【Objective】 The mRNA and lncRNA gene clusters and the key signal pathways that regulated cashmere growth induced by exogenous melatonin of skin in cashmere goats were analyzed, so as to provide a reference for resolving on the molecular mechanism of exogenous melatonin promoting cashmere growth from the perspective of gene clusters.【Method】 Six Hanshan White cashmere goats were selected and randomly assigned to control group and implanted group (melatonin treatment) with three replicates in each group.Take the natural year as the experiment period, skin samples were collected monthly for RNA-Seq analysis.Using Bowtie, TopHat, Cufflinks, Cuffmerge, Cuffdiff, Cuffcompare, CPAT and CPC software to analyze differentially expressed mRNA (DE-mRNA) and differentially expressed lncRNA (DE-lncRNA), Mfuzz package to mine the gene clusters regulating cashmere growth, GSVA and GGally package to analyze the signal pathways regulating cashmere growth.【Result】 A total of 2 024 DE-mRNAs and 329 DE-lncRNAs were identified, which were divided into 8 gene clusters with different dynamic modes in control and melatonin treatment group, respectively.The gene clusters in control group participated in regulating cashmere growth through Hippo signaling pathway, response to interleukin-1, keratin filament, intermediate filament, melanogenesis, cell cycle, FoxO signaling pathway, Wnt signaling pathway, Hh signaling pathway and p53 signaling pathway. The gene clusters in melatonin treatment group participated in regulating cashmere growth through intermediate filament, PI3K-Akt signaling pathway, polymeric cytoskeletal fiber, TGF-beta signaling pathway, growth factor activity, Wnt signaling pathway, Hippo signaling pathway, ECM-receptor interaction, p53 signaling pathway and gap junction.The correlation between DE-mRNA and DE-lncRNA showed that the co-expression network of 55 mRNAs and 10 lncRNAs (PCC>0.960) induced by exogenous melatonin regulating cashmere growth in goats.Gene set variation analysis (GSVA) visualized the dynamic matrix of FoxO, p53, PI3K-Akt, Wnt, Hh, Hippo and TGF-beta signaling pathways regulating cashmere growth at different stages of secondary hair follicle development in skin of cashmere goat.After exogenous melatonin induction, p53 signaling pathway was extremely significantly positively correlated with FoxO signaling pathway (r=0.843, P<0.01).Hh signaling pathway was extremely significantly or significantly positively correlated with Hippo (r=0.836, P<0.01), TGF-beta (r=0.868, P<0.01), Wnt (r=0.790, P<0.01) and PI3K-Akt (r=0.630, P<0.05) signaling pathways.Hippo signaling pathway was extremely significantly or significantly positively correlated with Wnt (r=0.846, P<0.01) and TGF-beta (r=0.806, P<0.05) signaling pathways.Wnt signaling pathway was extremely significantly positively correlated with the TGF-beta signaling pathway (r=0.866, P<0.01).【Conclusion】 The coexpression regulation relationship between 55 mRNAs and 10 lncRNAs regulating cashmere growth in skin of cashmere goats induced by exogenous melatonin had been revealed from the perspective of gene cluster at the omics level, the seven key signaling pathways of FoxO, p53, PI3K-Akt, Wnt, Hh, Hippo and TGF-beta regulating cashmere growth had been identified, which provided a reference for further investigations on the mechanism of melatonin promoting cashmere growth in goats.

【Objective】 Defensins are novel active peptides produced in vivo with broad spectrum antimicrobial activity.In-depth understanding of the basic information of β-defensins AvBD1 and AvBD2 in chickens will be helpful for further study and full application of their biological functions.【Method】 The sequences of AvBD1 and AvBD2 genes in Yellow-feather chickens were amplified using PCR, then cloned and sequenced, subsequently the CDS sequences obtained by sequencing were deduced as amino acid sequences for similarity alignment.The bioinformatics characteristics of the predicted proteins were analyzed by online software, and the expression of the corresponding encoding genes in different tissues of Yellow-feather chickens were compared.【Result】 The CDS length of AvBD1 and AvBD2 genes in Yellow-feather chickens were 198 and 195 bp, which encoded 65 and 64 amino acids, respectively.The similarity of CDS between AvBD1 and AvBD2 genes was 56%, and the similarity of amino acid sequences was 36%, indicating that the two were not homologous genes.The phylogenetic tree showed that Yellow-feather chickens had the closer genetic relationship with several other breeds of chickens, and AvBD1 and AvBD2 genes had the furthest genetic relationship with Mus musculus and Parus major, respectively.Bioinformatics analysis displayed that both AvBD1 and AvBD2 were hydrophobic proteins, and AvBD1 had stronger hydrophobicity and stability than AvBD2.They all contained signal peptides, however, they were located at different amino acid sites and there was no transmembrane domain.The secondary structure elements were mainly alpha helix and random coil, respectively, but the proportion of amino acids involved in the formation of the two conformations was significantly different.Real-time quantitative PCR results found that the overall expression trend of AvBD1 and AvBD2 genes were similar in heart, liver, lung, kidney and duodenum of Yellow-feather chickens, with the highest expression in lung and the lowest expression in duodenum.【Conclusion】 In this study, the CDS sequences of AvBD1 and AvBD2 genes in Yellow-feather chickens were cloned successfully, and the structure and function of the corresponding proteins were analyzed.Both of them were consistent at the mRNA level in visceral tissues of Yellow-feather chickens.The results laid a reference for further elucidating the biological function and application of AvBD1 and AvBD2 in production.

【Objective】 The purpose of this study was to obtain the CDS region sequence of ATP binding cassette subfamily D member 4 (ABCD4) gene in Bama Miniature pigs, predict the structure and function of ABCD4 protein, construct eukaryotic expression vectors, understand the tissue distribution of ABCD4 gene in Bama Miniature pigs, and provide a theoretical and molecular basis for exploring the effect of ABCD4 gene on the growth and development of Bama Miniature pigs.【Method】 Using cDNA of subcutaneous adipose in Bama Miniature pigs as the template, the CDS region of ABCD4 gene was amplified by RT-PCR and sequenced.The CDS region sequence of ABCD4 gene in Bama Miniature pigs was compared with different species by bioinformatics analysis software, and the phylogenetic tree was constructed.Meanwhile, the physicochemical properties and transmembrane structure of ABCD4 protein were predicted.The obtained target gene was ligated into pEGFP-N1 vector and transfected into IPEC-J2 cells, and then the fluorescence and gene expression changes were detected.The expression of ABCD4 gene in different tissues of Bama Miniature pigs were detected by Real-time quantitative PCR.【Result】 The CDS region of ABCD4 gene in Bama Miniature pigs was 1 818 bp in length, encoding 605 amino acids with 99.7% similarity to the porcine reference sequence on Ensembl(ENSSSCT00000037529).There were 6 base mutations and 1 base insertion.The similarity of ABCD4 sequence between Bama Miniature pigs and Homo sapiens, Gallus gallus, Pan troglodytes, Equus caballus, Camelus bactrianus and Bos taurus were 87.6%, 72.7%, 87.6%, 91.7%, 88.1% and 88.6%, respectively.The phylogenetic tree showed that the Bama Miniature pigs was closest distant to Equus caballus and the most distant to Gallus gallus.Bioinformatics analysis showed that the molecular weight of ABCD4 protein was 68.66 ku, the theoretical isoelectric point (pI) was 7.13.ABCD4 protein was predicted to have 5 transmembrane helical structures without signal peptide, which belonged to a transmembrane protein.The protein estimated 58 phosphorylation sites and 10 N-glycosylation sites.The secondary structure of ABCD4 protein was composed of 50.91% alpha helix, 3.64% beta turn, 28.43% random coil and 17.02% extended chain.The results of cell transfection showed that there were fluorescence in recombinant plasmid and empty plasmid control groups after transfection 30 h.The expression of ABCD4 gene in the cells transfected with the recombinant plasmid was extremely significantly higher than empty plasmid control group (P<0.01).Real-time quantitative PCR results showed that the expression of ABCD4 gene was the highest in subcutaneous adipose of Bama Miniature pigs, which was significantly higher than that in other tissues (P<0.05), the expression was the lowest in heart of Bama Miniature pigs.【Conclusion】 In this study, the CDS sequence of ABCD4 gene in Bama Miniature pigs was successfully amplified and obtained.ABCD4 protein was a hydrophobic transmembrane protein, which was mainly expressed in subcutaneous adipose.The results provided a molecular basis for exploring the effect of ABCD4 gene on backfat thickness of pigs.

【Objective】 This study was aimed to analyze the sequence conservation and target genes of hsa-miR-21-5p by bioinformatics, so as to provide ideas for further exploring its function and regulatory mechanism during oocyte maturation.【Method】 The gene expression profiles (GSE31681) of cumulus cell at different stages of human oocytes during maturation were obtained from GEO database.The differentially expressed genes (DEGs) were screened by R software.The miRNAs targeted with DEGs were predicted by FunRich software and found the DEGs targeting by hsa-miR-21-5p to obtain miRNA-mRNA relationship pairs.The similarity and phylogenetic tree were analyzed and constructed by BLAST program and Mega 11.0 software, respectively, and the secondary structure of pre-miR-21-5p were predicted by ViennaRNA Web Services.The target genes of hsa-miR-21-5p were predicted using miRTarBase website, and GO function and KEGG pathway enrichment analysis was carried out.The STRING online database was accessed to develop the protein interaction network and the result was visualized through Cytoscape software.【Result】 A total of 7 DEGs targeted by hsa-miR-21-5p in cumulus cells corresponding to GV/MⅡ stages during oocyte maturation were screened.The sequence analysis of miR-21-5p among species showed that thesimilarity of human pre-miR-21-5p with Mus musculus, Rattus norvegicus, Bos taurus, Ovis aries, Tupaia chinensis and Macaca mulatta was over 95%, and miR-21-5p was highly conserved during evolution.The ViennaRNA Web Services prediction results showed that the secondary structure of pre-miR-21-5p had a typical single stem-loop structure.Target genes prediction revealed that hsa-miR-21-5p had 723 potential target genes.GO function analysis results showed that the target genes were mainly enriched in regulation of cell proliferation, intrinsic apoptotic signaling pathway, positive regulation of protein kinase activity and so on.KEGG pathway analysis results showed that the target genes were mainly enriched in MAPK and p53 signaling pathways.Protein interaction analysis results revealed that the proteins encoded by the target genes MYC, FOXO3 and STAT3 had more targeted relationships with other proteins, and involved in PI3K/Akt, JAK-STAT and other signaling pathways.【Conclusion】 miR-21-5p was highly conserved in biological evolution and played a role in oocyte maturation mainly by regulating the expression of related genes to regulate signaling pathways such as MAPK and p53 in cumulus cells.

