ACP),lactate dehydrogenase (LDH),as well as the content of malondialdehyde (MDA) in jejunum,the level of cytosolic interleukin (IL-8) in jejunum and ileum (P＜0.05),and decreased the activity of superoxide dismutase (SOD),the content of monocyte chemotactic protein (MCP-1) and cytosolic interleukin 10 (IL-10) in jejunum and ileum at 1 d post infection (P＜0.05).At 3 d post infection,the activities of AKP,ACP and LDH in serum of broilers infected with high dose increased significantly (P＜0.05),the contents of MDA,glutathione (GSH),tumor necrosis factor (TNF-α),MCP-1,IL-10,cysteine protease (caspase-1) and the relative expression levels of caspase-1,IL-1β,NOD-like receptor thermoprotein structural domain-associated protein 3 (NLRP3) mRNA in jejunum were significantly or extremely significantly increased (P＜0.05 or P＜0.01), and the contents of MDA,GSH,TNF-α,MCP-1,IL-1β,IL-8 and IL-10,as well as the relative mRNA expression levels of caspase-1,IL-1β,NLRP3 and Gasdermin A in ileum were significantly or extremely significantly increased (P＜0.05 or P＜0.01).The activity of AKP in serum and the activity of SOD in jejunum of broilers in the medium dose group were significantly increased at 1 d post infection (P＜0.05),and the activity of ACP and AKP in serum and GSH content in ileum were significantly increased at 3 d post infection (P＜0.05).The activities of ACP and AKP in serum of low dose group were significantly increased at both 1 and 3 d post infection post infection (P＜0.05),and the activity of SOD and the content of IL-18 in jejunum were significantly decreased at 1 d post infection (P＜0.05).In the high-dose group,the jejunum and ileum mucosa of broilers were bleeding,the epithelial cells of intestinal mucosa were necrotic and exfoliated,and inflammatory cells were infiltrating.The villi of jejunum and ileum in the middle and low dose groups were slightly broken,and the histological structure of jejunum and ileum in the control group was normal.【Conclusion】 1×10⁹ CFU Clostridium perfringens infected 14 day-old Yellow-feathered broilers for 3 consecutive days,might increase the expression of inflammatory factor in jejunum and ileum of chicks by activating NLRP3 inflammatory pathway,reduce the antioxidant level of chicks,cause intestinal pathological damage and induce chicken necrotizing enteritis.

【Objective】 This study was aimed to explore the growth curve,pH,bile salt tolerance,antimicrobial activity,antibiotic sensitivity and other characteristics of 5 strains of self screening Bacillus amyloliquefaciens(YN-BA1,YN-BA2,YN-BA3,YN-BA4 and YN-BA5),and provide theoretical basis for the development of microecological agents in aquaculture.【Method】 In this test,the growth curve of Bacillus amyloliquefaciens was determined by turbidimetry.The 5 strains of Bacillus amyloliquefaciens were inoculated into LB broth prepared with hydrochloric acid,pH 2.0,3.0,4.0 and 7.0 for 2 h,and cultured 5 h in an environment with 0.3% bile salt concentration to evaluate their tolerance in artificial gastric juice intestinal juice.The antibacterial activity in vitro was determined by agar drilling method,and the antibiotic sensitivity was determined by KB paper method.【Result】 The growth rates of 5 strains of Bacillus amyloliquefaciens increased rapidly and entered the logarithmic growth phase after the slow growth 0-2 and 6-8 h.After 36 h,the growth decreased and entered the recession period.The 5 strains had different abilities of acid resistance and bile salt tolerance.Among them,the survival rates of YN-BA2 strain at pH 2.0,3.0 and 4.0 were 42.10%,51.50% and 74.67%,respectively,which were higher than the other 4 strains.After being treated for 5 h at 0.3% bile salt concentration,the survival rate of YN-BA2 was 51.00%,which was higher than the other 4 strains,indicating that YN-BA2 strain had the highest tolerance.The 5 strains of Bacillus amyloliquefaciens had antibacterial effect on Salmonella Enteritidis YN-S1 and YN-S2,Salmonella Pullorum YN-SP9 and YN-SP10,and Escherichia coli DO157.YN-BA2 had high sensitivity to Salmonella Pullorum YN-SP10.The other 4 strains of Bacillus amyloliquefaciens were moderately sensitive to intestinal pathogens preserved in the laboratory,indicating that YN-BA2 had the best antibacterial effect.The 5 strains of Bacillus amyloliquefaciens were resistant to tetracycline (except for YN-BA5) and norfloxacin,ofloxacin,peillin G and cephalexin to varying degrees,and were sensitive to erythromycin,kanamycin (except for YN-BA5),levofloxacin,ciprofloxacin,gentamicin,azithromycin (except for YN-BA1),ampicillin and lincomycin.【Conclusion】 Bacillus amyloliquefaciens could be activated rapidly,had good tolerance to bile salt and pH,had certain inhibitory effects on Escherichia coli,Salmonella Pullorum and Salmonella Enteritidis,and was resistant to tetracycline and norfloxacin.Therefore,Bacillus amyloliquefaciens had the potential to be used in the development of microecological agents for livestock and poultry production.

