【Objective】 The aim of this study was to compare the difference of gene expression profile during the developmental process of the backfat tissue in Sujiang pigs, and explore the key genes affecting the backfat tissue development.【Method】 The backfat tissue in Sujiang pigs at the age of 1, 3, 6 and 8 months (M1, M3, M6 and M8) was selected for high-throughput transcriptome sequencing, and the differentially expressed genes (DEGs) were screened for functional enrichment and expression trend analysis.【Result】 Transcriptome sequencing results showed that 1 869, 296 and 1 257 DEGs were identified in M1 vs M3, M3 vs M6 and M6 vs M8 groups, respectively.Functional enrichment showed that Wnt signaling pathway and ECM-receptor interaction were activated in M1 vs M3 group, while PI3K-Akt signaling pathway, AMPK signaling pathway, butanoate metabolism and citrate cycle (TCA cycle) were activated in M6 vs M8 group.Gene expression trend analysis showed that secreted protein, acid and rich in cysteine (SPARC), recombinant cathepsin S (CTSS), Kruppel-like factor 7 (KLF7) and other genes related to cell proliferation and development were highly expressed at 3 months old, while insulin-like growth factor 1 receptor (IGF1R), signal transducer and activator of transcription 1 (STAT1), fatty acid synthase (FASN) and other genes related to lipid metabolism were significantly expressed after 6 months old.【Conclusion】 This study obtained 4 gene expression profiles of backfat tissue in Sujiang pigs at different growth stages, the key genes related to cell proliferation and development were obtained, including SPARC, CTSS and KLF7, the key genes related to cellular lipid metabolism, including IGF1R, STAT1 and FASN, which provided basic data for genetic improvement of backfat thickness in Sujiang pigs.

【Objective】 The purpose of this study was to obtain the eukaryotic expression vector of CDS of interferon regulatory factor 7 (IRF7) gene in Cherry Valley ducks, to study the structure and biological characteristics of IRF7 protein, and to understand the expression level of IRF7 gene in different tissues, so as to lay a foundation for further study of duck innate immune system.【Method】 The CDS of IRF7 gene in Cherry Valley ducks was amplified by RT-PCR, and the eukaryotic expression plasmid was constructed.The protein expression was detected by Western blotting.The effect of Poly(I:C) on the nuclear translocation of IRF7 protein was observed by fluorescence microscope.The nucleotide similarity alignment was performed on the IRF7 gene sequence, and the phylogenetic tree was constructed, and the molecular and structural characteristics of IRF7 protein were analyzed and predicted online by bioinformatics method.Real-time quantitative PCR was used to detect the expression specificity of IRF7 gene in different tissues.【Result】 The CDS of IRF7 gene in Cherry Valley ducks was 1 536 bp and encoded 512 amino acids.Through the eukaryotic expression of the recombinant plasmid, the total protein was extracted and the protein expression was detected by Western blotting.The size of the recombinant protein was about 110 ku.The treatment of duck embryo fibroblasts with Poly (I:C) could cause in the localization of pCMV-C-EGFP-IRF7 in the nucleus.The results of nucleotide similarity showed that the nucleotide sequence similarity of the IRF7 gene between Cherry Valley ducks, Anas platyrhynchos and Gallus gallus was 99.1% and 80.8%, respectively, while the similarity with Xenopus laevis was only 41.8%.The phylogenetic tree results showed that Cherry Valley ducks had the closest genetic relationship with Anas platyrhynchos and Gallus gallus, and the most distant genetic relationship with Xenopus laevis.IRF7 protein was a structurally unstable hydrophobic protein, mainly composed of alpha helix, beta turn and random coil.IRF7 gene was expressed in various tissues, with the highest expression in gallbladder and the lowest in lung.【Conclusion】 The size of the CDS region of IRF7 gene in Cherry Valley ducks was 1 536 bp, encoding 512 amino acids.The IRF7 protein was a structurally unstable hydrophobic protein, and Poly (I:C) stimulation could cause nuclear translocation of IRF7.The expression of this gene varies greatly in different tissues.

【Objective】 This study was to obtain an immortalized porcine alveolar macrophages (iPAMs) with CD163 (cluster of differentiation 163) monoallelic expression by using CRISPR/Cas9 gene editing technique, and explore the characteristics of their mediating Porcine reproductive and respiratory syndrome virus (PRRSV) infection, which laid the foundation for further study on the mechanism of CD163 gene in PRRSV infection.【Method】 Eight single guide RNA (sgRNA) in exon 1 of porcine CD163 gene were designed and introduced into pX458 vector, and the editing efficiency of different sgRNA vectors was detected by T7E1 assay.Three highly active sgRNA vector was electrotransfected into iPAMs, and the monoclonal cells were isolated by flow cytometry.Then genotypic identification, protein expression detection, off-target analysis and PRRSV infection analysis were performed on the obtained monoclonal cells.【Result】 The editing rate of 3 of the 8 sgRNAs was more than 28%.The results of genotype identification showed that 4 iPAMs cell lines with expected CD163 monoallelic expression were selected, with an efficiency of 8%.The results of protein expression detection and off-target analysis showed that the expression of CD163 protein significantly decreased in CD163 single allele expression cell line (P<0.05), and there was no off-target event at the predicted site.PRRSV infection analysis showed that CD163 monoallelic expression cell lines could be infected with PRRSV normally, and there was no significant difference in virus copy number and the expression of PRRSV-N protein between the CD163 monoallelic expression cells and wild-type cells (P>0.05).【Conclusion】 In this study, an iPAMs cell lines normally infecting PPRSV with CD163 monoallelic expression was constructed using CRISPR/Cas9 editing system, which laid a foundation for elucidating the role of CD163 in PRRSV infection and breeding new disease-resistant pig breeds.

【Objective】 The sequence of bone morphogenetic protein-7 (BMP7) gene in Dolang sheep was cloned and bioinformatics analyzed, and the expression patterns of five tissues in three stages of puberty were detected, which provided reference for exploring the biological function of BMP7 gene in Dolang sheep during the puberty.【Method】 The sequence of BMP7 gene in Dolang sheep was amplified by PCR, and cloned and sequenced, the phylogenetic tree was constructed using Mega 7.0 software.The physicochemical properties and structural function of BMP7 protein was analyzed by bioinformatics software.The expression of BMP7 gene in hypothalamus, pituitary gland, ovary, oviduct and uterus of Dolang sheep at prepuberty, puberty and postpuberty were detected by Real-time quantitative PCR.【Result】 The BMP7 gene sequence of Dolang sheep was successfully cloned, which was 1 334 bp long, and the CDS sequence was 1 296 bp which encoded 431 amino acids.The BMP7 gene sequence of Dolang sheep had the closer genetic relationship with Ovis aries and Bos taurus, followed by Homo sapiens, Macaca mulatta and Equus caballus, and had the furthest genetic relationship with Mus musculus and Canis lupus familiaris.Bioinformatics analysis results showed that the molecular formula of BMP7 protein was C₂₂₀₀H₃₃₇₄N₆₃₂O₆₂₈S₁₇, the theoretical isoelectric point (pI) was 8.17, the total number of atoms was 6 851, the instability index was 53.18, the lipid solubility coefficient was 76.96, and the total average hydrophilicity was-0.419.BMP7 protein was an unstable hydrophilic basic protein.The secondary structure was mainly consisted of alpha helix and random coil, and the prediction result of tertiary structure was consistent with the secondary structure.Real-time quantitative PCR results showed that BMP7 gene was expressed in prepuberty, puberty and postpuberty of Dolang sheep, and the expression in hypothalamus was significantly higher than that in other tissues (P<0.05).The expression of BMP7 gene was significantly increased in hypothalamus and uterus from prepuberty to puberty (P<0.05), while decreased significantly from puberty to postpuberty (P<0.05).【Conclusion】 The BMP7 gene sequence of Dolang sheep was cloned successfully, and this gene was expressed in five tissues at three different stages of puberty.The results provided a reference for exploring the role of BMP7 gene in puberty of Dolang sheep.

【Objective】 The purpose of this experiment was to study the effects of lipopolysaccharide (LPS) on tissue injury and oxidative stress of bursae of Fabricius of Magang geese and to explore its regulatory mechanism.【Method】 One hundred 1-day-old Magang geese (half female and half male) were randomly divided into control and LPS group with 5 replicates in each group and 10 geese in each replicate.The goslings in LPS group were injected intraperitoneally with 2 mg/kg LPS at 24, 26 and 28 days, respectively, while the control group was injected with the same amount of saline.After 1 hour of injection at 28 days, the bursa of Fabricius was collected aseptically for histological observation and oxidative stress index detection and transcriptome library construction.Candidate genes related to LPS-induced bursa of Fabricius injury were screened by differentially expressed gene identification, GO function and KEGG pathway enrichment, and protein interaction network analysis.Ten differentially expressed genes were randomly selected and the expressions were verified by Real-time quantitative PCR.【Result】 Histological observation of bursae of Fabricius of Magang goose showed that the structural integrity of bursae of Fabricius was significantly decreased, the cell arrangement in vesicles was disordered and the number of vacuoles increased in LPS group.Compared with the control group, the activity of glutathione peroxidase (GSH-Px) of bursae of Fabricius was significantly decreased in LPS group (P <0.05).A total of 507 differentially expressed genes were obtained in LPS group, of which 277 genes were up-regulated and 230 genes were down-regulated.GO function and KEGG pathway enrichment analysis showed that differentially expressed genes were mainly concentrated in items related to inflammation and immune response, and related to Cytokine-cytokine receptor interaction pathway and Notch and Hedgehog signaling pathways. A total of 27 differentially expressed genes, including TNF superfamily member 10 (TNFSF10), CREB-binding protein (CREBBP), paracentric substance 1 (PCM1), protein tyrosine phosphatase non-receptor type 6 (PTPN6), were screened by binding protein cooperative network analysis.The results of Real-time quantitative PCR showed that the transcriptome sequencing results were accurate and reliable.【Conclusion】 This study explored the differential expression genes in bursa of Fabricius in Magang goose before and after LPS-induced, and identified the Cytokine-cytokine receptor interaction, Notch and Hedgehog signaling pathways and related genes such as TNFSF10, CREBBP and PTPN6 affecting bursa of Fabricius injury, which provide theoretical reference for understanding the molecular mechanism of LPS-induced bursae of Fabricius injury in geese.