【Objective】 This study was aimed to screen the key genes related to intramuscular fat (IMF) deposition, and provide reference for mining molecular markers to improve IMF deposition in buffaloes.【Method】 The GSE19586 dataset was downloaded from GEO database, the differentially expressed genes (DEGs) of longissimus dorsi muscle in Guangxi buffaloes and Qinghai yaks at 36 months of age were screened using limma R package, the up- and down-regulated DEGs were enriched for GO function and KEGG pathway using clusterProfiler R package, respectively, and the protein-protein interaction (PPI) network of DEGs were analyzed using STRING online.The results were visualized by Cytoscape 3.9.1, and the top 20 pivotal genes in terms were screened.【Result】 A total of 254 DEGs were screened in this study, including 61 up-regulated genes and 193 down-regulated genes.GO function enrichment analysis showed that up-regulated DEGs were mainly enriched in processes such as actin-myosin filament sliding, myosin complex, myosin binding, triglyceride metabolism, acylglycerol metabolism, neutral lipid metabolism, cell apoptosis signaling pathway regulation, etc.Down-regulated DEGs were mainly enriched in short-chain fatty acid metabolism, unsaturated fat metabolism, cell reaction to ketone, cell matrix connection, lipoprotein synthesis, protein maturation, processing, polymerization and other processes.KEGG pathway enrichment analysis showed that up-regulated DEGs were mainly involved in metabolic pathways, adipocytokines, insulin resistance, AMP-activated protein kinase (AMPK), thyroid hormone signaling pathways, carbohydrate digestion and absorption and other signal pathways.Down-regulated DEGs were mainly involved in metabolic pathway, phagosome, lysosome, adhesive spot, apoptosis and other signal pathways.There were 116 nodes and 205 links in PPI network, including 26 up-regulated proteins and 90 down-regulated proteins.The top 20 pivotal genes were ACTB, PPARGC1, NR3C1, FOS, GOT2, SERPINE1, RAC1, LEP, STAT5B, HPRT1, DDC, FABP1, HDAC1, RHOA, EEF1A1, PCK1, SOD2, PRKCB, GPX1 and CTGF.【Conclusion】 A total of 254 DEGs in longissimus dorsi muscle of Guangxi buffaloes and Qinghai yaks at 36 months of age were screened in this study.The candidate genes MYH3, GPX1, LDLR, LEP and PCK1 might be related to IMF deposition in buffaloes were mined, which provided a theoretical basis for finding molecular markers to improve the meat quality in buffaloes.

【Objective】 The aim of this study was to construct the lactic acid bacteria expression vector with different constitutive promoters, evaluate the activity of these promoters driving foreign gene expression, and screen out the enhanced expressed promoter with high stability, so as to provide a theoretical basis for the subsequent study that lactic acid bacteria expresses foreign antigens or functional proteins as viable bacterial carriers.【Method】 Using the Escherichia coli-lactic acid bacteria shuttle vector pPG as framework, the promoter sequences in the vector were respectively replaced by Pldh, PHH and Phce, enhanced green fluorescent protein (EGFP) and ampicillin resistance gene (Ampr) were amplified by PCR and the reporter genes luciferase (LUC), EGFP and Ampr were respectively inserted into the downstream polyclonal sites of the vector.The nine constructed lactic acid bacteria expression vectors were electroporated into Lactobacillus paracasei HLJ-27 competent cells to screen and obtain the nine recombinant lactic acid bacteria.Real-time quantitative PCR was used to detect the mRNA transcription level of reporter genes in recombinant bacteria, Western blotting and indirect ELISA were used to detect the protein expression, and the activity of the expressed protein was detected to evaluate the activity of the three promoters.【Result】 The 720 bp of EGFP gene and the 850 bp of Ampr gene were successfully amplified by PCR.Western blotting results showed that the LUC protein of 58 ku, EGFP protein of 26 ku and Ampr protein of 28 ku were successfully expressed in the nine recombinant lactic acid bacteria.Real-time quantitative PCR and indirect ELISA results showed that the level of exogenous gene transcription and exogenous protein expression driven by the promoter Pldh were significantly higher than those of promoters PHH and Phce (P<0.05 or P<0.01).The results of reporter gene detection showed that the promoter Pldh showed higher driving activity of the foreign proteins LUC, EGFP and Ampr than the promoters PHH and Phce (P<0.05 or P<0.01).【Conclusion】 Three promoter-driven reporter gene expression systems were successfully constructed, and the activity of three promoters was verified to be Pldh>PHH>Phce.Ampr reporter system was identified as a superior screening system for promoter activity screening.The results of this study provided a necessary basis for the establishment of lactic acid bacteria carrier system with high expression.

【Objective】 The purpose of this study was to amplify the CDS region of heme oxygenase 1 (HMOX1) gene in Wuliangshan Sooty chickens and carry out the bioinformatics analysis, and detect the tissue expression characteristics of HMOX1 gene, so as to lay a foundation for the subsequent functional research of HMOX1 gene.【Method】 The HMOX1 gene was amplified by PCR using the adipose tissue cDNA in Wuliangshan Sooty chickens as the template, and the PESI-T vector was connected for sequencing analysis.The obtained sequence was translated by DNAMAN software, and the sequence was compared with that of Gallus gallus.BLAST, Mega 7.0, SOPMA, ProtParam and ExPASy were used to carry out the similarity alignment, phylogenetic tree construction and the prediction of physicochemical properties and protein structure.STRING software was used to predict HMOX1 interacting proteins.The expression of HMOX1 gene in different tissues of Wuliangshan Sooty chickens were detected by Real-time quantitative PCR.【Result】 The length of CDS region of HMOX1 gene in Wuliangshan Sooty chickens was 891 bp, encoding 296 amino acids.Three mutations were found in the nucleotide of HMOX1 gene with that of Gallus gallus (accession No.:NM_205344.1), two synonymous mutations, G>C at 217 bp was a missense mutation, resulting in aspartic acid mutation at 73 to glycine.The higher amino acid similarity of HMOX1 gene were 99.6% and 95.0% with Gallus gallus and Coturnix japonica, respectively, and the lowest was 60.2% with Equus caballus.Phylogenetic tree analysis showed that Wuliangshan Sooty chickens had the closer genetic relationship with Gallus gallus and Coturnix japonica, and was far away from non poultry. HMOX1 protein was unstable and hydrophilic, was mainly located in cytoplasm, and there was no signal peptide cleavage site, it was a non secreted protein.There were 1 transmembrane helix structure and 47 phosphorylation sites.The secondary structure of HMOX1 protein was mainly alpha helix.HMOX1 protein might interact with 10 proteins such as HMOX2, BLVRA, HEPH, CP and COX10.The tissue expression analysis showed that HMOX1 gene was expressed in all tissues of Wuliangshan Sooty chickens, with the highest expression in liver and the lowest expression in heart.【Conclusion】 The CDS length of HMOX1 gene in Wuliangshan Sooty chickens was 891 bp, encoding 296 amino acids, predicting it interacted with 10 proteins.HMOX1 gene was highly expressed in liver.The results provided a theoretical basis of informatics for further exploring the role of HMOX1 gene in chicken fat deposition.

【Objective】 The aim of this study was to disclose whether the introduction of major royal jelly protein 1 (MRJP1) to vascular smooth muscle cells (VSMCs) could regulate mice blood pressure in vivo.【Method】 Based on the CRISPR/Cas9 strategy, the gRNA to Hipp11 site, Cas9, and donor vector containing honeybee Mrjp1 cDNA with the upstream Sm22α promoter were co-injected to embryos to produce the Mrjp1 gene knock-in mice that Mrjp1 gene specifically expressed in VSMCs, followed by the verification of knock-in correctness.The systolic and diastolic blood pressures of different days and Media/Lumen area ratios of thoracic aorta for wild type (WT) and homozygous (Mrjp1^+/+) mice with continuous angiotensinⅡ(AngⅡ) infusion by mini-osmotic pumps implanted subcutaneously on the back were compared.【Result】 The heterozygous (Mrjp1^+/-) mice were generated by crossing F0 chimeric with WT mice.PCR amplification and sequencing, as well as Southern blotting, for homology fragments showed that Mrjp1 gene was successfully integrated into the Hipp11 site of mouse chromosome.The WT, Mrjp1^+/- and Mrjp1^+/+mice were generated through Mrjp1^+/-mating.Mrjp1 gene was able to transcript and translate in thoracic aorta.Continuously induced by AngⅡ, the blood pressure, including systolic and diastolic, of the Mrjp1^+/+ mice showed the lower trend than that of the WT.To be exact, the systolic blood pressure at the days of 3, 6, 9 and 12 were significantly different(P<0.05), and the diastolic blood pressure at the days of 3 (P<0.05) and 6 (P<0.01) were significantly different.After the treatment of AngⅡ, the Media/Lumen area ratio of thoracic aorta of Mrjp1^+/+ mice was also significantly lower than that of the WT (P<0.05). 【Conclusion】 The specific knock-in of Mrjp1 gene to VSMCs inhibited the mice blood pressure stimulated by AngⅡ in vivo.The current work would cast light on the in-depth elucidation of MRJP1 function and the precise utilization of royal jelly.

【Objective】 The aim of this study was to screen the candidate genes related to the regulation of eggshell color in Jingyuan chickens by genome-wide association study and transcriptomic analysis, and explore the regulatory mechanism of eggshell color in Jingyuan chickens.【Method】 A total of 50 green shell laying hens and 50 pink shell laying hens at 180 days of age were selected to collect blood and perform simplified genome sequencing.Candidate genes were screened by genome-wide association study.The eggshell gland tissues of green shell hens and pink shell hens at 300 days of age were collected for transcriptome sequencing, differentially expressed genes were screened, and GO function and KEGG pathway enrichment analysis were performed.Through genome-wide and transcriptomic analysis, the genes related to the regulation of eggshell color were screened and the protein interaction network was analyzed.【Result】 The results of simplified genome sequencing showed that 232 465 and 241 008 SNPs were screened from pink shell laying and green shell laying hens, respectively.A total of 54 candidate genes related to eggshell color, including solsolic carrier organic anion transporter family members 1A2 (SLCO1A2), SLCO1C1 and phosphatidylinositol 4-phosphate 3-kinase catalytic subunit type 2 γ (PIK3C2G) were screened by genome-wide association study.277 differentially expressed genes were identified by transcriptome sequencing, of which 85 genes were up-regulated and 192 genes were down-regulated.GO function and KEGG pathway enrichment analysis showed that differentially expressed genes were significantly enriched in related pathways such as linoleic acid metabolism, linolenic acid metabolism, arachidonic acid metabolism, tyrosine metabolism, melanin production, adhesion plaques and calcium signaling.Genome-wide and transcriptomic analysis identified two genes that were significantly associated with eggshell color, which were endothelin-3 (EDN3) and zinc finger protein 831 (ZNF831) genes.【Conclusion】 Through genome-wide association study and transcriptomic analysis, it was found that EDN3 and ZNF831 genes might be candidate genes for regulating eggshell color, which provided a theoretical basis for the study of the regulation mechanism of eggshell color, and provided molecular genetic markers for shell color selection of Jingyuan chickens.