【Objective】 The purpose of this study was to explore the effect of the combination of traditional Chinese medicine with probiotics on the prevention of Eimeria tenella.【Method】 A total of 180 12-day-old Guinong golden chickens were divided into blank control group,infection group,traditional Chinese medicine group,probiotic group,traditional Chinese medicine + probiotic group and monensin group,with 3 replicates in each group and 10 chickens in each replicate.Blank control group and infection group were treated with fasting normal saline (200 μL/chicken),traditional Chinese medicine group was feed the basal diet with 2% Chinese medicine prescription,probiotics group was treated with fasting compound probiotics (200 μL/chicken),traditional Chinese medicine + probiotics group was treated with fasting compound probiotics (200 μL/chicken) and feed the basal diet with 2% Chinese medicine prescription.Monensin group was feed basal diet with 100 mg/kg monensin.At 14 days of age,except blank control group,all groups were orally administrated with 1×10⁵ oocysts/chicken sporoidized oocysts suspension of Eimeria tenella. The relative weight gain rate and survival rate were measured before inoculation and culturing.Blood samples were collected on days 0,4 and 8 after infection,and serum biochemical,immune and antioxidant indexes were determined by ELASA method.Cecal tissues were collected and histopathological changes were observed.【Result】 The results showed that the relative weight gain rate of each prophylactic administration group was higher than that of infection group,and the number of oocysts per gram of cecum was significantly lower than that of infection group (P<0.05). The relative weight gain rate of traditional Chinese medicine + probiotics group was the highest,the number of oocysts per gram of cecum was the lowest,and the anticoccidial index (ACI) was higher than that of monensin group.Compared with infection group,on days 4 of infection,the contents of serum albumin (ALB) and triglyceride (TG) in traditional Chinese medicine group,probiotic group and traditional Chinese medicine + probiotic group were significantly increased (P ＜0.05),and the content of creatinine (CREA) was significantly decreased (P＜0.05).The contents of total protein (TP),interleukin-2 (IL-2),IL-6 and interferon-γ (IFN-γ) of serum in traditional Chinese medicine group and traditional Chinese medicine + probiotic group were significantly increased (P ＜0.05),and the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly decreased (P＜0.05).On days 8 of infection,the contents of TP and ALB of serum in traditional Chinese medicine group,probiotic group and traditional Chinese medicine + probiotic group were significantly increased (P＜0.05),while the content of urea nitrogen (BUN) and AST activity were significantly decreased (P＜0.05).The contents of TG,IL-2,IL-6 and IFN-γ in traditional Chinese medicine group and traditional Chinese medicine + probiotics group were significantly increased (P＜0.05),the contents of CREA and the activity of ALT were significantly decreased (P＜0.05).The results of cecal histopathology showed that the infection group and the probiotics group had obvious pathological damage,while the traditional Chinese medicine group and the traditional Chinese medicine + probiotics group had slight tissue damage.【Conclusion】 This study showed that the combined use of traditional Chinese medicine and probiotics could improve the growth inhibition caused by Eimeria tenella infection,reduce the output of oocysts,improve the immunity and antioxidant capacity of the body,and effectively protect the cecum tissue. This study provided providing reference for the preventio and treatment of coccidiosis.