【Objective】 This study was aimed to clone the CDS sequence of RY-box transcription factor 6 (SOX6) gene in Xiannan cattle and carry out bioinformatics analysis, and explore the expression rules of SOX6 in different tissues of Xiannan cattle at different months and the differentiation of bovine primary skeletal muscle cells.【Method】 The CDS sequence of SOX6 gene was amplified by PCR using the cDNA template of leg muscle in Xianan cattle. Sequence similarity alignment and phylogentic tree contruction were carried out with other species, and the bioinformatics analysis of SOX6 protein was performed by the online software. The relative expression of SOX6 gene in different tissues of calves (0 month old), young cattle (12 months old) and adult cattle (24 months old) and different differentiation stages of bovine primary skeletal muscle cells were analyzed by Real-time quantitative PCR.【Result】 The CDS region of SOX6 gene in Xianan cattle was 2 526 bp in length, which encoded 841 amino acids.The results of phylogenetic tree showed that Xianan cattle had the closest relationship with yak and the farthest relationship with chicken.SOX6 protein was a weakly alkaline and hydrophilic protein with a molecular weight of 93.35 ku, which located in the nucleus without transmembrane structure and signal peptide.The secondary structure of SOX6 protein was mainly composed of alpha-helix, random coil, extended chain and beta-turn, accounting for 41.26%, 45.54%, 8.09% and 5.11%, respectively.Protein interaction analysis showed that SOX6 had interaction with type Ⅱ collagen α1 (COL2A1), chondroproteoglycan (ACAN), carboxy-terminal-binding protein 2 (CTBP2), and forkhead box 3(FOXJ3).The relative expression of SOX6 gene in leg muscle of Xianan cattle was the highest, and the expression in lung and spleen was the lowest.The expression level of leg muscle in 12 months old was significantly higher than that in 0 and 24 months old cattle (P<0.05).The expression of SOX6 gene increased gradually with the differentiation of bovine primary skeletal muscle cells, and the expression level of SOX6 gene on days 6 was significantly higher than that on days 0, 2, 4 and 8 (P<0.05).【Conclusion】 The CDS sequence of SOX6 gene in Xiannan cattle was successfully cloned, and its encoded protein was unstable, weakly alkaline and hydrophilic.The expression of SOX6 gene was significantly higher in leg muscle of different months than other tissues, and the expression in bovine primary skeletal muscle cells on days 6 of differentiation was significantly higher than that at other periods.The results provided a theoretical basis for further study on the regulatory role of SOX6 gene in the muscle development of Xiannan cattle.

【Objective】 The purpose of this study was to explore the histological expression rules of pleomorphic adenoma gene 1 (PLAG1) in Qinchuan cattle, clone the promoter sequences of PLAG1 gene, predict and analyze the key transcription factor binding sites, so as to provide theoretical reference for exploring the transcriptional regulation mechanism of PLAG1 gene.【Method】 The relative expression of PLAG1 gene in the heart, liver, spleen, lung, kidney, subcutaneous fat, longissimus dorsi muscle and rumen of three 20-month-old Qinchuan adult bulls were detected by Real-time quantitative PCR, and the sequences of the upstream promoter region of PLAG1 gene were cloned.Bioinformatics software was used to predict PLAG1 gene transcription start sites and promoter core regions, and to analyze and screen key transcription factor binding sites in core promoter regions.【Result】 PLAG1 gene was expressed in all tissues of Qinchuan cattle, and the expression of PLAG1 gene in longissimus dorsi muscle was significantly higher than that in other tissues (P<0.05).The full length of the PLAG1 gene promoter sequence was 1 861 bp, and bioinformatics prediction analysis found that the core promoter region of the PLAG1 gene was located at -297-+42 bp. There were highly conserved transcription factor binding sites for Krüppel-like factor 5 (KLF5), cAMP-responsive element-binding protein-1 (CREB1) and early growth response factor 1 (EGR1), and the CpG island was located in the core promoter region of PLAG1 gene.【Conclusion】 PLAG1 gene was highly expressed in muscle tissue, and its core promoter region was located at -297-+42 bp.Transcription factors KLF5, CREB1 and EGR1 might have a regulatory effect on transcriptional activity of PLAG1 gene.The results provided theoretical basis for further investigation of PLAG1 gene transcriptional regulation mechanism.

【Objective】 The purpose of this study was to use CRISPR/Cas9 technique to knock out the peroxisome proliferation-activated receptor γ (PPARγ) gene in porcine small intestinal epithelial cells (IPEC-J2), and to investigate the biological mechanism of PPARγ gene associated with intestinal inflammation at the cellular level.【Method】 According to porcine PPARγ gene sequence (GenBank accession No.:NM_214379), two pairs of sgRNA sequences were designed in exon 2 region.After annealing, DNA double strand was formed and connected to the eukaryotic expression vector gRNA_GFP-T1.The constructed PPARγ vector (forward target vector, reverse target vector) and Cas9-nickase plasmid were co-transfected into IPEC-J2 cells, after G418 drug screening, the monoclonal cell lines were sampled, DNA was extracted, PCR amplified, identified by gel electrophoresis and sequenced to select the cell lines with the best gene knockout effect, the expression of PPARγ protein was detected by immunofluorescence.【Result】 The target vector sequencing results showed that the target sequences were correctly connected.A total of 48 monoclonal cells were obtained after transfection and screening.The sequencing results showed that 6 monoclonal cells showed double peaks, namely KO-15, KO-17, KO-21, KO-25, KO-32 and KO-35, respectively.The KO-25 with higher basal peak was selected for immunofluorescence detection, and the results showed that the expression of PPARγ protein decreased.【Conclusion】 In this study, IPEC-J2 cells with PPARγ gene knockout were successfully obtained using CRISPR/Cas9 technology, which laid the foundation for further research on the function of PPARγ gene in inflammatory response.

【Objective】 This research was aimed to study the isolation, identification and culture characteristics of cannie hematopoietic stem cells (cHSCs) derived from bone marrow, establish the optimal conditions for in vitro culture, and lay a theoretical foundation for clinical application.【Method】 Mononuclear cells(MNCs)were isolated from 3-month-old Beagle dogs by Ficoll density gradient centrifugation.cHSCs were separated by flow cytometer and cultured in vitro.CCK-8 method was used to detect the cell activity of cHSCs in different media, different concentrations of fetal bovine serum (FBS), different cytokines, and different proportions of cHSCs to cBMSCs, and to screen the optimal in vitro culture scheme.Trypan blue staining counting method was used to calculate the proliferation of cHSCs after 1, 7, 14 and 21 days of co-culture in vitro.Flow cytometry and Real-time quantitative PCR were used to detect the positive percentage of CD34 molecules as surface markers of cHSCs in P0, P1, P2 and P3, and the mRNA expression of stemness genes sex determining region Y-box 2 (SOX2) and octamer binding factor 4 (OCT-4), respectively.【Result】 cHSCs were isolated and obtained successfully, and there was no significant difference of cell activity in different medium (P>0.05).In different concentrations of FBS, the cHSCs activity in 5% FBS group was significantly lower than that in 10%, 15% and 20% FBS groups (P<0.05), the cHSCs activity in 10% FBS group was significantly lower than that in 20% FBS group(P<0.05).Under different cytokine conditions, the cHSCs activity in TPO+FLT-3+SCF+IL-6 group was significantly higher than that in TPO+FLT-3 and TPO+FLT-3 group (P<0.05).Under different cHSCs and cBMSCs inoculation ratios, the cHSCs activity in 1:10 group was extremely significantly higher than that in 1:5, 5:1 and 10:1 groups (P<0.01), and the cHSCs activity in 5:1 group was extremely significantly higher than that in 1:5 and 10:1 groups (P<0.01).cHSCs entered the logarithmic growth stage and reached the peak on the 7th day after co-culture with the optimal culture scheme, and the number of cells in cHSCs+cBMSCs+cytokine group was significantly higher than that in cHSCs+ cytokine group (P<0.05).With the passage culture of cHSCs, the cell differentiation potential was changed.The positive percentage of CD34 molecule and the mRNA expression of SOX2 gene in P1, P2 and P3 were extremely significantly lower than those in P0 (P<0.01), and the mRNA expression of OCT-4 gene in P1 and P2 was extremely significantly higher than that in P0 group (P<0.01), the mRNA expression of OCT-4 gene in P1 group was significantly higher than that in P2 (P<0.01).【Conclusion】 In this study, cHSCs were successfully isolated from canine bone marrow.The optimal culture conditions for cHSCs were IMDM medium containing 10% FBS, cHSCs and cBMSCs inoculated at a ratio of 1:10, and TPO+FLT-3+SCF+IL-6 cytokines were added.P1 cHSCs were in logarithmic growth stage with a high positive percentage of CD34, which could provide reference for future clinical application.

In recent years, with the deepening of research in the field of in vitro modeling, it has been found that cell tests have some shortcomings, such as single cell, difficult to simulate complex tissue structure and normal physiological and pathological state of the body, and the high cost of animal tests and related physiological ethics and other issues, organoids have gradually become the focus of attention of scientists.Organoids are derived from the body's own tissues and stem cells, which are 3D structured cell clumps with original tissues and organs formed through 3D culture in vitro.They are highly consistent with the source tissues in terms of tissue structure, cell type and function.The construction of organoid model not only provides more methods for the research of replacing regenerated tissues and organs, but also plays an important role in tissue transplantation and repair.This review mainly introduces the culture methods for promoting organoid maturation, elaborates four methods, including microporous array method, microfluid method, bioreactor method and hydrogel matrix method, reviews the historical evolution of organoid culture system, and analyzes the limitations and future challenges in the field of organoid research in recent years.As well as the application prospect of organoids in emerging engineering technology, it aims to provide more theoretical basis and new ideas for further improvement of organoids culture methods and research and application in related fields.

【Objective】 To explore the effects of enterotoxigenic Escherichia coli (ETEC) on intestinal morphology, tight junction protein and extracellular matrix (ECM) expression of weaned piglets.【Method】 Twelve Duroc weaned piglets aged 35 days were randomly divided into 2 groups (6 replicates per group, 1 pig per replicate):Control group (fed with 20 mL normal saline) and enterotoxin-producing Escherichia coli group (K88 group, fed with 20 mL of 1×10⁹ CFU/mL ETEC K88 suspension), and continuous feeding for 3 days. All piglets were free to eat and drink.On day 1 and day 4, each pig was weighed by repetition and the feed consumption was recorded.The average daily gain (ADG), average daily feed intake (ADFI) and feed to gain ratio (F/G) of each group were calculated.On day 4, the piglets were slaughtered and the intestines were collected, and sliced to observe the intestinal tissue morphology of jejunum and ileum and the content of collagen fiber in the intestines.Real-time quantitative PCR was used to analyze the expression of intestinal compact junction protein gene.Real-time quantitative PCR and western blotting were used to determine the expression of collagen molecules and regulatory enzymes in ECM.【Result】 Compared with control group, the average daily gain of K88 group was significantly decreased (P<0.05), and the average daily feed intake was extremely significantly decreased (P<0.01).The villus height in jejunum and the ratio of villus height to crypt depth in K88 group were extremely significantly decreased (P<0.01), and the villus height in ileum was also significantly decreased (P<0.05).The mRNA expression of Occludin was significantly decreased (P<0.05), and the mRNA expression of Claudin-1 was extremely significantly decreased (P<0.01) in jejunum.At the same time, the mRNA expressions of Occludin and ZO-1 were extremely significantly decreased (P<0.01), and the mRNA expression of Claudin-1 was also significantly decreased (P<0.05) in ileum.The content of collagen fiber in both jejunum and ileum was significantly reduced (P<0.05).The mRNA expressions of type Ⅳ collagen α2 chain (COL4A2), COL5A1 and COL6A1 in jejunum were extremely significantly decreased (P<0.01), and the protein expression of COL4A2 and COL6A1 in the jejunum of K88 group were significantly decreased (P<0.05).The relative mRNA expression of MMP9 in jejunum was significantly increased (P<0.05), while the relative mRNA expression of PAs was highly significantly decreased (P<0.01), and the MMP9 protein content was extremely significantly increased in K88 group (P<0.01).【Conclusion】 In conclusion, feeding ETEC K88 reduced average daily gain and average daily feed intake, damaged intestinal morphology and reduced the expression of intestinal tight junction protein in weaned piglets.In addition, ECM changes could be induced by reducing the content of collagen fibers and the expression of collagen molecules and ECM regulatory enzyme system in the intestine.

