To understand the genetic characteristics of fowl adenovirus serotype 4 (FAdV-4) in Guangxi region,the whole-genome sequencing were performed on a FAdV-4 strain GX2019-010 isolated from Guangxi in this study.Genome-wide nucleotide sequences were analyzed for homology alignment and genetic evolution,and the amino acid sequences of the major structural proteins Hexon,Penton and Fiber were further analyzed.The results showed that GX2019-010 had a full length of 43 719 bp and GC content of 54.9%,mainly including 43 potential protein coding regions.Genome-wide nucleotide homology comparisons revealed that GX2019-010 was 95.8% homologous to the pathogenic strain MX-SHP95,and 95.0% and 95.2% homologous to non-pathogenic strains ON1 and KR5,respectively.Genetic evolutionary analysis showed that GX2019-010 was in the same branch as the popular strains of FAdV-4 in China in recent years,but not in the same branch as the foreign strains of FAdV-4.This results indicated that there were genetic differences in FAdV-4.The main differences was at the right end of the genome and all domestic strains isolated in China were 1 966 bp missing including ORF19,ORF48 and ORF27 genes.It showed that FAdV-4 popular in China was territorial.Analysis of the amino acid sequences of the major structural proteins of Hexon,Penton and Fiber revealed that all of the three major structural proteins had mutations in amino acid sites,of which Fiber-2 had the most mutations.This study enriched the gene pool of FAdV-4 in Guangxi region and laid the foundation for future research on the prevention and control of HHS and epidemiological investigation.

The aim of this study was to investigate the expression and biological characteristics of adiponectin (ADIPOQ) gene in longissimus dorsi muscle of Hanper sheep at different months of age.The ADIPOQ gene mRNA expression was detected in longissimus dorsi muscle of Hanper sheep at 3 different growth periods (1,7 and 13 months old) by Real-time quantitative PCR.The bioinformatics method was used to analyze the nucleotide and amino acid sequences of ADIPOQ gene,protein characteristics and network interaction protein.The results showed that ADIPOQ gene was expressed in longissimus dorsi muscle of Hanper sheep,which showed an increasing trend with the growth of the months age without significant difference (P>0.05).There was higher homology of ADIPOQ gene with Capra hircus (98.60%,99.00%) and Bos taurus (98.60%,87.00%) in nucleotide and amino acid sequences.The results of physical and chemical properties analysis showed that the molecular formula of ADIPOQ protein was C₁₁₇₂H₁₇₇₆N₃₁₄O₃₄₉S₅,the molecular mass was 26.00 ku,and the theoretical isoelectric point (pI) was 5.88,which suggested that the protein was acidic.The average value of ADIPOQ hydrophilic protein (GRAVY) was －0.403,which indicated that the protein was hydrophilic and didn’t belong to transmembrane protein.In addition,the signal peptide sequence was Met¹-Ala¹⁷,and the cleavage site was between Ala¹⁷ and Arg¹⁸.ADIPOQ existed collagen structure domain and C1Q conserved structure domain at position 45－101 and 101－237,respectively.The secondary structure mainly composed of random coil (66.95%).The construction of protein interaction network indicated that ADIPOQ might interact with ADIPOR1,ADIPOR2,CDH13 and LEP.The results provided a theoretical basis for further exploring the molecular biological functions of ADIPOQ gene in the process of muscle development in Hanper sheep.

The purpose of this study was to investigate the variation of Orf virus (ORFV) immune related genes after infection with different species.The ORFV genomes of sheep and camel were extracted and named ORFV-Y and ORFV-LT,respectively.Based on ORFV genome sequence published in GenBank (accession No.:KF234407.1),three pairs of specific primers were designed and synthesized to amplify the B2L,F1L and VIR gene fragments of ORFV-Y and ORFV-LT,respectively,and the amplified fragments were cloned into pMD19-T vector,transformed into E.coli DH5α competent cells.The recombinant plasmid was identified,and positive clones were selected for sequencing,DNAStar software was used to analyze the homology,amino acid sequence and phylogenetic tree of 13 ORFV genome sequences published on NCBI.The results showed that the nucleotide homology of B2L,F1L and VIR genes were 92.8% to 99.2%,95.7% to 99.5% and 77.6% to 100%,respectively.After comparing the amino acid sequence between the two genomes and the reference sequence,it was found that there were obvious differences in the immune related genes between the two genomes,and F1L gene had some rules to follow.The phylogenetic analysis of B2L,F1L and VIR genes showed that ORFV-Y was closely related to the Chinese Fujian goat strain,while ORFV-LT was far from the reference strains,and was a separate branch.The results showed that ORFV had obvious difference in immune related genes between sheep and camels,it provided a reference basis for further research on the changes of ORFV gene sequences in different species and the development of vaccines for different species in the future.