【Objective】 The molecular mechanism of leonurine in the treatment of intestinal inflammation was investigated using network pharmacology,molecular docking and kinetic simulation techniques.【Method】Pubchem,PharmMapper and SwissTargetPrediction database were used to collect the potential targets of leonurine and intestinal inflammation,and obtain the intersection targets of leonurine in the treatment of intestinal inflammation in the Venny 2.1 online mapping tool platform system.The protein-protein interaction (PPI) relationship was obtained through STRING and DAVID databases,and the PPI network diagram of the intersection target was drawn.The GO function and KEGG pathway enrichment analysis of the intersection target was carried out,and the core target protein was screened by Cytascape 3.8.2 software.The core target was verified by molecular docking technology and molecular dynamics simulation.【Result】 A total of 379 potential target proteins related to leonurine and 15 464 potential targets related to intestinal inflammation were collected.170 potential intersection targets of leonurine for the treatment of intestinal inflammation were obtained.16 core targets including serine/threonine protein kinase 1(AKT1),tumor suppressor genes TP53,prostaglandin-endoperoxide synthase 2(PTGS2),mechanistic target of rapamycin (mTOR),poly ADP-ribose polymerase 1 (PARP1),etc. were screened.The results of GO function and KEGG pathway enrichment analysis showed that leonurine regulated 144 biological processes such as protein phosphorylation,cell proliferation,negative regulation of apoptosis,proteolysis,and inflammation,as well as 55 signal pathways,such as Fc epsilon RI signaling pathway and Fc γ-R-mediated phagocytosis including MAPK signaling pathway and mTOR signaling pathway to treat intestinal inflammation.Molecular docking results showed that leonurine had good docking results with the core targets PTGS2,mTOR and PARP1,and had strong binding activity.Molecular dynamics simulations had found that leonurine was mainly bound to the three proteins by hydrogen and hydrophobic bonds.【Conclusion】 Leonurine exerted its therapeutic effect on intestinal inflammation through multiple targets such as mTOR,PARP1 and PTGS2,and multiple signaling pathways and multiple pathways.The result of this study laid a foundation for the clinical application of leonurine.

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious infectious disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV) that primarily causes reproductive disorders in sows and respiratory symptoms in piglets.Vaccination is the primary methods for preventing and controlling PRRSV,however,because of the high variability of the PRRSV strain,the vaccine’s effect on the disease is still suboptimal.Traditional Chinese medicine (TCM) contains several active components and physiological activities,such as antibacterial,antiviral,and antioxidant actions,which become a hot topic in anti-virus medicine research.Studies have proved that TCM can inhibit the proliferation,block the adsorption and kill the PRRSV in the way of monomer or compound,as well as affect the immune response of the body.TCM anti-PRRSV method comprises both direct and indirect antiviral actions.The direct antiviral mechanism is the direct action of the active components of TCM on the virus’s replication cycle,which is mostly manifested as preventing the virus’s adsorption,internalization,replication,assembly,and release in order to inhibit the virus.The active components of TCM inhibit the negative effects of viruses on host cells by regulating cytokine synthesis,inflammatory signaling pathways,cell apoptosis,oxidative metabolism,and the immune system,ensuring the generation of immune response and reducing body damage.Due to the complex composition of TCM and the unclear anti-PRRSV mechanism,the recent studies on anti-PRRSV of TCM were reviewed based on the antiviral mechanism,in order to provide a theoretical basis for the prevention and control of PRRSV with TCM.

【Objective】 Three-dimensional reconstruction and 3D printing techniques were used to construct a three-dimensional model of the liver in Beagle dogs to provide three-dimensional data and morphological basis for the diagnosis,treatment and related surgery of liver diseases in dogs,and at the same time,it laid a foundation for the dose reduction and use of experimental animals and improving animal welfare.【Method】 A female 8-month-old healthy Beagle dog weighing 14.5 kg was intravenously injected with iohexol after general anesthesia,and two-dimensional image data were collected from the liver of the dog by 128-slice spiral computed tomography (CT),followed by digital segmentation and three-dimensional reconstruction using IPS software,and images were measured using the Touch Viewer software.Finally,the reconstructed data were transmitted to a 3D printer to generate 3D printed models of portal vein,hepatic vein,hepatic artery and other tissues one by one,and finally the 3D physical model of the dog liver was obtained after coloring,layer optimization and assembly processing.【Result】 The three-dimensional reconstruction model of the liver constructed by CT enhanced angiography data had realistic shape and strong stereoscopic sense,could be arbitrarily scaled and rotated at an angle in the three-dimensional space,and could be mapped plane by adjusting the threshold to form a section of each site or separately display the direction and distribution of the hepatic artery,hepatic vein,portal vein,and bile duct,which was convenient to observe the spatial location of each vascular structure and display the complex local anatomical structure of the liver.Touch Viewer measurements showed the volume of the liver was 556.14 mL with a surface area of 600.85 cm² and the volume of the gallbladder was 12.67 mL with a surface area of 32.47 cm².The 3D printed model showed that the vascularity of each tissue was clear and bright in color,which was basically consistent with the digital three-dimensional model,and a more real and ideal liver model was obtained.【Conclusion】 Three-dimensional reconstruction and 3D printing technology could successfully construct a canine liver model,which could visually display the complex three-dimensional shape and spatial structure of the liver and could be used as a good simulation platform and practical tool for digital and precise pet medicine as well as veterinary anatomy and imaging teaching.