【Objective】 The aim of this experiment was to study the effects of different dietary energy and protein levels on growth performance, body size traits, serum biochemical indices and slaughter performance of Xueshan breeding hens during brooding period, and to determine the appropriate dietary energy and protein levels during brooding period.【Method】 A total of 2 250 one-day-old Xueshan breeding hens with close body weight and good health were randomly divided into 9 groups with 3 energy levels (11.7, 11.9 and 12.1 MJ/kg) and 3 protein levels (18.0%, 19.0% and 20.0%) according to a 3×3 two-factor experiment design.Each group had 10 replicates and each replicate had 25 chickens.The experiment lasted for 42 days.Growth performance indicators were measured at the end of the test.Two chickens were selected for blood collection and one chicken was slaughtered for sampling, and serum biochemical indexes and slaughtering performance were measured.【Result】 ①Dietary energy level had significant effects on average daily feed intake (ADFI), average daily gain (ADG), ratio of feed to gain (F/G) and terminal weight of chicks (P<0.05).With the increase of energy level, ADFI and F/G showed a downward trend, while ADG and final body weight showed an upward trend, but evenness had no significant change(P>0.05).Dietary protein level had a significant impact on ADFI (P<0.05), which was decreased with the increase of protein level, but had no significant impact on other growth performance indicators(P>0.05).Dietary energy and protein levels had no significant interaction on growth performance indexes of chicks(P>0.05).②Dietary protein level had significant effects on keel length, serum triglyceride content and abdominal fat percentage of chicks (P<0.05), but had no significant effects on other body size indexes, serum biochemical indexes and slaughter performance indexes(P>0.05). Dietary energy level had no significant effect on the above indexes of chicks (P>0.05), and dietary energy and protein levels had no significant interaction on these indexes(P>0.05).【Conclusion】 Considering the growth performance, body size traits, serum biochemical indices and slaughter performance, it was recommended that the optimal level of dietary energy and protein for breeding hens of Xueshan breeding hens was 12.1 MJ/kg and 20.0%.

【Objective】 The purpose of this study was to explore the effects of herbal tea residue and earthworm hydrolysate on the production performance, behavior and blood indexes of cattle under heat-moisture condition.【Method】 24 healthy fattening Simmental crossbred cattle of 22-24 months with similar body weight were selected and randomly divided into 3 groups:Control group(CON group, feeding basal diet), herbal tea residue group(HT group, feeding basal diet supplemented with 9.5% herbal tea residue) and earthworm hydrolysate group (EF group, feeding basal diet supplemented with 0.37% earthworm hydrolysate).The trial period was 45 days, pre-feeding period was 3 days, and the feeding period was 42 days.The air temperature and humidity of the cowshed were recorded daily, and the temperature-humidity index was calculated.The cattle were weighed on the first and last day of the feeding period.Feed intake was recorded every 7 days, and average daily gain (ADG), average dry matter intake (ADFI) and feed to gain ratio (F/G) were calculated.Serum cortisol (Cor) concentration was determined on the first day of the pre-feeding period, and respiratory rate (RR) was recorded on the first to third day of the pre-feeding period.The standing time, lying time, rumination time, feeding time and RR of beef cattle were recorded from days 39 to 41 of the feeding period.On the 42nd day of the trial period, blood samples were collected before morning feeding to determine serum biochemical indexes, hormone indexes and antioxidant capacity indexes.【Result】 During the trial, the cattle was in a heat-moisture condition, the air temperature was 19.1 to 39.9 ℃, and the humidity was 31.7% to 89.7%.Compared with CON group, ①the concentrations of Cor and heat shock protein 70 (HSP 70) in serum of cattle were significantly decreased (P<0.05), and RR of cattle were significantly decreased in HT and EF groups (P<0.05).②ADG of cattle was significantly increased (P<0.05), and F/G was significantly decreased (P<0.05) in HT group.While F/G in EF group had no significant difference (P>0.05).③The time of feeding and standing of cattle were significantly decreased (P<0.05), and the time of lying of cattle were significantly increased (P<0.05) in HT and EF groups.④The activity of superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) in serum of cattle in HT and EF groups were significantly increased (P<0.05), and the concentrations of malonaldehyde in HT group was significantly decreased (P<0.05).【Conclusion】 Under heat-moisture condition, the heat stress was effectively relieved, and comfort behavior expression could be improved in cattle fed diets supplemented with herbal tea residue or earthworm hydrolysate.The effects of herbal tea residue was better than earthworm hydrolysate on improving growth performance and antioxidant capacity of cattle.

【Objective】 The purpose of this study was to compare the protective effects of porcine bile acid and sheep bile acid on liver and jejunum of heat stressed mice.【Method】 Forty BALB/c healthy male mice aged 7 weeks were randomly divided into blank control group (MOCK group), heat stress group (HS group), heat stress-pig-bile acid group (PBAs group) and heat stress-sheep-bile acid group (SBAs group).Seven days before heat stress, mice in MOCK and HS groups were fed a basal diet, and those in PBAs and SBAs groups were fed the basal diet supplemented with 400 mg/kg pig bile acid and sheep bile acid, respectively.On day 8, mice in MOCK group were placed at room temperature, and mice in other 3 groups were placed in incubators at 36-38 ℃ and 60% relative humidity for 2 h.The blood, liver and jejunum tissues of mice were collected, and the pathological changes of jejunum tissues were observed by HE staining.The content of urea nitrogen (BUN), the activities of alkaline phosphatase (AKP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, and the contents of malondialdehyde (MDA), reduced glutathione (GSH) and the activities of superoxide dismutase (SOD) in liver and jejunum tissue were determined by ELISA.The mRNA expression of heat shock protein 60 (HSP60), HSP70 and inflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and IL-10 genes in liver and jejunum were detected by Real-time quantitative PCR.【Result】 Compared with MOCK group, the villus height of jejunum in HS group was extremely significantly decreased (P<0.01), and the crypt depth was significantly increased (P<0.05).The activity of ALT in serum and SOD activity in liver and jejunum were significantly decreased (P<0.05).The relative expressions of HSP60 and HSP70 genes in liver and jejunum were extremely significantly increased (P<0.01).Compared with HS group, villus height, and the ratio of villus height and crypt depth (V/C) in jejunum of PBAs and SBAs groups were extremely significantly increased (P<0.01).ALT activity of serum and SOD activity of jejunal in SBAs group were significantly or extremely significantly increased (P<0.05 or P<0.01). The contents of MDA and GSH, and the relative expressions of HSP60 and HSP70 genes in liver of PBAs and SBAs groups were extremely significantly decreased (P<0.01), while the relative expressions of IL-1β and TNF-α genes were extremely significantly increased (P<0.01).In addition, the relative expression of HSP60 gene in liver and jejunum of SBAs group was extremely significantly or significantly lower than that of PBAs group (P<0.01 or P<0.05), the relative expressions of IL-1β and TNF-α genes in liver were significantly higher than those in PBAs group (P<0.05).【Conclusion】 Under the condition of heat stress, dietary supplementation of sheep bile acid had better effects than that of porcine bile acid on increasing serum ALT activity, improving antioxidant capacity of liver and jejunum, reducing heat shock protein expression and repairing jejunum structural damage, but porcine bile acid had better anti-inflammatory effect than sheep bile acid.

【Objective】 The purpose of this study was to screen out the key genes, biological processes and signaling pathways affecting the development of ovarian follicles in Duolang sheep treated with superovulation, so as to provide reference for further understand the mechanism of ovarian growth and development in Duolang sheep under superovulation.【Method】 After superovulation treatment of 15 Duolang sheep aged 2-3 years old and in good physical condition, the ovarian tissues at the early (11 d), middle (13 d) and late (15 d) stages of superovulation were taken out for transcriptome sequencing by high-throughput sequencing technology.The reads sequence obtained by sequencing was aligned with the reference genome to screen out the differentially expressed genes.The protein interaction network of the differentially expressed genes was analyzed by STRING database, and the Cytoscape software was used for visual editing.GO function and KEGG pathway enrichment analysis were performed using eggNOG-mapper software.【Result】 A total of 223 differentially expressed genes were screened between days 11 and 13 of supernumerary ovulation ovaries, and 259 interactions were obtained.The differentially expressed genes were significantly annotated in entries such as cell growth, ribonucleoprotein complex, etc.Among them, the genes with significant differential expression included ITGB3 and SNAI1, but no significant enrichment pathways were found.A total of 694 differentially expressed genes were screened between day 13 and 15 of supernumerary ovulation ovaries, and 785 interactions were obtained.The differentially expressed genes were significantly annotated in initiation of DNA replication, DNA replication and DNA metabolic processes, etc.The genes with significant differential expression mainly included CASP3, NOTCH1, WNT2 and RUNX2.There were 2 enriched pathways of DNA replication and cell cycle signalling pathway.A total of 238 differentially expressed genes were screened between days 11 and 15 of supernumerary ovulation ovaries, and 151 interactions were obtained.The differentially expressed genes were significantly annotated in the entries of protein hydrolysis, intracellular signaling, etc.The genes with significant differential expression mainly included PRKAR2B, AQP5 and HEY2.There were 2 significantly enriched pathways of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) signaling pathways.GO function and KEGG pathway results were used to screen out 10 genes with direct or indirect functions in the development of ovarian follicles in sheep:ITGB3, SNAI1, CASP3, PRKAR2B, AQP5, NOTCH1, RUNX2, WNT2, DPP10 and HEY2.【Conclusion】 In this study, 10 differentially expressed genes were screened from different developmental stages of superovulation ovary, and found that their related functional roles were consistent with the mechanism of ovarian follicular development in different periods, indicating that these genes might be the key genes that directly or indirectly affect ovarian follicular development, in order to provide theoretical reference for exploring the mechanism of ovarian follicular development in superovulation.