【Objective】 The purpose of this experiment was to optimize the optimum enzyme production conditions of a strain of Aspergillus fumigatus producing heat-resistant cellulase, analyze the fiber degradation effect of the enzyme solution on straw, and provide the basis for its application in the biodegradation of cellulose.【Method】 3, 5-dinitrosalicylic acid (DNS) method was used to determine the activities of cellulase produced by Aspergillus fumigatus WL002 from the previous study, and its enzymatic properties were analyzed.The culture conditions and fermentation medium were optimized by the one factor at a time strategy combined with orthogonal test.The crude fiber degradation effects of the crude enzyme on roughage were analyzed by in vitro fermentation.【Result】 Enzymatic characterization analysis revealed that the optimum reaction pH of cellulase produced by WL002 was 7.0, and the residual enzyme activity was 74%-95% and 56%-90% after preservation at pH 6.0-8.0 for 0.5 and 1 h, respectively.The optimal reaction temperature was 60℃, and after holding at 70℃ for 0.5-1.5 h, the residual enzyme activity was 70%-80%.The maximum cellulase production by strain WL002 were achieved with peptone (1.0%) and wheat straw (2.0%) as the best carbon and nitrogen sources, respectively, at pH 7.0, 7% initial inoculum, the incubation time of 48 h and a culture temperature of 50℃, the average cellulase activity reached 3.11 U/mL.The degradation rates of wheat straw and corn straw were 6.1% and 4.7%, respectively, when treated with the crude enzyme produced by strain WL002 for 60 h.【Conclusion】 The cellulase produced by Aspergillus fumigatus WL002 had a wide pH range, strong thermostability and fiber degradability.The proper conditions for producing cellulase of strain WL002 were achieved, which laid a foundation for its follow-up exploitation and application.

【Objective】 The purpose of this experiment was to investigate the changes of the expression levels of intestinal alkaline phosphatase (IAP) and nuclear transcription factor-κB (NF-κB) in mice with the prolonged inflammatory time induced by lipopolysaccharide (LPS), and provide a theoretical basis for reducing intestinal mucosal stress damage.【Method】 A total of 35 healthy male ICR mice with similar body weight and age were randomly divided into 7 groups with 5 mice in each group.The mice were intraperitoneally injected with 1 mL/mouse LPS (5 mg/kg).Before (0 h) and after injection (3, 6, 12, 24, 48 and 72 h) of LPS, the mice in each group were killed by neck dissection after anesthesia, and duodenum, jejunum and ileum tissues were collected.Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression of IAP and NF-κB in each intestinal segment.The interaction between IAP and NF-κB was detected by co-immunoprecipitation.【Result】 Compared with 0 h, IAP gene expression in the small intestine of mice showed a wave change with the prolongation of LPS inflammatory time, the highest level was reached in duodenum and jejunum at 6 h (P < 0.01), and the highest level was reached in ileum at 48 h (P < 0.01).At 24 h, the expression of NF-κB gene in duodenum and jejunum were extremely significantly or significantly increased (P <0.01 or P < 0.05), there were no significant differences in expression levels at other time points (P > 0.05).After LPS treatment, the expression of IAP and NF-κB protein in each intestinal segment of mice showed a trend of first increasing and then decreasing, and the protein expression reached the highest level at 6 h (P < 0.05).The results of co-immunoprecipitation showed that NF-κB interacted with IAP.【Conclusion】 In the inflammatory process of LPS, the mRNA and protein expressions of NF-κB and IAP increased with the occurrence of intestinal inflammation, and reached the highest level at 6 h after inflammation, but gradually decreased with the extension of inflammatory time.There was a synergistic effect between IAP and NF-κB in the process of intestinal mucosal stress injury.

【Objective】 The effects of Hermetia illucens larvae meal (HILM) as dietary protein source on performance, antioxidant capacity and intestinal health of Sichuan White geese were studied to explore the application of HILM in the diet of Sichuan White geese.【Method】 A total of 180 1-day-old Sichuan White geese (half male and half female) with similar body weight were randomly divided into 3 groups, including control group, miscellaneous meal group and 15% HILM group.Each group consisted of 6 replicates with 10 animals per replicate.During the experiment, food and water were eaten and drank freely, and the feed intake was weighed weekly in the unit of repetitions.The feed intake was recorded, and the average daily feed intake (ADFI), average daily gain (ADG) and feed to gain ratio (F/G) were calculated for each group.Serum antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were detected at 4 weeks of age.The tissue morphology of duodenum, jejunum and ileum was observed by section.The villus height and crypt depth of each intestinal segment were measured, and the villus height/crypt depth was calculated.Real-time fluorescence quantitative PCR was used to analyze the expression of intestinal compact linking protein gene.【Result】 Compared with the control group, in 15% HILM group, the ADFI and ADG of Sichuan White geese were increased (P<0.05), the serum GSH-Px concentration was significantly increased (P<0.05), and the villus height of duodenum, jejunum and ileum was increased (P<0.05 or P<0.01), the crypt depth of duodenum and ileum was decreased (P<0.01), and the villus height/crypt depth of duodenum and ileum was increased (P<0.01).At the same time, the expression of Occludin gene in jejunal tissue in 15% HILM group was increased (P<0.05).Compared with miscellaneous meal group, in 15% HILM group, the serum SOD activity was increased (P<0.05), the crypt depth of duodenum was decreased (P<0.01), and the villus height/crypt depth of duodenum and ileum were increased (P<0.01 or P<0.05).【Conclusion】 As a dietary protein source, HILM could enhance serum antioxidant capacity of Sichuan White geese, improve the intestinal health and absorption function of goslings, and thus improve the production performance.

【Objective】 This study was conducted to evaluate the feeding effect of different plant tannin preparations on Litopenaeus vannamei.【Method】 A total of 600 shrimp were randomly selected and divided into 5 groups with 3 replicates per group and 40 shrimp per replicates.Five isonitrogen and isolipid diets were prepared containing 0 (G1), 0.1% (G2) and 0.2% (G3) Chinese gallotannin preparations, and 0.1% (G4) and 0.2% (G5) chestnut tannin preparations.The experiment was lasted for 42 days.At the end of the experiment, the weight, quantity, and feed intake of shrimp each tank were counted to calculate their growth performance.Blood was collected from the cardiocoelom of the sampled shrimp to separate serum for determination of serum biochemical and serum antioxidant and immune indicators, and the intestine of the shrimp was separated for determination of intestinal digestive enzyme activity.【Result】 Compared with G1, the feed coefficient of shrimp in G5 was significantly increased (P<0.05), while the intestinesomatic index and meat rate were significantly decreased (P<0.05).The content of serum triglyceride of shrimp in G3 was significantly decreased (P<0.05), and the contents of serum glucose, triglyceride and urea nitrogen of shrimp in G5 were significantly decreased (P<0.05).Compared with G1, the content of serum malondialdehyde in G3 was significantly decreased (P<0.05), while the content of serum immunoglobulin M in G5 was significantly increased (P<0.05).Compared with G1, intestinal trypsin activity of shrimp in G2 and G3 were significantly increased (P<0.05).【Conclusion】 Taking growth performance, antioxidant capacity and intestinal digestive enzyme activity as comprehensive evaluation indicators, 0.2% Chinese gallotannin preparation had a good application effect on Litopenaeus vannamei.

【Objective】 The experiment was to study the effects of dietary soy isoflavone (SIF) on laying performance, egg quality, reproductive organ development, hatching performance and plasma biochemical indices of Yellow-feathered broiler breeder hens during laying period, in order to provide data reference for application of soy isoflavone in broiler hens.【Method】 One hundred and ninety-two healthy 21-week-old LingnanYellow-feathered broiler breeder hens were randomly assigned to 4 groups.Each group had 6 replicates with 8 hens per replicate.Broilers in control group were fed basal diet, and groups 2 to 4 were supplemented with SIF in the basal diet, and the supplemental levels were 5, 15 and 25 mg/kg, respectively.The experiment lasted for 10 weeks from 25 to 35 weeks of age.During the experiment, the daily feed intake, egg number and egg weight were repeatedly recorded.At 34 to 35 weeks of age, 50 eggs in each group were selected for incubation to determine the hatching performance.At 35 weeks of age, 24 eggs were randomly selected from each group for egg quality measurement, and 2 chickens from each replicate were selected for blood and sample collection to determine reproductive organ development and plasma biochemical indices.【Result】 SIF had no significant effects on laying rate, average egg weight, daily egg weight and ratio of feed to egg of Yellow-feathered breeder hens (P>0.05).Dietary SIF significantly reduced the unqualified egg rate, and it was significantly lower in 25 mg/kg SIF group than that in control group (P<0.05).Dietary SIF significantly improved eggshell strength and yolk color, and yolk color in SIF 5 and 25 mg/kg groups were significantly higher than that in control group (P<0.05), the eggshell strength of SIF 5 mg/kg group was significantly higher than that of control group (P<0.05).Dietary SIF significantly reduced the percentage of abdominal fat, and it was significantly lower in SIF 25 mg/kg group than that in control group (P<0.05).Dietary SIF significantly reduced the contents of malondialdehyde (MDA) and cholesterol (CHO), while increased the activity of glutathioneperoxidase (GSH-Px) in plasma, and the content of MDA in 25 mg/kg SIF group was significantly lower than that in control group. The content of CHO in 5 and 15 mg/kg SIF group were significantly lower than that in control group, and the activities of GSH-Px in 5, 15 and 25 mg/kg SIF groups were significantly higher than that in control group (P<0.05).【Conclusion】 Dietary SIF could reduce unqualified egg rate, improve egg quality, reduce abdominal fat percentage, decrease the contents of MDA and CHO, increase the activity of GSH-Px in plasma.The suitable supplemental level of SIF in laying period of Yellow-feathered broiler breeder hens was 25 mg/kg.

【Objective】 The aim of this study was to establish a rapid method for the determination of antimicrobial peptide NZ2114 in rat powder feed by solid phase extraction and high performance liquid chromatography (SPE-HPLC), which was used to detect the content of the test substance NZ2114 in rat powder feed samples at low, medium and high concentrations (2, 10 and 50 g/kg), and to analyze whether the antimicrobial peptide NZ2114 was evenly distributed.【Method】 The experiment mainly optimized extraction conditions, purification column and purification method, which investigated the influence of extraction agent and extraction time on extraction efficiency, the effect of SPE cleaning agent and elutriation agent on recovery rate.Feed samples were extracted by ultrasonic centrifugation, and the supernatant was enriched via WCX solid-phase extraction column, followed by purification and elution with 3 mL methanol and 5 mL of 20% ammoniated methanol.ZORBAX Eclipse Plus C18 was used as the stationary phase, and 0.1% formic acid-acetonitrile and 0.1% formic acid-water were used as mobile phases.The column temperature was 35℃, the injection volume was 20 μL, and the flow rate was 1.0 mL/min.The content of NZ2114 in feed samples was quantitatively determined by external standard method at a wavelength of 276 nm, and the linearity, limit of detection and limit of quantification, matrix effect, recovery rate, intra-day and inter-day precision of the method were investigated.【Result】 The SPE-HPLC method had a good linear relationship in the concentration range of 62.5-4 000 μg/mL, and the linear equation was y=0.9353x-32.721, whose correlation coefficient (R²) was 0.9996.The recovery rate of the method was 88.05%-111.23% at low, medium and high concentrations.The intra-day and inter-day precision were 0.58%-4.45%, 3.18%-6.37%, respectively.And the matrix effects at 125, 500 and 2 000 μg/mL concentrations were 1.02, 1.01 and 0.99, respectively.【Conclusion】 In this study, the SPE-HPLC method for the determination of antimicrobial peptide NZ2114 in rat feed was established, which met the requirements of methodological investigation, with the advantages of high sensitivity and precision, good reproducibility and rapid analysis.Thus, the method could achieve accurate quantification of NZ2114 in feed samples at three concentration levels.

