【Objective】 This study was aimed to investigate the mechanism of growth and development in skin hair follicles of Jiangnan cashmere goats,enhance comprehension of the genetic regulation and function of candidate genes related to secondary hair follicle development,and provide guidance for future breeding of Jiangnan cashmere goats to improve their fleece production.【Method】 The high-throughput sequencing technology was used to carry out transcriptomic analysis of circular RNA (circRNA) in the skin tissues of secondary hair follicles in Jiangnan cashmere goats during anagen,catagen and telogen phases.The differentially expressed circRNA (DE circRNA) in different comparison groups was selected,and the host genes were analyzed by GO function,KEGG pathway and protein-protein interaction (PPI).The competitive endogenous RNA (ceRNA) network was constructed by combining the preliminary transcriptome data.【Result】 A total of 2 482 circRNAs were identified in 9 skin tissues of Jiangnan cashmere goats,of which 138 were DE circRNAs.In the comparison group of phases,there were 54,74 and 65 DE circRNAs were found in the comparison group of catagen and anagen,catagen and telogen,telogen and anagen phases,respectively.GO function and KEGG pathway analysis revealed that DE circRNA host genes were mainly concentrated in functional pathways such as cell adhesion,integrin-mediated signaling pathway,cell integrin complex,and some signaling pathways related to physiological processes of hair follicles such as MAPK,TGF-beta and Hedgehog.Combining with the PPI network,it was speculated that 14 circRNAs might participate in the regulation of growth and development of secondary hair follicles in Jiangnan cashmere goats.In addition,6 DE circRNAs,20 DE miRNAs,3 DE mRNAs and 2 DE lncRNAs were constructed in the ceRNA network of Jiangnan cashmere goats.【Conclusion】 In this study,transcriptome sequencing was used to screen essential circRNAs related to secondary hair follicle cycle development in Jiangnan cashmere goats,including novel-gene5238-2,novel-gene7855-1,novel-gene4455-1,etc.The results provided theoretical foundation for the biological function and regulatory property of circRNA in the development of secondary hair follicles in cashmere goats,and provided a new prospect for breeding of Jiangnan cashmere goats.

【Objective】 The aim of this study was to obtain the CDS region sequence of the secreted fizzled-related protein 1 (SFRP1) gene in buffalo,predict the structure and function of the encoded protein,construct the eukaryotic expression vector of SFRP1 gene,and detect the expression of SFRP1 gene in different tissues of buffalo,laying a foundation for exploring the role of SFRP1 gene in the growth and development of buffalo.【Method】 Using the cDNA of ovarian tissue as template,the CDS region sequence of SFRP1 gene was amplified by RT-PCR and sequenced,and the SFRP1 gene of buffalo was compared with different species by bioinformatics online analysis softwares, the phylogenetic tree was constructed,and the physicochemical properties,signal peptide,and transmembrane structure of SFRP1 protein were predicted.The obtained target gene was connected to pCMV-HAhyPBase-mcheery vector and transfected into buffalo granulosa cells. The fluorescence and gene expression were detected after transfection.The expression of SFRP1 gene in different tissues of buffalo was detected by Real-time quantitative PCR.【Result】 The CDS region of SFRP1 gene in buffalo was 927 bp,encoding 308 amino acids.Multiple sequence comparison results showed that the amino acid sequence of SFRP1 gene similarity between buffalo and Bos taurus,Bos mutus,Capra hircus,Orcinus orca,Pan troglodytes,Vulpes lagopus, Felis catuswas,Homo sapiens and Mus musculus were 100%,100%,99.65%,98.58%,98.23%,98.23%,98.58%,98.23% and 96.45%,respectively. There were CRD_FZ,NTR_like and DUF3367 domains.The nucleotide sequence of SFRP1 gene similarity between buffalo and Bos taurus,Ovis aries, Capra hircus,Bos mutus,Bison bison bison,Cervus elaphus,Camelus ferus,Sus scrofa and Homo sapiens were 97.3%,95.9%,95.8%,94.5%,94.2%,93.7%,80.4%,78.5% and 77.3%,respectively.Phylogenetic tree analysis showed that buffalo,Bos taurus,Bos mutus,and Bison bison bison converged into one group.Bioinformatics analysis showed that SFRP1 protein was alkaline and unstable.Signal peptide existed at amino acids 1-15, which was a secreted protein with a transmembrane structure and was mainly located outside the cell.There were 21 phosphorylation sites and 8 O-glycosylation modification sites.The secondary structure was mainly alpha-helix,extended chain and random coil.The tertiary structure of SFRP1 protein in buffalo,Bos taurus and Homo sapiens was highly similar.pCMV-mcheery-SFRP1 eukaryotic expression vector was successfully constructed and transfected into buffalo granulosa cells.After transfection 72 h,the expression of SFRP1 gene in pCMV-mcheery-SFRP1 recombinant plasmid group was extremely significantly higher than that in control group (P＜0.01).Real-time quantitative PCR result showed that SFRP1 gene was expressed in different tissues of buffalo,and the expression level in spleen was the highest,which was extremely significantly higher than that in other tissues (P＜0.01).【Conclusion】 SFRP1 gene was highly conserved in different species and in the process of genetic evolution,and was widely expressed in different tissue of buffalo.The results laid a foundation for future research for the function and molecular mechanism of SFRP1 gene in the growth and development of buffalo.

【Objective】 The aim of this study was to clone and analyze Ras homolog enriched in brain (Rheb) gene in Nubian goats,and construct its eukaryotic expression vector,which laid a foundation for further revealing the molecular mechanism of Rheb regulation on the skeletal muscle of Nubian goats.【Method】 Rheb gene was amplified from the longissimus dorsi muscle tissue of Nubia goats by RT-PCR method.After being verified by agarose gel electrophoresis and sequencing,the bioinformatics analysis was carried out.At the same time,the eukaryotic expression vector of Rheb gene was constructed.After transfecting cells,Real-time quantitative PCR was performed to verify the correctness of the constructed vector.【Result】 The length of coding region of Rheb gene in Nubian goats was 555 bp,encoding 184 amino acids.The molecular formula of Rheb protein was C₉₁₀H₁₄₅₆N₂₃₆O₂₇₉S₆,the molecular weight was 20 359.34 u,the total number of atoms was 2 887,and the theoretical isoelectric point was 5.93.Rheb protein was found to be a stable hydrophilic protein,which did not contain transmembrane domains.The results of protein secondary structure prediction showed that alpha helix,beta turn,extended chain and random coil accounted for 40.76%,7.07%,22.83% and 29.35%,respectively,in Rheb protein of Nubian goats.After transfecting the constructed eukaryotic expression vector pcDNA3.1-Rheb into goat skeletal muscle cells,the expression of Rheb gene was extremely significantly increased compared with the empty vector group (P＜0.01).【Conclusion】 This experiment successfully cloned the sequence of the coding region of Rheb gene and constructed the eukaryotic expression vector pcDNA3.1-Rheb,which provided theoretical support for the in-depth understanding of the role of Rheb gene in the muscle of Nubian goats.

【Objective】 Differential lncRNA and mRNA of pectoralis muscle development in pigeons of different day-olds were analyzed to identify the key candidate genes affecting the growth and development of pigeons,explore the molecular mechanism of growth and development of pigeons,and provide a theoretical basis for the research on the promotion of the growth and development of pigeons.【Method】 White King pigeons were weighed at 1 (D1),7 (D7),14 (D14) and 25 (D25) days of age,followed by slaughtering at D7 and D25.lncRNAs from the pectoralis muscles of the selected day-old White King pigeons were sequenced to predict the target genes using RNA-Seq technology,construct the lncRNA-mRNA interactions network,and the screened differentially expressed lncRNAs were analyzed by GO and KEGG enrichment,and the reliability of sequencing results was verified by Real-time quantitative PCR.【Result】 Pigeons gained the fastest daily weight at the stage from 7 to 14 days of age,and the weight of breast pigeons reached the market age at 25 days of age.A total of 56 421 lncRNAs and 30 208 mRNAs were obtained from the six libraries constructed,of which 2 335 lncRNAs were expressed differentially and 1 553 mRNAs were targeted in the two groups.The lncRNA-mRNA regulatory network showed that there was mutual regulation between lncRNA and mRNA.The results of KEGG and GO analyses showed that the differential genes were associated with GTpase activity,etc.The differential lncRNAs and their target genes were mainly enriched in signaling pathways such as MAPK signaling pathway,skeletal muscle tissue development,TGF-β signaling pathway,Notch signaling pathway and other signaling pathways,and the key lncRNAs might regulate the pectoralis muscle development of the breast muscle of the pigeon through these signaling pathways.Among them,LTCONS_00012834,LTCONS_00032767,LTCONS_00045211,and LTCONS_00002003 targeted XIRP1,PITX3,BMPR1B,and MYLK4,respectively,and played key roles.Real-time quantitative PCR results were consistent with the high-throughput sequencing results,indicating the reliability of the sequencing data.【Conclusion】 A total of 2 335 differentially expressed lncRNAs and 1 553 mRNAs were obtained in this study,and four differentially expressed lncRNAs:LTCONS_00032767,LTCONS_00012834,LTCONS_00002003,and LTCONS_00045211 targeted genes related to skeletal muscle development PITX3,XIRP1,MYLK4,and BMPR1B,respectively,and these genes were regulated by multiple lncRNAs.The theoretical basis was provided for elucidating the molecular mechanism of pectoralis muscle growth and development in pigeons as well as the molecular selection and breeding of White King pigeons.

【Objective】 The purpose of this study was to analyze the protein characteristics of chicken DDB1 and CUL4 associated factor 1(DCAF1) and construct an eukaryotic expression vector,with investigating the effect of DCAF1 on ALV-J replication.【Method】 The amino acid sequence of DCAF1 protein of related species were downloaded from NCBI and a phylogenetic tree was constructed using Mega 11.0 software.Online programs such as ProtParam,ProtScale,and TMHMM were utilized to analyze the amino acid sequence of DCAF1 protein in chicken,predict its biological information such as physicochemical properties,conserved domains,secondary and tertiary structures,and interacting proteins.The recombinant eukaryotic expression vector of DCAF1 gene in chicken was constructed,and the effects of ALV-J infection on the expression of DCAF1 mRNA and ALV-J Env protein,as well as the effects of overexpression of DCAF1 on ALV-J replication were detected using Real-time quantitative PCR and Western blotting techniques.【Result】 The DCAF1 protein of chicken was about 1 496 amino acids,and its molecular weight was about 170 ku. The results of phylogenetic tree showed the chicken was closet related to Oxyura jamaicensis and farthest related to Danio rerio.The bioinformatics prediction results indicated that DCAF1 protein of chicken had no transmembrane domain,no signal peptide expression,and contained three main structural domains:ARM folding,LisH and WD40 duplication,and one acidic domain at the end,and interacted with proteins such as DDB1,CUL4A and SAMHD1.The secondary structure of DCAF1 protein of chicken was mainly consisted of random coil and alpha helix,which accounted for 44.85% and 41.18%;followed respectively by extended chain and beta turn,which accounted for 9.76% and 4.21%.The tertiary structure results were basically consistent with the secondary structure,with a modeling consisitency of 93.79%.After 24 and 48 h of ALV-J infection,the protein and gene expression of DCAF1 were extremely significantly higher than those at 0 h of infection (P＜0.01).After overexpression of DCAF1,compared with the infection control group,the ALV-J Env protein was significantly increased (P＜0.05).【Conclusion】 The DCAF1 protein of chicken consisted of 1 496 amino acids with a molecular weight of about 170 ku.It was a stable hydrophilic protein and did not contain a transmembrane domain.This study successfully constructed eukaryotic expression vector of DCAF1 gene.In ALV-J infected cells,the expression of DCAF1 protein and gene were extremely significantly increased,while overexpression of DCAF1 significantly promoted ALV-J replication.This results laid the foundation for further elucidating the biological function of chicken DCAF1 and its molecular mechanism of interaction with ALV-J.

