【Objective】 The purpose of this study was to clone and analyze the CDS sequence of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) gene in buffalo,and its coding protein was analyzed by bioinformatics,so as to prepare for the study of effects of TRAIL protein on the development of follicle,proliferation and apoptosis of granulosa cells in buffalo ovary.【Method】 The CDS sequence of TRAIL gene in buffalo was cloned by RT-PCR.The similarity of nucleotide and amino acid sequences of TRAIL gene were analyzed,the phylogenetic tree was constructed,and the structure and function of TRAIL protein were analyzed by bioinformatics softwares.【Result】 The CDS sequence of TRAIL gene in buffalo was successfully cloned,with a length of 864 bp,encoding 287 amino acids.The similarity of TRAIL gene between buffalo and Bos mutus,Bos taurus,Sus scrofa, Capra hircus, Ovis aries,Equus caballus,Pan paniscus, Homo spiens,Mus musculus were 99.2%,99.3%,95.9%,96.3%,84.7%,84.8%,81.3%,81.3% and 70.0%,respectively.The phylogenetic tree results showed that buffalo had the closest genetic relationship with Bos mutus and Bos taurus,and the farthest genetic relationship with Mus musculus.Amino acid sequence alignment results showed that the transmembrane domain and TNF domain were highly conserved among different species.TRAIL protein was a hydrophilic protein and had a transmembrane domain,the 140-285 amino acids were TNF region.There was no glycosylation site and signal peptide of TRAIL protein,which were mainly located in the cytoplasm,and had 29 phosphorylation sites.The secondary structure of TRAIL protein was mainly random coil,accounting for about 51.57%,followed by extended chain (24.39%) and alpha helix (24.04%).The tertiary structure of TRAIL protein was consistent with the secondary structure,and the similarity with the model protein TRAIL protein in human was 75.53%.【Conclusion】 In this experiment,the CDS sequence of TRAIL gene was cloned in buffalo,with a size of 864 bp and encoding 287 amino acids.The buffalo had the closest relationship with Bos mutus and Bos taurus,and the TRAIL protein transmembrane domain and TNF domain were highly conserved among different species,which might be related to its function.

【Objective】 This study was aimed to understand the biological information of let-7a-5p,and initially explore its functional role in the muscle tissue of Jingyuan chickens.【Method】 The mature sequence of let-7a-5p in Jingyuan chickens obtained by transcriptome sequencing was conservatively analyzed by the bioinformatics method.The target genes of let-7a-5p were predicted by miRDB,TargetScan,and miRanda online softwares,and their intersection was used for GO function and KEGG pathway enrichment analysis.Real-time quantitative PCR was used to detect the expression of let-7a-5p in tissues of female and male in Jingyuan chickens.【Result】 The results showed that the mature sequence of let-7a-5p was highly conserved among species,and 38 gene sets were predicted.The GO items with rich target genes in biological process,cellular composition and molecular function were cellular process,biological regulation,cell,binding,and catalytic activity.Target genes were significantly enriched in ketoglycan biosynthesis,the role of AGE-RAGE signal pathway in diabetic complications,ubiquitin-mediated proteolysis,TGF-β signal pathway and vasopressin-regulated water reabsorption signal pathway,and the most enriched was metabolic pathway,the role of AGE-RAGE signal pathway in diabetic complications,ubiquitin-mediated proteolysis and MAPK signal pathway.The interaction of let-7a-5p-target genes mainly inhibited the expression or degraded the target mRNA by acting on its 3'-UTR.Tissue expression results revealed that let-7a-5p was expressed in heart,liver,spleen,lung,kidney,chest muscle, and leg muscle tissues of female and male in Jingyuan chickens,and the expression was higher in chest muscle and leg muscle.【Conclusion】 let-7a-5p might regulate the expression of target genes and participate in the muscle growth and development of Jingyuan chickens,which provided a theoretical basis for further exploring the function and mechanism of let-7a-5p.

Gene editing is a modern biological technology that relies on endonuclease for directional modification of target genes,it can achieve the addition and deletion of specific fragments and the insertion,deletion and replacement of specific bases.The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/associated nuclease 9) system is an acquired immune system found in bacteria and archaea to resist the invasion of foreign plasmids and phages.After modification,it has become a novel genome editing tool.Because of its advantages of simple operation,low cost and high cutting efficiency,it is widely used in genome editing of various animals,plants and microorganisms.CRISPR/Cas9 system can be used to analyze and verify gene functions,study genetic breeding and screen drug targets and vaccine molecules on the basis of gene editing,so as to reduce the pathogenicity of pathogenic microorganisms,enhance the disease resistance of animals and plants,and treat genetic diseases,viral infections and tumors.Due to the complex life history of parasites,larger genome and more complex structure compared with bacteria and virus,and lack of effective research tools,relevant research progress is slow.However,the emergence of CRISPR/Cas9 gene editing technology provides a new technical means for gene function research.In this review,the discovery of CRISPR/Cas9 system and its application in parasite genome editing were reviewed,and the application status and development of CRISPR/Cas9 system were summarized and prospeced.

【Objective】 The aim of the experiment was to clone different splice isomers of cyclin B2 (CCNB2) gene in chicken,and analyze their bioinformatics and tissue expression.【Method】 Reverse transcription PCR was used to amplify and clone the different splice isomers of CCNB2 gene in Taihang chickens.Partial nucleotide sequences of different splice isomers of CCNB2 gene were compared by bioinformatics software, the amino acid sequence similarity of different splice isomers of CCNB2 in different species was compared, the phylogenetic tree was constructed,and its subcellular localization and secondary structure were predicted.The relative expression of different splice isoforms of CCNB2 gene in ovaries and follicles of Taihang chickens at different developmental stages were analyzed by Real-time quantitative PCR.【Result】 In Taihang chickens,the CCNB2 gene had three splicing isomers:CCNB2-AS1,CCNB2-AS2 and CCNB2-AS3,which encoded 390,383 and 401 amino acids,respectively.The amino acid sequence similarity among the three splices CCNB2-AS1,CCNB2-AS2 and CCNB2-AS3 of Taihang chickens was 97.00%,the three spliceosomes had the highest similarity with Gallus,which were 99.71%,99.71% and 100%, respectively.The three splice isomers were evolutionarily closest to Gallus gallus,followed by Phasianus colchicus.Subcellular localization showed that the three splice isoforms were mainly localized in the cytoplasm.Protein secondary structure analysis showed that the three splice isomers were alpha helix.The relative expression levels of CCNB2-AS1,CCNB2-AS2 and CCNB2-AS3 in the follicles of Taihang chickens at different developmental stages were different,and the expression trends of different splice isomers in the follicles at the same stage were also different. CCNB2-AS1 was highly expressed in pregrade follicles.The expression of CCNB2-AS2 in great white follicles was significantly higher than that in other stages (P＜0.05).The expression of CCNB2-AS3 showed an opposite trend to that of CCNB2-AS1,and was highly expressed in graded follicles.【Conclusion】 In this study,three splice isoforms of CCNB2 gene in Taihang chickens were successfully identified,which were expressed in follicles at all stages,but there were differences in the expression trend in pregrade follicles.

【Objective】 This study was aimed to clone the galectin-1 (Gal-1) gene in chickens,analyze its encoded protein by bioinformatics techniques,and detect its expression in different tissues in White Leghorn chickens,in order to provide scientific basis for further elucidating its in antiviral effect.【Method】 PCR was used to amplify the complete CDS region of Gal-1 gene with the spleen cDNA as template,and the similarity alignment and phylogenetic tree construction were carried out.Bioinformatics softwares were used to predict the physicochemical properties,hydrophilicity/hydrophobicity,transmembrane domain,signal peptide,modified structure,conserved domains and advanced structures of Gal-1 protein.Real-time quantitative PCR was used to detect the expression of Gal-1 gene in heart,liver,spleen,lung,kidney,brain,glandular stomach,muscular stomach,duodenum,jejunum,cecum,rectum,pectorales and leg muscles tissnes.【Result】 The length of CDS region of chicken Gal-1 gene was 408 bp,encoding 135 amino acids.The similarity alignment results showed that the similarity of Gal-1 gene nucleotide sequence of White Leghorn chickens with Meleagris gallopavo,Anas platyrhynchos and Taeniopygia guttata were 97.1%,88.4% and 82.2%,respectively.The phylogenetic tree analysis showed that White Leghorn chickens had the closest relationship with Meleagris gallopavo.The relative molecular weight of Gal-1 protein was 15.06 ku,the isoelectric point was 6.57,the instability coefficient was 36.05 and the lipid coefficient was 74.30,and the average hydrophilic index was ―0.259.Gal-1 protein had no signal peptide and no transmembrane domain,while had two obvious hydrophilic domains,and the coding protein was stable and hydrophilic.The prediction of secondary structure showed that the Gal-1 protein was dominated by random coil (45.93%) and extended chain (41.48%),and the prediction of tertiary structure was consistent with that of secondary structure.Real-time quantitative PCR results showed that Gal-1 gene mRNA was widely expressed in tissues of White Leghorn chickens,with the highest expression level in the lung and the lowest in the brain.【Conclusion】 In this study,the CDS region sequence of Gal-1 gene was successfully cloned,and it was widely expressed in 14 tissues such as heart and liver of White Leghorn chickens,which could provide references for further study on the function of Gal-1 protein in chickens.

Galectin-1 (GAL-1) is a β-galactoside-binding protein with a molecular mass of about 14 ku,which is distributed in many different types of cells and tissues,and has a wide range of intracellular and extracellular biological activity,involved in the regulation of various physiological and pathological processes.GAL-1 regulates cell proliferation in a dose-dependent and cell-specific bidirectional manner,promotes angiogenesis through the vascular endothelial growth factor receptor 2 (VEGFR2) pathway,participates in the immune regulation of the body,induces T cell apoptosis,inhibits T cell proliferation and antigen-presenting cell activation.In addition,GAL-1 is considered as a marker of various types of tumors,and tumor cells secrete GAL-1 to promote angiogenesis and autoimmune escape in tumors.Deer antler is the unossified and young horns with densely hairy derived from the head of the deer,and it is also the only bone appendage that can achieve periodic holomorphosis in mammals.The regeneration of deer antler is based on the process of deer antler stem cells proliferation. GAL-1 is overexpressed in deer antler stem cells,and the regeneration process of deer antler is closely related to the biological function of GAL-1.However,the role of GAL-1 in deer antler regeneration remains to be further investigated.The authors reviewed the protein structure and biological functions of GAL-1,as well as its role in the regeneration of deer antler,and hopes to bring new enlightenment to the study of the function of GAL-1 and the mechanism of deer antler regeneration.