【Objective】 The aim of this experiment was to evaluate the effects of α-ketoglutaric acid (AKG) on rumen fermentation, nutrient digestion and metabolism, and blood biochemical parameters in sheep, to provide a reference for AKG as a regulator of rumen fermentation in ruminants.【Method】 Five healthy small-tailed Han rams with permanent rumen fistulas were used in a 5×5 Latin square design, with each animal receiving a basal diet (the ratio of concentrate to roughage was 55:45) supplemented with AKG at doses of 0, 25, 50, 100 and 200 mmol per day, respectively;The experimental consisted of 5 phases, each experimental phase lasted for 24 days, including a 14-day adaptation period, a 7-day digestion and metabolism test period, and a 3-day ruminal fluid sampling period.Jugular venous blood was collected 1.5 h after morning feeding on the first day of the ruminal fluid sampling period.All feces and urine were collected during the digestion and metabolism test period to determine apparent digestibility of nutrients and nitrogen, calcium, and phosphorus metabolism.Rumen fluid was used to determine fermentation parameters and protozoan counts, and blood was used to determine physiological and biochemical parameters.【Result】 AKG had no significant effect on NH₃-N, valerate, and isovalerate levels in rumen fluid (P>0.05).The rumen fluid pH decreased linearly with the increase of AKG addition (P<0.05).The number of protozoa and the ratio of acetate to propionate showed a quadratic effect with an increasing and then decreasing (P<0.05), and acetate, propionate, and total volatile fatty acids showed a quadratic effect with a decreasing and then increasing with the increased dose of AKG supplementation (P<0.05).The addition of AKG had no significant effect on the apparent digestibility of dry matter, organic matter, crude protein, crude fat, calcium, phosphorus, neutral detergent fiber, and acid detergent fiber, and the metabolism of nitrogen, calcium, and phosphorus (P>0.05).AKG had no significant effect on blood albumin and urea nitrogen concentrations, and the activities of aspartate aminotransferase and alanine aminotransferase (P>0.05).With the increased of AKG addition, the total protein concentration in blood showed a quadratic effect with increasing and then decreasing (P<0.05).【Conclusion】 The addition of AKG to sheep diets acted mainly in the rumen, lowering the pH of the rumen fluid, 50 mmol/d AKG reduced the production of propionate and total volatile fatty acids, improved the ratio of acetate to propionate, and increased the concentration of total protein in the blood.

【Objective】 The experiment aims to investigate the effect of supplementing different levels of ellagic acid on the diversity of fecal bacteria in foals, and to explore the changes in fecal microbiota, in order to provide reference for the diversity of gastrointestinal microbiota and intestinal health in lactating foals.【Method】 Fifteen three month old pure blood ponies with similar birth dates and a weight of (143.33±16.10) kg were selected for the experiment.All the foals were randomly divide into 3 groups with 5 foals in each group, named control group, group Ⅰ, and group Ⅱ.Under the same feeding conditions, the ponies in control group did not undergo any treatment and the ponies in group Ⅰ and Ⅱ were fed with 15 and 30 mg/kg BW ellagic acid every day for 60 days.On the 60th day of the experiment, fecal samples of the foal were collected by rectal sampling method before feeding, and the diversity of fecal microbiota was measured.【Result】 The OTUs results showed there was a total of 789 OTUs in all groups, and 182 unique was in group Ⅰ.There was no significant difference in alpha diversity between the experimental groups and the control group (P>0.05), but group Ⅰ showed a trend of increasing Shannon, Chao1, and ACE indices.In terms of microbial abundance, the top 10 dominant bacterial species in each experimental group and control group were the same, with the main being Firmicutes, Bacteroidetes, Actinobacteria, and Verrucomycetes, and the abundance accounts for more than 92.94% of the total.There was no significant difference in the abundance of phyla, family, and genus level microbial communities among each group (P>0.05).LefSe analysis showed that there were 9 species of differential bacteria in group Ⅰ and 2 species in group Ⅱ.The top 10 predicted functions include global and overview maps, carbohydrate metabolism, and amino acid metabolism, but the differences were not significant (P>0.05).【Conclusion】 Bacillota, Bacteroidota, Actinomycetota and Verrucomicrobiota were the dominant flora in the feces of pure blood horses during lactation.Supplement of 15 mg/kg BW ellagic acid could improve the fecal flora diversity and increase the species of fecal flora.

【Objective】 The purpose of this study was to reveal the genetic mechanism of microRNA (miRNA) on estrous activity in primiparous sows from the neuroendocrine system.【Method】 RNA-Seq technology was utilized to sequence small RNA from the hypothalamic-pituitary axis of anestrous and estrous primiparous sows in this study.The miRNA expression profiles in the hypothalamus and pituitary of sows were obtained and the differentially expressed miRNA were screened for functional analysis such as GO and KEGG.【Result】 A total of 1 096 miRNAs were identified from four libraries, of which 364 were known miRNAs and 732 were novel miRNAs.Compared with estrous primiparous sows, 16 differentially expressed miRNAs were identified in the hypothalamus of anestrous sows, including 6 up-regulated and 10 down-regulated miRNAs.9 differentially expressed miRNAs were identified in the pituitary of anestrous sows, including 2 up-regulated and 7 down-regulated miRNAs.A total of 5 212 target genes were predicted for 16 differentially expressed miRNAs in the hypothalamus, and 6 897 target genes were predicted for 9 differentially expressed miRNAs in the pituitary gland.Functional analysis of differentially expressed miRNA target genes in hypothalamus showed some GO entries and KEGG signaling pathways were significantly enriched, such as cell projection assembly, plasma membrane bounded cell projection assembly, negative regulation of neuron projection development, protein serine/threonine kinase activity, Ras signaling pathway, neurotrophin signaling pathway, ErbB signaling pathway, and mTOR signaling pathway, etc.These miRNA target genes in pituitary were also significantly enriched in some GO entries and KEGG signaling pathways, such as regulation of mRNA processing, Ras GTPase binding, ubiquitin-like protein ligase activity, epidermal growth factor receptor binding, protein kinase, sphingolipids signaling pathway, apoptosis, ErbB signaling pathway, and NF-κB signaling pathway, etc.Chr3_8404_mature, ssc-miR-9 and ssc-miR-34c were screened from miRNA regulatory network with target genes that were involved in regulation of reproduction, and these 3 miRNAs might play an important role in regulating estrous activity in the hypothalamic-pituitary axis of primiparous sows.8 differentially expressed miRNAs (4 in hypothalamus and 4 in pituitary) were selected for validation by Real-time quantitative PCR, and their expression trends were generally consistent with the sequencing results.【Conclusion】 The miRNA expression profiles of hypothalamic-pituitary axis of anestrous and estrous primiparous sows were successfully constructed and the functions of differentially expressed miRNA were deeply explored in this study.3 miRNAs that play an important role in the estrous activity of primiparous sows were screened.A theoretical basis for the analysis of the genetic mechanism of estrous activity in primiparous sows had been provided.

In the modern dairy industry, the multiple ovulation and embryo transfer (MOET) is the key technology, which can significantly accelerate the genetic improvement of dairy population.However, there is a large variation in superovulation response among different donor cows, which is one of the major limitations for the widely applying of superovulation.Therefore, there is an urgent need to explore the causes of variation in superovulation response and to predict the effect of exogenous hormone treatment for donor cows so that suitable donors can be selected.In previous studies, it had been widely reported that the superovulation response trait is heritable in dairy cows, and that genetic selection can substantially improve superovulation efficiency.In general, superovulation response trait is defined as the total number of embryos as well as the number of transferable embryos.Superovulation response trait can be influenced by several factors, including season, donor age, procedure and operator.As a novel reproductive trait with low to medium heritability, it was found to be associated with several genes that regulate the expression of reproductive hormones and cell growth and differentiation.At present, there are relatively few studies on genetics and breeding of superovulation response trait in China, and only a few studies reported candidate genes associated with superovulation response trait.The author systematically reviewed the advance of superovulation response trait in dairy cattle, including the trait definition, modeling of non-genetic factors, estimation of genetic parameters, and identification of candidate genes.In addition, we also discussed the research methods and application of superovulation response trait in the future.

【Objective】 This study was aimed to explore the protective mechanism of asperuloside on hippocampus in chronic unpredictable mild stress (CUMS) rats based on proteomics, and provide a basis for the development and utilization of asperuloside.【Method】 Forty-five male SD rats were chosen and randomly divided into three groups after one week pre-feed with 15 rats in each group, including the blank, model and asperuloside groups.In the blank group, 3 rats were raised in one cage, while in the other groups, one cage for one rat.The experimental period was 5 weeks, stimulated mice while medication administration starting from the 3rd week of the experiment.The rats in blank and model groups were given equal volume of saline by oral administration while in the asperuloside group was given a dose of 0.070 g/kg BW by intravenous injection once a day.The total hippocampus protein was extracted and the proteomics analysis of the hippocampus of depression rats was carried out by tandem mass tags (TMT)-LC-MS/MS technique to screen potential differential expression proteins.In addition, parallel reaction monitoring (PRM) was used to verify the expression of selected biomarkers in hippocampus.【Result】 A total of 6 up-regulated and 17 down-regulated proteins were screened between asperuloside and model groups.GO function and KEGG pathway enrichment analysis showed that the differentially expressed proteins were involved in signaling pathways such as neuroactive ligand-receptor interaction, complementary and synergistic regulation cascade and gastric acid secretion.PRM was used to verify the expression trend of bradykinin, immunoglobulin (IGHM and ILD) and proteasome activator complex subunit 1 (PSME1) were consistent with TMT quantitative results, it might be potential target proteins for the regulation of depression by asperuloside.Asperuloside could protect hippocampus through various pathways such as controlling key targets involved in regulating neuroactive ligand receptor interactions signaling pathways, exerting direct or indirect protective effect on neurons by down-regulating BK protein expression, up-regulating PSME1 protein levels significantly correlated with IgG leads to immunosuppression and normalization of immune protein concentration.【Conclusion】 Asperuloside might alleviate depression symptoms through multiple synergistic effects.The results could provide a references for further exploring the pathological mechanisms of the CUMS model and the proteins or pathways regulated by asperuloside in hippocampus.

【Objective】 This study was aimed to detect the antibiotic and disinfectant resistance-related genes of Yersinia pestis in Inner Mongolia natural plague foci, and carry out bioinformatics analysis, so as to provide scientific basis for plague prevention and control in this region.【Method】 PCR method was used to detect the streptomycin resistance genes (StrA and StrB), beta-lactam resistance genes (Tem and Ctx-m), sulfanilamide resistance gene (Sul1 and Sul2) and quaternary ammonium salt resistance gene (QacEdelta) in 60 strains of Yersinia pestis isolated from Inner Mongolia natural plague foci.At the same time, the structure, function and antigenic epitopes of StrA, Sul1 and QacEdelta proteins were predicted.【Result】 PCR amplification results showed that the negative and positive controls were established, and the amplification results of 60 strains of Yersinia pestis were negative.No genes related to streptomycin resistance, sulfonamides resistance, beta-lactam resistance and disinfectant resistance were found in the plague strains isolated from Inner Mongolia natural plague foci.Bioinformatics analysis showed that StrA, Sul1 and QacEdelta proteins were all hydrophobic outer membrane proteins that did not contain transmembrane regions, and were secreted proteins.The secondary structure of the three proteins was dominated by alpha helix, which accounted for 37.83%, 45.88% and 46.09%, respectively.Antigen epitope prediction showed that the three proteins contained 9, 8 and 3 B cell antigenic epitopes, and 9, 16 and 14 T cell antigenic epitopes, respectively.【Conclusion】 60 strains of Yersinia pestis isolated from Inner Mongolia plague foci did not have the characteristics of drug resistance and disinfectant resistance related genes.StrA, Sul1 and QacEdelta proteins contained more B and T cell antigenic epitopes and had good antigenicity.The results provided a reference for the rational use of antibiotics and disinfectants in plague prevention and control in these foci.