【Objective】 The aim of this study was to analyze the bioinformatics of Brucella BspF protein,and further explore the effect of BspF protein on the expression of inflammatory cytokines in macrophages.【Method】 The physical and chemical properties,hydrophilicity and hydrophobicity,signal peptide,transmembrane region,antigenic determinant and domain,secondary structure and tertiary structure of Brucella BspF protein were analyzed by bioinformatics online software.Constructed recombinant plasmid pET-32a-BspF and then transformed into Escherichia coli BL21 (DE3) competent cells.BspF protein was induced by IPTG and purified.The expression and purification of BSPF protein were detected by SDS-PAGE and Western blotting.The toxicity of purified BspF protein on mouse macrophages (RAW264.7) was detected by CCK-8 method,and the effects of BspF protein on mRNA and protein expression of inflammatory factors in cells were detected by Real-time quantitative PCR and Western blotting,respectively.【Result】 The results showed that BspF protein,with a molecular mass of 48.75 ku,was an unstable protein with 15 antigenic determinant,no transmembrane region and no signal peptide,and the secondary structure was dominated by alpha helix.The prokaryotic expression vector of Brucella BspF gene was successfully constructed,and the target protein with the size of 60 ku was obtained after induced expression.Compared with control group,after the purified protein was stimulated at the final concentration of 50 μg/mL for 24 h,the mRNA expression of NLRP3,Caspase-1,IL-1β and IL-18 in BspF group were significantly or extremely significantly increased (P＜0.05 or P＜0.01),the protein expression of NLRP3 and Pro-Caspase-1 were extremely significantly increased (P＜0.01).【Conclusion】 Brucella BspF protein could trigger the inflammatory response of host cells through the NLRP3 inflammatome and promote the expression of inflammatory factors in macrophages,which provided a theoretical basis for the study of the inflammatogenic mechanism of Brucella and the screening of anti-inflammatory targets.

【Objective】 This experiment was conducted to investigate the effects of phospholipid (PL) and soybean isoflavone (ISO) supplementation on growth performance,meat quality and fatty acid composition in the breast muscle of Yellow-feathered broilers,in order to improve growth performance and meat quality of broilers through nutritional regulation,and to provide theoretical basis and technical support for improving quality and efficiency of Yellow-feathered broilers.【Method】 The experiment was 2 (with or without ISO in diet)×3(control,soybean phospholipid (SL) or lysophospholipid (LPL) in diet) factorial design.A total of 900 48-day-old female medium-growing Yellow-feathered broilers were randomly divided into 6 groups with 6 replicates per group and 25 broilers per replicate.The broilers in group 1 were fed basal diet supplemented with 3.00% rapeseed oil (control group).On the basis of control group,1.00% SL were used to replace rapeseed oil in the diet in group 2 (SL group).On the basis of group 1,500 mg/kg LPL products was supplemented in group 3,and in groups 4-6,300 mg/kg ISO products were supplemented on the basis of groups 1-3,respectively.The experiment lasted for 32 days.At 80 days of age,the broilers were weighed with fasting and feed intake was analysed to calculate the growth performance.One broiler with average body weight was selected for blood collection and slaughter,and the slaughter performance and plasma biochemical indexes were determined.The breast muscle was taken to evaluate the meat quality,malondialdehyde (MDA) content,volatile basic nitrogen (TVBN) content and fatty acid composition.【Result】 Compared to control group, supplementation of PL and ISO in the diets of female medium-growing Yellow-feathered broilers aged 48 to 80 days could significantly reduce the mortality of broilers (P＜0.05),and there was an interaction between them.Dietary PL or ISO supplementation significantly decreased the contents of GSSG and MDA in plasma (P＜0.05),significantly increased the ratio of GSH/GSSG in plasma (P＜0.05).Dietary LPL or ISO supplementation significantly decreased cooking loss of breast muscle(P＜0.05).Dietary PL supplementation significantly increased a^*_{45 min} and the contents of monounsaturated fatty acid(P＜0.05),and significantly decreased the content of n-6 polyunsaturated fatty acid (n-6 PUFA) in breast muscle (P＜0.05).Dietary SL supplementation significantly decreased MDA and TVBN contents in breast muscle (P＜0.05).Dietary ISO supplementation significantly increased plasma HDL-C and GSH content and the contents of PUFA,n-3 PUFA,linoleic acid and linolenic acid in breast muscle (P＜0.05),and significantly decreased plasma TG content and n-6 PUFA/n-3 PUFA values in breast muscle (P＜0.05).【Conclusion】 The supplementation of PL or ISO in the diet of female medium-growing Yellow-feathered broilers of 48 to 80 days of age could increase the antioxidant capacity,optimize the fatty acid composition in breast muscle,and improve the meat quality.

Feathers are the key economic traits of waterfowl,and also important physical characteristics.Feathers play an important role in flight,heat preservation,swimming,protection,temperature control and so on.The structure of feathers is complex,feather types and composition are also very different,and the development of feathers requires three generations of progressive growth before final growth.The growth and development of hair follicles also play an important role in the growth and development of feathers.From the structure and composition of hair follicles,the development process of hair follicles,the renewal of hair follicle growth,degeneration and quiescence,the classification of primary and secondary hair follicles all show that hair follicle development is the key to the growth and development of feathers.The growth and development of feathers is an extremely complex process,in addition to being affected by multiple factors (Wnt/β-catenin signaling pathway,fibroblast growth factor (FGF) family,SHH(sonic hedge) hog signaling pathway,Notch signaling,Hox gene,epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)),are also regulated by various nutrients,such as proteins,amino acids,vitamins,and mineral elements.However,in recent years,with the intensive development of the poultry industry and the progress of the scale of dry farming,the problems of waterfowl feathers (such as slow growth,dull,abnormal shedding,etc.) have become increasingly prominent,seriously affecting the quality of their feathers and the appearance of slaughter,resulting in great economic losses.In this paper,the factors involved in feather growth and hair follicle development of waterfowl were reviewed from the perspective of feather growth,hair follicle structure and development,and the nutrients that have positive regulation on feather growth and development were summarized,hoping to provide theoretical basis for the advancement of waterfowl industry,especially the promotion of drought culture.

【Objective】 This study was aimed to analyze the contents of pesticide residues,heavy metal pollutants and aflatoxin B₁ (AFB₁) in cotton straw from the six major cotton producing areas in Xinjiang,in order to fill in the problem of missing basic data such as pesticide residues when using cotton straw as feed.【Method】 In this experiment,one cotton field was randomly selected from each of six major cotton planting areas in Xinjiang,with three areas in the front,middle and back.Then set up five 1 m² sample plots in each area,all cotton straw in the sample plots was collected with a stubble height of 15 cm.The cotton straw samples were cut it into small pieces with pruning pliers,mixed well,and collected 1 kg of sample according to the quartering method.The contents of 58 pesticide residues,AFB₁,deoxynivalenol,fluorine and five heavy metal pollutants were detected.【Result】 Only 3,5,3,3,2 and 1 pesticide residues were detected in the samples collected from six major cotton producing areas in Xinjiang.Most registered pesticides were not detected as residues in cotton straw samples.The maximum residual content of fenvalerate,triazophos,chlorfenapyr,chlorpyrifos,fenpropathrin,pyraclostrobin,cypermethrin,malathion and acetamiprid in cotton straw samples were 0.150,0.190,0.087,0.210,0.043,0.058,0.024,0.014 and 0.015 mg/kg,respectively,which were lower than the limit value of rice in the food safety standard GB 2763―2021.Four heavy metal pollutants of Pd,Cd,Cr and As were detected in cotton straw samples,the maximum content were 0.428,2.400,0.180 and 1.090 mg/kg,respectively,which were lower than the limit values in hygienical standard for feed GB 13078―2017,and met the feed standards.AFB₁ and deoxynivalenol were not detected in cotton straw samples,and the maximum content of fluorine detected was 20.67 mg/kg,which were lower than the limit value in the feed standard.【Conclusion】 Cotton straw fitted the limits of pesticide residues,AFB₁,deoxynivalenol,fluoride and heavy metal pollutants in hygienical standard for feed GB 13078―2017,and could be safely used as roughage for herbivorous livestock.

Silkworm excrement,a by-product of the sericulture industry,possesses a plethora of conventional nutrients such as crude protein,crude fiber,and crude fat,as well as essential minerals including calcium,phosphorus,copper,iron,zinc,and manganese.Additionally,this by-product contains valuable bioactive compounds such as flavonoids,polyphenols,vitamins,and alkaloids.Due to its abundant production and cost-effectiveness,silkworm excrement can serve as a high-quality unconventional feed ingredient.However,silkworm excrement includes a substantial amount of microorganisms,cellulose,nitrites,tannins,pectin,phytic acid,and other toxic,harmful,or anti-nutritional substances,which can adversely impact animal health when consumed directly.Consequently,this greatly hampers the potential utilization of silkworm excrement as feed.By implementing various technical approaches,especially microbial fermentation technology,it is possible not only to degrade pectin,cellulose,nitrites,lignin,phytic acid,tannins,and other anti-nutritional substances present in silkworm excrement but also to inhibit the proliferation of harmful microorganisms.Moreover,this process can enhance the content of crude protein,crude fat,and other nutritional substances,subsequently improving the nutritional structure and value of silkworm excrement.As a result,this bears great significance in promoting the feed utilization of silkworm excrement and alleviating the scarcity of feed resources in China.Within animal production,the appropriate inclusion or substitution of silkworm excrement in animal diets has been proven to enhance animal performance,improve the quality of animal products,and regulate the immune system and metabolism of the animal.This suggests that integrating silkworm excrement into animal feed holds promising prospects for development and application in the field of animal husbandry.The authors summarized the nutritional characteristics,compositional constituents,and processing methods of silkworm excrement,along with its application in animal production,aiming to provide a reference for the utilization of silkworm excrement as feed.