【Objective】 The lentiviral overexpression vector of porcine neuromedin B receptor (NMBR) gene was constructed and its expression was detected in porcine Leydig cells,in order to provide references for studying the function of NMBR gene overexpression in Leydig cells.【Method】 Specific primers were designed and synthesized according to the CDS sequence of porcine NMBR gene (GenBank accession No.:KM058699),and the full-length sequence of porcine NMBR CDS was amplified by RT-PCR.pMD19-T-NMBR was constructed.The plasmids of pCD513B-1 and pMD19-T-NMBR were digested by Xba Ⅰ and EcoR Ⅰ.pCD513B-1-NMBR plasmid was constructed.HEK-293T cells were co-transfected with pCD513B-1-NMBR and packaged plasmids,and the green fluorescence protein (GFP) was observed by fluorescence inversion microscopy after transfection for 48 h,and the porcine NMBR gene overexpression lentivirus was obtained from cell culture liquid supernatant,and the titer of the virus was measured by multiple dilution method.Then lentivirus was transfected into porcine Leydig cells,and GFP expression was observed after transfection for 48 h.The cells were collected and the expression of NMBR mRNA was detected by Real-time quantitative PCR.【Result】 The full-length sequence of porcine NMBR CDS was successfully amplified by RT-PCR,and the size was 1 173 bp.The pMD19-T-NMBR recombinant plasmid was cleaved into 2 specific bands,which were consistent with the size of pMD19-T plasmid and porcine NMBR CDS sequence fragment,respectively,indicating that pMD19-T-NMBR plasmid was successfully constructed.The linearized pCD513B-1 and NMBR sequences were obtained by double digestion.RT-PCR and sequencing results showed that the lentiviral overexpression vector pCD513B-1-NMBR was successfully constructed.48 h after pCD513B-1-NMBR transfection,HEK-293T cells showed a large amount of GFP,indicating that the packaged virus was successful,and the virus titer was about 4×10⁶ TU/mL.More than 80% of Leydig cells were expressed GFP after NMBR gene lentivirus transfection for 48 h,indicating that most of the cells were successfully transfected with lentivirus.The Real-time quantitative PCR results showed that the expression of NMBR mRNA was extremely significantly increased after transfection with lentivirus(P＜0.01).【Conclusion】 In this study,the lentivirus overexpression vector of porcine NMBR gene was successfully constructed,and it was confirmed that porcine NMBR gene overexpression lentivirus could increase the expression of NMBR gene in porcine Leydig cells.The results laid a foundation for studying the role of NMBR gene overexpression in Leydig cells.

【Objective】 The aim of this study was in order to find out the cause of the tumorigenicity difference between two different strains of Avian leukemia virus subgroup K(ALV-K),further consider the pathogenic mechanism of ALV-K,and realize the construction of infectious clones and virus rescue of two recombinant viruses of ALV-K.【Method】 The infectious clones of the previously constructed ALV-K HB2018003 and HB2015032 strains were used as templates,and the separate 5'-long terminal repeat(5'-LTR) gene fragments and the remaining target gene fragments of the two strains were obtained by PCR amplification.After the target fragment was recovered by cutting glue,the 5'-LTR of the two strains were exchanged,and the connecting product was obtained by homologous recombination.After transformed into E.coli DH5α competent cells,the ligation products were coated with Amp resistant plates for overnight culture.The positive colonies identified by PCR were selected to extract recombinant plasmids TOPO-003-032 and TOPO-032-003 and sequenced.The recombinant plasmids identified by PCR and sequencing were transfected into susceptible cells DF-1 for 3 generations blindly,and the rescued virus was detected by group-specific antigen P27 ELISA,multiplex PCR and indirect immunofluorescence.【Result】 The results of sequencing identification and recombinant infectious clone PCR identification showed that the 5'-LTR exchanged recombinant plasmid was successfully constructed.Multi PCR results showed that the expected band ALV-K appeared at the size of the target fragment,and ALV-A and ALV-J bands did not appear.The results of indirect immunofluorescence test showed that DF-1 cells inoculated with rescue virus showed obvious bright green fluorescence,while the negative control did not show fluorescence.Above all,the two recombinant viruses were successfully rescued and named rHB2022050(TOPO-032-003) and rHB2022051(TOPO-003-032),respectively.【Conclusion】 In this study,the 5'-LTR exchanged infectious clones TOPO-003-032 and TOPO-032-003 were constructed by homologous recombination,and the viruses rHB2022050(TOPO-032-003)and rHB2022051(TOPO-003-032) were successfully rescued.

【Objective)】 This study was aimed to explore the developmental changes of nutritional components and meat quality characteristics of Yuxi Black pigs at different weight stages.【Method】 5 group Yuxi Black pigs with average body weight of 60,75,90,105 and 120 kg were selected,there were 25 in total,5 in each group,3 in each group were randomly selected for slaughter,male and female random.After slaughter,the longissimus dorsi muscle were collected to measure eye muscle area,backfat thickness,fat rate,water loss rate,fatty acid,amino acid and cholesterol, and determine intramuscular fat content,lipid droplet diameter and lipid droplet number.100 g psoas major muscle were used to analyze the cooked meat rate.【Result】 Before the weight of Yuxi Black pigs reached 120 kg,the eye muscle area,backfat thickness,fat percentage,cooked meat rate,intramuscular fat content,meat color,shearing force,number and diameter of fat drop increased with the increase of slaughter live weight.The eye muscle area at 105 and 120 kg were extremely significantly higher than that at 60 and 75 kg (P＜0.01),and significantly higher than that at 90 kg (P＜0.05).The backfat thickness at 90,105 and 120 kg were extremely significantly higher than that at 60 kg (P＜0.01).The intramuscular fat content at 90,105 and 120 kg were significantly higher than that at 60 and 75 kg (P＜0.05).The diameter and number of lipid droplets at 75,90,105 and 120 kg were significantly higher than those at 60 kg (P＜0.05).The main saturated fatty acids were myristic acid,palmitic acid and stearic acid,while the main unsaturated fatty acids were palmitic acid,oleic acid and linoleic acid.There were abundant types of amino acids,and the contents of histidine,tyrosine,valine and isoleucine at 120 kg were significantly higher than those at 105 kg (P＜0.05).The body weight was extremely significantly or significantly positively correlated with cooked meat rate,crude protein,meat color and crude fat (P＜0.01 or P＜0.05).The intramuscular fat content was extremely significantly or significantly positively correlated with marbling,crude fat,meat color and lipid droplet diameter (P＜0.01 or P＜0.05).【Conclusion】 When the slaughter live weight up to 120 kg,the intramuscular fat content was moderate,the marbling pattern was rich,the ratio of unsaturated fatty acid to saturated fatty acid was suitable,the content of umami amino acid was increased,the content of cholesterol was lower,the meat color was fresh and tender,and the carcass quality and meat quality were the best.

【Objective】 The purpose of this experiment was to study the effects of feeding fermented feed in early growth stage (1-21 days old) on growth performance,nutrient utilization,intestinal health and meat quality of broilers.【Method】 A total of 240 1-day-old AA broilers were randomly divided into 4 groups with 6 replicates in each group and 10 chicks in each group.In the early growth stage (1-21 days old),the four treatment groups were fed with experimental feed supplemented with 0,5%,10% and 15% fermented feed respectively,while in the later growth stage (22-42 days old),all the chicks in four treatment groups were fed basic feed without fermented feed.On the 21st and 42nd day of the experiment,the growth performance in 1-21 days old,22-42 days old and 1-42 days old,nutrient utilization efficiency in 19-21 days old and 40-42 days old,the number of intestinal bacteria and meat quality on 42nd day of broilers were measured.【Result】 Compared with the control group,feeding 10% fermented feed significantly decreased the feed to gain ratio of 1-21 days old broilers (P＜0.05);Feeding 15% fermented feed significantly increased the average daily gain and average daily feed intake of broilers aged 1-42 days,the dry matter utilization rate and energy utilization rate of 19-21 days old broilers (P＜0.05).Feeding fermented feed at early stage had no significant effect on the number of cecal bacteria of broilers at 42 days old (P＞0.05).Feeding fermented feed significantly decreased the content of cholesterol in meat of broilers on the 42nd day compared to control group (P＜0.05).【Conclusion】 Feeding 10% fermented feed at early growth stage significantly reduced feed to gain ratio of 1-21 days old broilers,and feeding 15% fermented feed significantly increased the average daily gain,average daily feed intake,and dry matter and energy utilization rate of 19-21 days old broilers.Feeding fermented feed reduced cholesterol content on the 42nd day of chicken,but had no significant effect on the number of cecal bacteria of 42-day-old broilers

【Objective】 Response surface method was used to optimize the formulation of lyophilized nutritional protectants for Pichia pastoris recombinant bacteria containing active peptide CC34.【Method】 The optimum range of skimmed milk powder,trehalose,sorbitol and monosodium glutamate to improve the survival rate of recombinant Pichia pastoris was screened by single factor test. The regression equation of the survival rate of recombinant Pichia pastoris was established by Box-Behnken method,and the effect relationship of three protectant combinations on the survival rate was analyzed.【Result】 When the concentration of skimmed milk powder was 10%,the freeze-drying survival rate of recombinant Pichia pastoris was the highest,reaching 51.23%. When the concentration of trehalose was 15%,the freeze-drying survival rate was the highest,reaching 67.50%. When the concentration of sorbitol was 20%,the freeze-drying survival rate was the highest,reaching 52.19%.And when the concentration of sodium glutamate was 3%,the freeze-drying survival rate was the highest,reaching 36.92%.The concentrations of skimmed milk powder,trehalose and sorbitol in the freeze-drying protectant combination of Pichia pastoris recombinant strain obtained by response surface were 10.75%,15.70% and 20.19%,respectively. The survival rate of recombinant Pichia pastoris was predicted to be 77.00% by the optimal model.The freeze-drying test with the optimal combination of protectants showed that the survival rate of the cell was 78.98%.【Conclusion】 In this experiment, the formulation of compound freeze-drying protectant for recombinant Pichia pastoris was optimized by response surface methodology. Under the conditions of skim milk powder concentration of 10.75%, trehalose concentration of 15.70% and sorbitol concentration of 20.19%, a higher survival rate (78.98%) of recombinant Pichia pastoris could be obtained. The compound freeze-drying protectant could be used for the preservation of recombinant Pichia pastoris containing active peptide CC34 and subsequent experimental research.