【Objective】 This study was aimed to investigate the effects of coated folic acid (CFA) and coated taurine (CTau) on semen quality, antioxidant capacity and hormone level in serum and seminal plasma of the heat stressed rams, so as to provide a reference for the relief the heat stress and improvement of semen quality of rams in summer.【Method】 Twenty adult Dorper rams in good body condition with similar body weight and age were selected and randomly divided into 4 groups.The rams in control group were fed with the basal feed(0 mg/d CFA and CTau), the rams in CFA, CTau and CFA+CTau groups were fed the basal feed plus 291.6 mg/d CFA+0 g/d CTau, 0 mg/d CFA+2.916 g/d CTau, and 291.6 mg/d CFA+2.916 g/d CTau, respectively.The trial period was 75 d, with a pretrial period of 15 d and a regular trial period of 60 d.Blood and semen of ram were collected on the 60th day of the trial period, the semen quality indicators (sperm collection volume, sperm viability, sperm concentration, percentage of fast forward progressive motility of sperm, DNA integrity of sperm, plasma membrane integrity and sperm malformation rate), and the total antioxidant capacity (T-AOC), the activity of catalase (CAT) and superoxide dismutase (SOD), the content of malondialdehyde (MDA), and the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone(T), cortisol(COR), triiodothyronine(T₃) and thyroxine(T₄) in serum and seminal plasma were detected.【Result】 ①Dietary CFA and CTau had significant interaction effects on sperm viability (P<0.05), and had extremely significant interaction effects on sperm concentration, DNA integrity of sperm and plasma membrane integrity (P<0.01).Compared with control group, the sperm viability, sperm concentration, DNA integrity of sperm and plasma membrane integrity in CFA, CTau and CFA+CTau groups were extremely significantly increased (P<0.01), the sperm malformation rate in CTau and CFA+CTau groups were extremely significantly decreased (P<0.01).② Compared with control group, the MDA content of seminal plasma in CFA and CFA+CTau groups were significantly decreased (P<0.05), but the other antioxidant indexes had no significant effects (P>0.05).③ Dietary CFA and CTau had a significant interaction effect on COR level in seminal plasma (P<0.05).Compared with control group, the COR level of serum in CFA group and the T₄ level of serum in CFA+CTau group were significantly increased (P<0.05), respectively, the LH level of seminal plasma in CTau and CFA+CTau groups were significantly decreased (P<0.05), the T₃ level of seminal plasma in CFA, CTau and CFA+CTau groups were significantly increased (P<0.05).【Conclusion】 Dietary supplementation of CFA or CTau could improve the antioxidant capacity and hormone secretion levels, and improve the semen quality of heat stressed rams, and adding together were more effective.

【Objective】 The aim of this study was to investigate the polymorphism of myostatin (MSTN) gene and its effect on meat quality traits in sheep, in order to provide effective genetic molecular marker for the selection and breeding of meat performance in sheep.【Method】 The variation sites of MSTN gene in 91 sheep including Dupo sheep (D), Tan sheep (T) and Small-tailed Han sheep (XH) were obtained using liquid phase capture sequencing technology, and the single nucleotide polymorphism (SNP) with significant interspecific differences were screened.A total of 30 sheep (D, T, XH and Dupo×Tan×Small-tailed Han sheep (DTH)) were genotyped for the selected site using in-flight mass spectrometry to analyze its association with meat quality traits in sheep.【Result】 Three genotypes were detected for rs129059715 of MSTN gene in 4 sheep populations, including 15 CC genotypes, 9 CA genotypes and 6 AA genotypes, with C being the dominant allele (except for Dupo×Tan×Small-tailed Han sheep). rs129059715 was in Hardy-Weinberg equilibrium in Dupo, Tan and Dupo×Tan×Small-tailed Han sheep populations (P>0.05), and was in Hardy-Weinberg disequilibrium in Small-tailed Han sheep population (P<0.05).rs129059715 was low polymorphic in Dupo and Small-tailed Han sheep populations (PIC<0.25), and was moderately polymorphic in Tan and Dupo×Tan×Small-tailed Han sheep populations (0.25<PIC<0.50).Association analysis revealed that the backfat thickness and trans-linoleic acid content of CA genotype at rs129059715 were extremely significantly higher than that of CC genotype (P<0.01), the net meat percentage, kidney weight, the contents of fat, undecanoic acid, decanoic acid and decanoic acid of CA genotype were significantly higher than that of CC genotype (P<0.05).The muscle fibre diameter of CC genotype was significantly higher than that of AA genotype (P<0.05), pH of CC genotype was significantly higher than that of CA genotype (P<0.05).There were no significant differences with other traits among different genotypes (P>0.05).【Conclusion】 Through liquid phase capture sequencing technology, rs129059715 of MSTN gene with significant differences was obtained, which had significant effects on the slaughter performance, meat quality and fatty acid content of sheep.rs129059715 could be used as a potential candidate marker for meat production performance and meat quality.

【Objective】 The purpose of this study was to explore the effect of the expression N⁶-methyladenosine (m⁶A) demethylation enzyme FTO on the differentiation of porcine muscle satellite cells, and compare FTO expression and m⁶A methylation modification levels of pigs with different phenotypes.【Method】 Porcine muscle satellite cells on days 0, 2 and 4 of differentiation were collected, and the protein expressions of FTO and myosin heavy chain (MyHC)and mRNA expression of myogenic differentiation factors (MyoD)and myogenin(MyoG) genes related to muscle cell differentiation were detected by Western blotting and Real-time quantitative PCR.The expression of MyHC, a marker of myocyte differentiation, was detected by immunofluorescence assay.Dot blotting was used to detect the methylation level of m⁶A.Overexpressed vector (OE-FTO), blank control (NC), interference vector (siRNA-FTO) and negative control (siRNA-NC) of FTO gene were transfected into porcine muscle satellite cells and induced differentiation, respectively.The expression of FTO and myocyte differentiation related genes and the methylation modification level of m⁶A were detected.Immunofluorescence was used to detect the expression of MyHC and the formation of muscle tubes.At the same time, Western blotting and Dot blotting were used to detect FTO protein expression and m⁶A methylation modification level in different tissues of Large White pigs and Ningxiang pigs.【Result】 Compared with day 0, the expression of FTO and MyHC protein were extremely significantly increased on days 2 and 4 (P<0.01), and the mRNA expression of FTO, MyoD and MyoG genes were significantly or extremely significantly increased (P<0.05 or P<0.01).The methylation modification level of m⁶A was significantly decreased on days 4 (P<0.05).Compared with NC group, the expression of FTO protein in OE-FTO group was extremely significantly increased (P<0.01), and the mRNA expression of FTO and MyHC genes in OE-FTO group were extremely significantly increased on days 0, 1 and 2 of induction differentiation (P<0.01), and the mRNA expression of MyoD gene was extremely significantly decreased (P<0.01).On days 1, 2, 3 and 4, the methylation modification level of m⁶A in OE-FTO group was significantly or extremely significantly decreased (P<0.05 or P<0.01).Compared with siRNA-NC group, the expression of FTO protein in siRNA-FTO group was significantly decreased (P<0.05), and the mRNA expression of FTO and MyHC genes in siRNA-FTO group were extremely significantly or significantly decreased on days 0, 1, 2 and 3 of induction differentiation (P<0.01 or P<0.05).The methylation level of m⁶A was significantly or extremely significantly increased (P<0.05 or P<0.01);The mRNA expression of MyoG gene in siRNA-FTO group was significantly decreased on days 0, 2 and 3 of induction differentiation (P<0.05).The expression of FTO protein in longissimus dorsi muscle of Large White pigs was significantly higher than that of Ningxiang pigs (P<0.05), and the methylation level of m⁶A was significantly extremely lower than that of Ningxiang pigs (P<0.01).【Conclusion】 The expression of FTO had a significant effect on the differentiation of porcine muscle satellite cells, and the methylation level of m⁶A was negatively correlated with the differentiation degree of porcine muscle satellite cells.Interference with FTO inhibited the differentiation of porcine muscle satellite cells and increased the methylation level of m⁶A.Overexpression of FTO could promote the differentiation of porcine muscle satellite cells and reduce the methylation level of m⁶A.This results provided reference for further exploring the regulatory mechanism of FTO in muscle differentiation.

Polydactyly is a common hereditary limb malformations in vertebrates.Equine polydactyly presents additional digits on limbs, which can be classified into three categories:Unilateral polydactyly, bilateral polydactyly, and four limb polydactyly according to the number and positions of toes.The type of unilateral polydactyly is the most popular, accounting for 58% in all.Hereditary variations are the main factors attributed to polydactyly, and the inheritance pattern of polydactyly are relatively complex.Evidence had shown that polydactyly is associated with chromosomal variation in horses.However, due to the scarcity coupled with singleton and long generational intervals, it is difficult to construct large-scale lineages to identify mutations using genome-wide association study (GWAS) and forward-genetic methods.Consequently, the molecular mechanisms and genes of horses polydactyly are still not clear till now.The growing experimental evidence in human and other species illustrated that the signaling pathways controlling limb development are conserved and gene expression is temporal.A total of 199 polydactyly-associated genes are known in different species, and some of them have pleiotropy.Gene similarity analysis revealed 178 homologous genes in horses, of which 56% had >90% similarity and 96% had >80% similarity.Therefore, the polydactyly genes found in other species could be the candidate genes for equine polydactyly, offering valuable insights into the molecular mechanisms of this condition.

【Objective】 This study was aimed to explore the effect of leonurine on the cryopreservation effect of sheep semen, and provide theoretical basis for the preparation of appropriate diluent for cryopreservation of sheep semen in practical production.【Method】 Semen of three Duolang sheep were collected by pseudoginosis method.0 (control), 20, 40, 60, 80 and 100 μmol/mL leonurine was added to semen cryopreserved diluent, respectively.Semen quality indexes such as sperm viability, acrosome integrity and plasma membrane integrity, total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activities, and malondialdehyde (MDA) content were detected during semen cryoppreservation.The optimal concentration of leonuri during semen preservation was screened.After the optimal concentration of leonuri was added, the expression of TNF receptor superfamily member 6 (Fas), Caspase-3, B lymphoblastoma-2 (Bcl-2) and B lymphoblastoma-2-associated X protein (Bax) during cryopreserved semen were detected by Real-time quantitative PCR.The conception rate of ewe was calculated using the method of two intraventricular insemination.【Result】 Compared with control group, the sperm viability, plasma membrane integrity, acrosome integrity, T-AOC level, SOD, GSH-Px and CAT activities of 40 μmol/mL leonurine group were significantly increased (P<0.05), while the content of MDA was significantly decreased (P<0.05). Cryopreservation for 144 h, the indexes of 40 μmol/mL leonurine group were significantly different from those of other leonurine groups (P<0.05).Real-time quantitative PCR results showed that compared with control group, the expressions of Fas, Caspase-3 and Bax genes in 40 μmol/mL leonurine group were significantly decreased (P <0.05), while the expression of Bcl-2 gene was significantly increased (P<0.05).【Conclusion】 In this study, adding 40 μmol/mL leonurine to cryopreservation semen diluent could significantly improve the quality of cryopreservation of Duolang sheep semen, enhance the antioxidant capacity, and played an anti-apoptotic role.The conception rate of ewes with 40 μmol/mL leonurine cryopreservation for 72 h was 75%, which reached the average level of vaginal transfusion of fresh semen.