【Objective】 This study was aimed to determine the nutrient components of Galega orientalis and evaluate the antioxidant activity of its extracts,and explore the effects of different extraction solvents on the antioxidant activity of Galega orientalis,so as to provide theoretical reference for developing Galega orientalis-based functional products.【Method】 National standards were followed to determine the contents of conventional nutrient components in Galega orientalis.The extract A-E was obtained by extracting Galega orientalis with water and 30%,50%,70% and 90% ethanol solutions,respectively.The contents of total flavonoids,total polyphenols and total polysaccharides in the extracts of Galega orientalis were determined by aluminum trichloride colorimetry,Folin-Ciocalteu and phenol-sulfuric acid methods,respectively.In vitro antioxidant activity of Galega orientalis extracts was evaluated by DPPH and ABTS radical scavenge and total antioxidant capacity assays.Correlation analysis was performed between total flavonoid content,total polyphenol content,total polysaccharide content,DPPH free radical half inhibitory concentration (IC₅₀),ABTS free radical IC₅₀ and ferric ion reducing antioxidant power (FRAP) values of Galega orientalis extracts.【Result】 The contents of crude protein,crude fat,crude fiber and ash in Galega orientalis were 19.74%,2.53%,37.10% and 8.79%,respectively.The contents of total flavonoids in extracts B and C were 97.56 and 100.78 mg/g,and the contents of total polyphenols and total polysaccharides in extract C were 89.91 and 38.69 mg/g,respectively,which were significantly higher than those in other extracts (P＜0.05).The DPPH free radical IC₅₀ of extracts C and D were 0.13 and 0.11 mg/mL,and the ABTS free radical IC₅₀ were 0.73 and 0.74 mg/mL,respectively,which were significantly lower than those of other extracts (P＜0.05).The FRAP values of extracts C and D were 1 497.87 and 1 431.85 mmol/g,respectively,which were significantly higher than those of other extracts (P＜0.05).Correlation analysis showed that the contents of total flavonoids and total polyphenols in the extracts of Galega orientalis were extremely significantly negatively correlated with DPPH and ABTS free radical IC₅₀(P＜0.01),and were extremely significantly positively correlated with total antioxidant capacity (P＜0.01).【Conclusion】 Galega orientalis was rich in nutrients such as crude protein,crude fat and mineral elements,had high value as animal feed.The contents of total flavonoids and total polyphenols in Galega orientalis extracts had a strong correlation with antioxidant activity.Among them,the content of total flavonoids and total polyphenols in 50% ethanol solution extract was the highest,and the antioxidant activity was the strongest.

Rosmarinic acid is a natural phenolic acid compound widely found in many plants,which can be isolated from plants in the family of Comfrey,Cucurbitaceae,and Labiatae,etc.It possesses a variety of biological functions such as anti-inflammatory,antioxidant,bacteriostatic,anticancer,and antiviral,and it is widely used in the fields of food,medicine,and cosmetics.In veterinary clinics,rosmarinic acid and its source plants are widely used in the production of a variety of economic animals,such as pigs,poultry,ruminants,aquatic animals and rabbits,and have broad application prospects in regulating the intestinal microbial community,improving the immunity of the organism and production performance.In this paper,a systematic review of the biological functions of rosmarinic acid and its applications in the production of a variety of economic animals was conducted with the aim of providing references for the basic research on rosmarinic acid as well as its further application and promotion in animal production.

【Objective】 This experiment was conducted to explore the effects of restricted basal diet supplemented with hydroponic barley seedling on intestinal microflora of Zi geese using 16S rDNA high-throughput sequencing technology.【Method】 A total of 160 Zi geese at 28 days of age were randomly divided into 4 groups with 4 replicates in each group and 10 geese per replicate.Geese in group A were fed basal diet ad libitum.Geese in groups B,C and D were fed the basal diet with 85％,72.5％ and 60％ of the feed intake in group A,respectively,with free access to hydroponic barley seedlings.The experiment lasted for 42 days.Two goose was randomly selected and slaughtered from each replicate at 70 days of age,and the mixed contents of jejunum and ileum and cecum contents were collected as samples for high-throughput sequencing by Illumina MiSeq sequencing platform and bioinformatics analysis.【Result】 ① In terms of diversity analysis,there were no significant differences in Alpha diversity indexes and results of Beta diversity analysis among all groups (P＞0.05).② Intestinal microflora from 4 groups had 4 common dominant phyla (relative abundance＞1％):Firmicutes,Proteobacteria,Bacteroidetes and Actinobacteria,7 common dominant genera:Enterococcus,Pseudomonas,Bacteroides,Subdoligranulum,Oscillospira,Faecalibacterium and Helicobacter. Intestinal microflora in group D had significant lower Actinobacteria relative abundance than that of group A (P<0.05). Intestinal microflora in groups B,C and D had significant lower Spirochaetes,Fusobacteria, Corynebacterium,Staphylococcus and Aerococcus,and significant higher Enterococcus relative abundance than those of group A (P＜0.05).③ LEfSe analysis found that intestinal microflora in group A had 4 labeled species:Actinobacteria,Enhydrobacter,Lactobacillus,Lactobacillaceae.The microflora in group B had 3 labeled species:Chloroplast,Streptophyta and Intrasporangiaceae.The microflora in group C had 11 labeled species:Xanthomonadaceae,Xanthomonadales,Bradyrhizobiaceae,Oxalobacteraceae,Burkholderiales,Ralstonia,beta-Proteobacteria,TM7,TM7_3,Rs_045 and I025.No labeled specie was found in the intestinal microflora in group D.【Conclusion】 Under these experimental conditions,restricted basal diet supplemented with hydroponic barley seedling had no significant effect on diversity of intestinal microflora of Zi geese,while could affect relative abundance of microflora,and could inhibit the growth of some pathogenic bacteria and improve intestinal health.However,excessive low proportion of basal diet supply might cause intestinal flora imbalance.

Soybean meal,as a byproduct of soybean oil extraction,has become one of the main plant protein feed materials in China due to its high protein content,balanced amino acids,and comprehensive nutritional elements.However,soybean meal contains anti-nutritional factors (such as trypsin inhibitors,urease,soybean lectins and non-starch polysaccharides) that severely hinder the digestion and absorption of nutrients in animals.Soybean peptide,as an incompletely hydrolyzed product of soybean meal protein,has the characteristics of fast transport speed,low carrier saturation,low energy consumption,avoidance of amino acid competition,and multiple biological activities.Therefore,increasing the content of peptides in soybean meal can significantly improve its nutritional value.Currently,the main methods for preparing peptides from soybean meal include chemical methods,microbial fermentation methods,and enzymatic hydrolysis methods.Compared to the first two preparation methods,enzymatic hydrolysis has stronger specificity for the removal of anti-nutritional factors in soybean meal and the increase of peptide content.The author summarizes the nutritional characteristics of soybean meal and the main factors affecting its nutritional value,and analyzes the mechanisms of anti-nutritional factors in soybean meal.In addition,the advantages and disadvantages of chemical methods and microbial fermentation methods for peptide preparation are discussed,and the advantages and practical application effects of enzymatic hydrolysis of soybean meal for peptide preparation are summarized.The existing problems are also analyzed.This provides a reference for the enzymatic hydrolysis method to eliminate anti-nutritional factors in soybean meal and prepare peptides.

As a basic characteristic of dogs and cats,also being their most apparent features,coat hair serves important functions such as skin protection and ornamental values,and has always been of great concern to pet owners.When the coat hair of dogs and cats are in poor state,it can lead to abnormal conditions such as skin inflammation,pruritus,and even hair loss.The abnormal conditions significantly impacting the overall health of pets.Therefore,investigating how to maintain and improve the healthy state of coat hair in dogs and cats is a worthwhile research question in the field of pet nutrition.Polyunsaturated fatty acids (PUFA),a type of long-chain fatty acids containing two or more double bonds,have been shown to have positive improvement effects on coat hair health,making PUFA promising for the development of products aimed at improving coat hair health in dogs and cats.The authors summarized the mechanism of PUFA in maintaining and improving coat hair health in dogs and cats,and the application of PUFA from animal,plant and microorganisms sources in pet hair health products,with a view to providing a reference for the development and application of PUFA in coat hair health products of dogs and cats in the future.

【Objective】 The aim of this study was to analyze the integrated transcriptome sequencing data of Chinese indigenous pigs and lean pigs,identify the differentially expressed genes (DEGs) in longissimus dorsi muscle,and find out the potential candidate genes that contributed to the differences in meat quality traits between Chinese indigenous pigs and lean pigs,in order to provide a references for meat quality research in pigs.【Method】 The raw sequencing data of Chinese indigenous pigs and lean pigs were downloaded from the NCBI-SRA database and self-test data were integrated,including 32 samples from 8 Chinese indigenous pig breeds and 14 samples from 2 lean pig breeds,and the data were filtered,quality controlled,compared and counted,the DEGs were screened using DESeq2 software in R and subjected to gene enrichment analysis as well as protein-protein interaction (PPI) analysis.【Result】 In the results of differential expression analysis,there were 191 DEGs in Chinese indigenous pigs compared with lean pigs,of which 149 were highly expressed and 42 were lowly expressed.Nine significantly enriched pathways were obtained through gene enrichment analysis,and the DEGs might affect meat quality traits by regulating fat deposition and metabolism,glucose metabolism,insulin production,energy metabolism,pigmentation and other processes.The PPI analysis identified 21 genes that played an important role in the regulatory network,which FABP3,SLC38A4,KIT and LDHB genes were at the core of the PPI network.【Conclusion】 The experiment identified that genes such as FABP3,CTSL,PLIN5,SLC38A4,KIT,APPL1,IDNK, LDHB,ND6,SLC39A8, GPR27 and KCNJ13 were related to the differences of meat quality traits in Chinese indigenous pigs and lean pigs,which directly or indirectly regulated meat quality traits.The results provided a theoretical basis for the excavation of new genes as well as the study on genes of regulating meat quality traits in pigs.

【Objective】 The purpose of this study was to explore the difference of meat color and myoglobin content in Rongchang (RC),Yorkshire×RC (YR) and Duroc×Landrace×Yorkshire (DLY) pigs,verify the correlation between meat color and myoglobin,and provide basic data for the evaluation of meat color in commercial pigs.【Method】 15,20 and 20 longissimus dorsi muscle samples of three kinds of pigs were collected.Combined with meat values,visible absorption spectrum and formula calculation,the meat color and myoglobin content of three kinds of pigs at 45 min and 6,12 and 24 h after slaughter were measured, respectively,and the relationship between meat color and myoglobin content was explored through correlation analysis.【Result】 The redness value (a^*) of pork in RC pigs was significantly higher than that of DLY pigs (P＜0.05).At 45 min after slaughter,the lightness value (L^*) of pork in three kinds of pigs was significantly lower than that of other point of time (P＜0.05).The characteristic peaks of oxymyoglobin appeared at 543 and 580 nm in the scanning spectra of myoglobin in three kinds of pigs,and the peak height showed RC＞YR＞DLY.At 45 min after slaughter,the total myoglobin content in RC pigs was significantly higher than that in YR and DLY pigs (P＜0.05),the oxymyoglobin content in RC pigs was significantly higher than that in DLY pigs (P＜0.05),and the relative content of metmyoglobin in RC pigs was significantly lower than that in YR and DLY pigs (P＜0.05).At 24 h after slaughter,the metmyoglobin content,and the relative content of oxymyoglobin and metmyoglobin in RC pigs were significantly higher than that at 45 min and 6 h (P＜0.05).The correlation analysis results showed that there was a significantly high positive correlation among a^*,total myoglobin content and oxymyoglobin content (P＜0.05).【Conclusion】 The pork in RC pigs had bright red meat color due to high a^* and total myoglobin content and low metmyoglobin relative content.The meat color was closely related to myoglobin content and its redox states.