The gastrointestinal tract of animals is regarded as an important barrier against external pathogens,and the health of gastrointestinal tract is inseparable from the health of animal organism.The gastrointestinal tract of animals is inhabited by a large and diverse microbial population,the interactions between microorganisms are complex and diverse,and the function and species composition of microorganisms affect the homeostatic balance of gastrointestinal tract.By colonizing the gastrointestinal mucosa,the microbiota plays a crucial role in the development of mucosal immune system,it also plays an important role in the health and function of gastrointestinal tract.There are many ways to regulate the health of gastrointestinal tract,and at present,the main way to regulate the function of gastrointestinal tract and maintain the health of gastrointestinal tract is by adding non-nutrients and nutritional regulators such as prebiotics and probiotics.However,immunoglobulin regulation of gastrointestinal tract has received less attention compared to other nutritional regulators,is a new immunomodulation technology,which has the advantages of specificity and selectivity,easy and low-cost production,and significant effects.Immunomodulation is an efficient and relatively safe modality of regulation.The author introduces the main physicochemical properties of four immunoglobulins (IgG,IgA,IgY and nanobodies),synthesizes the current research on the regulation of gastrointestinal tract functions by immunoglobulins and provides a prospect for future development,so as to provide guidance for the application of immunoglobulins to regulate gastrointestinal tract health and promote nutrient metabolism in animals.

【Objective】 This study was conducted to explore the general characteristics and reveal influencing factors of the eating time in Holstein cows in Beijing area for the purpose of laying a foundation for genetic analysis of daily eating time,and providing reference for accurate management of Holstein cows.【Method】 The original (one record per hour) and daily records (one record per day) for eating time of 1 166 lactating Holstein cows from a large-scale dairy farm in Beijing area were collected from July 2019 to March 2021.The mixed linear model of SAS 9.2 software was used to analyze the impacts of parity,season,lactation stage,herd and test year on daily eating time.【Result】 The average daily eating time was (224.43±60.24) min,and the original eating time was (9.41±4.67) min/h for Holstein cows. According to the statistical analysis,the eating time was greatly influenced by feeding management and the peak of eating time always came up with the feeding time of TMR in dairy farm.Furthermore,season,parity and lactation stage had impacts on eating time. During the test period, the daily feeding time decreased generally with the increase of temperature and humidity index (THI), and it was relatively low in summer.The parity,season,lactation stage,herd and test year had significant effects (P＜0.01) on daily eating time.The daily eating time was the highest in spring (201.66 min±1.91 min) and the lowest in summer (158.85 min±1.92 min).As the increase of parity,a decrease trend for daily eating time was observed,and it dropped by 4.90 and 27.36 min in second and third parity,respectively.Daily eating time had a slightly downtrend during the whole lactating stage after late transition period and it was the highest at mid-lactation period(197.62 min±1.80 min).【Conclusion】 The population characteristics of eating time in Holstein cows were invetigated and showed that daily eating time could be influenced by environment and individual physiological effects.Based on the results from this study,dairy farms could improve the farm management using eating time records automatically obtained by the collar system.

【Objective】 This experiment was conducted to investigate the effects of Astragalus polysaccharide (APS)on milk yield,immune function and antioxidant performance of dairy cows.【Method】 Forty-four Chinese Holstein dairy cows with good body condition,similar body weight,milk yield,lactation days and parity were randomly divided into control group(CK),APS20,APS55 and APS90 groups,with 11 cows in each group.All experimental cattle were fed with basal diet.The control group was given 200 mL warm water,and the experimental groups were given 200 mL APS solution of 20,55 and 90 g/d,respectively,the pre-test period lasted for 10 days,and the formal test period lasted for 60 days.During the normal feeding period,milk yield was counted every 10 days.Milk samples were collected at 0,30th and 60th days of normal feeding period,and milk somatic cell count(SCC) was determined.The blood samples of tail root vein were collected before morning feeding on day 0,30th and 60th of normal feeding period,and the related immune indexes and antioxidant indexes in serum were determined.【Result】 During the whole experiment period,there was no significant difference in milk yield among all groups (P＞0.05).In terms of somatic cell count,compared with CK group,on the 30th and 60th days, the SCS of APS55 group was significantly decreased (P＜0.05),the SCS of APS20 and APS90 groups had no significant difference at all time points (P＞0.05),and the SCS of APS90 group was significantly higher than APS55 group (P＜0.01).In terms of immune indicators,compared with CK group,on the 30th day,the serum IL-2 content in APS20 group was significantly increased (P＜0.05),and the serum IL-2 contents in APS55 and APS90 groups were extremely significantly increased (P＜0.01).On the 60th day,the serum IL-2 contents in APS20,APS55 and APS90 groups were extremely significantly increased (P＜0.01),and the IL-2 contents in APS55 and APS90 groups were extremely significantly higher than that in APS20 group (P＜0.01).On the 60th day,the serum IL-6 content of APS55 group was significantly increased (P＜0.05).In terms of antioxidant indexes,compared with CK group,serum CAT activity in APS20,APS55 and APS90 groups was extremely significantly increased on the 30th and 60th days (P＜0.01);On the 60th day,the serum MDA concentration in ASP90 group was significantly decreased (P＜0.05),the serum GSH-Px concentration in APS20,APS55 and APS90 groups was significantly increased (P＜0.05),and the serum T-AOC activity in APS55 and APS90 groups was extremely significantly increased (P＜0.01) and was significantly higher than that in APS20 group (P＜0.05).【Conclusion】 APS 55 g/d could improve immune function and antioxidant capacity of dairy cows,and reduce milk somatic cell count,the results provided references for the application of APS in dairy production.

In recent years,the price of feed protein raw materials has continued to rise,and China is in short supply of feed protein raw materials and relies on imports for a long time.Therefore,finding and developing new protein sources has become a research hotspot to solve the shortage of feed protein raw materials.Hermetia illucens is a kind of saprophytic insect of diptera,Stratiomyidae family.It can transform organic waste into biological matter with high added value quickly,and has obvious advantages of low breeding cost,fast breeding speed and high output.It is rich in amino acids,antimicrobial peptides (AMPs),chitin and other bioactive substances,and its high proportion of protein and fat content lays a foundation for it to be a high-quality animal feed.A large number of studies have shown that Hermetia illucens partly replaces feed protein composition can effectively enhance animal immune and antioxidant capacity,at the same time through positive regulating intestinal beneficial microbial population and increase the way bacteria abundance,have the effect of improving animal intestinal health,as a kind of promising protein feed raw materials is expected to find a new breakthrough for animal husbandry and aquaculture.The authors have briefly described nutrition characteristic and main active ingredient of Hermetia illucens,systematically summarized the latest research progress on the effects of feeding Hermetia illucens on intestinal barrier,immune function and intestinal flora of pigs and poultry.The future research ideas and development direction of Hermetia illucens are prospected,in order to provide references for the further development and research of Hermetia illucens and its popularization and application in animal breeding production.

【Objective】 This study was aimed to explore the polymorphism of fucosyltransferase 8 (FUT8) gene g.74522417 C＞T and its effect on the growth traits in sheep,and screen the new genetic markers that could be used for molecular breading in sheep.【Method】 The g.74522417 C＞T of FUT8 gene in Hu sheep (n=992) and Ujumqin sheep (n=333) were typed by Sequenom MassARRAY? SNP typing technology,Illumina OvineSNP 600K BeadChip and Illumina OvineSNP50K BeadChip chip detection technology,and analyzed the association between g.74522417 C＞T and growth traits in Hu and Ujumqin sheep.【Result】 There were three genotypes (CC,TC and TT) of FUT8 gene g.74522417 C＞T in Hu and Ujumqin sheep,respectively.Population genetic analysis showed that g.74522417 C＞T was in moderate polymorphism in Hu and Ujumqin sheep (0.25＜PIC＜0.50).The result of χ²test showed that g.74522417 C＞T was in Hard-Weinberg equilibrium in two sheep population (P＞0.05).Association analysis revealed that FUT8 gene g.74522417 C＞T was significantly associated with body length at 4 months of age and body height at 6 months of age in Ujumqin sheep (P＜0.05),was significantly or extremely significantly associated with shin circumference at 3 months of age,waist angle width and shin circumference at 5 months of age,and loin muscle area and shin circumference at 6 months of age in Hu sheep (P＜0.05 or P＜0.01).【Conclusion】 FUT8 gene g.74522417 C＞T could be used as a potential molecular marker for growth and development in sheep,providing a reference for the application of FUT8 gene in molecular breeding of sheep.

【Objective】 The purpose of this study was to analyze the effects of farms,birth season,birth month,birth type and sex on the birth weight trait of Hu sheep,and estimate the heritability of this trait.【Method】 The non-genetic factor analysis of 8 352 lambs produced by a total of 4 570 ewes in three large-scale Hu sheep breeding enterprises in Xinjiang in 2021 was carried out using SAS 9.2 software,and the variance components of the birth weight traits of Hu sheep was analyzed by DMU software.The heritability of the trait was estimated using two single-trait animal models,and the optimal fixed-effects combination and animal model were determined according to Akaike’s information criterion (AIC).【Result】 The farms,birth season,birth month,birth type and sex had extremely significant effects on the birth weight of Hu sheep (P＜0.01).There was no significant difference in the mean birth weight between farm 1 and farm 2 (P＞0.05),and both were extremely significantly higher than that of farm 3 (P＜0.01).The birth weight of lambs born in winter was generally large,and the birth weight of lambs born in January was the largest.The more abundant the birth type were,the smaller the birth weight of lambs was,and the birth weight of lambs among different birth type showed extremely significant difference (P＜0.01).According to the calculation of single trait animal model (considering maternal effect),the direct,maternal and total heritability of birth weight traits of Hu sheep were 0.251,0.668 and 0.211,respectively,which belonged to medium heritability.【Conclusion】 When analyzing the production data of the first year of the sheep farm,it was more appropriate to choose the month of birth as the time factor than the birth season.It could not only analyze the production of Hu sheep in each month to make the corresponding breeding plan adjustment,but also select the month as the fixed effect to increase the accuracy of the estimated genetic parameters of traits.The heritability estimation of the birth weight trait of Hu sheep would be too large without considering the maternal effect,so the maternal effect should be given priority in the early stage which was greatly affected by the maternal effect.