Because of its strong toxic effect on spermatogonium, busulfan is often used to prepare spermatogonium stem cell transplantation recipients, but the specific toxic mechanism is still unclear, which affects its scientific use and effect improvement.Studies have shown that busulfan can cause oxidative stress and inflammatory response of spermatogenic cells, inhibit autophagy, damage blood-testis barrier, and poison spermatogenic function of testis, but the relationship between them and the regulatory pathway remain unclear.Based on the important regulatory role of nuclear transportation factor κB(NF-κB) pathway in inflammatory response, the author reviewed the research results of oxidative stress, inflammatory response, damage of blood-testis barrier and inhibition of testicular cell autophagy caused by busulfan, and analyzed the possible regulatory role of NF-κB signaling pathway in testicular toxicity of busulfan.The aim of this study was to reveal the toxic mechanism of busulfan to spermatogenesis, and to provide reference for scientific use of busulfan and to avoid environmental toxins.

【Objective】 The purpose of this study was to investigate the effect of catalpol on the inflammatory injury of rat liver sinusoidal endothelial cells (RLSECs) induced by advanced glycation end products (AGEs) via affecting the interaction between Galectin-3 and CD146.【Method】 After incubating RLSECs with 0, 0.1, 1 and 10 μmol/L catalpol for 48 h, the effect of cell proliferation was observed by CCK-8 method.Incubating RLSECs with 10 μmol/L catalpol for 0, 12, 24, 48 and 96 h, and the effect of cell proliferation was observed by the same method as above.Set Control (blank control group), AGEs (AGEs treatment), Cat1 (1 μ mol/L catalpol), Cat10 (10 μ mol/L catalpol) and positive control GB1107 (1 μmol/L GB1107) groups.After Cat1, Cat10 and GB1107 groups were incubated with drugs for 30 min, then all groups were stimulated by 200 μg/mL AGEs except for Control group, the morphological changes of RLSECs in the above groups were observed, and the degree of cell damage was detected by lactate dehydrogenase (LDH) method.The secretion of monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1) were measured by ELISA.The macrophages RAW264.7 were divided into groups and administered with the same method as above.After 48 h, the amount of nitric oxide (NO) released to the cell supernatant in each group was observed with Griess method.RAW264.7 cells were seeded in the Transwell chamber and RLSECs were seeded at the bottom of the well plate.Control, AGEs, Cat10, Cat10+LV Galectin-3-GFP and Cat10+LV Galectin-3-shRNA groups were set up.The last two groups were transfected with lentivirus for 48 h, and then administered with drugs for 30 min respectively, AGEs were added to the culture medium with a final concentration of 200 μg/mL for stimulation except for Control group, and the number of migrated macrophages was observed by crystal violet method 48 h later.After incubation of RLSECs in Control, AGEs, Cat10 and GB1107 groups for 48 h, the co-localization of Galectin-3 and CD146 was observed by immunofluorescence.The protein samples from Control, AGEs and Cat10 groups were used to detect the interaction between Galectin-3 and CD146 and their respective expression levels by Western blotting and Co-IP methods.【Result】 Compared with Control group, there was no significant difference in cell proliferation rate of RLSECs in Cat0.1 group (P>0.05), while the cell proliferation rate of RLSECs in Cat1 and Cat10 groups was significantly increased (P<0.05).Compared with the group incubated for 0 h, the cell profiferation rate of RLSECs was significantly increased in the group with 10 μmol/L catalpol incubated for 48 h. Therefore, cells were incubated with 10 μmol/L catatpol for 48 h for subsequent experiments. Compared with AGEs group, in the Cat1 and Cat10 groups the cell damage induced by AGEs was improved, and the LDH activity of RLSECs supernatant, the release of MCP-1 and ICAM-1 were significantly decreased (P<0.05).Compared with AGEs group, in the catalpol groups the activation of macrophage RAW264.7 was inhibited and the release of NO from cells was significantly reduced (P<0.05).By overexpression of lentivirus vector and knockdown of Galectin-3 of RLSECs and RAW264.7, it was proved that catalpol could significantly improve the infiltration of macrophages by inhibiting the expression of this molecule (P<0.05).Compared with AGEs group, in Cat10 group the binding of Galectin-3 and CD146 was significantly inhibited (P<0.05) and the respective expression of the two molecules did not affect (P>0.05).【Conclusion】 Catalpol could promote the uncoupling effect of Galectin-3 and CD146 molecular complexes induced by AGEs, improved the damage of sinusoidal vascular endothelial cells induced by AGEs and the release of proinflammatory factors, reduced the activation of macrophages and the secretion of NO, and played a protective role in sinusoidal vascular endothelial cells.The mechanism of catalpol to improve the liver injury caused by AGEs deposition in diabetes was preliminarily proved through in vitro experiments.

【Objective】 To provide important experimental materials for the research of African swine fever virus (ASFV) detection method, the monoclonal antibody against p30 protein of ASFV was prepared.【Method】 Recombinant ASFV p30 protein was expressed by Escherichia coli and used to immunize female BALB/c mice aged 6-8 weeks.The splenocytes of the immunized mice were fused with SP2/0 cells by cell fusion technique.The positive hybridoma cells secreting specific antibody against p30 protein were screened by indirect ELISA, and the hybridoma cells were intraperitoneally inoculated into mice to produce ascites antibodies.The class/subclass of the monoclonal antibodies were identified by ELISA, and the reactivity of the monoclonal antibody with p30-N terminal (1-105 amino acids) and p30-C terminal (100-194 amino acids) of p30 protein were identified by Western blotting and indirect immunofluorescence assay (IFA).Different peptides were synthesized for the p30 protein region recognized by the antibody, and the epitopes recognized by antibodies were identified by ELISA and Dot blotting.Indirect ELISA was used to analyze the cross-reactivity of the monoclonal antibodies with ASFV p72 protein, Porcine circovirus type 2 (PCV2) capsid (Cap) protein, Porcine epidemic diarrhea virus (PEDV) S protein, and the blocking effect of ASFV antiserum on the antigen binding activity of monoclonal antibodies.【Result】 Two hybridoma cells (C7 and G10) were obtained, and the monoclonal antibodies (C7 and G10) secreted by the two cell lines belonged to the IgG1 subclass and κ type (IgG1κ).The titers of C7 and G10 in hybridoma cell culture supernatant and ascites were 1:1 280, 1:640, and 1:10⁷, 1:10⁶, respectively.The two monoclonal antibodies were specifically bound to the p30-C terminal (100-194 amino acids) region, and recognized the same epitope ¹¹⁵CTSSFETLFEQEPSSEVPKD¹³⁴.There was no cross reaction with ASFV p72, PCV2 Cap and PEDV S proteins, and anti-ASFV positive serum could effectively block the two monoclonal antibodies from binding to p30 protein.【Conclusion】 Two hybridoma cell lines secreting p30 monoclonal antibody were obtained, and the epitopes recognized by monoclonal antibodies were identified, which enriched the information of the epitopes of p30 protein, and laid an foundation for further research on the pathogenesis of ASFV.

【Objective】 This study was aimed to understand and master the biological characteristics and genetic variation of Bamboo rat parvovirus (BRPV) in Guangxi, and to provide theoretical basis and technical support for the control of BRPV.【Method】 The tissue supernatant was prepared from the diseased bamboo mice from farms, and the virus was isolated by Vero cell synchronous inoculation method.Identification was carried out through methods such as cytopathic observation, PCR, transmission electron microscopy observation, and hemagglutination test, and specific amplification primers were designed and synthesized for sequencing analysis of the whole genome sequence of BRPV.【Result】 The isolated virus could grow and proliferate stably on Vero cells, and the infected cells became long, spindle or thread-like.After purification, virions with a diameter of 20-25 nm were observed by electron microscopy with stereosymmetric, membraneless virions.They were identified as BRPV by PCR, agglutination test and whole genome sequencing.In this study, a total of 3 BRPV (GenBank accession No.:MF497824, MF497825 and MF497826), the total length of the genome of this strain was about 4 758 bp, and the whole genome sequence comparison showed that it was closely related to Bat-derived arvovirus.The amino acid genetic characteristics of BRPV gene have strong correlation with its host specificity.The amino acid variation of NS1 gene was more similar to that of bat and Rat parvovirus, which was in the same branch.Nucleotide homology was 96.9% to 97.6%, and amino acid homology was 97.5% to 98.4%.Amino acids 257 and 360 belonged to high mutation sites.The phylogenetic tree of VP2 gene encoded amino acids showed that the phylogenetic tree was located in an independent branch, with the nucleotide homology of 58.3% to 66.8% and the amino acid homology of 51.6% to 63.0% with rat, dog, cat and other animal parvoviruses.Amino acids at sites 8, 99, 114, 118, 247 and 253 of VP2 peotein showed host bias, and amino acids at sites 53 and 386 were highly mutated.【Conclusion】 The BRPV strains isolated in this study were Parvovirus.The results clarified the genetic characteristics and morphological characteristics of BRPV in China, which laid a foundation for the epidemiological investigation and control of BRPV.

【Objective】 The purpose of this study was to understand the epidemic status of infectious diarrhea pathogens in calves from 13 counties (cities, districts) of Tangshan, and to provide clinical data for the effective prevention and control of diarrhea in calves in this area.【Method】 788 diarrhea fecal samples from 38 dairy farms in Tangshan were used to detect 9 kinds of pathogens Bovine viral diarrhea virus (BVDV), Bovine rotavirus (BRV), Bovine coronavirus (BCV), Escherichia coli, Salmonella, Clostridium perfringens, Coccidia, Cryptosporidium and Giardia lamblia, and the positive rate and mixed infection were analyzed.【Result】 Among 788 calf diarrhea feces samples, the highest positive rate was Escherichia coli, which was 43.53% (343/788).In all tested areas, Luannan county had the highest infection rate of BVDV, with a positive rate of 45.38% (59/130).The infection rates of BRV and BCV in Hangu district were the highest, with the positive rates of 55.00% (22/40) and 37.50% (15/40), respectively.The infection rates of E.coli and G.lamblia in Guye district were the highest, with the positive rates of 68.75% (11/16) and 18.75% (3/16), respectively.The infection rate of Salmonella in Lunan district was the highest, with a positive rate of 35.89% (14/39).The infection rate of C.perfringens in Qian'an county was the highest, with a positive rate of 20.21% (19/94).The infection rates of Cryptosporidium and Coccidia in Lubei district were the highest, with the positive rates of 48.48% (16/33) and 57.57% (19/33), respectively.From the sampling season, the infection rate of BRV was the highest in spring and summer, with the positive rates of 27.71% (46/166) and 39.58% (114/288), respectively.The infection rate of E.coli was the highest in autumn, with the positive rate of 54.08% (106/196).In winter, BVDV infection was the main infection, with the positive rate of 52.17% (72/138).Judging from the mixed infection situation, the double mixed infection of BRV+BCV was the main, and the positive rate was 8.37% (66/788) and there was no mixed infection of five or more pathogens.【Conclusion】 There were 9 kinds of pathogenic infections in diarrhea dairy calves from 13 counties (cities, districts) of Tangshan and the highest positive rate was bacterial pathogen E.coli, followed by viral pathogens BVDV and BRV.