With the constant variation and spread of viruses, the research on virus detection methods is also developing and deepening.When viral diseases break out, it is particularly important to detect viruses timely and accurately.Traditional virus detection methods include virus isolation and identification, PCR and ELISA, but these methods all have problems such as complicated operation, long detection time and low sensitivity.In recent years, some emerging technologies and optimized methods have been applied to the detection of various viruses, which have brought more accurate and efficient virus detection.For example, the sensitivity of virus detection based on droplet digital PCR can be as much as 16 times higher than Real-time quantitative PCR.There is also a virus detection method based on gene sequencing technology, which attracts more and more attention, it can not only detect the types and quantities of viruses, but also directly and accurately obtain virus gene sequences, and even discover new viruses.In addition, detection technologies based on CRISPR/Cas, biosensors and flow cytometry have also been widely used in the field of virus detection.Although these new technologies have obvious advantages in specificity and sensitivity, they still face some challenges, such as high detection cost and the need for laboratory environment, which limit the popularization and promotion of virus detection technology.Therefore, the future research will focus on improving the accuracy and practicability of detection and reducing the cost, and developing more perfect virus detection technology.The author compares the optimized and emerging detection technology with the traditional detection technology, and summarizes its advantages and disadvantages, in order to provide basis and reference for virus detection and virus disease prevention and control.

【Objective】 The objective of this study was to investigate the essential functional genes of Enterococcus faecium SWUN5732, a strain isolated from yak, through an analysis of its genome sequence detection outcomes.【Method】 Through Illumina PE150 sequencing system, the genome of Enterococcus faecium SWUN5732 was sequenced, and the basic functional characteristics of the genome were annotated in GO, KEGG, COG and NR databases.Combined with the annotation of NR database, the potential antibacterial, antioxidant and antibacterial peptide related genes in the genome were excavated.【Result】 The genome size of Enterococcus faecium SWUN5732 was 2 824 168 bp, and the effective average GC-content was about 38.09%.The number of detectable coding genes was approximately 2 919, and the total length of all coding genes was approximately 2 387 787 bp, with an average length of approximately 818 bp.In the GO database, a total of 8 851 genes in the SWUN5732 genome were annotated, with 45 entries in three major categories:Molecular function, cellular component and biological process.In the KEGG database, there were a total of 1 534 genes annotated in the SWUN5732 genome, including 38 pathways for six major functions:Cellular processes, environmental information processing, genetic information processing, human diseases, metabolism, and biological systems.In the COG database, a total of 2 118 genes had been annotated in the SWUN5732 genome, which also included four functional sequences with ABC type amino acid transport systems (osmotic enzyme components) and sequences of ABC transport proteins with repetitive ATPase domains.According to the functional annotation of Enterococcus faecium SWUN5732 in NR database, a total of 2 844 genes were annotated, and it was found that the genome of SWUN5732 contained antibacterial substances, antioxidant substances and antibacterial peptides related genes.The phylogenetic tree analysis results indicated that the strain had the closest evolutionary relationship to ATCC 19434.【Conclusion】 In this study, the genome of bacteriocin-producing Enterococcus faecium SWUN5732 from yaks was sequenced and potential antimicrobial substances were excavated, which confirmed that the strain had acid and bile salt tolerance environmental adaptability and explored the genes related to antioxidant, immune regulation, bacteriocin-production and antimicrobial substances, providing a reliable basis for the strain as an antibiotic substitute or feed probiotic additive.

【Objective】 This study was aimed to investigate the origin of J-avian leukosis of a black-finned egg farm in Hubei and the evolutionary trend of its envelope gene, and provide information for the epidemiological investigation of avian leukosis in Hubei.【Method】 Pathological examination, ELISA and Multi-PCR were used for laboratory diagnosis of suspected cases of hemangioma J-Avian leucosis virus (ALV-J).Viral fluid was prepared and inoculated with DF-1 cells for virus isolation and indirect immunofluorescence (IFA) detection, and the gene encoding the envelope protein of this strain was analyzed by DNAStar and other softwares.【Result】 The case presented typical hemangioma typical symptomatic lesions, the p27 antigen test was positive, the Multi-PCR results showed ALV-J specific bands, the IFA results showed specific fluorescence, which indicated that 1 strain of ALV was isolated successfully, named HB2021017.The amino acid similarity were increased progressively between the envelope membrane protein ENV, membrane surface glycoprotein SU, transmembrane protein TM of the isolate HB2021017 and the corresponding proteins of the cited ALV-J strains of myeloblastoma, mixed myeloblastoma and hemangioma, and hemangioma-type ALV-J strains.Phylogenetic tree analysis showed that the env gene of the isolate HB2021017 was most closely related to the hemangioma-inducing ALV-J strain isolated from local breed chickens in China, and was in the same branch;While it was more distantly related to the hemangioma-inducing or myeloblastoma-inducing ALV-J strain isolated from other chickens, and was in different branches.【Conclution】 A strain of hemangioma-inducing ALV-J, named HB2021017, was isolated.The envelope genes of the hemangioma-inducing ALV-J strain isolated from local breed chickens had formed a relatively independent evolutionary branch.

Bacteria resistance is closely related with the development of animal husbandry industry, human public health safety and food safety.The mechanism of bacteria resistance is complex.In addition to the complex interaction among human, animal and environment, the use of antibiotics has played a very important impact on the generation and spread of bacterial resistance.Therefore, it is necessary to develop a variety of strategies to cope with bacterial resistance.The newly developed drug theory and the rise of molecular biology technology have greatly promoted the understanding of bacterial drug resistance mechanism, the discovery of new antibacterial drugs and the rational use of drugs, which provided important theoretical basis and technical means for the control of bacterial drug resistance.Presently, bacterial drug resistance has been a serious problem which can not be ignored and has arouse much concern.Due to the complexity of bacterial drug resistance mechanism and the difficulty of new drug development, it is necessary to carry out research through multiple methods, approaches and perspectives to prevent further development of bacterial resistance.Based on the current research, the author summarized a variety of strategies and methods to deal with bacterial drug resistance, focusing on the development of new antimicrobial drugs, drug combination, pharmacokinetic-pharmacodynamic modelling and multi-omics technology to elaborate their role in dealing with bacterial drug resistance, which would provide reference and guidance for slowing and controlling the development of bacterial resistance and clinical treatment of bacterial infection.

【Objective】 The aim of this experiment was to construct a colloidal gold immunochromatography for the rapid detection of Staphylococcus aureus.【Method】 The colloid gold was prepared and characterized, and then connected with aptamer, characterized and sprayed on the binding pad.The test strip was constructed with polyclonal antibody as the test line and the complementary sequence of aptamer as the control line.The test conditions of sample pad and binding pad treatment solution, sample dilution solution, suspension solution, T-line antibody concentration, and aptamer connection concentration were optimized to obtain the best conditions.At the same time, the sensitization effect of the test strip was studied, and the sensitivity and specificity of the test strip were tested under the best conditions.Finally, the artificial contaminated sample was prepared to evaluate the effect of the test strip in the actual sample detection.【Result】 The particle size of colloidal gold was about 20 nm, and the results showed that the colloidal gold was successfully connected to the aptamer.The optimal conditions for the sample pad, the binding pad and the sample diluent were SELEX buffer with different components, the resuspension with gold standard was HEPES resuspension, and the T-line antibody concentration was 2 mg/mL.The aptamer binding concentration was 1 μmol/L, and 40 mmol/L hydroxylamine was sensitized with 0.5% gold chloride aqueous solution, and the detection sensitivity was 1×10⁴ CFU/mL with good specificity.For artificially contaminated meat and milk samples, the sensitivity was 1×10⁵ CFU/mL.【Conclusion】 The test strip detection method for Staphylococcus aureus was established successfully, and had good specificity, which provided a theoretical basis for further exploring the application of nucleic acid aptamer in the detection of pathogenic bacteria.

【Objective】 This study was aimed to explore and detect the immunogenicity of aquaporin 3 in Dermacentor marginatus (DmAQP3), and provide materials for the subsequent development of anti-tick immunity test.【Method】 PCR technology was used to amplify the Dmaqp3 gene and construct an amino acid sequence phylogenetic tree of the Dmaqp3 gene.The bioinformatics analysis of DmAQP3 protein was carried out.The best region of antigenicity was selected to construct the truncated recombinant plasmid pET-32a-jdDmaqp3, and the recombinant protein DmAQP3 (rDmAQP3) was expressed.The reactogenicity of the recombinant protein was detected by Western blotting.Kunming mice were immunized with the purified recombinant protein rDmAQP3 to prepare polyclonal antibodies, and the titer of polyclonal antibodies was detected by indirect ELISA.【Result】 The PCR amplification fragment size of Dmaqp3 gene was 879 bp, with a similarity of 98.37% to the aqp3 gene of Dermacentor silvarum (XM_049662329.1).The amino acid sequence of the DmAQP3 protein was closest to Dermacentor silvarum (AQP-9 subtype X2) and Dermacentor andersoni (AQP-9 like subtype X2).DmAQP3 was a stable protein with a theoretical isoelectric point of 8.65, it had 6 transmembrane regions and 2 Asn-Pro-Ala (NPA) structures.There were 7 B cell epitope and 18 phosphorylation sites, which was hydrophobic protein.The secondary structure of the protein consisted of alpha helix, beta turn, random coil and extended chain, which accounted for 31.27%, 3.58%, 40.07% and 25.08%, respectively.The tertiary structure prediction showed that the protein was composed of 4 subunits.The truncated recombinant plasmid pET-32a-jdDmaqp3 was successfully constructed and the recombinant protein rDmAQP3 was obtained.Western blotting results showed that the recombinant protein rDmAQP3 reacted with positive serum with a target band of 27 ku in size, indicating that the protein had good reactogenicity.The results of indirect ELISA showed that the titer of the prepared polyclonal antibodies against recombinant protein rDmAQP3 was as high as 1:409 600, indicating that the protein had good immunogenicity.【Conclusion】 In this experiment, Dmaqp3 gene was cloned, the prokaryotic expression vector of DmAQP3 was constructed, and the recombinant protein rDmAQP3 was induced and expressed.The protein had good reactogenicity and immunogenicity, which provided conditions for further study of its biological characteristics and establishment of model animal immune test against ticks.