【Objective】 In order to study the polymorphism of major facilitator superfamily domain containing 2a (MFSD2A) gene of buffalo and screen the SNP associated with milk production traits of buffalo.【Method】 The blood DNA was collected from 383 healthy buffaloes,and the SNP were screened and genotyped by PCR amplification and Se-quenom MassARRAY SNP time-of-flight mass spectrometry.The genetic diversity,linkage disequilibrium (LD) and haplotype analysis were analyzed,and the association between different genotypes and haplotypes and milk production traits was analyzed.【Result】 Seven SNPs were detected in MFSD2A gene of buffalo,and the polymorphism information content (PIC) ranged from 0.25 to 0.40,which belonged to moderate polymorphism.Except g.8290 T＞C,all the other sites were in Hardy-Weinberg equilibrium(P＞0.05).The results of association analysis showed that the seven SNPs were significantly associated with 305 d milk yield (P＜0.05),and the 305 d milk yield of mutant heterozygote was the lowest among the three genotypes.g.568 C＞G and g.701 A＞G were significantly associated with milk fat percentage (P＜0.05).The order of milk fat percentage genotypes was mutant homozygote＞wild homozygote＞mutant heterozygote.g.568 C＞G,g.3326 G＞A,g.8290 T＞C and g.8523 C＞G were significantly associated with milk protein percentage (P＜0.05),but these seven SNPs were not significantly associated with milk urea nitrogen (P＞0.05).The results of LD and haplotype analysis showed that g.568 C＞G,g.701 A＞G and g.3203 T＞G could form a haplotype module (Block 1),while g.8523 C＞G and g.9138 G＞T could form another haplotype module (Block 2).The r² of g.568 C＞G and g.701 A＞G,g.568 C＞G and g.3326 G＞A,g.8523 C＞G and g.9138 G＞T were 0.87,0.37 and 0.48,respectively,which were in a state of strong linkage disequilibrium.The milk protein percentage of H1 (CAG) haplotype in Block 1 was significantly higher than that of H2 (CAT) and H3 (GCT) haplotype (P＜0.05),and the 305 d milk yield of D3 (GG) haplotype in Block 2 was significantly higher than that of D1 (CG) and D2 (CT) haplotype (P＜0.01).【Conclusion】 The seven SNPs of MFSD2A gene were significantly associated with 305 d milk yield,g.568 C＞G and g.701 A＞G were significantly associated with milk fat percentage,g.568 C＞G,g.701 A＞G,g.3326 G＞A,g.8290 T＞C and g.8523 C＞G were significantly associated with milk protein percentage were significantly associated,and none of these seven SNPs were significantly associated with milk urea nitrogen.Homozygous genotypes had more advantages in the selection of high 305 d milk yield and milk fat percentage.H1 (CAG) and D3 (GG) haplotypes were favorable haplotypes to increase 305 d milk yield and milk protein rate in buffalo.

High-throughput sequencing (HTS) is the basis for studying the genetic mechanisms of complex biological traits.With the continuous optimization and improvement of high-throughput sequencing technology,some genomic variations related to biological phenotype traits have been accurately excavated,including single nucleotide polymorphism (SNP),insertion/deletion (Indel),copy number variation (CNV),and structure variation (SV) as the representative molecular markers.Compared with the traditional breeding method,molecular genetic markers exhibit high polymorphism throughout the genome.The detection method is simple,quick,and economic.By detecting genome-wide molecular markers and using genome-wide genetic information to assess individual or population genetic resources,the generation interval can be shorten and the accuracy of seed selection can be improved,thus achieving great genetic progress in a short period of time.From the perspective of high-throughput sequencing technology mining molecular genetic markers,this paper summarizes the development history and application fields of third-generation sequencing technology and the application of third-generation molecular genetic marker detection technology in the innovation of laying hens breeding industry.Moreover,The precise application of high-throughput sequencing technology combined with molecular genetic markers in genetic diversity and evolutionary classification of laying hens,construction of population genetic map and mapping of functional genes,genetic mechanism analysis of quantitative traits and genetic mechanism analysis of quality traits were discussed in detail,in order to provide scientific basis and guidance for laying hens genome selection into practical application stage.

【Objective】 This study was aimed to explore the effects of breeds,boar birth parities,number of boar piglets in the same litter,number of boar nipples,sperm collection seasons,sperm collection month ages and sperm collection intervals on boar semen quality,as well as the effect of different breeds on the stability of semen quality.【Method】 A total of 27 408 semen records were collected from 909 Duroc,Landrace and Yorkshire boars in a boar station from April 2021 to April 2022.The records were analyzed by the mixed linear analysis model and variance analysis to explore the effects of breeds,boar birth parities,number of boar piglets in the same litter,number of boar nipples,sperm collection seasons,sperm collection month ages and sperm collection intervals on semen volume,semen concentration,sperm motility,proportion of straight moving sperm,sperm malformation rate,total sperm count and stability of semen traits.【Result】 From the effect of different breeds on semen quality,the results show that semen volume and total sperm count of Landrace were significantly higher than those of Yorkshire and Duroc pigs(P＜0.05),semen concentration of Duroc pigs was significantly higher than that of Yorkshire and Landrace pigs (P＜0.05),sperm motility of Duroc and Yorkshire pigs were significantly higher than that of Landrace pigs (P＜0.05),and proportion of straight moving sperm of Yorkshire pigs was significantly higher than that of Landrace and Duroc pigs (P＜0.05),sperm malformation rate of Landrace and Yorkshire pigs was significantly lower than that of Duroc pigs (P＜0.05).From the effect of different boar birth parities on semen quality,boars born from 1st~3rd parities had higher semen quality.From the effect of different semen collection seasons on semen quality,semen concentration,sperm motility and total sperm count in autumn and winter were significantly higher than those of spring and summer(P＜0.05).From the effect of different month ages of semen collection on semen quality,boars aged from 16 to 25 months old had higher semen quality.From the effect of different semen collection intervals on semen quality,4 to 5 days was the best semen collection interval,and long semen collection interval would lead an increasing of sperm malformation rate.From the total number of nipples of different boars,when the number of nipples was 13 to 16,the quality of each semen character was at a medium level,which was conducive to the production and application of boars.The stability trends of semen traits were different from three breeds,semen volume and sperm motility of Duroc and Yorkshire pigs were significantly higher than that of Landrace pigs (P＜0.05).Semen concentration,sperm malformation rate and total sperm count of Duroc pigs were significantly higher than that of Yorkshire pigs (P＜0.05).proportion of straight moving sperm in Yorkshire pigs was significantly higher than that of Landrace and Duroc pigs (P＜0.05).Among the breeds,the stability of Landrace pigs was poor.The stability of sperm motility was the best among all semen traits.【Conclusion】 The breeds,boar birth parities,sperm collection seasons,sperm collection month ages and sperm collection intervals would affect boar semen quality.These factors must be highlighted,which could help to formulate more perfect selection schemes of boar stations in different breeds,improve semen quality and accelerate boar genetic improvement.

【Objective】 The aim of this study was to investigate the effects of limonin (Lim) on the development potential of in vitro maturation (IVM) mouse oocytes and subsequent in vitro fertilization (IVF) embryos,in order to provide reference for the optimization of in vitro maturation culture system.【Method】 Different concentrations of Lim (0,10,20,50 μmol/L) were added into the culture medium of mouse IVM culture medium.After 12 h of maturation culture,the excretion rate of the first polar body (PBI) of mouse oocytes was calculated,and the optimal concentration of Lim was selected.The optimal concentration of Lim was added to IVM culture medium,0 μmol/L Lim was used as control group,after mature culture for 12 h,the levels of reactive oxygen species (ROS),glutathione (GSH) and mitochondrial membrane potential (MMP) were detected by immunofluorescence staining.The mRNA expression levels of antioxidant and apoptosis-related genes in oocytes were detected by Real-time quantitative PCR.The embryo cleavage rate and blastocyst rate were calculated at 24 h and 3.5 d after in vitro fertilization.The total number of blastocyst cells and the ratio of apoptotic cells in blastocysts were detected by Fluorescein-dUTP staining and Hoechst 33342 staining.【Result】 Compared with 0 μmol/L Lim group,PBI excretion rate of oocytes in 20 μmol/L Lim group was significantly increased (P＜0.05),and 20 μmol/L Lim was used in the subsequent experiments.Compared with control group,ROS levels in oocytes of mice in 20 μmol/L Lim group were significantly decreased (P＜0.05),GSH and MMP levels were significantly increased (P＜0.05).The expression of antioxidant related genes (GPx3,CAT and Prdx3) and anti-apoptotic related genes (Bcl-2 and Bcl-xl) were significantly up-regulated (P＜0.05),while the expression of pro-apoptotic related gene (Caspase-3) was significantly down-regulated (P＜0.05).After in vitro fertilization,the cleavage rate,blastocyst rate and blastocyst total cell number of embryos were significantly increased (P＜0.05),and the apoptosis rate of cells in blastocyst was significantly decreased (P＜0.05).【Conclusion】 Adding 20 μmol/L Lim to in vitro maturation medium could improve the quality of mouse oocytes by inhibiting oxidative stress and cell apoptosis and increasing MMP level,thus improving the development potential of in vitro fertilization embryos.

【Objective】 The purpose of this study was to screen out the key genes for growth and development of Guangxi Ma chickens through transcriptome sequencing (RNA-Seq) of leg muscles at 1- and 60-day-old.【Method】 In this study,three healthy 1- and 60-day-old hens were selected,and leg muscle tissues were collected respectively.Illumina Novaseq^TM 6000 platform was used for mRNA transcriptome sequencing.DESeq2 software was used to analyze the differentially expressed genes,and GO function and KEGG pathway enrichment analysis of the differentially expressed genes were performed.The genes related to birth growth and development were further screened,and the transcriptome sequencing results were verified by Real-time quantitative PCR.【Result】 Transcriptome sequencing reaults showed that 34 296 198-41 647 642 clean reads were obtained from 6 samples.Compared with 1-day-old leg muscle,a total of 2 304 differentially expressed genes were obtained at 60-day-old leg muscle,of which 998 genes were up-regulated and 1 306 genes were down-regulated.Go function enrichment analysis found that 572 enrichment items were obtained,including 381 biological processes,70 cell components and 121 molecular functions.The enrichment analysis of KEGG pathway found that a total of 34 signal pathways were obtained.Myocardial contraction,MAPK signal pathway,tight junction,actin cytoskeleton signal pathway and adrenergic signal transduction in cardiomyocytes were related to growth and development.Through GO function and KEGG pathway enrichment analysis,5 growth and development related genes were obtained,which were myosin heavy chain 10 (MYH10),myosin heavy chain 15 (MYH15),fibroblast growth factor 10 (FGF10),fibroblast growth factor 16 (FGF16) and myostatin (GDF8) genes, respectively.MYH10,MYH15 and FGF10 were up-regulated differentially expressed genes,FGF16 and GDF8 were down-regulated differentially expressed genes.The results of Real-time quantitative PCR of the selected differentially expressed genes were consistent with the gene expression levels of transcriptome sequencing,indicating that the transcriptome sequencing results were reliable.【Conclusion】 By analyzing the transcriptome data of 1- and 60 day-old Guangxi Ma chickens,5 genes related to growth and development were obtained,including MYH10,MYH15,FGF10,FGF16 and GDF8,which provided a reference for further exploring the regulation mechanism of growth and development of Guangxi Ma chickens.