【Objective】 This study was aimed to prepare the polyclonal antibody of Toll-like receptor 7(TLR7) protein of Larus ridibundus, and analyze the expression characteristics of TLR7 protein in DF-1 cells, so as to provide materials for the in-depth understanding of the function and mechanism of TLR7 protein in Larus ridibundus.【Method】 Primers were designed to amplify the CDS region of TLR7 gene in Larus ridibundus, and its prokaryotic and eukaryotic expression vectors were constructed.The recombinant prokaryotic expression plasmid pET32a-TLR7 was converted to Escherichia coli Transetta (DE3) competent cells for in vitro induction of recombinant protein expression, and the conditions for inducing temperature, inducing time and IPTG concentration were optimally screened.New Zealand White rabbits were choosed to prepare polyclonal antibody, the serum antibody valence was detect by indirect ELISA method, and the antibody specificity was identified by Western blotting.The eukaryotic expression plasmid pcDNA3.1-TLR7 was overexpressed in DF-1 cells, and the expression characteristics of TLR7 protein in Larus ridibundus at different time points after transfection of DF-1 cells were detected by indirect immunofluorescence technology.【Result】 The CDS region of TLR7 gene in Larus ridibundus was 1 182 bp.The double digestion results showed two sets of fragments of different sizes of 5 900 and 1 182 bp (prokaryotic expression vectors), 5 428 and 1 182 bp (eukaryotic expression vector), respectively.Sequencing results showed that the prokaryotic and eukaryotic expression vectors were successfully constructed.When the induction temperature in vitro was 30 ℃ and the final concentration of IPTG was 1.0 mmol/L, a large number of recombinant proteins in the form of inclusion bodies were obtained by culture for 5 h, and the protein size was 63 ku.The antiserum valence of indirect ELISA method was more than 1:256 000.The Western blotting assay results showed that the prepared polyclonal antibody had good specificity.The indirect immunofluorescence results showed that green fluorescence could be observed 2 h after transfection of DF-1 cells, the green fluorescence density gradually increased with time, most of the green fluorescence gathered in and around the nucleus, and a small part was scattered in the cytoplasm.【Conclusion】 The prokaryotic expression of TLR7 protein in Larus ridibundus had good immunogenicity, the prepared polyclonal antibody had high reactivity and specificity, and TLR7 protein was successfully expressed in DF-1 cells.

【Objective】 This study was aimed to explore the species and morphological characteristics of the Dactylogyrus parasitized in the Abramis brama orientalis in Irtysh River, and the phylogeny of Dactylogyrus parasitized in Cyprinidae, so as to provide theoretical basis for the phylogeny of Dactylogyrus and its co-evolutionary relationship with host.【Method】 From July 2021 to July 2022, morphological and molecular biological methods were used to identify and phylogenetically analyze Dactylogyrus parasitized in the gills of Abramis brama orientalis.【Result】 According to the morphological observation and chitin structure characteristics, the Dactylogyrus parasitized in the Abramis brama orientalis were identified as Dactylogyrus zandti and Dactylogyrus wunderi, and the structure of their copulatory organ was significantly different.By amplifying the 18S-ITS1-5.8S rDNA and 28S rDNA sequences of two species of Dactylogyrus, and constructing the phylogenetic tree by using Bayesian method (BI) and maximum likelihood method (ML), respectively, the Dactylogyrus zandti and Dactylogyrus wunderi were clustered into one branch, and were located in the same branch as the branchial Dactylogyrus parasitized in the subfamily of Leuciscinae.The phylogenetic trees constructed by different ribosomal sequences were basically the same.Most of the Dactylogyrus parasitized in different subfamily fishes were clustered together, and the Dactylogyrus parasitized in Cyprininae fishes were located at the base of the phylogenetic tree.In the phylogenetic tree constructed by 28S rDNA sequence, the Dactylogyrus parasitized in Cyprininae and Labeoninae fishes were clustered into one branch and located at the base of the phylogenetic tree.【Conclusion】 This experiment described the morphological characteristics of two species of Dactylogyrus parasitized in the Abramis brama orientalis, and supplemented the morphological data of Dactylogyrus.The phylogenetic trees with different ribosomal sequences indicated that there were host transfer and intra-host speciation phenomena in the co-evolution between the Dactylogyrus and its hosts.This study supported Cyprininae fishes as the early hosts of Dactylogyrus, and the phylogeny of Dactylogyrus might be influenced by host specificity and geographical location.

Fatty liver hemorrhagic syndrome (FLHS) is a nutritional metabolism disease that often occurs in high-laying hens, and its clinical manifestations are mainly liver lipid deposition, sudden drop in egg production and acute death caused by layer hen lipid metabolism disorders, resulting in serious economic losses, which is a disease that needs to be focused on in intensive breeding at present.Gut microbiota, an important role in the growth and development of the body and immune metabolism, participate in the lipid synthesis and transport of the host, maintain the integrity of the intestinal barrier to avoid harmful substances from damaging the liver and affecting its function through blood circulation, and the amount and transformation direction of intestinal microbial metabolites will also have a good or bad impact on liver health.At present, it is believed that the pathogenesis of FLHS mainly includes nutrition, genetics, hormones, gut microbiota and other factors, and the "gut-liver axis" axis has also received extensive attention in the current treatment of liver diseases.Taking the gut microbiota as the starting point, by summarizing the common gut microbiota of laying hens and their functions related to lipid metabolism, the relevant research of gut microbiota in lipid metabolism and FLHS was reviewed, and the possible mechanism of action of gut microbiota and their metabolites in the process of FLHS development was introduced, in order to explain the possible pathogenesis of FLHS from the perspective of gut microbiota, find ways to prevent or treat FLHS by regulating gut microbiota in production practice, and promote the development of poultry farming.

【Objective】 The polyclonal antibody of cyclophilin A (Cyp-A) was prepared, in order to provide a basis for revealing the mechanism of mucosal immune response in Bactrian camel.【Method】 The coding region was selected from the Cyp-A gene sequence of Bactrian camel in GenBank (accession No.:XM_010969543.1).The extracellular sequence without signal peptide was intercepted and optimized its base, and the recombinant plasmid pET-28a-Cyp-A was constructed by connecting it with pET-28a (+) vector.The recombinant plasmid pET-28a-Cyp-A was transformed into E.coli BL21 (DE3) competent cell to induce expression.After optimizing the induction conditions, the expression of the recombinant protein was identified by SDS-PAGE.Then the recombinant protein was purified and used to immunize rabbits to prepare rabbit anti-Bactrian camel Cyp-A polyclonal antibody.The antibody titer and specificity were detected by ELISA and Western blotting, respectively.HE and SABC immunohistochemical staining methods were used to observe the location of Cyp-A using self-made polyclonal antibody.【Result】 The recombinant plasmid pET-28a-Cyp-A was mainly expressed in the form of inclusion body.The size of Cyp-A protein was about 17 ku.The optimal induction conditions were 0.7 mmol/L IPTG and 8 h.The optimal elution concentration of the protein was 5 mmol/L imidazole.By ELISA and Western blotting detection, the titer of rabbit anti-Bactrian camel Cyp-A polyclonal antibody was 1:64 000, which could specifically recognize the recombinant protein Cyp-A.HE and SABC immunohistochemical staining results showed that Cyp-A polyclonal antibody could only specifically stain M cells in ileum follicular associated epithelium (FAE).【Conclusion】 The rabbit anti-Bactrian camel Cyp-A polyclonal antibody with high titer and specificity was successfully prepared, which could be used to specifically recognize M cells in ileum of Bactrian camel.

【Objective】 The purpose of this experiment was to explore the prospects of Brucella outer membrane vesicles (OMVs) for use in subunit vaccines, prepare subunit vaccines of Brucella OMVs, and assess the immunogenicity of Brucella OMVs.【Method】 OMVs of Brucella melitensis were prepared by high-speed centrifugation, and the morphological and fractional analyses of the prepared OMVs were performed by transmission electron microscopy, SDS-PAGE and bioinformatics analysis.Mice were immunized after emulsification of OMVs using nanoadjuvant (LDH) and Freund's adjuvant, and the specific antibodies were detected by indirect ELISA method for OMVs at 7, 14, 21, 28 and 35 d after immunization, and the IgG antibody levels in serum of mice post-immunization were detected using the mouse IgG ELISA kit, so as to assess the level of humoral immunity induced by OMVs.The level of cellular immunity induced by OMVs was assessed by isolation of mouse spleen lymphocytes with cytokine (IL-4 and IFN-γ) assay and flow cytometry assay of T lymphocyte subpopulations in the peripheral blood of mice.【Result】 The OMVs were successfully extracted and purified, with diameters ranging from 20 to 160 nm.Bioinformatics analysis showed that the main constituent proteins of OMVs, OMP25, OMP31, OMP16, OMP19, BP26 and SOD were hydrophilic and antigenic proteins, and there were several B and T cell dominant.Compared with PBS group, after immunization with OMVs, the different adjuvant groups induced high levels of specific and IgG antibodies in mice (P<0.01).Meanwhile, OMVs significantly induced IFN-γ production in mouse spleen lymphocytes (P<0.05), and promoted the expression of CD4⁺ T cells (P<0.05).【Conclusion】 This study confirmed that Brucella OMVs of sheep species had strong immunogenicity and could induce strong humoral and cellular immune responses in mice, laid a theoretical foundation for the development of a new subunit vaccine for Brucella.