Animal parasitic nematodes are one of the most diverse and abundant groups of animal parasites and pose severe threats to humans, domestic and wild animals.The mitogenome is the only extranuclear genetic material of animal parasitic nematodes and is an important information carrier for studying nematode species identification, genetic evolution and phylogenetics, as well as the prevention and control of nematodosis.In recent years, with the rapid development of next-generation sequencing and bioinformatics technology, more and more mitogenomes of animal parasitic nematodes were decoded, and up to February 28, 2022, there are 283 mitogenomes of animal parasitic nematodes deposited in GenBank of NCBI, including 213 nematode species, belonging to two subclasses of Secernentea and Adenophorea.Despite this, there is still a lack of systematic and comprehensive generalization about the characteristics and applications of animal parasitic nematode mitogenomes.Thus, this review aimed to summarize the structure and content, base preference, variations in tRNA/AT-enriched regions of the existing animal parasitic nematode mitogenomes which are sequenced so far and propose new gene arrangement (GA) divisions which contain 39 Gas, and also further concluded the mitogenome applications in population genetics, phylogeny, molecular epidemiology and diagnostics of animal parasitic nematodes, aiming to provide an informative reference for the prevention and control of animal nematodosis.

【Objective】 The experiment was aimed to confirm the pathogen of gill rot disease in a goldfish farm in Beijing.【Method】 The moribund goldfishes with clinical symptoms were collected, and wet mount of gill tissue were prepared for observing parasites and fungal hypha.Liver, kidney, spleen and gill tissues were collected to detect Goldfish hematopoietic necrosis virus (GFHNV).At the same time, the pathogenic bacteria were isolated from the gill, and the morphological characteristics, physical and chemical characteristics, taxonomic status and drug sensitivity of the pathogenic bacteria were analyzed.The experiments of artificial infection of pathogenic bacteria were carried out by the methods of gill tissue scratch soaking and non-scratch soaking.【Result】 There was a small amount of trichomyces parasitica on the gill filaments of naturally occurring fish, no fungal hypha was observed, and GFHNV nucleic acid test was negative.A large number of rhizoid colonies were isolated from gill tissue.The strain was Gram-negative longobacterium, with a body size of (0.5-0.7) μm×(4-8) μm.It was positive for oxidase and catalase, degraded gelatin and chondroitin sulfate, produced hydrogen sulfide, and did not fermentation glucose and other sugar alcohols.The phylogenetic tree constructed based on 16S rDNA sequence was polymerized with Flavobacterium columnare.The isolated bacteria was identified as Flavobacterium columnare, genotype Ⅰ.All groups of goldfish soaked in scratches experienced death, and the symptoms were similar to those of the naturally infected fish.No death occurred in scratch control group and non-scratch soaking group.The calculated median lethal dose (LD₅₀) of the bacteria to the goldfish was 8.8×10⁶ CFU, which was a mild strain.The bacterium was sensitive to enrofloxacin, doxycycline hydrochloride and sulfonamides, but not to the other antimicrobials tested.【Conclusion】 The weakly virulent strains of Flavobacterium columnare isolated in this study could cause gill tissue damage and death in goldfish.Based on practical production, many goldfish had been infected with a small amount of Trichodina spp., causing gill tissue damage but no deaths.It was determined that this chronic and low mortality rate of goldfish gill rot disease was caused by a mixed infection of Trichodina spp.and weakly virulent strains of Flavobacterium columnare, and the secondary infection of weakly virulent strains of Flavobacterium columnare was the main cause of goldfish death.

【Objective】 The purpose of the test was to establish an indirect ELISA method for detection of antibodies to Mycoplasma bovis, and provide a method for testing the efficacy of inactivated vaccine against Mycoplasma bovis.【Method】 The prokaryotic expression plasmid pET-32a-P48 was constructed and transformed into E.coli BL21(DE3) Plyss competent cells to screen the positive plasmid and perform double digestion verification.The recombinant protein was induced to express with IPTG with a final concentration of 1 mmol/L, and purified with nickel column.The protein purity and molecular weight were verified by SDS-PAGE, and the specificity and reactogenicity of the recombinant P48 protein were detected by Western blotting.The polyclonal antibodies serum was prepared by immunizing rabbits with the recombinant P48 protein.An indirect ELISA antibody detection method for Mycoplasma bovis was established and the conditions of the method were optimized, the critical value was determined.The sensitivity, stability, specificity and coincidence rate of the established method with plate agglutination test were tested.【Result】 The molecular weight of P48 protein was 62.68 ku and its purity was over 90% by SDS-PAGE.Western blotting test results showed that P48 protein was only bound to the corresponding antibody and showed a single band, which showed good specificity and reactivity.The positive serum titer was 1:64 by agar diffusion method.The optimized results of the established indirect ELISA detection method were as follows:The antigen-coated concentration was 0.3 μg/mL, 1% BSA was blocked at 37℃ for 120 min, the serum to be tested was diluted at 1:1 280, incubated at 37℃ for 60 min, the secondary antibody dilution was 1:5 000, incubated at 37℃ for 60 min, and o-phenylenediamine was colored at room temperature for 10 min.D_{492 nm} value>0.3196 was positive, D_{492 nm} value<0.3101 was negative, 0.3101 ≤ D_{492 nm} value ≤ 0.3196 was suspicious.The results of sensitivity test showed that when the dilution of positive sample was 1:2¹⁸, the interpretation of positive serum was still positive.The stability test results showed that the coefficient of variation of the 96-well plates of 3 batches of P48 protein coated was less than 10%.There was no cross reaction with positive serum of Mannheimia haemolytica and Pasteurella.Compared with plate agglutination test, the coincidence rate of positive serum and negative serum was 100%(18/18) and 97.5%(39/40), respectively.【Conclusion】 The indirect ELISA antibody detection method for Mycoplasma bovis P48 established in this study had good sensitivity, specificity, and stability, providing a detection method for Mycoplasma bovis antibody detection and Mycoplasma bovis vaccine efficacy detection

Bovine viral diarrhea virus (BVDV) is one of the important pathogens affecting the cattle industry in the world, which has caused huge economic losses to animal husbandry.BVDV consists of two biological types and a variety of different genetic subtypes, which have different antigenicity, cytopathology and pathogenicity.Although countries around the world have made many efforts to control and prevent the occurrence and prevalence of BVDV, there are still reports of BVDV infection in animals.The author mainly discussed the molecular biology, pathogenesis and host immune response to viral infection, especially immune escape strategy, immune prevention and control of BVDV.Some immune escape strategies of BVDV by hijacking the host immune system to ensure successful replication of the virus were described, in order to provide reference for curbing the emergence and spread of BVDV, so as to achieve purification and control of the virus.

【Objective】 The aim of this experiment was to express and purify African swine fever virus (ASFV) p72 protein and prepare the monoclonal antibodies.【Method】 The prokaryotic expression vector pET-28b-p72 was constructed and transformed into Escherichia coli BL21(DE3) competent cells for inducing expression and protein purification.The purified p72 protein was used to immunize BALB/c mice, and the spleen of the immunized mice was fused with myeloma cells to prepare monoclonal antibodies.The positive hybridoma cells secreting antibodies were screened by indirect ELISA and subcloning.Antibody types were detected by mouse antibody type detection kit.Ascites of monoclonal antibody against ASFV p72 protein was prepared by in vivo induction method, purified by saturated ammonium sulfate precipitation method, and the titer and specificity of monoclonal antibody were detected by indirect ELISA, indirect immunofluorescence assay (IFA) and Western blotting.【Result】 The recombinant protein of ASFV p72 was expressed in the form of inclusion bodies in Escherichia coli.Four monoclonal cell lines secreting IgG1 antibody were obtained, named 6F8, 7C3, 8H7 and 9G2, respectively.Indirect ELISA results showed that the titer of the antibody secreted by 6F8 and 8H7 cell lines was 1:6 400, and the titer of the antibody by 7C3 and 9G2 cell lines was 1:28 000.In addition, the antibody secreted by four cell lines was positive in reaction with p72 protein, but negative for Pseudorabies virus (PRV), Classical swine fever virus (CSFV), Porcine parvovirus (PPV), Porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine epidemic diarrhea virus (PEDV). The antibodies secreted by four monoclonal cell lines could react specifically with p72 eukaryotic protein.【Conclusion】 Four cell lines that could secrete monoclonal antibodies specifically bound to ASFV p72 protein were successfully prepared, which laid a foundation for further investigation of the function of ASFV p72 protein and the prevention and diagnosis of African swine fever.

At present, cattle reproduction barrier is one of the important factors affecting the development of cattle industry.The uterus is the heart of new life.Uterine microecological imbalance can not only damage endometrial function, but also affect ovarian function, leading to infertility and causing huge losses to animal husbandry economy.Recent studies have found that there is a microbial community in bovine uterus, which contains pathogenic bacteria and opportunistic bacteria in addition to the normal flora, and these microorganisms will dynamically change with different growth cycles and different clinical conditions of cattle.The pathogenic microorganisms inducing bovine reproductive diseases mainly include bacteria, fungi and viruses, which destroy the homeostasis of the reproductive tract by infecting epithelial cells or secreting toxic metabolites.The normal microbiota can maintain the microecological balance of the reproductive tract by interacting with the immune system to prevent the colonization of pathogenic microorganisms.Therefore, microbiota plays an important role in cattle production and reproduction.Although the research on the factors affecting the changes of microbiota is not deep enough, it has been confirmed that hormones, health status, and environmental factors have different effects on the abundance and diversity of microorganisms in bovine uterus.The author compared the microbial composition of healthy and diseased bovine uterus, and further analyzed the factors of uterine microbial imbalance, so as to provide reference basis for the prevention and treatment of bovine uterine related diseases.

【Objective】 The purpose of this experiment was to study the protection of Macleaya cordata leaf extract (MCLE) on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) mice, and provide a theoretical basis for resource utilization and drug development of Macleaya cordata leaves.【Method】 Sixty C57BL/6 mice were randomly divided into 6 groups:Control group (CON), dextran sodium sulfate (DSS) model group, mesalazine (MES) positive control group (200 mg/kg), and MCLE high (MCLE-H, 450 mg/kg), medium (MCLE-M, 150 mg/kg) and low (MCLE-L, 50 mg/kg) dose groups.The test lasted for 14 days.The therapeutic effect of MCLE on DSS-induced colitis in mice was evaluated by general signs, body weight change, disease activity index (DAI), spleen index, colon length and histopathology.The expression levels of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α) in serum and colon tissues were detected by ELISA and Real-time quantitative PCR, respectively.The contents of myeloperoxidase (MPO), nitric oxide (NO), catalase (CAT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) in colonic tissue were determined by colorimetry.The effect of MCLE on intestinal flora was determined by analyzing 16S rRNA gene sequence.The content of short chain fatty acids (SCFAs) in colonic contents was determined by gas chromatography.【Result】 MCLE treatment significantly inhibited the weight loss and colon length shortening of UC mice (P<0.01), and significantly reduced DAI, spleen index and colon histopathological score (P<0.05).Moreover, MCLE significantly down-regulated the expression of IL-1β, IL-6 and TNF-α in colon and serum (P<0.05), decreased the content of MPO, NO and MDA in colon tissue (P<0.05), and increased the content of GSH-Px, SOD and CAT (P<0.05).At the same time, compared with DSS group, the abundance of beneficial bacteria such as Bacteroides, Alistipes and Akkermansia increased in MCLE administration group, and the proportion of pathogenic bacteria such as Desulfovibrio decreased.In addition, MCLE treatment also significantly increased the levels of SCFAs (acetic acid, propionic acid, butyric acid) in intestinal contents of UC mice (P<0.05).【Conclusion】 MCLE might play an anti-colitis role by regulating the level of inflammatory factors, reducing the effects of oxidative stress, maintaining intestinal flora homeostasis and restoring the production of SCFAs.