【Objective】 The aim of study was to explore the effect of acetochlor on the maturation of mouse oocytes in vitro,in order to provide a basic reference for judging the toxicity standard of acetochlor.【Method】 The collected cumulus-oocyte complexes (COCs) were placed in the in vitro maturation solution containing different concentrations (0 (control group),10,50,100,130,200 μmol/L) of acetochlor for 14 h,the first polar body excretion rate of oocytes was calculated.After in vitro fertilization(IVF),the blastocyst rate of IVF embryos was counted,suitable acetochlor concentrations for oocyte culture in vitrowereselected out.After COCs were cultured in mature medium of control group and selected appropriate concentrations acetochlor group for 14 h,the ROS levels of oocyteswere detected by DCFH-DA.The GSH levels of oocytes were detectedby CellTracker^TM blue dye.The mitochondrial membrane potential (MMP) of oocytes was detected by JC-1 staining method.The expression levels of apoptosis-related genes in oocytes were detected by Real-time quantitative PCR.The total number of blastocyst cells of IVF embryos was detected by Hoechst 33342 staining,and the apoptosis rate of blastocyst cells was detected by Fluorescein-dUTP.【Result】 Compared with control group,the first polar body excretion rate of oocytes in 100 and 130 μmol/L acetochlor groups was significantly decreased (P＜0.05),and the blastocyst rate of VIF embryos in 100 and 130 μmol/L acetochlor groups was significantly decreased (P＜0.05),and IVF embryos in 200 μmol/L acetochlor group did not develop to blastocyst stage,therefore,100 and 130 μmol/L acetochlor were selected for follow-up tests.Compared with control group,in 100 and 130 μmol/L acetochlor groups,the level of ROS in oocytes was significantly increased (P＜0.05),the level of GSH and mitochondrial membrane potential were significantly increased (P＜0.05),the relative expression of the pro-apoptotic gene Caspase9 in the oocytes was significantly increased (P＜0.05),while the relative expression of the anti-apoptotic genes Bax and Bcl-2was significantly increased(P＜0.05).In 100 and 130 μmol/L acetochlor groups,the total number of blastocyst cells was significantly decreased (P＜0.05),the blastocyst cell apoptosis rate was significantly increased (P＜0.05).【Conclusion】 Acetochlor reduced the quality of oocytes by increasing ROS and promoting apoptosis of oocytes,thus affecting the developmental potential of oocytes after fertilization.

Rabbit industry is the representative industry of grain saving,environmental protection and low consumption herbivorous animal husbandry,as an important part of animal husbandry in China.The research on rabbit genetics,breeding and reproduction is an important basis and guarantee to promote the development of rabbit industry.Which are mainly attributed to the interdisciplinary integration and continuous progress of genetics,molecular biology,statistics and computer science have provided more advanced technical means for rabbit breeding.In this paper,the research progress on genetic breeding and reproduction of rabbits in 2021 are reviewed from the traditional breeding,molecular breeding and reproduction of rabbits.For abroad,lots of researches mainly focuses on the effects of selection and environment on the growth and reproductive performance of rabbits.In China,there are more studies on rabbit genetic breeding and reproduction than abroad,and the researches concentrate mainly on molecular breeding,including varieties and genetic diversity,functional genes related to fur traits,lipid and meat quality traits and reproductive performance.In addition,there are lots of studies on the effects of selection and environmental effects on growth and reproductive performance of rabbits and production performance measurement in traditional breeding.This review could provide references for rabbit breeding and production.

【Objective】 To study the changes of heart rate variability (HRV) between aborted Yili horse and normal delivery Yili horse during pregnancy,so as to prevent adverse pregnancy outcomes.【Method】 21 Yili horses (5 aborted and 16 delivered normally) with pregnancy for 29 weeks and 7 Yili horses non-pregnancy were selected in the third trimester of pregnancy,24-hour heart rate variability (HRV) was collected at the 30th gestational week in the second trimester,the 45th gestational week in the third trimester as well as 1 day before and after delivery.Including time domain indexes:Mean of all R-R intervals (Mean RR),standard deviation of R-R intervals (SDNN),mean heart rate (Mean HR),root mean square of successive differences (RMSSD),normal- normal intervals that differ by＞50 ms in the total number of RR intervals (NN50) and percentage of normal- normal intervals that differ by＞50 ms in the total number of RR intervals (pNN50);Frequency domain index:very low frequency (VLF),low frequency power (LF) and high frequency power (HF);Nonlinear index:Standard deviation of all R-R spacing (Y) (SD1) and standard deviation of all R-R spacing (X) (SD2).【Result】 In time domain indexes,Mean RR,SDNN,RMSSD,NN50 and pNN50 in abortion group and normal delivery group were extremely significantly or significantly lower than those in non-pregnancy group (P＜0.01;P＜0.05),Mean HR was extremely significantly higher than that in abortion group and normal delivery group (P＜0.01).In frequency domain indexes,HF and LF in abortion group and normal delivery group were extremely significantly lower than those in non-pregnant group (P＜0.01),VLF in abortion group and non-pregnant group were extremely significantly higher than that in normal delivery group (P＜0.01).In the nonlinear indexes,SD1 and SD2 in abortion group and normal delivery group were extremely significantly or significantly lower than those in non-pregnant group (P＜0.01;P＜0.05),SD2/SD1 in normal delivery group was extremely significantly higher than that in abortion group and pregnant group (P＜0.01).【Conclusion】 HRV of Yili horses fluctuated in different gestational states.Mean HR,Mean RR and SD2/SD1 values increased in pregnant mares.The parasympathetic nerve dominated the mare during gestation,and the autonomic nervous system was inhibited.The VLF index of aborted mares increased.Before and after the occurrence of abortion sympathetic nerves dominate,the autonomic nervous system produces excitement.

【Objective】 This study was aimed to analyze the prevalence,genetic difference of Hexon gene and pathogenicity of Fowl adenovirus-4 (FAdV-4) in Yunnan province,so as to provide reference for the epidemiology of FAdV-4 and the influence of Hexon gene on virus virulence.【Method】 158 samples of poultry liver tissue suspected of FAdV-4 infection and 310 fresh feces of wild birds were collected from January 2020 to June 2021 in Kunming,Qujing,Chuxiong,Dali,Honghe and Yuxi of Yunnan province,FAdV-4 nucleic acid was detected by PCR,representative positive samples were isolated and identified,and pathogenic and Hexon gene sequence of isolated strains were analyzed.【Result】 In 158 poultry tissue samples,19 samples were positive for FAdV-4 pathogenic nucleic acid,with a positive rate of 12.03%,and 1 sample (Black-necked cranes) was positive for 310 fresh feces of wild birds,with a positive rate of 0.3%.7 strains of FAdV-4 were isolated from positive samples.The results of pathogenicity study showed that all strains of FAdV-4 in Yunnan could cause chicken embryo dysplasia and pericardial effusion-hepatitis syndrome in 4-week broilers.The embryo median lethal dose (ELD₅₀) was 10^―6.17 to 10^―4.32/mL.The 7 strains were clustered in the branches of FAdV-C subgroup,and Hexon protein had the same characteristics of ¹⁸⁸R,¹⁹³R and ¹⁹⁵Q as domestic highly pathogenic strains.The strains infected with SPF chickens showed obvious clinical symptoms,highly pathogenic,high lethality and significant hepatocellular lesions.【Conclusion】 The prevalence of FAdV-4 in Yunnan was preliminarily understood,FAdV-4 pathogenic nucleic acid was detected in the feces of Black-necked cranes for the first time,7 strains of FAdV-4 were isolated,and some strains of Hexon protein had amino acid mutations.

【Objective】 This study was aimed to investigate the drug resistance and virulence characteristics of Salmonella carried in slaughtered pork in Maoming,Guangdong province,and provide a basis for hazard assessment and formulation of prevention and control measures for foodborne Salmonella in this area.【Method】 Nineteen strains of Salmonella isolated from pork,spleen and liver samples collected from slaughterhouses in Maoming,Guangdong province were tested for resistance to β-lactams,fluoroquinolones,aminoglycosides,tetracyclines,aminoglycols and sulfonamides by the K-B drug-sensitive paper method,and the β-lactam resistance genes (bla_TEM,bla_OXA-1 and bla_SHV),fluoroquinolone resistance genes (qnrA,qnrS and qnrB),aminoglycoside resistance genes (aadA1,aac(6')-Ⅰb and rmtB),tetracycline resistance genes (tetA,tetB and tetC),aminol resistance genes (Cat1 and floR),sulfonamide resistance genes (SulⅠ,SulⅡ and SulⅢ) and 10 virulence genes (mogA,sseL,mgtC,bcfA,araB,spvR,spvA,spvB,spvC and spvD) were detected by PCR method.【Result】 19 strains of Salmonella were highly resistant to tetracycline,doxycycline,chloramphenicol,florfenicol and sulfaisoxazole,and the resistance rates were more than 50%,with the highest resistance rates of 89.5% (17/19) to tetracycline,doxycycline and chloramphenicol,and 89.5% to 3 or more antimicrobial drugs,with the highest resistance to all 11 antimicrobial drugs.The detection rate of bla_TEM,qnrA,aadA1, aac(6')-Ⅰb,Cat1,floR,SulⅠ and SulⅢ resistance genes was high (≥50.0%),the highest detection rate of bla_TEM resistance gene was 84.2%,73.7% of strains carried 5 or more resistance genes at the same time,and the highest strain carried 12 drug-resistant genes;mogA,mgtC,bcfA,araB virulence genes were all detected and the detection rate was above 70%,of which mogA virulence gene had a detection rate of up to 100%,and the remaining 6 virulence genes did not detect out.【Conclusion】 Salmonella from the pork of Maoming slaughterhouse in Guangdong province was seriously multidrug-resistance,carrying multiple drug-resistance genes with complex gene combinations and multiple virulence genes.