【Objective】 This study was aimed to investigate the ectoparasite infection of Myotis chinensis (M.chinensis) in Yunnan province, to analyze the factors affecting ectoparasite infection of M.chinensis in Yunnan province, and to provide a scientific basis for the prevention and control of related vector-borne zoonoses.【Method】 The M.chinensis were captured at three sampling sites in Yunnan province between July 2021 and August 2022, and its surface parasites were collected and identified based on identification data.The χ² test (Chi-square Test), independent-samples t-test, linear correlation, and SPSS 26.0 software were used to statistically analyze the infection status of ectoparasite infections in M.chinensis.【Result】 Of the 53 M.chinensis captured, 49 were infected with ectoparasites, with a high rate of ectoparasite infection (92.45%).All 2 841 ectoparasites collected from the surface of M.chinensis belonged to the Gamasina and Nycteribiidae, with S.myoti, S.kolenatii and M.zhijinensis as the dominant species.The mean abundance (t=-4.566, P<0.01) and mean intensity (t=-4.58, P<0.01) of parasite infections were extremely significantly higher in females than in males, while there was no correlation between body size (forearm length and body weight) and mean abundance of parasites.【Conclusion】 The prevalence of ectoparasite infections in M.chinensis in Yunnan province was high, and there were differences in ectoparasite infection between the sexes of M.chinensis, with a sex-bias.There was no significant correlation between the body size of M.chinensis and the mean frequency of ectoparasite infection.The experimental results would fill in the scarce research on bat ectoparasites in China and provide important theoretical guidance for the prevention of certain vector-borne zoonoses.

【Objective】 The purpose of this study was to prepare polyclonal antibody against Bluetongue virus (BTV) NS4 protein and lay a foundation for studying the role of BTV NS4 gene in virus proliferation and antagonizing the innate immune response of host cells.【Method】 The pCzn1-NS4 prokaryotic expression vector was constructed, and the positive plasmid was transformed into Escherichia coli BL21(DE3) competent cells after identification by PCR and sequencing.The recombinant NS4 protein was induced by IPTG and purified with His-tag Ni-sepharose purification, and then identified by SDS-PAGE and Western blotting.Then, New Zealand White rabbits were immunized with the purified protein to prepare its polyclonal antibody.The titer of polyclonal antibody was determined by SDS-PAGE and indirect ELISA.The expression of NS4 protein in BHK-21 cells infected with BTV was detected by indirect immunofluorescence assay (IFA) using NS4 polyclonal antibody as the primary antibody.【Result】 The prokaryotic expression plasmid pCzn1-NS4 was successfully constructed.At 37 ℃ and the induced concentration of IPTG was 0.6 mmol/L, a large amount of recombinant NS4 protein was obtained when induced 8 h.SDS-PAGE and Western blotting results showed that the molecular weight of the recombinant protein was about 18 ku, which could detect by rabbit His-tag antibody.The titer of polyclonal antibody prepared by immunizing New Zealand white rabbits was more than 1:512 000 detected by indirect ELISA.SDS-PAGE and IFA results showed that the polyclonal antibody had good purity and could specifically recognize the NS4 protein expressed in BHK-21 cells infected by BTV.【Conclusion】 In this study, a specific polyclonal antibody was prepared using the prokaryotic expression of NS4 protein, and the polyclonal antibody was preliminarily used for the IFA, which provided a powerful tool for the function and application study of BTV NS4 gene.

Porcine epidemic diarrhea (PED) is an intestinal infectious disease caused by Porcine epidemic diarrhea virus (PEDV), which has caused huge economic losses to the pig industry.At present, there is no effective treatment, and vaccination is still the main preventive measure.Since the first discovery of PEDV in 1971, the virus has continuously mutated and spread widely.Research on PEDV vaccines has also been continuously improved, but the immune effect is not ideal.Safety and efficacy are the primary factors in vaccine development.Traditional inactivated vaccines are safe, but require multiple doses.The immunogenicity of attenuated vaccine is strong, but the virulence is easy to atavism.The new subunit vaccine is similar to the inactivated vaccine, and the focus of future research and development is to ensure the natural conformation of antigenic proteins and improve the immunogenicity.The vector in recombinant live vector vaccine itself acts as an "immune booster".On the other hand, whether the weakened vector can stabilize the inheritance and its safety needs further study.In the face of Coronavirus with rapid mutation rate, the third-generation nucleic acid vaccine can be put into use quickly by simply modifying the corresponding gene.However, there is a big gap in this kind of vaccine, so we need to focus on the research and development of nucleic acid vaccines in the future.Using transgenic plants to produce vaccines can avoid contamination of other animal pathogens, without the need for low temperature storage, and plant cell walls can protect antigenic proteins and avoid digestion by enzymes, with good stability.But mass production of such vaccines requires attention to gene contamination, which can lead to a loss of natural diversity by transferring foreign genes to other plants through pollen.Compared with other kinds of vaccines, nano vaccines have a longer immune protective effect and can induce a good mucosal immune response, but the cost and preparation process problems need to be solved.In this paper, the traditional inactivated vaccine, attenuated vaccine and new subunit vaccine, virus live vector vaccine, bacterial live vector vaccine, nucleic acid vaccine, transgenic plant vaccine and nano vaccine were reviewed, and the advantages and disadvantages of various vaccines were discussed, so as to provide reference for the development of PEDV vaccines in the future.

【Objective】 The production collection of high-purity, high-concentration LwaCas13a protein capable of excellent trans-cleavage activity from Leptotrichia wadei was undertaken in vitro, which could provide an important biological tool for the development of new methods in animal virus RNA detection based on CRISPR-LwaCas13a system.【Method】 The recombinant plasmid pET15b-SUMO-LwaCas13a was constructed by PCR, double digestion as well as ligand reaction, and prokaryotic expression and condition optimization were performed.Ferment 100 mL of Escherichia coli BL21(DE3) was used to obtain the expression product and assess the purification procedure, the induced expression and purified products were concentrated by ultrafiltration, and the protein concentration was detected by BCA.The purified LwaCas13a protein was co-incubated with CRISPR RNA (CrRNA), target RNA from Porcine epidemic diarrhea virus (PEDV) and fluorescent-labeled single stranded RNA (ssRNA).A full-wavelength fluorescence analyzer was used to verify the trans-cleavage activity of CRISPR-LwaCas13a, which was also compared with commercial Cas13a protein.The optimal binding ratio between LwaCas13a and CrRNA was optimized for further research.【Result】 The recombinant plasmid pET15b-SUMO-LwaCas13a was successfully constructed.The optimal IPTG concentration of LwaCas13a recombinant protein was 0.4 mmol/L, and the optimum induction temperature and time was 18 ℃ for 16 h.After a series of purifications, LwaCas13a protein was produced at a purity of 95% and concentration of 7.5 mg/mL.The purified LwaCas13a protein was verified to have readily detectable trans-cleavage activity of ssRNA probes, and found its trans-cleavage activity was better than that of commercial Cas13a protein by about 1.25 times at the same concentration.The optimal binding ratio between LwaCas13a protein and CrRNA was 1:2.【Conclusion】 In this study, LwaCas13a protein at high purity and concentration was successfully obtained, and the purified LwaCas13a had excellent trans-cleavage activity than commercial Cas13a in vitro with the optimal binding ratio between LwaCas13a and CrRNA, which provided a basis for the development and use of CRISPR-LwaCas13a for animal virus RNA detection.

【Objective】 The aim of this study was to analyze the anti-inflammatory ingredients and mechanism of Sophora alopecuroides (S.alopecuroides) based on network pharmacology and experimental verification.【Method】 The effective ingredients of S.alopecuroides were screened from the HERB database and the corresponding targets were retrieved through SIB online tool.Cytoscape 3.6.1 software was used to construct an active ingredient-target network diagram.The anti-inflammatory related targets of S.alopecuroides were obtained from NCBI, GeneCards and OMIM databases, and the core target genes were uploaded to STRING platform to build a protein-protein interaction (PPI) network.Finally, DAVID database was used to analyze key targets by GO function and KEGG pathway enrichment.The target with the highest degree of PPI was selected for molecular docking with key pharmacodynamic components.The cytotoxicity of sophoridine and matrine of S.alopecuroides to RAW264.7 cells was detected, and the effects of sophoridine and matrine on the relative production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells were verified.【Result】 The results showed that there were ten active ingredients in S.alopecuroides, including sophoridine, matrine, cytisine, sophoramine, sophocarpine, etc.They could act on 55 key anti-inflammatory targets, including inhibitor of nuclear factor kappa B kinase subunit beta (IKBKB), NLR family pyrin domain containing 3 (NLRP3), mitogen-activated protein kinase 14(MAPK14), matrix metallopeptidase 2 (MMP2), etc.GO function enrichment analysis yielded 121 items (P<0.01), involving 61 biological processes, such as signal transduction, protein phosphorylation, response to drug, inflammatory response, etc.;37 molecular functions, such as protein binding, protein kinase activity, enzyme binding, etc.;And 23 cellular components, such as plasma membrane, cell surface, macromolecular complex, etc.104 related signaling pathways mainly included PI3K-Akt, MAPK, NF-kappa B (NF-κB), TNF and chemokine, etc.Molecular docking showed that sophoridine could bind tightly to target HSP90AA1 and matrine to target HDAC11.The cytotoxicity results showed that 0.02-1.28 mg/mL of sophoridine and matrine had no toxicity to RAW264.7 cells.Moreover, different concentrations of sophoranine and matrine could inhibit relative NO production in LPS-stimulated RAW264.7 cells.【Conclusion】 Sophoridine, matrine and other active ingredients of S.alopecuroides might have anti-inflammatory activity by regulating multiple targets such as IKBKB, NLRP3, MAPK14, MMP2, etc., and pathways such as PI3K-Akt, MAPK, NF-κB, TNF and other signaling pathways, etc., which provided a reference for further elucidation the anti-inflammatory mechanism of S.alopecuroides.

Klebsiella pneumoniae(K.pneumoniae) is an opportunistic pathogen that exists widely in water, soil, plants, and the mucosal surfaces of mammals.When the body was weakened, the immunity of the body was decreased, or antimicrobial agents were prolonged use, which can cause various clinical infections in humans and animals.Although cases of K.pneumoniae infection in poultry, livestock, and wildlife have been reported with a high case-fatality rate, the infection caused by K.pneumoniae from animals has not received sufficient attention.K.pneumoniae has a wide virulence spectrum, mainly including capsule polysaccharide, lipopolysaccharide, fimbriae and siderophores.Various factors interacting with each other result in pathogenicity.K. pneumoniae often causes pneumonia, mastitis, metritis, cystitis, meningitis and septicemia in animal at veterinary clinics.In order to cope with the economic losses caused by Gram-negative bacteria infections such as K.pneumoniae, antibiotics such as beta-lactams, polymyxins, tetracyclines, aminoglycosides, and quinolones were widely used in animals, leading to the emergence of multiple-resistant strain from animal and the increasing resistance of K.pneumoniae.In addition, animals and their products were considered to be the reservoir of multi-resistant strains.And K.pneumoniae can spread between humans and animals or between animals.This paper reviewed the pathogenicity, virulence factors, drug resistance and control measure of K.pneumoniae, providing recommendations for further control and delay of the emergence and spread of drug-resistant bacteria, and providing instructions for the development of novel veterinary drugs or non-antibiotic alternatives.