【Objective】 Tetracycline-resistant Escherichia coli was isolated and identified from duck samples collected in Qingdao, Shandong province, investigating their epidemiological distribution pattern and conducting drug sensitivity testing and identification of drug resistance genes for drug-resistant strains, and investigating the multi-drug resistance rate of tetracycline-resistant Escherichia coli and the carriage of drug resistance genes, so as to provide a theoretical basis for the rational application of antibiotics.【Method】 Cloacal swabs and cecum samples of broiler ducks were used as test materials to isolate Escherichia coli by tetracycline selective medium.The sensitivity of drug-resistant strains to 12 kinds of antibacterial drugs was determined by agar dilution method.PCR technology was used to detect the drug-resistant gene carried by tetracycline-resistant Escherichia coli.Further the transferability of tet(X) gene was assessed by conjugation experiments, furthermore, whole genome sequencing was performed on some tet(X) gene positive strains, at the same time, the genetic environment characteristics of drug resistance gene tet(X) was explored.【Result】 After isolation and identification, 48 strains of tetracycline-resistant Escherichia coli were obtained from 63 samples, with an isolation rate of 76.19%.The drug sensitivity test results showed that all strains were resistant to tetracycline and oxytetracycline, and the resistance rates for doxycycline and florfenicol were 93.75% and 89.59%, respectively, and the strains showed different levels of resistance to polymyxin, tigecycline and meropenem, which were 64.58%, 16.67% and 4.17%, respectively, and all strains had multiple drug-resistant phenomena, of which 7 drugs were most resistant.The results of drug resistance gene detection basically corresponded to the drug resistance situation, and the corresponding resistance genes of drugs with high resistance rates showed higher carriage rates, among them, the prevalence rates of floR, tet(A), qnrS, mcr-1, and bla_CTX-M genes were higher, which were 93.75%, 83.33%, 79.17%, 77.08% and 47.92%, respectively.Among 8 tigecycline-resistant strains, only 2 strains carried the known gene tet(X4), of which 1 tet(X4) gene had the transferability.Similarly, the carbapenem resistance gene bla_NDMwas detected in only one of the two meropenem-resistant strains, and no known resistance gene was detected in any of the other strains, suggesting that unknown tigecycline/carbapenem resistance mechanisms might exist in these strains.In contrast, 6 of the 37 mcr-1 gene-positive strains were present and still showed sensitivity to polymyxin, indicating that the mcr-1 gene resistance function in these bacteria was not successfully exerted.【Conclusion】 Tetracycline-resistant Escherichia coli had been widely prevalent in broiler ducks, and the phenomenon of multiple drug resistance mediated by multiple drug resistance genes had been very serious, and the drug resistance mechanisms were complex and diverse, further increasing the risk of food safety and threatening human health.Therefore, the monitoring and research of drug-resistant bacteria of duck origin should be strengthened to improve the level of rational drug use in breeding.

【Objective】 The aim of this study was to investigate the effect of yersiniabactin (Ybt) on duodenum inflammation induced by Escherichia coli (E.coli) in mice, and provide new theoretical basis for the mechanism of intestinal inflammation induced by E.coli from pigs.【Method】 The R package was used to mine the gene expression omnibus (GEO) datasets of E.coli infection cells for differential gene volcano plot, heatmap enrichment, and KEGG pathway analysis.Molecular docking software was used to predict the binding of Ybt with Toll-like receptor 4 (TLR4).Mice injected with lipopolysaccharide (LPS) were used as positive control, and mice were infected with E.coli ZB-1 and previously constructed Ybt-deficient strain ZB-1ΔHPI.Duodenum damage after infection was observed by HE staining.Real-time quantitative PCR was used to detect mRNA levels of TLR4, nuclear factor kappa-B (NF-κB), interleukin 18 (IL-18), and IL-1β.ELISA was performed to measure the secretion levels of pro-IL-18, pro-IL-1β, IL-18 and IL-1β in the intestines.Immunohistochemistry (IHC) staining was used to observe the expression and localization of inflammatory factors IL-18 and IL-1β in the intestines.【Result】 Volcano plot and heatmap analysis of public databases showed significant upregulation of TLR4, NF-κB, IL-1β and chemokines (CXCL1, CXCL3, CXCL5, and so on) in response to E.coli infection (P<0.05).KEGG enrichment analysis revealed the involvement of TLR4/NF-κB pathway in E.coli infection.Additionally, molecular docking showed that Ybt could bind to TLR4.HE staining demonstrated that E.coli infection with Ybt induction resulted in more severe duodenum inflammation and damage in mice.Real-time quantitative PCR results showed that mRNA levels of TLR4, NF-κB, IL-18, and IL-1β were significantly higher in ZB-1 group compared to ZB-1ΔHPI group (P<0.05).ELISA results showed that ZB-1 group significantly promoted the release of pro-IL-Iβ, IL-Iβ, pro-IL-18 and IL-18 compared to ZB-1ΔHPI group (P<0.05).Furthermore, IHC results showed abundant expression of IL-18 and IL-1β in duodenum villous epithelial cells, and Ybt producing E.coli infection significantly enhanced this result (P<0.05).【Conclusion】 Ybt exhibited a strong pro-inflammatory effect and promotes E.coli-induced duodenum inflammation in mice through the TLR4/NF-κB pathway.These finding providde new theoretical evidence for the mechanism of duodenum inflammation induced by porcine-derived E.coli.

【Objective】 The purpose of this study was to isolate and identify the pathogenic bacteria that caused a large number of acute deaths in carp, determine their virulence genes and drug sensitivity, and provide reference for the subsequent clinical treatment of sick carp.【Method】 The pathogenic bacteria were isolated and cultured from the viscera of diseased carp.Morphological observation, Gram staining, physiological and biochemical identification and 16S rRNA molecular identification were performed.The drug sensitivity and pathogenicity of the pathogen were analyzed by virulence gene detection, K-B drug sensitive disk method and artificial regression infection test.【Result】 The isolated bacteria showed round or oval colonies on LB solid medium.Microscopic examination showed short rod-like Gram-negative bacteria with certain locomotor ability.The results of physiological and biochemical tests showed that the isolated strains showed positive reactions in D-glucose, gelatin, urease, oxidase, arginine, ornithine, lysine and citric acid tests, while the other indexes showed negative reactions, which was consistent with the physiological and biochemical characteristics of Pseudomonas aeruginosa.The phylogenetic tree of 16S rRNA gene sequence showed that the isolated bacteria showed high homology with Pseudomonas aeruginosa NR_114471.1, with 99% similarity.Based on the above test results, the isolated bacteria was identified as Pseudomonas aeruginosa and named HB2210.Virulence gene detection showed that the strain contained several virulence genes, such as flagellin structure gene fla gene, aerolysin aer gene, cytotoxic enterotoxin act gene, outer membrane protein ompA gene and heat stable enterotoxin AST gene. The strain was found to be sensitive to nine antibiotics including ceftazidime, ceftriaxone, cefoperazone, norfloxacin, ofloxacin, ciprofloxacin, polymyxin B, levofloxacin and piperacillin by drug sensitivity testing.And it was found to be acutely virulent to common carp by artificial regression infection tests, with mortality rates of up to 85% in the high concentration group.【Conclusion】 In this experiment, Pseudomonas aeruginosa HB2210 isolated from diseased common carp tissues had strong virulence, and nine antibiotics such as ceftazidime, ceftriaxone, cefoperazone, norfloxacin, ofloxacin, ciprofloxacin, polymyxin B, levofloxacin, and piperacillin, etc.screened out by sensitization test, and could provide the theoretical basis of the precise medication for common carp aquaculture enterprises.

【Objective】 The purpose of this experiment was to understand the antibiotic resistance present status and virulence factor carrier of swine origin Enterococcus, provide data support for the monitoring of Enterococcus antibiotic resistance status and provide scientific basis for formulating reasonable treatment plan.【Method】 In this study, 156 swine small intestine samples from a large slaughterhouse in Guangzhou were isolated, cultured, Gram stained, and identified by PCR.The minimum inhibitory concentration of 14 kinds of antibiotics and 2 kinds of disinfectants were determined by agar diffusion method, and the carrying of drug resistance genes, virulence genes, and antiseptic resistance genes were detected by PCR technology.【Result】 The results showed that 84 strains of Enterococcus were isolated in this research (the separation rate was 53.85%), including 44 E.faecalis and 40 E.faecium.Isolation and culture showed that the colony edges were smooth and neat, round or oval arranged, and the medium around the colony showed black colonies.Suspected colonies of Gram positive cocci that appear circular after Gram staining, arranged individually or in piles.The DNA of the suspected colony was amplified by PCR.The bands of about 941 bp were amplified by E.faecalis primer, and the bands of about 1 500 bp were amplified by 16S rRNA primer.Drug sensitivity test results showed that 44 strains of E.faecalis were highly resistant to clindamycin, doxycycline, ceftiofur, gentamicin, erythromycin, benzalkonium chloride and flufenicol, and antibiotic resistance rates were 97.7%, 90.9%, 88.6%, 88.6%, 81.8%, 81.8% and 77.3%, respectively.40 strains of E.faecium were resistant to clindamycin, ceftiofur, doxycycline, gentamicin, flufenicol and erythromycin, and the resistance rates were 90.0%, 90.0%, 85.0%, 77.5%, 72.5% and 67.5%, respectively.The frequency of efa, gelE and fsrC genes were found to be high in the E.faecalis virulence genes, and the detection rates were 95.5%, 84.1% and 84.1%, respectively.The detection antiseptic resistance gene rates of emeA and gsp65 genes were 95.5% and 90.9%, respectively.Only fsrB virulence gene was detected in E.faecium, the detection rate was 2.5%, while the isolates were negative for any antiseptic resistance genes.【Conclusion】 The isolated Enterococcus showed high resistant to most antibacterial drugs, and a part of Enterococcus were resistant to linezolid, so swine-derived Enterococcus epidemiology needed to be monitored.