【Objective】 The purpose of this study was to establish a stable production process of Foot-and-mouth disease virus(FMDV) infection and immune differential diagnosis and immune evaluation test paper,and promote its industrial production and clinical application.【Method】 In this study,the colloidal gold immunoprobe was combined with the epitope polypeptide coupling carrier protein of porcine IgG,FMDV structural protein (SP) and non structural protein (NSP) to prepare artificial antigen,and two detection lines were set to accurately intercept the detection mode of antibody. Taking the specificity and sensitivity of the test strip as the evaluation index,the parameters such as protein for colloidal gold immunoprobe,polypeptide antigen for test strip interception,detection line position,spray film buffer,gold standard protein preservation solution,sample pad buffer and sample diluent were optimized.The optimized test paper was used to detect 266 field pig serum samples,and the results were compared with the results of foot-and-mouth disease O antibody liquid phase blocking ELISA (LPB ELISA) and 3ABC blocking ELISA antibody detection kit (3ABC ELISA).The coincidence rate between the test paper and commercial ELISA kit was calculated.【Result】 After optimization,the parameters of the dual test strip for differential diagnosis of FMDV infection and immunity and immune evaluation were as follows:Staphylococcus aureus A protein (SPA) labeled with colloidal gold was used as immunoprobe,the mixed BSA-peptides were used as test lines.BSA-NSPs and BSA-SPs were dispensed on NC membrane as the test line 1 and test line 2,respectively.0.1 mol/L Tris-HCl was used as membrane spraying buffer.ddH₂O containing 15 mg/mL BSA,13.25 mg/mL Na₂HPO₄·12H₂O,15.9 mg/mL NaH₂PO₄·2H₂O,10% Trition X-100 and 0.3 mg/mL NaN₃ were used as gold standard protein preservation solution.0.02 mol/L Na₂B₄O₇·10H₂O containing 10 mg/mL casein,5% TritionX-100,0.3 mg/mL NaN₃ were used as sample pad buffer.Normal saline containing 1.0% Tween-20 were used as sample dilution.After optimization,the coincidence rate of the optimized strip with FMDV 3ABC ELISA and FMDV LPB ELISA was 96.20% and 94.36%,respectively.【Conclusion】 By optimizing the production process,a stable production technology was developed.The color of the test lines of the optimized strip were clearly visible with high recognition by the naked eyes.The specificity and sensitivity of the strip were markedly improved.This study laid the basis for the mass production and industrial application of the strip and provided a stable,specific,sensitive and accurate method for FMDV detection.

【Objective】 This study was aimed to express NP protein of an epidemic H9N2 subtype Avian influenza virus (AIV) strain isolated from South China using prokaryotic expression system,and prepare its polyclonal antibody.【Method】 The NP gene of a H9N2 strain was cloned into pET-32a(+) vector to construct the prokaryotic expression plasmid of NP protein.The recombinant plasmid was transformed into both the Escherichia coli(E.coli) BL21(DE3) and Rosetta (DE3) strains for expression.The recombinant protein was induced by IPTG.Coomassie bright blue staining and Western blotting were used to analyze and compare the expression of NP protein in the two kinds of expressing bacteria.Furthermore,the concentration of IPTG,induction time,and induction temperature were optimized to improve the protein production.By affinity chromatography,the recombinant protein was purified using a Ni-NTA column,and the concentration was determined using BCA assay.The polyclonal antibody was obtained from New Zealand,which had been inoculated with the purified protein.Western blotting and indirect immunofluorescence assay were used to identify the polyclonal antibody,the antibody titer was determined using indirect ELISA.【Result】 The prokaryotic expression plasmid pET-32a-H9N2-NP was constructed and the combinant NP protein was expressed successfully.Coomassie bright blue staining and Western blotting results revealed that the recombinant protein was expressed at a much higher level in E.coli Rosetta(DE3) than that in E.coli BL21(DE3),and its size was about 73 ku.The optimal induction conditions showed that the expression of recombinant NP protein reached the maximum at 37 ℃ and IPTG concentration of 1 mmol/L for 7 h.The results of Western blotting and indirect immunofluorescence assay demonstrated that the prepared polyclonal antibody could bind specifically to H9N2 subtype AIV at a high titer.By indirect ELISA detection,the titer was about 1∶409 600.【Conclusion】 The polyclonal antibody against NP protein prepared from rabbits had a higher titer,indicating that NP protein had good immunogenicity.The results laid the foundation for the preparation of monoclonal antibodies against NP protein and establishing the ELISA detection method.

【Objective】 This study was aimed to obtain enolase gene from Xinjiang strain of Theileria annulata (T. annulata),and analyze the biological characteristics and reactionogenicity of the protein.【Method】 The enolase gene of T.annulata was amplified and cloned,and the prokaryotic expression vector pGEX-4T-1-enolase was constructed.The recombinant enolase protein was induced and purified.The reactivity of the recombinant enolase protein was verified by Western blotting.Bioinformatics methods were used to predict the physicochemical properties,hydrophilicity and hydrophobicity,transmembrane domain,signal peptide,phosphorylation,subcellular localization and protein interaction network of enolase gene encoded protein.【Result】 A 1 248 bp enolase gene fragment was amplified by PCR.The size of enolase recombinant protein was about 70 ku.The results of Western blotting showed that the recombinant protein reacted with the positive serum of T.annulata.enolase gene encoded 416 amino acids with a theoretical isoelectric point of 5.91 and 38 phosphorylation sites.The secondary structure was mainly composed of alpha helix (43.03%) and random coil (33.17%).It had 17 B cell epitopes,and the subcellular localization was mainly located in the cytoplasm.enolase protein interacted with phosphoglycerate mutase (PGAM),nucleoside diphosphate kinase (NDK),phosphoglycerate kinase (PGK) and heat shock protein 70 (HSP70).【Conclusion】 In this study, the enolase gene of T. annulata from Xinjiang was successfully cloned. The protein-protein interaction network predicted the protein interaction of enolase gene with glycolysis and energy metabolism. The results of this study laid a foundation for the related research on the energy metabolism pathway genes of T. annulata.

【Objective】 The purpose of the experiment was to find out the cause of reproductive failure of sows in a pig farm.【Method】 The feed being eaten in the sow feed trough was collect to detect the content of aflatoxin B₁.The oral and nasal swabs,anal swabs and serums of returning sows were collect to detect Porcine circovirus 2 (PCV2),PCV3,Porcine pseudorabies virus (PRV),Porcine parvovirus (PPV),Porcine reproductive and respiratory syndrome virus (PRRSV),Classical swine fever virus (CSFV) by PCR and RT-PCR methods.13 returning sows were randomly dissected,and the spleens,lymph nodes,spinal cords,uterus,ovaries were collected to detect failure virus. ORF2 gene sequence analysis was performed on some positive samples of PCV2 and PCV3. The pathological changes of sows’ ovaries were observed,the results were summarized and analyzed,and the reasons for the low conception rate and continuous estrus return of sows in this farm were diagnosed.【Result】 The content of aflatoxin B₁ in the feed of the pig farm was 4.5-9.0 μg/kg,all within the allowable range of aflatoxin B₁ in feed specified in GB 13078－2017 (＜20 μg/kg).The results of PCR and RT-PCR showed that PCV2,PCV3,PRV,PPV,PRRSV,CSFV in oral and nasal swabs,anal swabs and serums were negative.The positive rate of PCV2 and PCV3 were 69.23% (9/13) and 38.46% (5/13) respectively,and the mixed infection rate of PCV2 and PCV3 was 30.76% (4/13).The results of Real-time quantitative PCR showed that the viral load of PCV2 in spinal cord was the highest,followed by spleen, and the lowest in lymph nodes.The viral load of PCV3 was the highest in spleen,followed by spinal cord,and the lowest in ovary.ORF2 gene sequence analysis showed that PCV2 was subtype 2d and PCV3 was subtype 3a.Ovarian pathological sections showed that the number of follicular cells decreased,and the follicular cavity was irregular,collapsed and flat.【Conclusion】 After diagnosis,it was determined that the main reason for the continuous return to estrus of the sows in this farm was co-infection with PCV2 and PCV3.According to the diagnosis results and the situation of the farm,all of the sows were vaccinated with PCV2 vaccine,meanwhile,the whole farm was disinfected to enhance biosafety prevention.Eventually,the farm achived the normal production.

Porcine deltacoronavirus (PDCoV) is a newly discovered enteropathogenic porcine Coronavirus causing severe diarrhea,vomiting,and dehydration,which leads to death eventually,posing a severe threat to the pig industry.Further research on PDCoV is of great significance for the prevention and control of this disease.The genome encodes four structural proteins,fifteen non-structural proteins and three accessory proteins play important roles in the virus replication,translation and the host pathogenic process.The pathogenesis of PDCoV is complicated.In this review,we summarized the pathogenesis from the aspects of its invading host cells,antagonising interferon response,inducing apoptosis,regulating autophagy,inhibiting pyroptosis to evade host immune response,and other molecular mechanisms affecting PDCoV replication.PDCoV shows a global epidemic trend,and S,M, N genes of PDCoV are often used as nucleic acid detection and antibody detection targets for diagnosis.So far,there are no effective vaccines and therapeutic methods available for PDCoV.With the development of biotechnology,the research on inactivated vaccines,live attenuated vaccines,recombined vector vaccines,virus-like particle vaccines and lactic acid bacteria carries oral vaccines have made considerable progress.In addition,broad spectrum antiviral drugs,antiviral proteins and new antiviral technologies have shown good application prospects in PDCoV antiviral research.Therefore,this review comprehensively expounded its coding protein function,pathogenicity,detection methods,vaccine development and antiviral therapy,hoping to provide reference for further systematic research and effective prevention and control.