To confirmed the core regions of uncoupling protein 3 (UCP3) and myosin heavy chain 1 (MYH1) genes promoter and the promoter activity in C2C12 and 3T3-L1 cells,PCR,double luciferase and liposome transfection methods were used to amplify of the promoter of UCP3 and MYH1 genes,construct recombinant vectors,and analyze promoter activity in this study.The results showed that pGL3-Basic-UCP3-pro and pGL3-Basic-MYH1-pro were successfully constructed and transfected into C2C12 and 3T3-L1 cells,respectively.The core promoter region of UCP3 and MYH1 genes were UCP3-P6 (－385 to +3 bp) and MYH1-P5 (－255 to +15 bp),respectively.The promoter activity of pGL3-Basic-MYH1-P2 to pGL3-Basic-MYH1-P5 in C2C12 cells were higher than that in 3T3-L1 cells,indicating that the promoter activity of MYH1 gene in C2C12 cells was higher.This study clarified the core region and promoter activity of UCP3 and MYH1 genes promters,provided a scientific basis for further study on the transcriptional regulation mechanism of UCP3 and MYH1 genes.

This study intended to construct an oocyte-specific expression vector (GDF9-ASMT) for melatonin (MLT) synthetase acetylserotonin methytransferase (ASMT),and granule (luteal) cell-specific expression vector (Cyp17-MTNR1A,MTNR1B) for MLT receptors MTNR1A and MTNR1B,the construction of the above vectors provided carrier materials for further production of transgenic sheep with high fertility.First,the goat GDF9 and Cyp17a1 promoter sequences were amplified separately.Then,the full-length CDS sequences of ASMT,MTNR1A and MTNR1B genes were amplified,and ASMT gene was connected to the downstream of GDF9 promoter by enzyme-cutting,with MTNR1A and MTNR1B respectively connected to the downstream of Cyp17a1 promoter to prepare Pro/GDF9-ASMT,Pro/Cyp17-MTNR1A and Pro/Cyp17-MTNR1B expression elements.Finally,the above-mentioned expression elements were integrated into the pcDNA3.1(+) vector by enzyme digestion,thereby constructing pcDNA3.1-Pro/GDF9-ASMT,pcDNA3.1-Pro/Cyp17-MTNR1A and pcDNA3.1-Pro/Cyp17-MTNR1B eukaryotic expression vectors.After sequencing verification,the GDF9 and Cyp17a1 promoters amplified in this study were 2 496 and 2 497 bp in length respectively,and they were 98% homologous to the 2 500 bp region upstream of the start codons of the goat GDF9 and Cyp17a1 genes in GenBank database.The amplified ASMT,MTNR1A and MTNR1B CDS sequence lengths were 1 037,1 100 and 1 130 bp,and the homology with the standard sequences submitted by GenBank were 98%,98% and 99%.The amino acid sequence homologies was 97%,98%,99% respectively.The construction of the above vectors provided references for further production of melatonin transgenic sheep with high fertility.

The purpose of this experiment was to study the correlation between exon 1 and 4 polymorphisms of DRB1 gene and brucellosis in Kazakh sheep.Using RBPT serological tests to try sheep serum,reference in GenBank sheep MHC Class Ⅱ area DRB1 gene sequences (Accession No.:NC_040271.1),the exon 1 and 4 pieces designed primers,using PCR-SSCP and DNA sequencing technology to 230 Kazak sheep DRB1 gene polymorphism detection,analyze its polymorphism loci and the relationship between the Kazak sheep Brucella susceptibility.The results showed that 66 Kazakh sheep were positive for Brucella in RBPT test,and the positive detection rate was 28.7%.There was one SNP locus (F1-G22A) in exon 1 fragment,and sequencing determined two genotypes (GG and GA),the dominant allele and genotype were G and GG respectively,and the susceptibility genotype of the polymorphisms of F1-G22A was GA.Chi-square test showed that there was no significant correlation between the polymorphisms of DRB1 gene F1-G22A and Brucella susceptibility in Kazakh sheep (P>0.05).According to the analysis of bioinformatics online software,the F1-G22A polymorphic sites lead to the change of RNA secondary structure and the decrease of minimum free energy,and lead to the change of protein secondary structure.No SNPs were found in DRB1 exon 4 fragment.Therefore,there might be a certain correlation between the polymorphisms of DRB1 gene F1-G22A and Brucella susceptibility in Kazakh sheep.