【Objective】 The purpose of this experiment was to explore the pharmacological activity and overall mechanism of compound plant essential oils (oregano essential oil,cinnamaldehyde,Eucalyptus oil) based on network pharmacology.【Method】 The targets of compound plant essential oils were screened using TCMSP database.The drug-ingredient-target network was constructed by Cytoscape 3.7.1 software,and PPI network was constructed by STRING and visualization.The targets were annotated by GO function and KEGG pathway enrichment analysis through DAVID database.And AutoDock Vina software was used for molecular docking of selected active components and key targets.【Result】 Compound plant essential oils acted on the key targets including interleukin 6 (IL6),prostaglandin-endoperoxide synthase 2 (PTGS2),Toll-like receptors 4 (TLR4),transcription factor p65 (RELA),nitric-oxide synthase,endothelial (NOS3),aryl hydrocarbon receptor (AHR),cytochrome P450 1A1 (CYP1A1),NF-kappa-B inhibitor alpha (NFKBIA),gamma-aminobutyric-acid receptor alpha-2 subunit (GABRA2) and so on with 8 active ingredients.The GO function enrichment analysis showed that 269 items were obtained,181 items were related to biological processes,involving adenylate cyclase-activating adrenergic receptor signaling pathway,regulation of postsynaptic membrane potential,etc.,59 items related to cell components,involving integral component of plasma membrane,neuron projection,etc..29 items related to molecular functions,involving neurotransmitter receptor activity,excitatory extracellular ligand-gated ion channel activity,etc..KEGG pathway analysis showed that 72 items related to signal pathways,and participated in the regulation of neuroactive ligand-receptor interaction,lipid and atherosclerosis,cGMP-PK signaling pathway,calcium signaling pathway,TLR signaling pathway,Th17 cell differentiation,etc..It was also associated with alcoholic liver disease,Kaposi sarcoma-associated Herpesvirus infection and other diseases.The results of molecular docking showed that the core components such as eugenol,caryophyllene,thymol were closely bound to the key targets such as IL6 and PTGS2,and had good affinity.【Conclusion】 The compound plant essential oils might act on key targets such as eugenol,caryophyllene,thymol,D-limonene,cinnamaldehyde and other active ingredients,played an important role mainly through regulating of nerves and immune systems,anti-bacterial,anti-inflammatory and anti-tumor signaling pathways acting on IL6,PTGS2,TLR4,RELA and other key targets.The compound plant essential oils had a multi-component,multi-target,multi-pathway action mechanism.

【Objective】 The aim of this study was to investigate the anti-inflammatory effect of leonurine on lipopolysaccharide (LPS)-induced endometritis in mice.【Method】 Sixty female C57 mice aged 6 to 8 weeks were randomly divided into 6 groups:Blank control group,model group,leonuri low-dose,medium-dose and high-dose groups and dexamethasone positive control group,with 10 mice in each group.Mice in the blank control group was injected with 50 μL PBS buffer vaginally, and the other groups were injected with 50 μL LPS (1 mg/mL) vaginally to establish an animal model of mouse endometritis. After modeling,the low-dose,medium-dose and high-dose groups were intraperitoneally injected with 7.5,15.0 and 30.0 mg/kg leonurine,respectively,while the positive control group was intraperitoneally injected with dexamethasone at a dose of 5.0 mg/kg,once every 6 hours,for 3 consecutive times.The mice were sacrificed 24 hours after modeling,and the uterine index of each group was calculated.The pathological changes of uterine tissue were observed by HE staining.The contents of interleukin-1β (IL-1β),IL-6 and tumor necrosis factor-α (TNF-α) and the activity of myeloperoxidase (MPO) in uterine tissue were determined by ELISA.【Result】 Compared with blank control group,the uterine index,the contents of inflammatory cytokines TNF-α,IL-1β and IL-6 in uterine tissue and the activity of MPO in model group were extremely significantly increased (P＜0.01).Compared with model group,different doses of leonurine could reduce the uterine index,the contents of inflammatory cytokines TNF-α,IL-1β and IL-6 and the activity of MPO in uterine tissue of LPS-induced endometritis (P<0.01) and showed dose-dependent.Compared with model group,uterine index,contents of inflammatory cytokines TNF-α,IL-1β and IL-6 and MPO activity in positive control group were extremely significantly decreased (P＜0.01).【Conclusion 】 Leonurine could reduce the contents of TNF-α,IL-1β,IL-6 and the activity of MPO in mice with LPS-induced endometritis and alleviate inflammatory symptoms,showing a good anti-inflammatory effect.