【Objective】 This study was aimed to construct a novel Porcine epidemic diarrhea virus (PEDV) live Lactobacillus vaccine vector.【Method】 The S-layer protein (SLP) gene of Lactobacillus acidophilus and the B cell epitope (EpitopeS2) of PEDV S2 gene fusion gene (SLP-EpitopeS2) were cloned into Lactobacillus sp.expression vector pTRK892 by DNA recombination technique,and the recombinant vector pTRK-SLP-EpitopeS2 was constructed.The recombinant plasmid was introduced into the Lactobacillus paracasei by electric transformation to obtain the recombinant Lactobacillus paracasei.The expression of the target protein in Lactobacillus paracasei was identified by SDS-PAGE,Western blotting and immunofluorescence assay (IFA),respectively.【Result】 A target band of 1 400 bp was successfully amplified by PCR,which was consistent with the size of the inserted fusion gene.Two bands of 1 400 and 4 700 bp were found in the double enzyme digestion assay,which were consistent with the size of the inserted fusion gene and vector fragment.The gene sequencing results showed no base loss or mutation,etc.Thus,the construction of the recombinant plasmid pTRK-SLP-EpitopeS2 was confirmed to be correct.The results of SDS-PAGE and Western blotting showed that the target protein band consistent with the theoretical value appeared at 48 ku,indicating that the fusion gene SLP-EpitopeS2 was effectively expressed in Lactobacillus paracasei.The results of IFA showed that,compared with the control group Lactobacillus paracasei,recombinant Lactobacillus paracasei could be stimulated to give a specific green fluorescent signal,which were consistent with the results of the supplementary identification test for bacterial membrane protein samples eluted by high concentration of lithium chloride (LiCl).These results indicated that the fusion protein SLP-EpitopeS2 might be expressed on the surface of Lactobacillus paracasei.【Conclusion】 The PEDV EpitopeS2 and Lactobacillus acidophilus SLP embedded fusion expression vector pTRK-SLP-EpitopeS2 was successfully constructed,which laid a foundation for the related research on live Lactobacillus carrier vaccine.

【Objective】 The purpose of the experiment was to explore the epidemic characteristics of Parvovirus from palm civets and the genetic variation of VP2 gene,so as to provide theoretical basis and technical support for Parvovirus control.【Method】 CRFK cells were used to isolate the virus from the intestines of palm civets with enteritis.The virus was identified by hemagglutination test and immunofluorescence test.The VP2 gene of the virus was amplified by PCR and its genetic variation was analyzed.【Result】 Typical cytopathic effects (CPE)of Parvovirus such as cell abscission,disintegration and fragmentation were observed in CRFK cells.The results of hemagglutination test showed that the virus could agglutinate porcine erythrocytes,and the hemagglutination titer was 1∶512.The results of immunofluorescence test showed that bright green fluorescence could be observed in the CRFK cells inoculated with the isolated strain,while no fluorescence was observed in the control cells.The isolated strain was named MPCPV-SX.Phylogenetic tree analysis of VP2 gene showed that it was in the same branch as Canine parvovirus,located between CPV-2 and CPV-2a.At the same time,the 87 and 101 amino acids of VP2 were CPV-2,while the 300 and 305 amino acids were consistent with CPV-2a.【Conclusion】 In this study,a strain of Parvovirus MPCPV-SX was successfully isolated from the intestines of palm civets.Its VP2 gene was an intermediate type between CPV-2 and CPV-2a,indicating that Parvovirus spreaded across the host through palm civets and other wild animals,which might play a transitional role in the genetic evolution of parvovirus.

【Objective】 Through genome sequencing and bioinformatics analysis of GD2107 strain of multi-drug resistant Actinobacillus pleuropneumoniae (APP),the genome database information of APP was enriched,the phylogenetic tree based on ApxⅣ gene was constructed,and the evolutionary relationship of the strain was analyzed,so as to provide reference for exploring the pathogenic mechanism of APP and clinical prevention and control of porcine infectious porcine contagious pleuropneumia (PCP).【Method】 The drug resistance spectrum of the isolated strains was determined by drug sensitivity test. The whole genome of bacterial DNA was sequenced by whole genome sequencing (WGS) method.Based on Illumina NovaSeq and PacBio SequeI sequencing platforms,the whole genome sequencing results were analyzed by gene function annotation and bioinformatics analysis (including basic genome information,functional element analysis and subsystem analysis,etc.). Phylogenetic tree was constructed based on ApxⅣ gene.【Result】 The results of drug sensitivity test of GD2107 strain showed that it was resistant to 14 kinds of antimicrobials including penicillin,cefradine (pioneer Ⅵ),kanamycin and so on.The whole genome of GD2107 strain was sequenced to obtain a circular chromosome of 2 271 987 bp (GC content was 41.21%) and two circular plasmids of 5 027 and 3 497 bp,respectively.A total of 2 290 coding genes were predicted,including 19 rRNA (7 5S rRNA,6 16S rRNA,6 23S rRNA),21 tRNA genes,20 ncRNA;23 gene islands,4 prophages and 2 groups of CRISPR related sequences.2 112,1 549 and 1 866 genes were annotated in the databases of COG,KEGG and GO,respectively,and the related proteins were mainly distributed in the metabolic process of APP.In addition,48 virulence genes and 22 drug resistance genes were annotated in VFDB and CARD databases (only floR gene was located on the plasmid).The whole genome circle map of the strain was drawn and the genome information was submitted to NCBI.The chromosome GenBank accession No. was CP097377 and the plasmids accession No. were CP097378 and CP097379, respectively. Phylogenetic tree analysis showed that the evolutionary relationship between this strain and APP strain from China (CP063424.1) was the closest.【Conclusion】 In this study, we completed the whole genome sequencing and bioinformatics analysis of the multi-drug resistant strain GD2107, fully understood the structure and function of the genome of the strain, and explored the related genes in drug resistance and pathogenic mechanism. The evolutionary relationship showed that the strain had a certain regional epidemic, which provided a reference for preventing the epidemic of PCP and exploring the pathogenic mechanism of APP.

【Objective】 A new enrofloxacin (ENR) salt was prepared and its solubility was investigated,aiming to improve the solubility of ENR and enrich its crystal form.【Method】 The new ENR salt was prepared by slurry method,and the single crystal was cultivated by slow evaporation method.The new salt was characterized and analyzed by powder X-ray diffraction (PXRD),single crystal X-ray diffraction (SXRD),Fourier transform infrared spectroscopy (FTIR),differential scanning calorimetry (DSC),thermogravimetric analysis (TGA) and scanning electron microscopy (SEM).The dissolution rates of ENR and new salt in pH 6.8 phosphate buffer at 37 ℃ were investigated by powder dissolution rate test.【Result】 The prepared new ENR salt was ENR-2,6-dihydroxybenzoic acid hemihydrate (ENR-2,6-DHBA·1/2H₂O).According to the analysis of single crystal structure,ENR-2,6-DHBA·1/2H₂O belonged to triclinic crystal system,P-1 space group,and the unit cell parameters were a=13.2495(7)Å,b=13.8266(11)Å,c=16.0672(8)Å,α=114.301°(6),β=90.322°(4),and γ=114.438°(7).The asymmetric unit of new salt contained two ENR cations,two 2,6-DHBA anions and one H₂O molecule in which a proton was transferred from the carboxyl group of 2,6-DHBA to the nitrogen atom of the piperazine in ENR and formed N⁺-H…O^-,confirming that the new form was salt.ENR and H₂O were connected by O-H…O hydrogen bonds.FTIR results indicated that the C=O absorption peak on 2,6-DHBA shifted from 1 680 to 1 725 cm^-1 due to proton transfer.The melting point of the new salt was 253 ℃,different from those of ENR and 2,6-DHBA.The morphology of the new salt was smooth rod-like shape.The powder dissolution rate curve showed that the apparent solubility of ENR-2,6-DHBA·1/2H₂O at equilibrium was 0.65 mg/mL,which was 1.86 times that of ENR.【Conclusion】 The results showed that ENR-2,6-dihydroxybenzoic acid hemihydrate salt was successfully prepared.The new salt improved the solubility of ENR,which provided reference basis for improving the bioavailability of ENR and enhancing the antibacterial effect.

【Objective】 The aim of this study was to investigate the preventive and therapeutic effects of Moringa oleifera leaf flavonoids (MOLF) on ulcerative colitis (UC) in mice induced by dextran sodium sulfate (DSS).【Method】 50 BALB/c mice were randomly divided into 5 groups:control group,DSS group (model group),MOLF-L(25 mg/kg),MOLF-M(50 mg/kg) and MOLF-H(100 mg/kg) groups.Each group contained 10 mice.Mice were induced UC by drinking 4% DSS, and MOLF-L,MOLF-M and MOLF-H groups were given the corresponding doses of MOLF 0.2 mL by gavaged,the control and DSS groups were gavaged with an equal volume of normal saline once a day for 7 d.The disease activity index (DAI) of each group was recorded daily. After 7 d of treatment,the blood was collected from the transorbital venous plexus,and the serum was separated from the blood samples for the determination of IL-1β,IL-10,TNF-α,HMGB1 and LPS.Mice were sacrificed and their colon tissues were harvested for HE staining to observe tissue morphology,and PAS staining to observe goblet cell numbers in tissues. The mRNA expression levels of IL-1 β,IL-10,TNF-α and HMGB1 in colonic tissue were respectively detected by Real-time quantitative PCR,the expressions of zonula occludens-1 (ZO-1) and Occludin were detected by immunofluorescence assay,and the expression levels of apoptosis related proteins cleaved-caspase 3,Bcl-2 and Bax were analyzed by Western blotting.【Result】 UC was successfully established in mice.Compared with the control group,the levels of LPS and inflammatory factors,the expressions of cleaved-caspase 3 and Bax in the DSS group were extremely significantly different (P＜0.01).Different doses of MOLF could improve the inflammation in UC mice,especially in the MOLF-H group:Compared with the DSS group, the DAI score and LPS content were extremely significantly reduced(P＜0.01), the mRNA expression of pro-inflammatory factors IL-1β,TNF-α and HMGB1 in serum and colon tissues were extremely significantly reduced (P＜0.01),the expression of anti-inflammatory factor IL-10 was extremely significantly increased(P＜0.01),besides,cleaved-caspase 3 and Bax were up-regulated and Bcl-2 expression level was down-regulated extremely significantly(P＜0.01).In addition,MOLF could improve colonic mucosal edema and inflammatory cell infiltration,protect the number and morphology of goblet cells,and increase the expression of ZO-1 and Occludin proteins,with a dose-effect relationship between MOLF.【Conclusion】 MOLF could protect against UC by regulating the levels of inflammatory factors,ameliorating DSS induced impairment of ZO-1 and Occludin expression and abnormal expression of apoptosis related proteins in the colon tissues of mice.