The purpose of the test was to analyze the role of the glycosyltransferase-encoding gene WadC in affecting the intracellular survival of Brucella.Using the Brucella sheep Rev.1 genome as template,the fusion fragments of the homologous arms of the upper and lower arms of WadC gene were obtained by homologous recombination,and ligated to the vector pUC19-SacB to construct the pUC19-SacB-ΔwadC recombinant vector,which was transferred to sheep species Brucella Rev.1,constructing a ΔwadC deletion strain (Rev.1ΔwadC),testing the genetic stability of the strain Rev.1ΔwadC,comparing and analyzing the growth characteristics of the parental strain Rev.1 and the deletion strain Rev.1ΔwadC and the BMDC and RAW264.7 viability of cells.The results showed that the gene-deficient strain was successfully constructed in the experiment,and no genetic back mutation was found in 30 consecutive passages.Under the same culture conditions in vitro,the growth trend of the deleted strain Rev.1ΔwadC was similar to that of the parental strain Rev.1,and both reached logarithmic growth period at 20 h and reached plateau period at 44 h.When the BMDC cells were infected at 48 and 72 h,the intracellular survival rate was significantly lower than that of the parent strain (P<0.05).The RAW264.7 macrophage test of infected mice showed that the parent strain had no significant difference with the gene deletion strain (P>0.05).To sum up,this experiment successfully constructed and obtained a strain of Brucella WadC gene with good genetic stability.The deletion strain had similar growth trend with the parent strain under in vitro culture conditions;However,the survival ability of the deletion strain in BMDC cells was significantly weakened.This study laid a foundation for further study on the function of WadC gene of Brucella.

This study was aimed to systematically analyze the gene sequences and proteins of the importin subtype genes (importin α1,importin α3,importin α4,importin α5,importin α6,importin α7,importin α8 and importin β),and the change of transcription level of importin subtype of F81 cells infected CPV.The full length of importin genes were amplified by RT-PCR techniques from F81 cells and the amino acid sequence and the structure and function of importin protein were subsequently analyzed by bioinformatics software.Meanwhile,the dynamic change of expression of importin genes in CPV-infected F81 cells were investigated at 24,48 and 72 h postinfection by Real-time RT-PCR.The results showed that full length of the importin α subtype genes were about 1 500 bp and the importin β was 2 600 bp.The physical chemical properties of importin proteins showed the theoretical isoelectric was about 4.60 and the importin α1,importin α5,importin α6,importin α7 and importin β belonged to instable proteins.Meanwhile,there were no signal peptide,no transmembrane structures and no cellular localization in importin proteins.The secondary structure of importin proteins were predicted by Predictprotein software and mainly composed of α-helix and random coil.Real-time RT-PCR results showed that the importin α1 (P<0.01) and importin β mRNA level were significantly reduced in CPV-infected F81 cells,while other importin mRNA level were markedly up-increased,the importin α8 gene had the highest expressional level (P<0.01).The study laid a foundation for further study on the transport mechanism of CPV into the nucleus of the cell and developing drugs.