【Objective】 This study was aimed to explore the effect of miR-455-3p on milk fat metabolism in chemically induced mammary epithelial cells (CiMECs) of dairy goats,so as to provide a theoretical basis for the study of microRNA (miRNA) regulation of mammary gland function in goats.【Method】 Goat fibroblasts (GEFs) were induced by a single small molecule compound RepSox (TGFβR-1/ALK5 inhibitor),and the morphological changes of the cells were observed after 8 days of induction,and immunofluorescence staining and oil red O staining,which were specific markers,were used to identify whether the mammary epithelial cells were successfully induced.The overexpression vectors (miR-455-3p mimics and miR-455-3p mimics-NC) and interference vectors (miR-455-3p inhibitors and miR-455-3p inhibitors-NC) for miR-455-3p were transfected into CiMECs using liposomes,which were analyzed by the cell proliferation and toxicity assay (CCK-8) kit,triglyceride assay kit and Real-time quantitative PCR to detect the proliferation rate,triglyceride secretion and the expression of milk fat metabolism genes of miR-455-3p for CiMECs.【Result】 GEFs induced the formation of insular,multinucleated and cobblestone cell shapes similar to goat mammary epithelial cells after 8 days of induction.The immunofluorescence results showed that CiMECs could express epithelial-specific marker antigens,including cytokeratin 14 (CK14) and CK18.The results of oil-red O staining showed that CiMECs possessed the function of secretion of lipid droplets.The CiMECs transfection results showed that compared with control group,miR-455-3p mimics extremely significantly promoted the expression of miR-455-3p (P＜0.01),and miR-455-3p inhibitors significantly inhibited the expression of miR-455-3p expression (P＜0.05).The results of CCK-8 and triglyceride assay showed that compared with control group,the cell proliferation rate and triglyceride secretion were extremely significantly decreased in miR-455-3p mimics group (P＜0.01),and the cell proliferation rate and triglyceride secretion were significantly increased in miR-455-3p inhibitors group (P＜0.05).Real-time quantitative PCR results showed that compared with control group,the expression of HADHB gene was extremely significantly decreased in CiMECs after induction (P＜0.01),was extremely significantly increased in miR-455-3p mimics group (P＜0.01),and was significantly decreased in miR-455-3p inhibitors group (P＜0.05).【Conclusion】 The inhibition of miR-455-3p promoted CiMECs proliferation and reduced the expression of the fat metabolism gene HADHB in dairy goats,thereby increasing the fatty acid content of goat milk.The results could provide a basis for the subsequent use of miRNA to improve the milk quality in goats.

Single-cell sequencing is a high-throughput sequencing technique based on obtaining all genetic information at the level of a single cell.It reveals the intracellular dynamics of individual cells and answer biological questions with high-dimensional catalogs of millions of cells,including genomics,transcriptomics,chromatin accessibility,epigenomics,and proteomics data across species.Within the domain of animal reproduction,single-cell sequencing has emerged as a crucial scientific tool with its powerful ability to decipher cellular and molecular landscapes at single-cell resolution.It is of great significance in solving scientific problems such as the cell classification,intercellular interactions,intercellular communication connections,and the mining of key reproductive genes in animal reproduction.This invaluable technique greatly contributes to advancing our understanding of reproductive mechanisms and enhancing animal reproductive performance.The authors briefly introduce the principles of single-cell sequencing and three types of sequencing,and summarize its role in animal reproductive physiology research,aiming to provide theoretical basis for future research endeavors.

【Objective】 The aim of this study was to study the changes of lipid deposition and lipase activity during the development of liver of Wuliangshan Black-boned chicken and its correlation with lipoprotein lipase(LPL) gene expression.【Method】 In this study,four stages of roosters and hens at 1(D1),42(D42),84(D84) and 126 days of age(D126) were used as the research objects.The body weight and liver weight were measured and the liver index was calculated.HE staining was used to identify the morphological changes of liver.The content of triglyceride (TG) in liver was detected by TG detection kit (single reagent GPO-PAP method).Colorimetric method was used to detect LPL and hepaticlipase (HL) activities.Real-time quantitative PCR was used to detect LPL gene mRNA expression.Pearson correlation analysis was used to determine the correlation between LPL gene expression and liver indexes.【Result】 There were significant differences in liver weight between roosters and hens at different growth stages (P＜0.05).The liver indexes of D42 and D84 were significantly different from those of D1 and D126(P＜0.05).TG content in the liver tissues of both roosters and hens peaked at D84.The activities of LPL and HL in hens reached the peak at D126 and were significantly higher than those at other stages (P＜0.05),and the activities of LPL and HL in roosters reached the peak at D42.The total esterase activity of roosters and hens reached the peak at D42 and D126 respectively and were significantly higher than that at other stages (P＜0.05).The peak of LPL mRNA expression in liver of roosters and hens were at D42 and D1 respectively,and the expression of LPL gene mRNA in hens was significantly correlated with HL activity at three developmental stages of D1,D42 and D126 (P<0.05).【Conclusion】 The liver weight,liver index,TG content,lipase content and LPL gene mRNA expression of Wuliangshan Black-boned chicken varied greatly at different developmental stages.The lipase activity of adult chickens were mainly affected by gender,and the expression of LPL gene mRNA in hens was significantly correlated with HL activity.This results provided a scientific basis for further study on the regulation mechanism of liver fat metabolism in Wuliangshan Black-boned chicken.

【Objective】 The objective of this experiment was to explore the effect of nucleoporin 98 (Nup98) and ribonucleic acid export 1 (Rae1) on meiotic maturation of mouse oocytes in vitro.【Method】 The oocytes of 4-week-old SPF female Kunming mice were isolated and cultured in vitro.The expressions of Nup98 and Rae1 genes in mouse oocytes were detected by Real-time quantitative PCR. The colocalization of Nup98 and Rae1 was detected by immunofluorescence.The Nup98 and Rae1 genes in mouse oocytes were knocked down by electrotransfer siRNA interference technology.The interference efficiency was detected by Real-time quantitative PCR,and germinal vesicle breakdown (GVBD) and first polar body extruction (PBE) were observed.The spindle assembly and chromosome arrangement of mouse oocytes after Nup98 and Rae1 genes knocked down were detected by immunofluorescence method.The rate of chromosome aneuploidy was detected by chromosome climbing film technique.【Result】 Both Nup98 and Rae1 genes were expressed in mouse oocytes. During the germinal vesicle (GV) stage,Nup98 and Rae1 were co-located at the nuclear membrane margin.During meiosis,Nup98 and Rae1 were concentrated on the kinetosomes of chromosomes.The results of siRNA interference test showed that,compared with control group,knockdown of Nup98 and Rae1 gene alone had no significant effect on the expression of the other gene (P＜0.05).After double-knockdown of Nup98 and Rae1 genes,there was no significant effect on the meiosis recovery rate of oocytes (P＞0.05),the PBE rate of mouse oocytes was extremely significantly increased after 9.5 h development (P＜0.01),and the chromosome separation rate and aneuploidy rate were extremely significantly or significantly increased (P＜0.01 or P＜0.05).【Conclusion】 The Nup98 and Rae1 genes were involved in chromosome separation during mouse oocyte meiosis,influencing the occurrence of aneuploidy and thus the developmental potential of fertilized embryos.

【Objective】 The experiment was aimed to investigate the effect of adding Astragalus polysaccharide into the diluent on the cryopreservation of Dorper ram semen.【Method】 The semen was collected from six adult Dorper rams by artificial vagina method,and semen with a viability rate ＞80% was mixed.The fresh semen of sheep was gradually diluted using the base diluent and the cooling diluent,and it was slowly cooled down to 4 ℃,then the semen was diluted with the added Astragali polysaccharides of 0,0.01,0.05,and 0.10 mg/mL and cryo-equilibrated.The balanced semen was dispensed into 0.25 mL freezing tubule and fumigated above liquid nitrogen before semen were frozen at liquid nitrogen.The sperm quality indicators (vitality and viability) and sperm motility parameters (velocity average path,velocity curvilinear,velocity straight line,wobble,linearity,and straightness) were detected through CASA after freezing-thawing.The plasma membrane integrity was detected by HOST,and the sperm DNA integrity and mitochondrial activity were detected using dual fluorescence staining.【Result】 Before and after semen freezing,the sperm motility,viability and motility of sheep were significantly reduced.Sperm vitality,viability,velocity average path,linearity,wobble and mitochondrial activity were significantly higher in 0.01 mg/mL Astragali polysaccharides -added group than in the rest of the groups after semen freezing and thawing (P＜0.05).The sperm velocity curvilinear,velocity straight line,straightness,plasma membrane integrity,and DNA integrity of 0.01 mg/mL Astragalus polysaccharide group were significantly higher than those of control group and 0.10 mg/mL Astragalus polysaccharide group (P＜0.05),but had no significant difference with 0.05 mg/mL Astragalus polysaccharide group (P＞0.05).【Conclusion】 The addition of 0.01 mg/mL Astragalus polysaccharide to the freezing diluents was able to protect the structure of sheep spermatozoa,effectively increase sperm viability,viability and motility after freezing-thawing,and improve the effect of sheep semen freezing.

【Objective】 This study was aimed to perform the bioinformatics analysis of non-structura (NSs) protein of novel Akabane virus (AKAV) and prepare anti-polyclonal antibody,so as to provide necessary materials for detection and functional study of novel AKAV.【Method】 The novel AKAV NSs protein was systematically analyzed using some online analysis website for protein bioinformatics.The full-length NSs gene was cloned and the recombinant NSs protein was expressed by the expression system of Escherichia coli.The induction conditions of the recombinant protein were optimized and the solubility of the recombinant protein was analyzed.The crushed Escherichia coli expressing NSs protein was collected,denatured by 8 mol/L urea,and purified by Ni²⁺-NTA affinity chromatography.After dialysis and concentration,the purified NSs protein was identified by SDS-PAGE and Western blotting.The polyclonal antibody was prepared by immunizing New Zealand White rabbits with purified recombinant NSs protein,then the titer and specific reactivity of the polyclonal antibody were determined by indirect ELISA,Western blotting and indirect immunofluorescence assay (IFA).【Result】 Bioinformatics analysis results showed that the NSs protein was composed of 91 amino acid residues,with an isoelectric point of 12.10,belonging to basic protein.Its instability index was about 79.04,and the average hydrophilicity index was ―0.059,indicating that it was an unstable hydrophilic protein.There was no transmembrane region and signal peptide,the NSs protein was a non-secreted protein.The NSs protein prokaryotic expression vector pET-32a-NSs was successfully constructed.The results of SDS-PAGE and Western blotting showed that the recombinant NSs protein was successfully expressed,with a molecular weight of 29.3 ku and mainly existed in the form of inclusion body,which was consistent with the results of bioinformatic analysis.The titer of the prepared polyclonal antibody reached 1∶16 384 000.The results of Western blotting and IFA showed that the prepared antibody could react specifically with NSs protein,having good specificity.【Conclusion】 The novel AKAV NSs protein was successfully purified and showed good immunogenicity.The polyclonal antibody with high titer and good specificity was prepared,which laid a foundation for the development of vaccine,disease detection and the function study of NSs protein.