【Objective】 The aim of this study was to isolate lactic acid bacteria with both antibacterial and antioxidant activities from traditional fermented horse milk wine in Kezhou, Xinjiang, and then obtain excellent strains that could be used for the development of micro-ecological agents.【Method】 MRS plate was used to isolate and culture lactic acid bacteria.Dominant strains were screened, morphological characteristics of bacteria were observed, and molecular identification was conducted by 16S rRNA gene sequence analysis.Then the growth curve, acid production ability, acid resistance, bile salt tolerance and bacteriostatic ability of the strain were studied. In vitro antioxidant function of the strain was evaluated by detecting the hydrogen peroxide resistance, the DPPH free radical scavenging ability, superoxide anion scavenging ability and hydroxyl radical scavenging ability of the strain's fermentation broth, cell-free extract and cell suspension.And the safety of the strain was evaluated by drug sensitivity test and feeding test in mice.【Result】 The results showed that a Gram-positive bacterium with round colony shape, white translucent and irregular edges was screened out and named JK-24.The strain was identified as Lactobacillus kefiranofaciens by 16S rRNA cloning and sequencing.From 6 to 18 h, D_600nm value increased rapidly and entered the logarithmic growth phase, and the pH of JK-24 fermentation broth decreased obviously.JK-24 could grow in pH 2.5 to 6.5 and tolerate 0.3% bile salt.The diameters of bacteriostatic rings of fermentation broth against Escherichia coli and Staphylococcus aureus were 15 and 20 mm respectively.It could tolerate 3.0 mmol/L H₂O₂ and promote the growth of the strain at the concentration of 1.0 and 2.0 mmol/L.The DPPH free radical scavenging ability, superoxide anion scavenging ability and hydroxyl radical scavenging ability of the strain's fermentation broth and cell-free extract were significantly higher than those of MRS medium (P<0.05).It was sensitive to 19 antibiotics such as penicillin.Intragastric administration of JK-24 had no significant effect on growth performance and organ index of mice(P>0.05).The hematological parameters were in the normal range, and there were no pathological changes in organs.【Conclusion】 The Lactobacillus kefiranofaciens JK-24 isolated from horse milk wine was safe, non-pathogenic, and had good bacteriostatic and antioxidant properties, which could be used as a strain source of biological feed additives.

【Objective】 The experiment was aimed to investigate the effects of sodium butyrate on the growth and virulence of Aeromonas hydrophila, to assess the antibacterial activity of sodium butyrate against Aeromonas hydrophila, and to explore its mechanism of action.【Method】 The minimum inhibitory concentration (MIC) of sodium butyrate on Aeromonas hydrophila was determined by micro-broth dilution method, the effect of sodium butyrate on the motility of Aeromonas hydrophila was examined by swimming and cluster motility assay, the effect of sodium butyrate on lipase, protease and hemolysis of Aeromonas hydrophila was examined by using enzyme activity and hemolysis assay, the effect of sodium butyrate on the biofilm of Aeromonas hydrophila was examined by biofilm assay, and the fluorescence quantitative PCR was used to detect the effect of sodium butyrate on the virulence of Aeromonas hydrophila and the expression of genes related to the population sensing system.【Result】 The MIC of sodium butyrate was >512 mg/L for all three strains of Aeromonas hydrophila, and the growth of the three strains of Aeromonas hydrophila was not affected when the concentration of sodium butyrate was ≤ 512 mg/L.When the concentration of sodium butyrate was ≥ 64 mg/L, the growth of the three strains of Aeromonas hydrophila was not affected.When the concentration of sodium butyrate was ≥ 64 mg/L, it significantly inhibited the motility of the three Aeromonas hydrophila strains (P<0.05), except for the cluster motility of GZ-SS2019 (P>0.05), which also significantly inhibited the biofilm formation of the three Aeromonas hydrophila strains at this concentration (P<0.05).At the concentration of 128 mg/L, sodium butyrate had less effect on the lipase activity of the three Aeromonas hydrophila strains (P>0.05), but significantly inhibited the protease activity of the three Aeromonas hydrophila strains (P<0.05). When the concentration of sodium butyrate was ≥ 32 mg/L, it significantly reduced the hemolysis rate of the remaining two Aeromonas hydrophila strains(P<0.05), except for the inhibition of GZDX-01, which was not significant (P>0.05).The fluorescence quantitative PCR results showed that when the concentration of sodium butyrate ≥ 128 mg/L, it significantly reduced the expression of luxS and ahyR genes in the population induction system (P<0.05), the expression of aerA, ompW, ompA and fur genes were also significantly reduced (P<0.05).【Conclusion】 It was shown that sodium butyrate with the concentration was ≤ 512 mg/L did not affect the growth of Aeromonas hydrophila, but could effectively interfere with the motility status, protease activity, hemolysis, and biofilm formation and expression of virulence genes and population sensing system of Aeromonas hydrophila, and when the concentration was ≥ 128 mg/L, it was more effective.

Viola yedoensis Makino is a plant of Violaceae, also known as Viola liao, Viola wild, Viola glabra, etc.It has a long history of drug use and it is a traditional Chinese herbal medicine plant in China.It tastes bitter, pungent, cold, and has the functions of clearing heat, detoxification, cooling blood, detumescence, clearing heat and dampness.It is mainly used to treat boils, carbuncle, scrofula, jaundice, dysentery, diarrhea, etc.In recent years, it has been shown that Viola yedoensis Makino has anti-inflammatory, antibacterial, antioxidant and other effects through modern pharmacological research and clinical treatment of humans and animals.In addition, since 2020, China has stipulated that the addition of antibiotics and some antibiotic veterinary drugs are prohibited in feed, so as to reduce the harm caused by the abuse of antibiotics and maintain the safety of animal derived food and public health.Chinese veterinary medicine products are derived from natural plants and animals, and have the advantages of multi-component, multi-function and multi target.They are also used for the whole animal body, rather than purely for pathogens.Therefore, the author reviewed the latest research progress of the pharmacological effects of Viola yedoensis Makino, such as antibacterial, anti-inflammatory, anti-oxidation, anti-virus, immune regulation, anti-cancer and so on, and it provides a theoretical basis for the subsequent research of Viola yedoensis Makino as a Chinese veterinary drug or feed additive.

【Objective】 The study was to determine the related substances of aspirin eugenol ester (AEE) chewable tablets by high performance liquid chromatography (HPLC), and establish a more convenient and accurate detection method for the quality control of AEE chewable tablets.【Method】 The specificity, linear range, limits of detection and quantification, precision, repeatability, stability, recovery, destructive testing, durability testing, and sample determination were investigated by HPLC, in order to comprehensively evaluate the determination method of related substances in AEE chewable tablets, and calculate the content of related substances by self-contrast method.The chromatographic separation was performed using a Phenomenex Luna C18 column (150 mm×4.6 mm, 5 μm) at 35 ℃.The mobile phase consisted of water containing 0.5% phosphoric acid (A) and acetonitrile (B) with gradient elution.The flow rate was 1.0 mL/min, the detection wavelength was 279 nm, and the injection volume was 10 μL.【Result】 The results showed that the HPLC method of AEE chewable tablets had high specificity, and the detected peaks were well separated from other peaks.The linear relation between peak area and concentration of the AEE was good in the range of 0.26-26.00 μg/mL, R²=1 (n=7).In addition, the linear relation between peak area and concentration of the impurity A was well in the range of 0.25-25.50 μg/mL, R²=0.9996 (n=7).The limits of quantification of AEE and impurity A were 0.52 and 0.51 μg/mL, and the limits of detection were 0.26 and 0.25 μg/mL, respectively.The relative standard deviation (RSD) of precision was less than 1%.RSD of intraday repeatability, daytime repeatability and stability were 0.88%, 2.50% and 1.84%, respectively.Average recovery was 100.14% (n=9, RSD=1.66%).Destructive test showed that material was basically conserved under different destruction conditions, and the material conservation was between 95% and 105%.The durability test showed that the durability of the detection conditions was good, RSD was 1.87%.In the three batches of samples, the content of impurity A in AEE chewable tablets was less than 0.5%, and the total impurity content was less than 1.0%.【Conclusion】 The method was validated to be simple, specific, accurate and suitable for the determination of related substances in AEE chewable tablets.

【Objective】 This experiment was aimed to investigate the pathogenicity and drug resistance of Staphylococcus chromogenes isolated from yaks in Lhasa, Tibet, so as to provide some reference for the prevention and treatment of Staphylococcus chromogenes infection in yaks.【Method】 Bacterial isolation and culture, biochemical test identification, 16S rDNA sequencing and similarity comparison analysis were used to identify Staphylococcus chromogenes from 65 yak samples collected from Lhasa, Tibet, and to study their drug resistance and pathogenicity.【Result】 A total of 9 isolates were obtained from 65 samples, which were named XZ1-XZ9 and the isolation rate was 13.85%.All the isolates showed small yellow colonies on 5% sheep blood plate and were positive by Gram staining.Biochemical test showed that the isolated strains were positive for mannose, fructose, sucrose and nitrate reduction, and the results were consistent with the characteristics of Staphylococcus chromogenes.Through 16S rDNA sequencing and similarity comparison analysis, it was found that the 9 isolates had the highest homology with Staphylococcus chromogenes, which was more than 96%.The results of drug sensitivity test showed that 9 strains were most resistant to three antibiotics and at least resistant to one antibiotic.The bacterial challenge test was carried out on SPF mice with the isolated strains.It was found that the isolated bacteria had strong pathogenicity to mice, resulting in congestion and swelling of the lungs and kidneys, intestinal lesions and yellow thin stools.【Conclusion】 The results of this study showed that Staphylococcus chromogenes isolated from yaks in Lhasa area had a certain pathogenicity and strong drug resistance, which should be paid more attention.

Glyphosate is an organophosphorus herbicide, which is widely used due to its low cost, strong systemic conductivity, and broad herbicidal spectrum.The extensive use of glyphosate not only destroys the ecosystem, but also accumulates in animals through bioaccumulation and spreads along the food chain, producing toxic effects on non-target organisms.Combined with domestic and foreign research progress, the authors overviewed the action mechanism, use and pollution status of glyphosate, and analyzed the toxic effects of glyphosate abuse on non-target organisms.The toxic effects of glyphosate on aquatic, terrestrial and amphibian animals were mainly discussed, such as neurotoxicity, oxidative toxicity, reproductive toxicity, genotoxicity, teratogenicity and carcinogenicity.In addition, the authors further summarized the main toxicological mechanisms of glyphosate toxicity in animals (such as causing oxidative stress, promoting cell apoptosis, damaging nerve conduction), initially put forward the key issues that need to be solved in this research field, and looked forward to the reasonable use of glyphosate and related research on animal toxicity in the future.The purpose of this paper was to provide reference basis for the in-depth study of glyphosate toxicity in animals and the environmental risk assessment in its use.