The purpose of this study was to explore the expression and potential functions of circular RNA (circRNA) in the skin,subcutaneous fat,and longissimus dorsi muscle of Xiang pig.In this study,RNA-Seq technology and bioinformatics methods were used to analyze the expression of circRNAs in skin,subcutaneous fat,and longissimus dorsi muscle of Xiang pig,GO and KEGG enrichment analysis were performed for the host genes of three tissue-specific circRNAs,then target miRNA prediction was performed for the circRNAs that host genes associated with tissue structure and function.A total of 6 483 circRNAs were identified from the three tissues of Xiang pig,4 575,5 180 and 5 219 circRNAs were identified respectively from skin,subcutaneous fat and longissimus dorsi muscle respectively,among which 83,234 and 586 circRNAs expressed specifically.circRNAs distributed on chromosomes widely and mainly from exons,a few from introns and intergenic regions.The expression levels of most circRNAs were low.The host genes of skin-specific circRNAs were mainly involved inproteolysis,RNA transport,and gap junctions.Among them,host genes such as NF1 and MAPK1 were related to cutaneous diseases and might play a role in the form of circRNAs.The circRNA (circ-MAPK1) that the host gene MAPK1 could bind to miR-18a,miR-132,miR-411,and which might be involved in regulating the formation of collagen.The host genes of circRNAs expressed specifically in subcutaneous fat were enriched in adipocyte growth,development and cancer signaling pathways.ABHD5 and PTPN11 genes were related to fat metabolism,miR-103,miR-107 and miR-199a-5p bound by circ-PTPN11 could be involved in regulating adipocyte proliferation,differentiation and lipidosis.The host genes of circRNAs only expressed in longissimus dorsi muscle associated with cancer,infection,and muscle development.ROCK2 and PPP1CC were involved in myocyte differentiation and muscle contraction,while miR-339,miR-181a and miR-181b bound by circ-PPP1CC were involved in the regulation of myocyte differentiation,proliferation and growth.In conclusion,the circRNAs selected in this study and their combined miRNAs might be involved in skin collagen formation,fat deposition,and muscle cell differentiation and maturation.

Lactic acid bacteria can promote host health by adhering to host cells or mucosa and inhibiting pathogen growth,maintaining microecological balance and enhancing host immunities.It also plays an important role in food industry,antimicrobial production and in vivo fermentation.However,the specific molecular mechanism of probiotics in lactic acid bacteria is rarely described.In order to explore the new function and regulation mechanism of lactic acid bacteria itself and lactic acid bacteria in its host,omics technology can be used for research.The omics methods derived from the central dogma include genomics,transcriptomics,proteomics,and metabolomics.The application of monoomics technology in the study can further explore the function and mechanism of lactic acid bacteria.The application of multi-group technology can break through the limitation of single group technology,the potential function of lactic acid bacteria can be analyzed synthetically from different dimensions,and the lactic acid bacteria itself and its role in the host were further predicted.Several omics techniques which are commonly used in the field of lactic acid bacteria isolation and identification and functional research were reviewed in this paper.The application of multi-omics in the study of function and mechanism of lactic acid bacteria were reviewed.It provided a feasible technical method and direction to further explore the potential function of lactic acid bacteria.

The purpose of this experiment was to explore the influence of the sample loading volume,flow rate and protein concentration of protein on the removal efficiency of endotoxin and protein loss in porcine visfatin recombinant lipoprotein solution at 4 ℃.In this study,commercial and modified polymyxin B was used as the medium to specifically remove endotoxin from the protein solution.At 4 ℃,different loading volume (6,8,10,12 mL),flow rate (0.125-0.75 mL/min) and protein concentration (0.9,1.2,1.3,1.7 mg/mL) were used for endotoxin removal,the content of endotoxin was determined by a quantitative method using endotoxin reagents,and the BCA protein concentration assay kit was used to determine the protein concentration.The results showed that at 4 ℃ endotoxin removal was the best when protein loading volume was 12 mL and flow rate was 0.125-0.25 mL/min,the removal rate was 99.5%,the residual amount of endotoxin (1 EU/mL) was within the biosafety range,and the protein recovery rate was as high as 90% to 95%.The experimental results showed that the endotoxin was successfully removed from the solution of porcine lipin recombinant protein,which provided references for the related functional research and clinical application of the protein.