【Objective】 This study was aimed to explore the impact of the coexistence of LIPI-3 and LIPI-4 on the virulence of Listeria monocytogenes (LM),reveal their potential regulatory effects on bacterial virulence,and lay a foundation for further research on the pathogenic mechanisms of LM.【Method】 LM928 (possessing LIPI-4 present) and LM873 (possessing both LIPI-3 and LIPI-4) were used as research objects to monitor the adhesion and invasion of two strains by infecting HCMEC/D3 cells.The effect of two strains on the median lethal dose (LD₅₀) of chicken embryos were evaluated by chicken embryo infection test.The hemolytic activity of two strains were determined using hemolysis test.The apoptosis of HCMEC/D3 cells was analyzed using flow cytometry to evaluate the ability of two strain to induce cell apoptosis.The transcriptional differences of virulence genes between two strains under BHI culture conditions and post-infection of HCMEC/D3 cells were compared using Real-time quantitative PCR.【Result】 There was no significant difference of adhesion rates between LM928 and LM873 strains (P＞0.05).The invasion rate of LM928 strain was extremely significantly higher than that of LM873 strain (P＜0.01).The LD₅₀ of LM873 strain was approximately 1 000 times that of LM928 strain.The hemolytic values of LM928 and LM873 strains were 2⁴ and 2³,respectively.The apoptosis rate induced by LM928 strain at 24 and 48 h post-infection were significantly or extremely significantly higher than that of LM873 strain (P＜0.05 or P＜0.01),while there was no significant difference of the apoptosis rate between LM873 strain and control group (P＞0.05).When cultured in BHI culture,compared with LM928 strain,the transcription levels of virulence genes mpl,inlB,inlC,inlP,actA,plcA,plcB and sigB in LM873 strain were significantly or extremely significantly upregulated (P＜0.05 or P＜0.01),and the transcription levels of hly,inlA and iap genes were extremely significantly or significantly downregulated (P＜0.01 or P＜0.05).After infecting HCMEC/D3 cells,compared with LM928 strain,the transcription levels of virulence genes inlP,actA,plcA,plcB and prfA in LM873 strain were significantly or extremely significantly upregulated (P＜0.05 or P＜0.01),and the transcription levels of hly,mpl,inlA,inlB,inlC and iap genes were extremely significantly downregulated (P＜0.01).【Conclusion】 The coexistence of LIPI-3 and LIPI-4 reduced the virulence of LM,which holded significant implications for further research into the molecular mechanisms and complex interactions of LIPI-3 and LIPI-4 in bacterial virulence regulation.

【Objective】 Malassezia is one of the causes of external otitis in cats,which is lipid-dependent.The lipase SMG1 gene can regulate the synthesis of Malassezia globosa lipase.The purpose of this study was to construct the expression vector of lipase SMG1 gene in Pichia pastoris,obtain the recombinant expression product of SMG1 gene,and analyze the activity of lipase enzyme,so as to provide basis for the subsequent research on the function of Malassezia lipase SMG1 gene.【Method】 The pathogenic bacteria of a suspected case of Malassezia feline otitis externa were isolated and identified, and the fungi type was determined by similarity comparison. Lipase SMG1 gene was optimized by DNAworks software,the cloning vector of lipase SMG1 gene was constructed and identified.The eukaryotic expression vector of SMG1 gene was constructed using Pichia pastoris expression system,and the recombinant expression vector was screened and identified.The recombinant carrier lipase activity was determined.【Result】 A strain of Malassezia was isolated from the ear canal in cats which was suspected to have Malassezia external otitis,and the isolate was identified as Malassezia globosa.By optimizing the codon of Malassezia lipase SMG1 gene,the codon adaptation index (CAI) was increased to 0.95.The cloning vector of lipase SMG1 gene was successfully constructed.The recombinant plasmid pGAPZαA-SMG1 was successfully constructed and transferred into Pichia pastoris X33.After the addition of methanol for induced expression,the expression product was analyzed by SDS-PAGE,the expression product of recombinant lipase SMG1 protein was 35 ku,and its enzyme activity was determined to be 0.023 U/mL.The enzyme activity of recombinant lipase SMG1 was the highest at 30 ℃ (0.027 U/mL),which was extremely significantly different from that of 40 and 45 ℃ group (P＜0.01).【Conclusion】 Malassezia globosa had higher lipase activity near the temperature of ear canal in cats,and the lipids promoted the growth of Malassezia.The expression system of Pichia pastoris could express and secrete recombinant lipase SMG1.The results laid a foundation for the pathogenesis of Malassezia externa otitis in cats,the development of lipase inhibitors,and the development of rapid detection technology.

【Objective】 The emergence of multidrug-resistant pathogens poses a serious threat to public health security.The biological and genomic characteristics of a lytic phage of multidrug-resistant Klebsiella pneumoniae from sheep were analyzed in order to provide reference for the search of phage preparations for the prevention and treatment of Klebsiella pneumoniae infection.【Method】 Multidrug-resistant Klebsiella pneumoniae strain YH-2 from sheep was used as host bacteria to isolate and purify phage from fecal samples by double agar plate method,then observed the morphology by transmission electron microscope.The host spectrum,optimum multiplicity of infection (MOI),one-step growth curve,thermal stability,acid and base tolerance of phage were measured.The genome characteristics were analyzed based on whole genome sequencing.【Result】 A lytic phage from Klebsiella pneumoniae,named vB_KpnP_PYH-2 (PYH-2),was successfully isolated,which belong to the Podoviridae family.There was a halo around it,the halo spread around as the culture time went on.Phage PYH-2 could not only lyse the Klebsiella pneumoniae strains of sheep origin,but also lyse Klebsiella pneumoniae strains derived from cow mastitis.The optimal MOI was 0.001,the latent period was 15 min,the burst period was 10 min,and the amount of cleavage was 63 PFU/cell.Phage PYH-2 could maintain stable activity at 40-50 ℃ and pH 4.0-12.0.The full length of the phage PYH-2 genome was 45 958 bp,with 51.86% GC content,and it did not contain any drug resistance genes and virulence genes.There were 52 open reading frames (ORFs) in the genome of PYH-2,only 21 ORFs were known functional genes,including 6 tail-associated proteins.【Conclusion】 In this study,a phage with short incubation period,stable biological characteristics and high bactericides was isolated,which could be used as a candidate phage strain of cracking animal derived Klebsiella pneumoniae,laying a foundation for the prevention and treatment of multi-drug resistant Klebsiella pneumoniae infection.

【Objective】 The aim of this study was to provide more advanced evidence for a comprehensive understanding of the epidemic infection of Classical swine fever virus (CSFV) in Chinese pig herds and for the later epidemiological investigation of CSFV.【Method】 In this study,the relevant literature on the prevalence of CSFV in Chinese pig herds was searched in six major databases at home and abroad,such as CNKI,Wanfang,VIP,PubMed,Web of Science and Science Direct.The search time range was from January 1,2005 to January 1,2023.Through literature screening,literature quality score and data extraction of CSFV prevalence,Meta-analysis and systematic review of the overall prevalence of CSFV were performed using StataMP17 software.Meta-regression analysis and subgroup analysis were used to analyze the heterogeneity sources and influencing factors of the overall prevalence of CSFV.【Result】 A total of 55 literatures related to the prevalence of CSFV were screened from 6 major databases and included in the Meta-analysis and systematic review.Among them,18 literatures were included in the high-quality literature,and 37 literatures were included in the medium-quality literature.The results of Meta-analysis showed that the overall prevalence of CSFV in Chinese pig herds from 2005 to 2023 was 12% (95% CI:0.09-0.15).The results of Meta-regression analysis and subgroup analysis showed that the sources of heterogeneity of the literature did not include sampling time and pig species (P＞0.05),while the factors affecting the prevalence of CSFV included diagnostic methods,seasonal changes,climatic factors,altitude,latitude,longitude and regional distribution (P＜0.05).【Conclusion】 This study determined the overall prevalence of CSFV in pigs in China from the data.The overall prevalence rate was not high,and the epidemic trend was still scattered in the region,mainly chronic and atypical strains.The results had certain reference significance for grasping the global situation of CSFV epidemic in pig herds,effective prevention and control,and purification of classical swine fever (CSF).

【Objective】 This study was aimed to investigate the impact of the hypoxia inducible factor-1α (HIF-1α) signaling pathway and mitochondrial function on inflammatory responses in chicken macrophages (HD11) under lipopolysaccharide(LPS)-induced condition.【Method】 HD11 cells were treated with LPS at different concentrations and cultured for 24 h to detect cell viability and screen the optimal concentration.At the same time,the optimal action time of LPS was screened under the optimal concentration.HD11 cells were divided into control and model groups.The HD11 cells in model group were cultured with LPS at the optimal concentration,while the cells in control group were received an equivalent amount of complete culture medium.After culture according to the optimal time of LPS action,total RNA was extracted for transcriptome sequencing,and GO function annotation and KEGG pathway enrichment analysis were performed for differentially expressed genes.mRNA and protein expression of HIF-1α signaling pathway related factors were detected by Real-time quantitative PCR and Western blotting.Mitochondrial membrane potential and reactive oxygen species (ROS) were detected by flow cytometry.【Result】 The optimal condition of LPS-induced HD11 cell inflammation model was 1 μg/mL LPS culture for 12 h,and the cell inflammation model was successfully established under this condition.Compared with control group,a total of 2 063 differentially expressed genes were identified in model group,with 1 319 genes upregulated and 744 genes downregulated.GO functional annotation analysis showed that differentially expressed genes were significantly enriched in processes such as immune system response,response to external stimuli and cytokine receptor binding.KEGG pathway enrichment analysis showed that differentially expressed genes were significantly enriched in 50 signaling pathways,mainly involved in immune cell-mediated inflammatory response and Toll-like receptor,nuclear transcription factor-κB (NF-κB),HIF-1α and other signaling pathways.Among them,13 significantly upregulated genes were concentrated in HIF-1α-related signaling pathways,including Toll-like receptor 4 (TLR4),NF-κB,HIF-1α,and vascular endothelial growth factor (VEGF) genes. Flow cytometry determination results showed that compared with control group,mitochondrial membrane potential was extremely significantly decreased in model group (P＜0.01),and ROS level was significantly increased (P＜0.05).【Conclusion】 The LPS-induced inflammation model of chicken macrophages was established successfully.The crosstalk of HIF-1α and NF-κB signaling pathway was involved in the LPS-induced inflammation process.At the same time,the mitochondrial function of cells decreased,resulting in the increase of ROS production,and then promoted the expression of HIF-1α,and jointly aggravated the inflammatory response and metabolic disorders.

【Objective】 This study was aimed to investigate the changes of interferon (IFN) and interferon-stimulating genes (ISGs) expression in chicken Leghorn male hepatoma (LMH) infected with Fowl adenovirus serotype-4 (FAdV-4),and provide reference basis for the study of the interaction between FAdV-4 and ISGs.【Method】 The experiment was conducted by infecting LMH cells with FAdV-4,observing the cellular lesions and collecting cell samples at 0,12,24,36,48,60,72,84 and 96 h post-infection,and the dynamic change patterns of the expression of IFN-α and IFN-β and ISGs genes at the transcriptional level at different time points after infection were detected by Real-time quantitative PCR.【Result】 The results showed that after FAdV-4 infection of LMH cells,LMH cells showed typical cytopathic lesions at 48 h.It was detected that FAdV-4 proliferated rapidly at 12 h,and peaked at 60 h. Compared with control group,the expression of IFN-α at the transcriptional level was significantly down-regulated after infection (P＜0.05).Compared with control group,the expression of IFN-β was significantly down-regulated at 12 and 24 h after infection (P＜0.05),and then rapidly and significantly up-regulated to the peak at 36 h (P＜0.05),and remained at a high level at 48 h.After that,the expression declined and stabilized,but was still significantly up-regulated compared with control group (P＜0.05).The expressions of ISG12,IFIT5 and DDIT4 genes did not change much in the early stage of infection,and reached the peak at 96 h after infection (P<0.05).ZFP313,IFITM3 and Viperin genes did not change much in the early stage of infection,and then rapidly and up-regulated to peak at 36 h (P＜0.05),and remained at a high level at 48 h.After that,the expression declined,but was still significantly up-regulated compared with control group(P＜0.05).CD47,OAS and PKR genes showed down-regulated expression compared with control group.【Conclusion】 After FAdV-4 infection of LMH cells,IFN and a variety of ISGs showed regular changes at the transcriptional level,which were associated with the replication of the virus in LMH cells,suggesting that these natural immune factors might play an important role in the anti-FAdV-4 viral response.