【Objective】 The aim of this study was to explore the protective effect of self-constituted prescription Zhen-Ju decoction on mice with acute liver injury caused by three different causative factors.【Method】 The main components and contents of Zhen-Ju decoction were determined by high-performance liquid chromatography (HPLC).The antioxidant and anti-inflammatory effects of Zhen-Ju decoction were investigated using 1, 1-diphenyl-2-picrylhydrazyl radical(DPPH) scavenging rate, hydroxyl radical scavenging rate, total reducing capacity and inhibition of 5-lipoxygenase(5-LOX) activity.42 mice were randomly divided into 7 groups to perform 24 h oral acute toxicity test and 21 d accumulation toxicity test to evaluate its safety.An additional 113 mice were selected, of which 8 were randomly selected as the blank control group (Control), and the remaining 105 were randomly assigned equally to three drug-induced acute liver injury trials, with 35 mice in each trial.Each acute liver injury test group was randomly divided into Model group, positive drug Silybum marianum group (Silybin) and high, medium and low dose Zhen-Ju decoction groups, with 7 rats in each group.Control and Model groups were oral administrated with saline for 14 d, Silybin group was oral administrated with 50 mg/kg Silybin for 14 d, the high, medium, and low dose groups were oral administrated with 10.0, 5.0, and 2.5 g/kg of Zhen-Ju decoction for 14 d, respectively. On the 15th day, acute liver injury models were induced with carbon tetrachloride (CCl₄), acetaminophen (APAP), and alcohol (Alcohol), respectively. Mice were executed at 16 or 20 d, then detected changes in liver pathology, serum biochemical parameters, antioxidant parameters, inflammatory factors and liver-specific gene miRNA (miR-122).【Result】 According to the determination by HPLC, each 1 g of Zhen-Ju decoction formula contained 0.27 mg of rutin, 2.72 mg of lignan, 0.26 mg of isochlorogenic acid A and 0.28 mg of berberine hydrochloride.The DPPH radical scavenging half maximal inhibitory concentration(IC₅₀) and hydroxyl radical scavenging IC₅₀ of Zhen-Ju decoction were 2.394 and 104 mg/mL, respectively, with a total reducing capacity of 3.73±0.04 and an anti-inflammatory rate of 75.97%±3.11%.The oral acute toxicity test and accumulation toxicity test showed that Zhen-Ju decoction was safe and non-toxic.In the pathological model of acute liver injury caused by CCl₄, APAP and Alcohol, the liver tissues of mice in Model group showed different degrees of lesions, and different doses of Zhen-Ju decoction and positive drug Silybin group showed some degree of improvement.Compared with Model group, after the high-dose intervention of Zhen-Ju decoction, the liver serum biochemical indicators glutamic oxalyl transaminase (AST), glutamic alanine transaminase (ALT), triglyceride (TG) and total cholesterol (T-CHO) were significantly lower (P<0.05), the antioxidant index malondialdehyde (MDA) was significantly lower (P<0.05) and superoxide dismutase (SOD), total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px) were significantly higher (P<0.05), the inflammatory factors interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) and miR-122 were significantly lower (P<0.05).【Conclusion】 Zhen-Ju decoction was a safe and non-toxic formula, and oral administrated with 10.0 g/kg Zhen-Ju decoction could significantly improve liver lesions and increase the antioxidant capacity of mouse liver, and had a good protective effect against acute liver injury caused by CCl₄, APAP and Alcohol.

Salbutamol (SAL) is a selective beta stimulant that can reduce animal fat deposition and improve carcass lean meat percentage, but the accumulation of SAL in animal products can cause food poisoning, a serious threat to human health.Since 2002, SAL has been listed as a prohibited drug in the aquaculture industry in China, but there are still frequent reports of poisoning caused by SAL residue in pork, so it is of great significance to detect the residue of SAL in animal-derived food.In recent years, with the emergence of new materials and the rapid development of laboratory detection technology, the detection methods of SAL residue have made progress in terms of sensitivity, specificity and detection efficiency.The author reviewed the progress of SAL detection technology in recent years, including chromatography, immunology analysis, surface enhanced Raman spectroscopy, sensor analysis, electrospray extraction ionization mass spectrometry, etc., in order to provide help for rapid and accurate detection of SAL residue in large-scale aquaculture industry.

【Objective】 This study was aimed to investigate the protective effect of Poria cocos polysaccharide (PCP)on lipopolysaccharide (LPS) induced inflammation in crandell rees feline kidney (CRFK) and its anti-inflammatory mechanism.【Method】 MTT method was used to detect the changes of cell activity by different concentrations of PCP (5, 10, 15, 25, 35 and 45 μg/mL) and LPS (20, 40, 60, 80, 100, 125 and 150 μg/mL) on CRFK.The LPS-induced CRFK inflammation model was treated with different concentrations of PCP to observe the morphological changes of cells.Nitric oxide (NO) release and cell apoptosis in CRFK were detected by nitric reductase method and flow cytometry, respectively.Real-time quantitative PCR and Western blotting were used to detect the expression levels of genes and proteins related to cell inflammation and apoptosis.【Result】 5-25 μg/mL PCP was non-toxic to CRFK and could reverse the cell morphological changes of CRFK after LPS stimulation.25 μg/mL PCP significantly decreased NO release and apoptosis rate in CRFK after LPS stimulation (P<0.05).Real-time quantitative PCR results showed that different concentrations of PCP could significantly reduce the mRNA expressions of IL-6, TNF-α, Caspase3, Caspase8, Caspase9, Bid and Bax in LPS-induced CRFK, and significantly up-regulate the mRNA expression of Bcl-2 (P<0.05).Western blotting results showed that the expression levels of TLR4, NF-κB, Caspase3 and Caspase9 proteins in LPS-induced CRFK were significantly increased (P<0.05), but after PCP treatment, the expression levels of TLR4, NF-κB, Caspase3 and Caspase9 proteins were significantly decreased (P<0.05).【Conclusion】 PCP could inhibit LPS-induced cell inflammatory damage of CRFK by regulating apoptosis, and its mechanism might be related to the inhibition of TLR4-NF-κB signaling pathway.The results provided a new target and experimental basis for the treatment of CRFK inflammation by PCP.

【Objective】 The purpose of this study was to investigate the pathogenic mechanism of Duck enteritis virus (DEV) and Clostridium perfringens type A (CpA) co-infection.【Method】 Healthy non-immune ducklings at 37 days of age were randomly divided into control and co-infected groups.The co-infected group were gavaged once a day in the morning and evening, with 8 mL of CpA bacterial solution (1×10⁸ CFU/mL) for 5 consecutive days, and after 72 h of initial gavage, the DEV-GZ strain virus solution (0.2 mL, TCID₅₀=3.16×10^-9 TCID₅₀/0.1 mL) was inoculated into the leg muscles, and control group was inoculated with sterilized saline 0.2 mL.The clinical lesions and ileal dissection lesions of the ducks were observed.Bacterial isolation and identification and PCR were used to detect whether the co-infection model was successfully established by DEV and CpA.The ileum of control and co-infected ducks was collected at 90 h of infection, and liquid chromatography-mass spectrometry (LC-MS) was used for metabolomic analysis of the ileum of control and co-infected ducks at 90 h of infection to screen and analyze potential differential metabolites and related signaling pathways.【Result】 A duck DEV and CpA co-infection model was successfully constructed after testing. A total of 36 different metabolites were found in the ileum of ducks in control and co-infected groups.16 different metabolites were found in the positive ionization mode, mainly including 2'-deoxyinosine, putrescine, 8(z), 11(z), 14(z)-eicosatrienoic acid, cytidine, and 1-oleoyl racemic glycerol, and 20 different metabolites were found in the negative ionization mode, mainly including oleic acid, arachidonic acid, 11(z), 14(z)-docosa-2-dienoic acid, docosa-2-trienoic acid and linoleic acid.The main metabolic pathways enriched in differential metabolites were 10, mainly tryptophan metabolism, pyrimidine metabolism, purine metabolism and so on.【Conclusion】 11(z), 14(z)-docosadienoic acid, docosatrienoic acid, and arachidonic acid could be sensitive biomarkers in the ileum of ducks co-infected with DEV and CpA, suggesting that the co-infection of DEV and CpA exacerbated the inflammation of the ileum in ducklings, and that tryptophan metabolism pathway suggested a link between the changes in the immunity in ducks co-infected with DEV and CpA.This study laid a foundation for elucidating the pathogenic mechanism of DEV and CpA infection.

【Obgective】 The aim of this study was to investigate the therapeutic clinical effect of canine umbilical cord mesenchymal stem cells (UC-MSCs) combined with trochlear groove reconstruction in the treatment of patellar dislocation.【Method】 Twenty dogs with patellar dislocation were randomly divided into stem cell treatment group and conventional surgery group, with 10 dogs in each group. On the day of surgery, 0.5 mL UC-MSCs (10⁶/kg) suspension was injected into the joint cavity of the stem cell treatment group, and the same amount of normal saline was injected into the conventional surgery group.The therapeutic effect of UC-MSCs was evaluated by collecting the basic information of the affected dogs, follow-up records, blood routine and blood factor content detection, and imaging methods. 【Result】 Compared with the conventional surgery group, the interleukin-6 (IL-6) content in the stem cell treatment group was extremely significantly or significantly decreased at the 1st and 7th day after surgery (P<0.01 or P<0.05).There were no significant differences in transforming growth factor-β1 (TGF-β1), matrix metalloproteinase 13 (MMP-13), tumor necrosis factor α (TNF-α), total number of white blood cells and neutrophils between two groups (P>0.05). Upon reassessment 30 days postoperatively via digital X-ray, the stem cell treatment exhibited pronounced cartilage and bone regeneration at the bone lesion location, with no complications such as recurrence and arthritis observed.In the conventional surgery group, the bone growth at the bone injury site was slow, and 2 patients suffered from re-dislocation of patella (2/10).【Conclusion】 After trochlear groove reconstruction, intraarticular injection of UC-MSCs could promote cartilage and bone tissue growth and reduce the risk of recurrence under conventional post-operative care (anti-inflammatory, antibacterial, and pain reliever).UC-MSCs combined with trochlear groove reconstruction could improve the clinical treatment of patellar dislocation in dogs.

【Objective】 This experiment was conducted to investigate the effects of astaxanthin (AST) on the expression of antioxidation and inflammation related genes and glucocorticoid receptor (GR) proteins in liver of Channa argus induced by lipopolysaccharide (LPS).【Method】 500 healthy Channa argus (23.40 g±0.53 g) were selected and randomly divided into 5 groups, with 5 replicates in each group, and 20 in each replicate.The control group was fed with basic feed, and the test groups were fed with test feed containing 0 (LPS group), 50, 100 and 200 mg/kg astaxanthin, respectively.The test period was 56 days.At the end of the test period, the test group was intraperitoneally injected with 4 mg/kg BW LPS the next day, and the control group was injected with the same volume of PBS.After 24 h, liver samples were collected to detect the expression of antioxidant and inflammatory genes and glucocorticoid receptor(GR) protein.【Result】 ① Compared with the control group, the levels of interleukin-6 (IL-6) and nuclear factor-κB p65(NF-κB p65) genes in the liver of Channa argus were significantly upregulated in LPS group (P<0.05).Compared with LPS group, the liver IL-6 and NF-κB p65 genes expression levels were significantly downregulated in the 50, 100 and 200 mg/kg astaxanthin groups (P<0.05), while IL-10 and IκBα genes expression were significantly upregulated (P<0.05).② Compared with the control group, the relative expression levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione sulfotransferase (GST) genes in the liver of Channa argus in the LPS group were significantly lower (P<0.05).Compared with the LPS group, the expression levels of SOD, CAT, GST and GPx genes in the 50, 100 and 200 mg/kg astaxanthin groups increased, except for the non significant difference in relative expression levels of SOD gene in the 50 mg/kg astaxanthin group, all other differences were significant (P<0.05).③The expression level of phosphorylated NF-κB p65(p-NF-κB p65) protein in the liver of Channa argus in LPS group was significantly higher than that of control group (P<0.05).Compared with LPS group, the expression of p-NF-κB p65 protein in 50, 100 and 200 mg/kg astaxanthin group was significantly decreased (P<0.05), and the expression of phosphorylated glucocorticoid receptor (p-GR) protein was significantly increased (P<0.05).【Conclusion】 During the process of LPS induced liver inflammation and oxidative damage in Channa argus, astaxanthin exerts anti-inflammatory and antioxidant effects through the GR-NF-κB pathway at a dose of 50-200 mg/kg.