This study was conducted to investigate the effects of carvacrol and thymol on the relative abundance of colonic microorganisms in weaned piglets and their metabolic pathways.A total of sixty four 28-day-old (8.5 kg±1.0 kg) weaned piglets were divided into 4 groups with 16 replicate of 1 piglet:Group A (control group,basal diet),group B (basal diet＋20 g/T carvacrol),group C (basal diet＋20 g/T thymol),and group D (basal diet＋20 g/T carvacrol-thymol blend (mass ratio 2:1)).The experiment lasted 30 d.The sequencing was taken based on the Illumina HiSeq sequencing platform,and using paired-end sequencing (Paired-End) method.Perform species annotation and abundance were analysized by clustering OTUs,and alpha diversity analysis、beta diversity analysis、significant species difference analysis and functional gene prediction analysis were further conducted.The results showed as follow:①The colonic microorganisms biodiversity of the experimental groups were significantly higher than that of control group (P<0.05).②The relative abundance of Firmicutes in the colon of group D was extremely significantly higher than that of control group (P<0.01),significantly higher than groups B and C (P<0.05),the relative abundance of Bacteroidetes was significantly higher than that of control group and group C (P<0.05),and the relative abundance of Epsilonbacteraeota,Spirochaetes and Proteobacteria were extremely significantly lower than that of control group (P<0.01).The relative abundance of Campylobacter in the colon of group D was extremely significantly lower than that of control group (P<0.01),that of Rikenellaceae_RC9_gut_group in group D was significantly higher than that of group B (P<0.05),that of Roseburia in group D was extremely significantly higher than that of group A (P<0.01),that of Helicobacter was extremely significantly lower than that of group A (P<0.01).③The lipid metabolism level of colonic microorganisms in group D was extremely significantly higher than that of control group (P<0.01),and colonic microbial carbohydrate metabolites,biosynthetic of other secondary metabolites and other amino acid metabolism level were significantly higher than that of control group (P<0.05),and energy metabolism level of colonic microorganisms was extremely significantly lower than that of control group (P<0.01).In conclusion,under the condition of this experiment,dietary supplementation with 20 g/t carvacrol-thymol improved the microbial community structure of colonic microorganisms in weaned piglets.The relative abundance of colonic beneficial bacteria was increased,the relative abundance of colonic harmful bacteria was decreased,and positively affected the metabolic function of colonic microorganisms.

The purpose of this experiment was to study the usage and proportion of fermented feed in broiler feed.By adding 0,10% and 20% fermented complete formula feed to the broiler feed,and granulating normally,the influence of the usage mode of the fermented feed on the production performance,slaughter performance,intestinal tract and other related indexes of the broiler feed were studied,so as to provide reference for the application of the fermented feed in the broiler feed.180 1-day-old healthy AA male broilers were randomly divided into 3 groups with 6 replicates in each group and 10 chickens in each replicate.The three treatment groups were fed with 0,10% and 20% of the total fermented feed for 42 days.The results showed that:From the whole period (1-42 days old),the 10% fermentation feed substitution group significantly reduced the feed consumption and weight gain ratio (P<0.05),and the average daily weight gain increased (P=0.05).At 42 days old,there was no significant effect of fermented feed on slaughter performance (P>0.05).The ileal crypt depth of broilers in the 20% fermentation feed replacement group was significantly higher than that in the control group and the 10% replacement group (P<0.05).The length of duodenal villi and V/C in 20% and 10% fermentative feed substitution group was significantly higher than that in control group (P<0.05).The results showed that the replacement of 10% fermented feed in broiler feed could reduce the ratio of feed to weight gain,increase the average daily gain,and have a certain impact on intestinal development.