【Objective】 Avian metapnemovirus (aMPV) is a member of Metapnemovirus genus in the family Paramyxoviridae.The swollen head syndrome and decreased egg production caused by aMPV had caused serious harm to the breeding industry.In order to effectively prevent aMPV infection and further study its pathogenesis,enhanced green fluorescent protein (EGFP) was inserted into aMPV subtype C (aMPV/C) to construct recombinant aMPV expressing EGFP.Meanwhile,this study used different vectors and promoters to construct aMPV minigenome to optimize the construction of aMPV reverse genetic system (RGS).【Method】 EGFP was inserted into the non-coding region between P and M proteins in pBluescript-aMPV complete RGS,and rescued to observe the expression of EGFP.The titer and genetic stability of the recombinant virus were also determined.In order to optimize the construction of RGS,pBluescript SK(＋) with T7 promoter and pcDNA3.1 with T7 and CMV promoters were used to construct aMPV minigenome.Firstly,the leader and trailer regions at both ends of the aMPV/C genome and the cDNA encoding EGFP were amplified by PCR and gelled for recovery.Secondly,each glue recovery fragment was seamlessly cloned and connected with the pre-designed homologous arm in the order of leader-EGFP-trailer,and sequenced for verification.Finally,the fully connected minigenome was reverse-inserted into the two vectors,and the aMPV/C minigenome (pBR-aMPV/C and pc-aMPV/C) were constructed.In the RGS rescue experiment,two aMPV/C minigenomes were co-transfected into BHK-21 cells with three plasmids expressing N,P and L proteins,and the expression of EGFP was detected by inverted fluorescence microscopy.【Result】 The rescued aMPV-EGFP recombinant virus was sequenced,and the results showed that EGFP had been successfully inserted into the aMPV genome and was stably expressed in the aMPV-EGFP recombinant virus within three generations.The virus titer of aMPV-EGFP recombinant virus reached 10^5.5 TCID₅₀/mL at 96 h after infection.The sequencing results of two recombinant plasmids,pBR-aMPV/C and pc-aMPV/C,also proved that minigenome containing EGFP had been constructed.The three recombinant plasmids were then co-transfected into the cells with the helper plasmids.After transfection for 24 h,the expression of green fluorescence was observed in the cells,which proved that the aMPV-EGFP recombinant virus and the two minigenomes,pBR-aMPV/C and pc-aMPV/C,successfully expressed EGFP.【Conclusion】 In this study,two aMPV minigenomes were successfully constructed and the aMPV-EGFP recombinant virus was rescued.The recombinant virus had good genetic stability.When CMV and T7 were used as promoters in aMPV/C RGS,the rescue efficiency was the same.Both pBluescript SK(＋) and pcDNA3.1 could be used for aMPV/C RGS construction,and the result of this study provided a variety of methods for aMPV/C RGS construction.

【Objective】 The purpose of this experiment was to investigate the coccidial infection of yak in some areas of Sichuan and Tibet,and provide theoretical reference for the prevention and control of yak coccidiosis in this area.【Method】 A total of 376 fecal samples of yaks aged 2 to 6,7 to 12 and over 12 months were collected from 8 yak farms in some areas of Sichuan and Tibet.Qualitative examination of coccidium oocysts in fecal samples was carried out by saturated saline flotation method.The oocyst per gram (OPG) in coccidioid positive fecal samples was counted by MacMaster’s method,and to understand the coccidial infection of yaks in different farms and at different ages.【Result】 A total of 85 positive samples were detected in 8 yak farms,with an average infection rate of 22.6% (85/376) and an average OPG of 1 643.A total of 9 Eimeria species were detected,the dominant species was Eimeria alabamensis,and the infection rate was 37.6% (32/85),most of which were mixed infections.The average infection rate was the highest in yaks aged 2 to 6 months (37.9% (11/29)).The infection rate was 31% (57/184) in yaks aged 7 to 12 months.The infection rate of coccidia in yaks over 12 months was 10.4% (17/163).【Conclusion】 There were different degrees of coccidial infection in yaks in some areas of Sichuan and Tibet,and the mixed infection was the main infection.The infection rate,infection intensity and dominant species of coccidia in yaks were different in different regions and at different ages.

【Objective】 This study was aimed to investigate the improvement effect and anti-inflammatory protective mechanism of Bushen Tongfu decoction(BTD) on the learning and memory abilities of LPS-induced cognitive impairment model rats.【Method】 SPF grade male SD rats of 6-week-old were selected.The rat models of cognitive impairment were established by LPS lateral ventricle injection method.All the successful modeled rats were divided into model group,sodium oligomannate capsules (SOC) group,BTD high-dose,middle-dose and low-dose groups randomly.At the same time,the sham operation control group was set.Drugs were administered to rats by gastric perfusion once a day for 8 weeks.BTD high-dose,middle-dose and low-dose groups were give the drug at 15.12,7.56,3.78 g/kg.SOC group was given the drug at 0.094 g/kg.The model and sham operation control groups were administered with equal volumes of pure water.On the 8th weekend,all the rats were detected the spatial learning and memory ability by Morris water maze.The pathological changes in the hippocampus of rats were detected by hematoxylin eosin (HE) staining.The activation of microglia in the hippocampus was detected by immunofluorescence.The expression levels of tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6),IL-1β,N-methyl-D-aspartate receptor(NMDA),γ-aminobutyric acid (GABA) and acetylcholine (Ach) in hippocampus neurons were detected by Western blotting analysis.【Result】 Compared with sham operation group,the escape latency and swimming distance of rats in model group were significantly prolonged,the number of across the platform after remove platform were decreased significantly (P＜0.05).The cells were arranged irregularly,sparse,and there were cell vacuoles by the HE staining,the expression of inducible nitric oxide synthase (iNOS)/ ionized calcium binding adapter molecule 1 (Iba1) were increased and the expression of arginase-1(Arg1)/Iba1 were decreased significantly (P＜0.05),the expression levels of TNF-α,IL-6 and IL-1β were increased significantly and the expression levels of NMDA,GABA and Ach were decreased significantly in hippocampus neurons (P＜0.05).Compared with model group,the escape latency and swimming distance in BDT high-dose,middle-dose,low-dose groups and SOC group were significantly shortened,and the number of times of crossing the original platform after removing the platform was increased significantly (P＜0.05),the cells were arranged tightly and neatly,and the cell morphology tended to be normal by the HE staining,the expression of iNOS/ Iba1 were decreased significantly (P＜0.05),the expression of Arg1/Iba1 were decreased significantly only in the BTD high-dose (P＜0.05),the expression levels of TNF-α,IL-6,IL-1β were decreased significantly and the expression levels of NMDA,GABA,Ach were increased significantly (P＜0.05).【Conclusion】 BTD could improve the learning and memory ability of rats with LPS-induced cognitive impairment model.The possible mechanism was inhibiting the activation of proinflammatory microglia and decreasing the levels of TNF-α,IL-6,IL-1β and increasing the levels of NMDA,GABA,and Ach in the hippocampus.

【Objective】 The aim of this experiment was to investigate the effects of epigallocatechin gallate (EGCG) on lipopolysaccharide (LPS) induced inflammation and apoptosis in bovine mammary epithelial cells (BMECs) and its potential protective mechanisms.【Method】 The cell inflammation model was constructed by LPS-induced BMECs.The cell viability was detected by CCK-8 and the optimal concentration of EGCG was screened.BMECs were divided into control group,LPS treatment group and EGCG＋LPS co-treatment group.Real-time quantitative PCR was used to detect the mRNA expression levels of inflammatory factors,apoptosis factors and anti-oxidation-related genes.Mitochondrial membrane potential and reactive oxygen species (ROS) levels of BMECs were detected by kit.The apoptosis of BMECs was detected by flow cytometry.【Result】 After LPS-induced BMECs,the mRNA expressions of inflammatory factors interleukin-6 (IL-6),IL-8 and IL-1β genes were extremely significantly or significantly increased at all time periods compared with 0 h (P＜0.01 or P＜0.05),the cell inflammatory damage model of BMECs was successfully constructed.CCK-8 assay showed that the optimal concentration of EGCG was 5 μmol/L.Real-time quantitative PCR results showed that,compared with control group,the mRNA expressions of IL-6,IL-8, MDA, Bax and Caspase-3 genes in LPS-induced BMECs were extremely significantly increased (P＜0.01),while the mRNA expressions of GSH-Px and Bcl-2 genes were extremely significantly decreased (P＜0.01).Compared with LPS group,mRNA expressions of IL-6,IL-8,IL-1β,MDA,Bax and Caspase-3 genes were significantly or extremely significantly decreased in EGCG＋LPS group (P＜0.05 or P＜0.01),and the mRNA expressions of GSH-Px,SOD and Bcl-2 gene were significantly or extremely significantly increased (P＜0.05 or P＜0.01).The results of mitochondrial membrane potential showed that compared with control group,LPS significantly weakened the red fluorescence and enhanced the green fluorescence of BMECs,while the co-incubation of EGCG and LPS had no significant effect on the red fluorescence of BMECs,and the green fluorescence was relatively weakened.ROS level detection results showed that compared with control group,the green fluorescence of cells in LPS group was significantly enhanced,while the green fluorescence of cells was weakened after co-incubation of EGCG and LPS.Flow cytometry showed that compared with control group,death cells and apoptotic cells increased significantly after LPS treatment,the apoptosis rate was extremely significantly increased (P＜0.01).After co-incubation with EGCG and LPS,the apoptotic cells were significantly decreased,and the apoptotic rate was significantly decreased (P＜0.05).【Conclusion】 EGCG could alleviate inflammatory response and inhibit cell apoptosis by inhibiting ROS release induced by LPS in BMECs and alleviating mitochondrial damage.The results provide a theoretical basis for the prevention and treatment of mastitis in dairy cows with EGCG.

【Objective】 The experiment was aimed to prepare enteric formulations of temicoxacin using solid dispersion technology,improve its stability in acid,enable its targeted release in small intestine,improve its dissolution rate and bioavailability,and provide theoretical references for the development and clinical application of new dosage forms of the drug.【Method】 Acrylic resin Eudragit L100 and polyvinylpyrrolidone (PVP) were used to form a binary carrier,and the intestinal pH-triggered localized release formulation was prepared by the co-precipitation method.With in vitro cumulative solubility as the evaluation index,the optimal preparation conditions were screened by orthogonal test,and X-ray diffraction,Fourier infrared spectroscopy and scanning electron microscopy were used for the identification of the physical phase.【Result】 The optimal preparation process of tilmicosin solid dispersion enteric formulation was Eudragit L100∶PVP= 10∶1,drug-carrying ratio of 1∶2,stirring time of 2 h,curing time of 12 h.The dissolution of this formulation was ＜10% in an acidic environment for 2 h,and the degree of solubility reached 75.02% in buffer solution at pH 6.8 for 2 min,and it was completely dissolved in 10 min,which significantly improved the dissolution rate of tilmicosin.The dissolution rate of tilmicosin was significantly improved.X-ray diffraction result showed that the diffraction intensity of the solid dispersion at 17°and 23°was higher than that of tilmicosin API.Fourier infrared spectroscopy result showed that the sharp absorption peaks of solid dispersion of tilmicosin at 1 593 and 1 457 cm^-1 were disappeared.Scanning electron microscopy diagrams showed that tilmicosin had been uniformly dispersed in the carrier,the solid dispersion of tilmicosin was formed.【Conclusion】 Tilmicosin solid dispersion enteric formulation was prepared successfully by choosing the combined carrier,which was stable in acid and dissolved in pH 6.8 buffer with a good dissolution degree.