【Objective】 The purpose of this study was to investigate the digestive tract infection and environmental pollution parasites of sheep in large-scale sheep farms and free-range sheep farms,and provide reference for the comprehensive prevention and control of digestive tract parasites of sheep in large-scale sheep farms in China.【Method】 1 025 sheep feces samples and 712 environmental samples collected from 4 scale sheep farms and 12 free-range households in Henan and Ningxia were examined for parasite infection by centrifugation,Lugol’s iodine staining and saturated sucrose solution floatation methods,and the number of Coccidia oocysts per gram of feces (OPG) in coccidia-positive samples was counted by McMaster’s method.The seasonal dynamic survey was conducted in large-scale sheep farms of Ruzhou to understand the digestive tract infection and environmental pollution parasites of sheep farm in different seasons.【Result】 The total infection rate of digestive tract parasites in sheep was 82.83% (849/1 025),and 8 species (classes) of parasites were detected,including Coccidia,Amoeba,Giardia,Cryptosporidium,Moniezia,Whipworm, Nematodirus and Other Nematodes.The dominant worm species was Coccidia (71.90%),followed by Amoeba (49.07%),and Coccidia and Amoeba were more likely to have mixed infections.The total parasite positivity rate of environmental samples was 38.48% (274/712),and 6 species (classes) of parasites were detected,including Coccidia,Amoeba,Giardia,Whipworm,Nematodirus and other Nematodes,with the dominant species being Nematodes (22.75%).Infection rates of digestive tract parasites and parasite detection rates of environmental samples in Henan scale sheep farms were significantly lower than Ningxia scale sheep farms (P＜0.05).Compared with free-ranging sheep,sheep were infected with fewer worm species and lower infection rate.The infection rate of digestive tract parasites in lactating lambs was significantly lower than that of other physiological stages (P＜0.05).In Ruzhou large-scale sheep farms,the infection rate of digestive tract parasites of sheep in winter was significantly lower than that in other seasons (P＜0.05),The infection rate of Coccidia in suckling lambs was the lowest (14.95%),but the infection intensity was the highest (the average OPG was 105 203),the infection rate of fattening lambs was the highest (88.99%).【Conclusion】 The infection rate of digestive tract parasitic in sheep on large-scale sheep farms was higher,the mixed infections of Coccidia and Amoeba were common,and environmental parasitic contamination was serious.Hygiene measures should be strengthened to prevent environmental parasitic contamination and pathogen dispersal,and planned deworming should be done.

Composting technology is an important way for the harmless treatment of chicken manure,but the waste gas produced in the composting process has adverse effects on the composting quality,and causes harm to the ecological environment and human health.Therefore,the control technology of the waste gas produced by chicken manure composting is one of the restrictive factors for the development of composting technology.These exhaust gases often have complex components,and heavy smell,the exhaust gas containing both nitrogen and sulfur is the key research subject,including NH₃,NO_X,H₂S,and VOC_S of nitrogen and sulfur-containing alcohols,ketones,ethers,etc.The paper introduced the production mechanism of the specific components of the exhaust gas,the relationship between the law of the emission intensity and the properties of chicken manure,composting environment and process conditions was briefly described,reduce or removing exhaust gas emissions from compost by optimizing composting process conditions (temperature,oxygen content,pH,carbon-nitrogen ratio and water content),biological methods (adding microbial agents,biofilter and biological combined compost),regulators (adding biochar and other additives) and end technology.Reviewing the academic research achievements of scholars at home and abroad in recent years,analyzing the removal effect of different exhaust gas and the problems in practical application.The prospect of the future production practice was discussed,in order to provide a reference for the waste gas control research in the process of chicken manure and other waste composting.