【Objective】 The purpose of this study was to identify the pathogen of diarrhea in a large-scale pigeon farm in Guangzhou and to explore the in vitro bacteriostatic effect of tea polyphenols on the pathogens.【Method】 The feces of sick pigeons in a large-scale pigeon farm in Guangzhou were collected,and the pathogen was identified by isolation and purification,staining and microscopic examination,16S rRNA gene sequencing and phylogenetic tree construction.The drug sensitivity of pathogen was detected by drug sensitivity test,and the virulence genes carried by pathogen were detected by PCR technology.At the same time,the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration(MBC) of tea polyphenols against pathogen and the effects of different concentrations (0 (the control group),125,500,and 1 000 μg/mL) of tea polyphenols on the growth,movement and virulence gene expression of the pathogen were determined by in vitro bacteriostatic test,so as to comprehensively evaluate the in vitro bacteriostatic effect of tea polyphenols on the pathogen.【Result】 From the pathologic materials,a pathogenic strain was isolated and named GG20210604,which biological characteristics were basically consistent with the Escherichia coli.16S rRNA gene sequencing and phylogenetic tree results further confirmed that the strain was Escherichia coli.After detection,GG20210604 was resistant to ciprofloxacin and ampicillin,while carrying a total of four virulence genes,including fliC,fimH,iroN and hlyF.The results of in vitro bacteriostatic test showed,the MIC and MBC values of tea polyphenols against GG20210604 were both 4 000 μg/mL.Compared with the control group,the growth and movement of GG20210604 were significantly inhibited in 250,500 and 1 000 μg/mL of tea polyphenols (P＜0.05);the GG20210604 genes expression of fliC,iroN and hlyF were significantly decreased in 250 and 500 μg/mL of tea polyphenols (P＜0.05),and the genes expression of fimH was also significantly decreased in 250 μg/mL of tea polyphenols (P＜0.05).【Conclusion】 A strain of Escherichia coli was isolated from pigeon.The strain was resistant to ciprofloxacin and ampicillin,while carrying a total of four virulence genes,including fliC,fimH,iroN and hlyF.Tea polyphenols had positive bacteriostatic effect on this strain,which could significantly inhibit its growth,movement and virulence gene expression.This study provided a new idea for preventing and alleviating pigeon diarrhea caused by Escherichia coli,and provided a theoretical basis for the application of tea polyphenols as a plant bacteriostatic agent in pigeon production.

【Objective】 This study was conducted to investigate the synergistic effects of Chinese herbal monomers xanthohumol,isorhapontigenin,deoxyshikonin and calycosin and sodium butyrate on the expression of porcine host defense peptide (HDPs).【Method】 Porcine intestinal epithelial cells (IPEC-J2) and porcine alveolar macrophages (3D4/31) were treated with Chinese herbal monomers xanthohumol,isorhapontigenin,deoxyshikonin and calycosin and sodium butyrate alone or together.HDPs gene expression,cytokine gene expression and antibacterial effect of cells on enterotoxigenic Escherichia coli (ETEC) were measured to evaluate the synergistic effects of Chinese herbal monomers and sodium butyrate.【Result】 Xanthohumol and sodium butyrate had no synergistic effect on the expression of HDPs.The expression of pEP2C and pBD3 genes in IPEC-J2 cells was synergistically induced by isorhapontigenin and sodium butyrate (P＜0.01).Deoxyshikonin combined with sodium butyrate could synergically increase the expression of pBD3 and PG1-5 genes in IPEC-J2 cells (P＜0.05;P＜0.01).Treatment with calycosin and sodium butyrate could synergically promote PG1-5,pEP2C,pBD2 and pBD3 genes expression in IPEC-J2 cells (P＜0.05;P＜0.01) and pBD3 gene expression in 3D4/31 cells (P＜0.05).The co-treatment of Chinese herbal monomers and sodium butyrate had no significant cooperative effect on cytokine expression (P＞0.05).Except xanthohumol,other Chinese herbal monomers and sodium butyrate treatment alone or together could improve the antibacterial activity of cells to ETEC.Calycosin and sodium butyrate treatment showed the best synergistic effect on the inhibition of ETEC.【Conclusion】 Chinese herbal monomers isorhapontigenin,deoxyshikonin and calycosin and sodium butyrate had a certain synergistic effect on inducing HDPs expression,improving immune and antibacterial activity of the host.

【Objective】 Through physical crosslinking method and characterization analysis,a kind of ready to use hydrogel with adding water and stirring was developed for chicks.【Method】 The hydrogel was prepared,and the formula was optimized by pre-experiment and Box-Behnken response surface method.Scanning electron microscope (SEM) was used to observe the micromorphology.Fourier transform infrared spectroscopy (FTIR) was used to judge the interaction between molecules according to the position shift of chemical group absorption peak,and the changes of physical and chemical properties were studied by differential scanning calorimetry (DSC).【Result】 The optimal formula of the hydrogel carrier was 4% sodium polyacrylate super absorbent resin,10% maltodextrin,1.5% guar gum,and water.SEM results showed that both super absorbent polymer and hydrogel had porous network structure,maltodextrin and guar gum had tighter structure and smaller cavity.FTIR showed that there were hydrogen bonding and intermolecular entanglement,and no new chemical groups were formed.DSC results showed that they were interacted with each other,their endothermic peaks were wider,their melting point was higher,and their thermal stability was improved.【Conclusion】 A hydrogel carrier for chicks was prepared with simple preparation method,low cost and convenient use.

【Objective】 The aim of this study was to screen the optimal Chinese herbal formula that could inhibit the main pathogen of strangles Streptococcus equi subspecies equi,and provide a new reference for the prevention and treatment of strangles.【Method】 27 kinds of herbs including Radix Paeoniae Rubra,Rhubarb and Radix Sophorae Sinensis etc.were used as materials in this experiment,antibacterial Chinese herbal medicines was preliminary screened by in vitro antibacterial test (Oxford cup method,minimum inhibitory concentration(MIC),minimum bactericidal concentration(MBC),combined drug susceptibility test) and L₈(2⁶) orthogonal test,and then the formula for inhibiting Streptococcus equi subspecies equi was composed.Secondly,the optimal ratio of basic formula was further screened by L₁₈ (3⁶) orthogonal test.Finally,under the premise of basic formula,according to the theory of compatibility theory of Chinese herbal formula,two tonifying drugs were added,the ratio of modified formula was screened by in vivo bacteriostasis test of strangles mice model,and the antibacterial effect of modified formula was verified in combination with formula protection rate and pathological changes of tissues and organs.【Result】 In the experiment,seven herbs (Fritillaria thunbergii,Radix Paeoniae Licorice root,Rhubarb,Nymphaea candida,Sophora root,Sophora flavescens) were selected,which were super sensitive and highly sensitive to Streptococcus equi subspecies equi,The MIC of Rhubarb,Nymphaea candida,Sophora root,Radix Paeoniae Rubra,Sophora flavescenswere 0.078,0.078,0.125,0.156,0.156 and 0.25 mg/mL,respectively.The MBC of Nymphaea candida,Radix Paeoniae,Sophora root,Licorice root,Rhubarb,Sophora flavescens,Fritillaria thunbergii were 0.078,0.125,0.125,0.156,0.156,0.156 and 0.25 g/mL,respectively.According to the MIC values of each single herb and the MIC values after pairwise combination,it was calculated that the combined herb sensitivity index of Fritillaria thunbergii and Nymphaea candida was＞2,which was antagonistic,so Fritillaria thunbergii was deleted.Then the optimal ratio of the basic formula was obtained by two orthogonal tests as follows:Rhubarb∶Sophora flavescens∶Sophora root∶Radix Paeoniae Rubra∶Licorice root∶Nymphaea candida= 4∶2∶2∶4∶1.Combined with the in vivo bacteriostasis test of modified formula,the protection rate was 87.5% when the compatibility concentration of Astragalus membranaceus∶Angelica sinensis = 1∶1 was 0.5 mg/mL,and Chinese herbal formula could repair inflammatory injury of lung induced by Streptococcus equi subspecies equi in mice.【Conclusion】 In conclusion,the study successfully screened out a new Chinese medicine formula with good antibacterial and tissue protection effects on Streptococcus equi subspecies equi.The ratio was Rhubarb∶Sophora flavescens∶Sophora root∶Radix Paeoniae Rubra:Licorice root∶Nymphaea candida∶Astragalus-Angelica(1∶1)= 4∶2∶2∶4∶4∶1∶1.

【Objective】 The purpose of the test was to determine the main pathogenic bacteria in the suspected Salmonella disease materials sent by a laying hens farm in Yunnan,and to determine their pathogenicity and drug sensitivity.【Method】 The strains were identified by bacterial isolation and culture,morphological observation,biochemical test,serological identification and molecular methods.The pathogenicity of the isolated bacteria was analyzed through the pathogenicity test of chicken and the detection of virulence genes.The drug resistance of strains was studied by antibiotic sensitivity test.【Result】 The isolated strain formed purple colony on the Salmonella chromogenic medium,and was Gram-negative Brevibacterium and suspected to be Salmonella,named S2.The results of biochemical test showed that the results of lysine decarboxylase,hydrogen sulfide and dulcite were positive,and indol,urease,KCN,sorbitol,salicin,β-galactoside and malonate were negative,and S2 was judged to be a typical Salmonella.The results of serological identification showed that S2 belonged to Salmonella Enteritidis group D,and its antigen formula was 1,9,12:g,m.The results of genetic evolution analysis showed that S2 and Salmonella Enteritidis MT621365 were in the same branch,and the self-development value was 99%.The results of pathogenicity test showed that the isolated strain had strong pathogenicity to chickens,and all chickens died after 6 days of infection (10/10).The results of virulence gene detection showed that S2 carried invJ,hila,ssaB,misL,mgtC,orf319,sopB,spvA,spvB,spvC,spvR,stn and fimA virulence genes,but did not carried sopA and flic virulence genes.The isolated strain S2 was highly sensitive to most antibiotics,and it was sensitive to 12 antibacterial drugs including amoxicillin-clavulanic acid,cefoxitin,tetracycline and so on,but it was resistant to methoxypyrimidine,compound sulfamethoxazole and lincomycin.【Conclusion】 A strain of Salmonella Enteritidis was isolated in this study.It had strong virulence which could cause high mortality of chickens,and it was sensitive to most commonly used antibiotics,but was resistant to methoxypyrimidine,compound sulfamethoxazole and lincomycin.This study provided a certain reference basis for the clinical diagnosis of salmonellosis and the evaluation of drug effectiveness.