【Objective】 The purpose of this experiment was to determine the pathogen in the tissue samples of pigs with suspected piglet meningitis caused by Streptococcus suis in a pig farm in Guangdong,and to determine its drug sensitivity and pathogenicity,so as to provide medication for pig farms and explore the mechanism of meningitis caused by this pathgen.【Method】 Bacterial isolation and culture,morphological observation,biochemical testing,and molecular identification were performed on the samples of piglets suspected to have meningitis caused by Streptococcus suis.Drug sensitivity testing,virulence gene testing,mouse infection testing,and histopathological diagnosis were also conducted.【Result】 The isolate from the piglet brain tissue showed hemolysis on the blood plate.Gram staining microscopy showed that bacteria were stained purple,with a circular shape and some arranged in a chain shape.Biochemical characteristics showed that the isolate could ferment raffinose,lactose,inulin and esculin,but could not ferment sorbitol,mannitol,arabinose,melezitose and D-ribose.The hippurate reduction test was negative,which were consistent with Streptococcus suis,and was determined to be Streptococcus suis after sequencing analysis.The serotype identification results showed that the isolate was Streptococcus suis type 9,named GDS209.The results of drug sensitivity test showed that GDS209 was resistant to 10 antibacterials such as cefalotin (pioneer Ⅰ),cefalexin (pioneer Ⅳ) and ampicillin/sulbactam,and was sensitive to ticarcillin/clavulanic acid,florfenicol and amoxicillin.The detection results of virulence genes showed that GDS209 carried gdh,gapdh,fbps,orf2,srtA and ccpA virulence genes,but did not carry mrp,sly,luxS and epf virulence genes.The mouse infection test showed that the LD₅₀ of GDS209 was 1×10⁹ CFU.Histopathological observation showed that GDS209 could cause congestion and inflammation in Kunming mice’s heart,liver,spleen,lung and brain to varying degrees.【Conclusion】 In this study,a strain of Streptococcus suis serotype 9 was isolated,which was resistant to commonly used antimicrobials in most pig farms and sensitive to ticarcillin/clavulanic acid,florfenicol and amoxicillin.The results of this study could provide some reference for the clinical diagnosis and drug treatment of Streptococcus suis serotype 9.

【Objective】 Based on network pharmacology and molecular docking,the active ingredients and targets of Euphorbiae humifusae herba in the prevention and treatment of ulcerative colitis (UC) were analyzed,and its potential mechanism was explored to protect the intestinal tract of animals.【Method】 The active ingredients and corresponding drug targets of Euphorbiae humifusae herba were obtained by TCMSP database.Disease targets were obtained from GeneCards,DisGeNET,TTD,PharmGKB and DrugBank databases.Potential targets were obtained by intersecting drug targets and disease targets,and the protein-protein interaction (PPI) analysis and GO function and KEGG pathway enrichment analysis of potential targets were performed by STRING and DAVID databases,respectively.AutoDock 1.5.7 software was used for molecular docking verification,and PyMOL software was used to visualize the docking results.Sodium dextran sulfate (DSS)-induced UC model was established in 30 BALB/c mice in vivo.Control group,model group,Euphorbiae humifusae herba low (5 mg/mL),high dose group (15 mg/mL) and mesalazine group (52 mg/mL) were set up.Except for control group,mice in the other groups were free to drink 3% DSS to induce UC model for 7 d. Mice in the drug treatment group was intragastrically administered with 0.2 mL of the corresponding dose once a day.Mice in control and model groups were intragastrically administered with the same volume of sterile normal saline for 7 d.The changes of body weight,disease activity index (DAI) score and colon length were measured,and the contents of tumor necrosis factor-α(TNF-α),interleukin-6 (IL6) and IL10 were detected.【Result】 The main active ingredients of Euphorbiae humifusae herba were kaempferol,4’-5-dihydroxyflavone and ellagic acid,with 256 corresponding drug targets,4 265 disease targets,128 potential targets and 7 core targets.GO functional enrichment involved response to reactive oxygen species,response to oxidative stress,inflammatory response,membrane raft,cave,kinase regulator activity,etc.KEGG signaling pathway involved PI3K-Akt and TNF signaling pathways,and so on.Molecular bonding results showed that kaempferol and peroxisome proliferator-activated receptor γ (PPARG),4’,5-dihydroxyflavone and PPARG,ellagic acid and serine/threonine protein kinase 1 (AKT1),ellagic acid and IL6 had strong binding potential.In vivo experiments showed that compared with control group,mice in model group had severe weight loss,severe watery bloody stools,and an extremely significant increase in DAI score(P＜0.01).A large number of inflammatory cell infiltrations were found in the mucosa and submucosa of the colon tissue,and even small ulcers appeared,indicating that the mouse UC model was successful.Compared with model group,the infiltration of inflammatory cells in the colonic mucosa of each administration group were reduced,the DAI score was extremely significantly reduced (P＜0.01),the colonic contracture was improved,the contents of TNF-α and IL6 were significantly reduced (P＜0.05),the content of IL10 was significantly increased (P＜0.05),and the inflammation was inhibited.【Conclusion】 The main active ingredients such as kaempferol,4’-5-dihydroxyflavone and ellagic acid in Euphorbiae humifusae herba might participate in inflammation-related signaling pathways and biological functions by acting on core targets such as AKT1,PPARG and IL6,inhibited inflammation and reduced intestinal damage,thus exerting the effect of preventing and treating UC.

【Objective】 The aim of this study was to improve the solubility of baicalein (BAI),enhance its dissolution and bioavailability,and expand its clinical application.【Method】 Solid dispersion of baicalein (BAI-ASD) was prepared by rotary evaporation using polyethylene caprolactam-polyethylene acetate-polyethylene glycol graft copolymer (Soluplus) as the carrier,and it was characterized including that:Using the position and intensity of the characteristic diffraction peaks in powder X-ray diffraction (PXRD) to preliminarily determine whether the solid dispersion was successfully prepared;Differential scanning calorimetry (DSC) was used to study thermal properties;Fourier transform infrared spectroscopy (FT-IR) was used to analyze intermolecular interactions;Scanning electron microscopy (SEM) was used to observe the microscopic topography.The in vitro release performance was investigated by dissolution test,and the pharmacokinetic characteristics of oral BAI-ASD in chickens were investigated.【Result】 There was no crystal diffraction peak of BAI baicalein in PXRD and no obvious endothermic peak in DSC,indicating that the baicalein solid dispersion BAI-ASD was successfully prepared,and BAI baicalein existed in an amorphous form in the solid dispersion ASD.The results of in vitro dissolution showed that the cumulative dissolution of baicalein BAI within 240 min was only 6.62%,and the cumulative dissolution of solid dispersion ASD was significantly increased when the ratio was 1∶9 and 2∶8,and the cumulative dissolution within 240 min was 29.89% and 52.67%,respectively.The stability study results showed that BAI-ASD had good stability within 90 days at 45 ℃ and 75% RH.The pharmacokinetic results showed that the peak concentration (C_max(0-24)) and area under the curve (AUC_(0-24)) of BAI-ASD within 24 h of intragastric administration were (5.20±0.82) μg/mL and (17.03±0.67) μg·h/mL,respectively,which were 1.91 and 2.64 times of BAI,respectively.【Conclusion】 BAI-ASD,a solid dispersion of baicalein prepared by rotary evaporation with Soluplus as the carrier,had good stability,which could effectively improve the cumulative dissolution and oral bioavailability of BAI.

【Objective】 The purpose of the experiment was to develop an ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method,investigate the pharmacokinetic characteristics of two carprofen formulations in Beagle dogs,and evaluate the bioequivalence of two formulations.【Method】 Twenty-four beagle dogs were randomly allocated to two equal-sized treatment groups in a randomized dual-cycle and dual-sequence cross-over design.The test and the reference (RIMADYL^) formulations were subcutaneously administered in a single dose of 4.4 mg/kg BW.Blood samples were collected from brachiocephalic vein at 0,0.5,1,2,3,4,6,8,12,24,36,48 and 72 h after administration.The specificity,linearity,detection limit,quantification limit,accuracy,precision and stability of the UPLC-MS/MS method were investigated.The concentration of carprofen in plasma was determined by UPLC-MS/MS.The pharmacokinetic parameters (T_max,C_max,T_1/2 and AUC_0-t) and bioequivalence were calculated and analyzed by WinNonlin 8.1.【Result】 The methodological results showed good correlation over the concentration range of 10-5 000 ng/mL with a correlation coefficient(R²)≥0.99.The limit of detection and the limit of quantification were 5 and 10 ng/mL,respectively.Relative recoveries of high,medium,low and quantification limit concentrations were between 91.45%-111.61%.Coefficient variations of intra-assay and inter-assay were both less 15%.The pharmacokinetic parameters of two formulations were as follows:T_max were(2.27±1.09) and (2.33±1.10) h;C_max were (20.02±5.24) and (19.92±5.42) μg/mL;T_1/2 were (8.54±3.71) and (8.65±2.64) h;AUC_0-t were (246.78±55.94) and (249.22±53.33) μg·h/mL.The plasma profiles of carprofen following the administration of both formulations were similar.And the 90% CI of the average C_max,AUC_0-t and AUC_0-∞of the test/reference preparation were all fell between 80.00%-125.00%.【Conclusion】 The UPLC-MS/MS method established in this experiment was accurate and reliable,and could be used to determine the concentration of carprofen in plasma samples.The two formulations were bioequivalent for carprofen,and could be used clinically for the treatment of related diseases.The result of this study provided theoretical basis for clinical rational administration.

【Objective】 Clostridium perfringens (CP) is a Gram-positive anaerobic bacterium that can cause sudden calf death,hemorrhagic necrotizing enteritis,enterotoxemia,etc.,which has a huge impact on calf health. The experiment was aimed to understand the prevalence and drug resistance of Clostridium perfringens in Angus calves in a large-scale beef cattle farm in Kashgar prefecture,Xinjiang,so as to provide a scientific basis for the treatment of diseases caused by Clostridium perfringens in this region.【Method】 Twenty tissue samples and six enclosure manure samples were collected for isolation and identification of bacteria by separation and culture,morphological observation,staining microscopy,biochemical test,etc., the drug susceptibility test was carried out by K-B paper method,and the virulence type and drug resistance genes were detected by PCR.【Result】 The isolates appeared turbid in FTG liquid medium and produced a large amount of gas,round colonies with neat black edges grew in TSC medium,and colonies with typical double hemolytic rings grew on sheep blood plates.Microscopic examination of Gram staining showed that the bacteria were coarse and short,in single or double arrangement,and the morphology and microscopic examination results were consistent with the characteristics of Clostridium perfringens.A total of 10 strains of Clostridium perfringens were isolated in 26 samples,with a detection rate of 38.46%.After toxin genotyping,9 strains of Clostridium perfringens type A were identified with a detection rate of 90.0%,and 1 strain of Clostridium perfringens type D was identified with a detection rate of 10.0%.Susceptibility test results showed that the isolates had the highest resistance rate of 80.0% to kanamycin,50.0% to gentamicin and lincomycin,and were sensitive to meropenem,cefepime,florfenicol and doxycycline.The results of drug resistance genes showed that the detection rate of bla_TEM gene was the highest,which was 100.0%,and the detection rates of bla_SHV,qnrA and aac(6')-Ⅰb-cr resistance genes were 20.0% (2/10),10.0% (1/10) and 20.0% (2/10),respectively,and the two resistance genes of bla_CTX-M and qnrS were not detected.【Conclusion】 Clostridium perfringens infection in Angus calves in Kashgar prefecture,Xinjiang was mainly type A,with serious drug resistance and multiple drug resistance.The results of this study could provide a scientific basis for epidemiological research and prevention and control of Clostridium perfringens in the later stage.