The purpose of this experiment was to study the effect of compound enzyme and compound enzyme bacteria preparation on the degradation rate of diet in vitro and milk production performance of dairy cows at different lactation stages.In in vitro experiment,the basic diet (group Ⅰ) and the basic diet added with compound enzyme (group Ⅱ) and compound enzyme probiotics preparation (group Ⅲ) were used as fermentation substrate respectively.After 72 h of culture in vitro,the dry matter degradation rate (DMD) and crude protein degradation rate (CPD) of the diet were measured at different time (6,12,24,36,48 and 72 h).In feeding experiment,24 dairy cows with similar body weight,parity and condition were divided into three groups at early lactation (30 d±5 d),middle lactation (100 d±5 d) and late lactation (250 d±5 d),respectively.The cows in control group (group Ⅰ) were fed with TMR,in compound enzyme group (group Ⅱ) and the compound enzyme probiotics group (group Ⅲ) were fed with the TMR added with 1 kg/t compound enzyme and compound enzyme probiotics respectively.The trial period was 74 days,including 14 days of pre trial period and 60 days of normal trial period.`The results showed that:①In vitro DMD and CPD of the two groups increased with the prolongation of treatment time.At each time point,the values of DMD and CPD in group Ⅲ were higher than those in group Ⅱ,the difference of DMD was significant at 6 and 72 h (P<0.05),CPD was significant at 36 and 72 h (P<0.05),and there was no significant difference at other time points (P>0.05).②In the early stage of lactation,the average daily milk production of groups Ⅱ and Ⅲ increased by 3.68% and 4.10% respectively compared with group Ⅰ,the difference was significant (P<0.05),and the difference between groups Ⅱ and Ⅲ was not significant (P>0.05).In mid-lactation,the average daily milk production of groups Ⅱ and Ⅲ increased by 7.53% and 10.66% respectively compared with group Ⅰ (P<0.05),and the difference between groups Ⅱ and Ⅲ was not significant (P>0.05).In the later stage,the average daily milk production of groups Ⅱ and Ⅲ increased by 7.06% and 5.16% respectively compared with group Ⅰ (P<0.05),and the difference between group Ⅱ and Ⅲ was not significant (P>0.05).In conclusion,both compound enzymes and compound enzyme probiotics preparations could improve the in vitro DMD and CPD of the diet and the milk production of dairy cows in different periods to a certain extent.Among them,the compound enzyme preparation had a better effect on increasing milk production of dairy cows in the late lactation,while the compound enzyme probiotics preparation has a better effect on increasing milk production in mid-lactating dairy cows.

The objective of the experiment was to investigate the effect of Chinese herb additive on production performance and blood biochemical indices in transition dairy cows.Thirty six Holstein dairy cows were randomly assigned to three treatments,supplemented with 0,150 and 300 g/d Chinese herb additive,respectively.Experimental duration was 7 weeks and the first week was the preliminary period.After delivery,feed,blood and milk samples were collected once a week for the measurement of dry matter intake,blood biochemical indexes and milk components.The results showed that cows fed 150 and 300 g/d Chinese herb additive tended to increase milk production (5.1% and 10.6% respectively) compared to control group,but the difference was not significant (P<0.05).Meanwhile,the somatic cell count in milk significantly decreased (P<0.05),which decreased by 62.7% and 76.6%,respectively.Feeding Chinese herb additive had no significant effect on dry matter intake and other milk composition (P>0.05).Compared to control group,cows received Chinese herb additive significantly decreased the content of serum NEFA (P<0.05),which decreased by 29.0% and 35.1%,respectively.There was no significant difference on other indexes in serum (P>0.05).The results showed that dietary supplementation with 300 g/d Chinese herb additive had a good effect for transition dairy cows,which could significantly reduce the somatic cell count in milk and the content of NEFA in blood,and would improve the health status of dairy cows,and the postpartum production performance of dairy cows.

To study the effects of concentrate supplementation on serum biochemical indexes,40 nine-month-old Oura-type Tibetan sheep (30.21 kg±1.42 kg) were randomly divided into 4 groups.Group A grazed naturally,group B,C and D supplemented with 0.10,0.20 and 0.30 kg mixed concentrate per sheep every day after grazing,respectively.The trial lasted 210 days.The results showed that the content of total serum protein (TP),albumin (ALB),globulin (GLB),calcium (Ca),Ca/IP(inorganic phosphorus) value,creatine kinase (CK),lactate dehydrogenase (LDH),aspartate transaminase (AST),alaine transaminase (ALT) and γ-glutamine transferase (GGT) of the sheep increased with the concentrate supplemented.The content of serum glucose (GLU),cholesterol (CHO) and triglyceride (TG),the activities of superoxide dismutase (SOD) and catalase (CAT),the concentrations of growth hormone (GH),insulin-like growth facior-1 (IGF-1),cholecystokinin (CCK),IgA,IgG and IgM increased with the increasing of the amount of concentrate supplementation.But the IgG concentration of group D decreased a little compared with group C (P>0.05).Supplementary feed significantly increased the activity of alkaline phosphatase (AMP),the concentration of urea nitrogen (BUN) and the value of BUN/CRE (P<0.05),and significantly decreased the concentrations of creatinine (CRE) and uric acid (UA) (P<0.05).The results showed that supplemented with proper amount of concentrate after returning to grazing in cold season could improve the energy and fat metabolic capacity,enhance the synthesis of liver protein,improve the immunity and antioxidant capacity of the body,and regulate the metabolism of calcium and phosphorus of Oura-type Tibetan sheep.