【Objective】 This study was conducted to investigate the relationship between hoof disease and blood indicators and intestinal microbiota composition.【Method】 According to clinical examination,6 British thoroughbred mares aged (12±3) years old with hoof disease were selected as the experimental group (group S),and 6 healthy British thoroughbred mares with similar weight and age were selected as the control group (group C).Clinical examination and evaluation were performed for 3 days.The cervical venous blood and rectal feces of the two groups were collected,and blood routine and blood biochemical examination were performed.16S rRNA sequencing was used to detect the composition of intestinal flora in horses.【Result】 Clinical examination showed that horses in group S had lameness,hoof nail deformation and other symptoms.Blood routine test showed that there were no significant differences in blood indexes between the two groups (P＞0.05), among which the leukocyte count and neutrophil count in the blood of group S were higher than the normal range, and the hemoglobin concentration was lower than the normal range.Blood biochemical tests showed that compared with group C,the contents of triglyceride,total protein,urea,creatinine in group S showed an upward trend,while the contents of albumin,serum total bilirubin and other indicators showed a downward trend (P>0.05).16S rRNA sequencing results showed that at the phylum level,compared with group C,the relative abundance of Elusimicrobia in group S was significantly decreased (P＜0.05),and relative abundance of SR1 was extremely significantly decreased (P＜0.01).At the genus level,compared with group C,the relative abundance of Weissella,Pedomicrobium,Pontibacter and Kurthia in group S was significantly increased (P＜0.05),and the relative abundance of Escherichia,Candidatus_Endomicrobium,Microvirga,Peptococcus and Prevotella were significantly decreased (P＜0.05).There were 10 bacteria with significant difference in intestinal flora between the two groups. 【Conclusion】 After the occurrence of hoof disease,there would be inflammation in the body,resulting in damage to liver and kidney function,changes in the abundance of intestinal flora,and reduced digestive capacity.The results laid a foundation for further exploration of the relationship between intestinal flora and metabolic disorders and hoof disease,and provided clues for the prevention and treatment of hoof disease by regulating intestinal flora.

【Objective】 Rhodococcus equi infection can cause fatal suppurative pneumonia in the host.In order to provide guidance for clinical diagnosis and treatment,the biological characteristics of Rhodococcus equi isolate from lung pus of a dead foal in Xinjiang was analyzed.【Method】 The lung pus samples of dead foal were collected for bacterial isolation and culture.The species were identified by colony morphology observation,Gram staining,biochemical reaction,16S rRNA PCR amplification and sequencing,and the genetic evolution relationship was analyzed.The drug sensitivity of the isolate was determined by drug-sensitive disk diffusion method.The pathogenicity of the strain was evaluated by mouse infection test.【Result】 The isolated strain grew well on the NANAT plate,and the colonies were black and viscous,and the TSA medium was milky and slime.Blunt circles at both ends or rod-shaped Gram-positive bacilli were detected by Gram staining microscopy.The results of biochemical reaction showed that the isolated strain did not ferment carbohydrate and produce indigo matrix,did not liquefy gelatin,oxidase negative,decomposition of urea.16S rRNA sequencing results showed that the isolated strain and Rhodococcus equi strain WGC11 were in the same branch and were closely related.The results of drug sensitivity test showed that the isolated strain was resistant to polymyxin B and β-lactam drugs,and sensitive to other selected antibiotics.The results of pathogenicity test in mice showed that the mortality rate of mice was 33.3% (2/6) within 4 days of abdominal attack.Necropsy of the remaining surviving mice showed abscess and hemorrhage in lungs,swelling,bleeding and stasis of the liver and spleen.Histopathological observation showed that spleen,liver and lung tissues had different degrees of pathological changes. 【Conclusion】 A pathogenic Rhodococcus equi strain was isolated and identified from the lung samples of foal in Xinjiang.The strain had strong pathogenic ability and natural drug resistance.The results laid a theoretical foundation for the research and control of the pathogenic mechanism of Rhodococcus equi.

Immunomagnetic separation technology is a pre-treatment technology that separates the target adsorbed on the surface of the magnetic bead from the sample under the magnetic field.After the immune magnetic bead captures the target,the immunemagnetic bead-target complex is magnetically fixed on the tube wall,and the target can be obtained by elution of impurities.The enrichment effect of immunomagnetic separation technology is mainly affected by antibody specificity,magnetic bead diameter,magnetic bead addition amount,incubation time and sample pH,in which antibody specificity is the key to affect the enrichment effect of magnetic beads.Using the characteristics of immunomagnetic beads to adsorb antibodies,the coating step can be replaced in the enzyme-linked immunosorbent assay method,which can greatly reduce the detection time.As a pre-treatment method,immunomagnetic separation technology can improve the sensitivity and efficiency of the detection method when combined with other detection methods.In this review,the technical principle,general separation steps,common influencing factors and the application in enzyme-linked immunosorbent assay,immunochromatography,PCR,Real-time quantitative PCR,loop-mediated isothermal amplification reaction,flow cytometry,electrochemical sensors,liquid chromatography were described,so as to provide reference for the research and application of immunomagnetic separation technology.

Triterpenoids are a class of natural compounds of multiple isoprene units.They have various biological activities such as anti-inflammatory,antioxidant properties and potential therapeutic effects in relieving intestinal inflammation.Firstly,triterpenoids exert their anti-inflammatory effects by suppressing the secretion of inflammatory mediators (interleukins,prostaglandins,tumor necrosis factor) and their related signaling pathways,thereby reducing the production of intestinal inflammation.Secondly,triterpenoids improve the generation of reactive oxygen species,balance the redox system,and reduce oxidative stress damage,leading to their anti-inflammatory properties.Moreover,triterpenoids regulate the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome and multiple signaling pathways (NF-κB/MAPK/Nrf2 signaling pathways) to improve the levels of immunoglobulins and complement,so achieving immune balance.Fourthly,triterpenoids promote the expression of tight junction proteins,repair damaged intestinal mucosal epithelium,enhance intestinal barrier function,and reduce the penetration of irritants,thus inhibiting intestinal inflammation.Furthermore,triterpenoids exhibit a close relationship with the gut microbiota.They can maintain gut microbiota stability,preserve the integrity of the intestinal epithelial barrier,and reduce the occurrence of intestinal inflammation.In conclusion,triterpenoids protect against intestinal inflammation by regulating inflammation,oxidative stress,immune balance,mucosal barrier and gut microbiota.Although there are certain limitations and challenges in the current research on triterpenoids,this review provides an important reference for further exploration,development and utilization of triterpenoids,and offer new ideas for their application in the field of intestinal inflammation.

【Objective】 This experiment was aimed to study the antibacterial effect and mechanism of a novel antibacterial peptide LL-1.【Method】 The minimum inhibitory concentration (MIC) of antibacterial peptide LL-1 against Klebsiella pneumoniae was detected,and the in vitro antibacterial effect of antibacterial peptide LL-1 against Klebsiella pneumoniae was evaluated through the antibacterial curve.Leakage of alkaline phosphatase (ALP),β-galactosidase,nucleic acid and protein were determined to evaluate the effect of antibacterial peptide LL-1 on the cell membrane and cell wall of Klebsiella pneumoniae.The binding of antibacterial peptide LL-1 to Klebsiella pneumoniae DNA was detected by nucleic acid gel electrophoresis.The effect of antibacterial peptide LL-1 on the morphology of Klebsiella pneumoniae was observed through scanning electron microscopy.【Result】 Antibacterial peptide LL-1 had good antibacterial activity against Klebsiella pneumoniae,with a MIC of 50 μg/mL.The antibacterial curve showed that 2 MIC of antibacterial peptide LL-1 could completely inhibit the growth of Klebsiella pneumoniae within 6 h.Antibacterial peptide LL-1 led to the leakage of ALP and β-galactosidase of Klebsiella pneumoniae,and 1/4 MIC of antibacterial peptide LL-1 resulted in a extremely significantly higher leakage rate than the control group(P＜0.01).The detection of nucleic acid and protein leakage further confirmed that antibacterial peptide LL-1 might cause the efflux of intracellular contents in Klebsiella pneumoniae.In addition,antibacterial peptide LL-1 could bind to the DNA of Klebsiella pneumoniae.The results of scanning electron microscope observation suggested that antibacterial peptide LL-1 caused the surface of Klebsiella pneumoniae to become rough,bacterial deformation.【Conclusion】 The results showed that antibacterial peptide LL-1 increased the permeability of the cell wall and membrane in Klebsiella pneumoniae,and bound with the DNA of Klebsiella pneumoniae,and thus exerted antibacterial effects.This would lay the foundation for in-depth research on the antibacterial mechanism of antibacterial peptide LL-1 against Klebsiella pneumoniae.

【Objective】 This study was aimed to develope a comprehensive evaluation method that considered the interaction of host cells,internalized bacteria and antimicrobial agents to accurately compare internalized bacteria-mediated antimicrobial resistance.【Method】 Using IEC-6,RIMVECs and RAW264.7 cells as host cells,Staphylococcus aureus and Salmonella as internalized bacteria,seven chemical drugs including ampicillin,ciprofloxacin,tetracycline,and four traditional Chinese medicine standards including quercetin and kaempferol as antibacterial drugs,the minimum extracellular bactericidal concentration (MBC_EX) of antibiotics was detected,and the bacteria-cell interaction model was established.The minimum intracellular bactericidal concentration (MBC_IN) of antibiotics was detected by bacterial colony count.The percentage of endobacteria-infected cells (N) and the percentage of endobacteria-infected cells (N_D) after drug treatment were measured.The drug tolerance value (Tr) was calculated to evaluate the antimicrobial resistance mediated by internalized bacteria.【Result】 Staphylococcus aureus was resistant to ampicillin,ciprofloxacin,erythromycin,polymyxin,rifampin,tetracycline,vancomycin and baicalin (Tr＞1).Among them,the Tr value of ciprofloxacin was the highest and the tolerance was the most serious.It did not produce tolerance to quercetin,kaempferol and catechin (Tr≤1).The results of tolerance of different internalized bacteria to tetracycline showed that the Tr value of resistance of Staphylococcus aureus to tetracycline was greater than that of Salmonella.The influence of different host cells on internalized bacteria-mediated antimicrobial resistance showed that Staphylococcus aureus had the highest tolerance to tetracycline in IEC-6 cells.【Conclusion】 The Tr value evaluation method of tolerance could not only effectively evaluate the tolerance degree of internalized bacteria to different antibiotics,but also compare the influence of different host cells on the resistance to antibiotics mediated by internalized bacteria,and visually and data-present the tolerance of internalized bacteria to antibiotics,providing guidance for clinical precision medicine.