The study was conducted to investigate the effects of different dietary calcium levels on nutrient digestion and metabolism of Yunnan semi-fine wool sheep during the empty pregnant period,and to provide basis for formulating the standard of calcium requirement of Yunnan semi-fine wool sheep.Fifty Yunnan semi-fine wool sheep with healthy body condition,close body weight and 2 births were randomly allocated to 5 groups,which were groupsⅠ (0.44% calcium),Ⅱ (0.67% calcium),Ⅲ (0.82% calcium),Ⅳ (1.00% calcium) and Ⅴ (1.13% calcium).The experiment was divided into pre-trial period (14 days) and formal period (30 days).On the 7th day of the formal period,five sheep were randomly selected from each experimental group and placed in separate metabolic cages for digestive and metabolic tests.The experimental results showed that:With the increase of dietary calcium content,the dry matter intake of Yunnan semi-fine wool sheep in empty pregnant period decreased at first and then increasing (P>0.05).The higher the dietary calcium level of Yunnan semi-fine wool sheep in empty pregnant period,the greater the excretion of fecal calcium (P<0.05),and the greater the excretion of calcium (P<0.05).Regression analysis showed that the calcium maintenance requirement of Yunnan semi-fine wool sheep in empty pregnant period was 7.93 g/d and R²=0.5827 according to the linear relationship between different calcium levels of diets and average daily gain.Linear regression analysis between calcium intake and deposited calcium showed that the maximum deposited calcium of Yunnan semi-fine wool sheep during empty pregnant period was 3.64 g/d.In conclusion,the increase of dietary calcium level of Yunnan semi-fine wool sheep during empty pregnant period had a certain effect on dry matter intake,but had no significant effect on the apparent digestibility of each nutrient.Fecal calcium and excreted calcium of Yunnan semi-fine wool sheep in empty pregnant period increased with the increase of dietary calcium level.Under different dietary calcium levels,the calcium intake level had little correlation with the daily gain of Yunnan semi-fine wool sheep in empty pregnant period.

With the fetal growth and mammary gland development during late gestation,the metabolic of sows is gradually increased.Owing to imbalance of glucose and lipid metabolism,the metabolic disorder appears.The physiological changes and environmental stress experienced by sows in the reproductive cycle are of great significance to glycolipid metabolism.Energy accounts for more than 70% of the dietary composition.It is direct and effective to develop appropriate dietary energy sources during gestation and lactation for improving the reproductive performance of sows by regulating the balance of glycolipid metabolism.Starch and fat are the main energy sources of sows’ dietary.Previous studies have shown that dietary energy sources have a significant effect on puberty initiation of gilts or improvement of reproductive performance of sows.However,it is rarely reported that summary on the effects of different sources and levels of starch and fat on the reproductive performance of sows based on the glycolipid metabolism characteristics of sows.This paper firstly reviewed the characteristics of glycolipid metabolism during pregnancy and the possible mechanism behind metabolic changes,summarized the adverse effects of metabolic disorders during pregnancy on reproductive performance of sows,and then introduced the absorption and metabolism characteristics of starch and fat in dam,compared and analyzed the effects of different levels and sources of starch and fat on the reproductive performance of sows.Finally,to maximize the economic benefits of the pig farm,the basis for selecting the appropriate energy source was summarized according to the actual production purpose.The purpose of this paper was to offer data and theoretical support for the selection of appropriate levels and sources of starch and fat to improve the reproductive efficiency of sows.

In order to identify the molecular markers related to alive litter size of Bama Xiang pigs,the genome-wide association study (GWAS) was used to map and screen the candidate genes affecting the alive litter size trait.Ear tissue samples of 297 Bama Xiang pigs with multiple parity records were collected,and DNA was extracted and genotyped by porcine 50K SNP beadchip.After quality control and genotype imputation,the alive litter size of Bama Xiang pigs were GWAS by Tassel.The results showed that the average number born alive per litter of Bama Xiang pigs increased gradually with the increasing of parity in the range of 1-9 parities.A total of 32 816 SNPs were obtained after quality control and filtration.8 SNPs related to alive litter size of Bama Xiang pigs were screened by genome-wide association analysis,which were significant at genome or chromosome level.Based on the enrichment analysis of the coding genes in the region between 500 kb upstream and downstream of the associated significant SNP loci,and the QTL regions and gene functions related to porcine reproductive traits,4 genes (CAPZB,MSH3,CITED2 and HSD17B7) were finally identified to be candidate genes related to alive litter size of Bama Xiang pigs.

The rabbit is a kind of grain-saving and small herbivore animal.Rabbit breeding which is low investment and obvious efficiency has become an advantageous industry of increasing income of rural farmers and poverty alleviation.In recent years,with the development of life science,great progress has been made in rabbit genetic breeding at home and abroad.In this paper,the research progress in genetic breeding and reproduction of rabbits in 2019 are reviewed from the traditional breeding,molecular breeding and reproduction of rabbits.For abroad,lots of researches involved in traditional breeding,molecular breeding and reproduction were done.Effects of selection,environment and additives on productive performance had been analyzed in traditional breeding.Molecular breeding mainly focused on reproductive performance among which there were more studies on litter size and spermatine.Secondly,the related genes of growth,meat quality and hair color were studied.The research on reproduction mainly focuses on the preservation method of male rabbit semen and the effects of additives on rabbit semen quality,litter size and conception rate.In China,genetic breeding and reproduction research on rabbit also mainly included traditional breeding,molecular breeding and reproduction,but the focus of domestic research was molecular breeding,among which breed and genetic diversity and functional genes of fur trait were mainly researched using high-throughput sequencing,gene cloning and gene editing to screen the important functional genes and regulatory networks related to research traits.However,compared with molecular breeding,there are fewer studies on the traits evaluation of traditional breeding and rabbit reproduction.This review can provide references for rabbit breeding and production.

The purpose of this study was to clone the prostaglandin D synthase (L-PGDS) gene and identify its expression pattern in various tissues and testis at different development stages in cattle-yak,in order to provide experimental basis for studying the mechanism of L-PGDS gene in testis development of cattle-yak.The total RNA of heart,liver,spleen,lung,kidney,large intestine,small intestine,gastric,brain and testis at different development stages (fetal,calve tuberty and old) of healthy cattle-yak were extracted by Trizol method.The L-PGDS gene sequence was amplified and cloned by RT-PCR,and analyzed by related biological software.The expression of L-PGDS gene mRNA in different tissues and testicular tissues at different development period of in cattle-yak were detected by Real-time quantitative PCR.The results showed that the length of L-PGDS gene was 624 bp,including the ORF of 576 bp,which encoded 191 amino acids.Homology alignment results showed that L-PGDS gene in cattle-yak had high homology with Bos taurus and Ovis aries,which were 99.28% and 94.00%,respectively.Phylogenetic tree results showed that the relation between cattle-yak and Bos taurus was the closest,followed by Ovis aries and Equus caballus.The L-PGDS protein was an unstable hydrophobic protein,the molecular weight was 21.229 ku,the molecular formula was C₉₅₃H₁₄₇₁N₂₅₁O₂₈₁S₉,the isoelectric point was 6.43,and the instability index was 47.25.The L-PGDS protein had no transmembrane region and no signal peptide.The secondary structure of L-PGDS protein contained alpha helix,random coil and extended chain,the rats of them were 25.65%,53.40% and 20.94%,respectively.L-PGDS gene was highly expressed in testis of cattle-yak,which was extremely significantly higher than that in other tissues (P<0.01).The expression of L-PGDS gene mRNA in testis of cattle-yak increased firstly and then decreased along with age,and with the highest expression in puberty stage,which was extremely significantly higher than that at other periods (P<0.01).This study provided basic data for further study on the mechanism of L-PGDS gene in the development of testis in cattle-yak.

The purpose of this study was to explore the polymorphism of exon 3 of fibroblast growth factor 14 (FGF14) gene and its effect on growth traits in Yanbian Yellow cattle.The third exon of FGF14 gene was sequenced and analyzed by DNA direct sequencing method,and the genotyping of FGF14 gene was detected by HRM typing technique with high resolution melting curve,so as to explore the relationship between different genotypes of FGF14 gene and growth traits of 16 and 24 months of age in Yanbian Yellow cattle.The results showed that one A>G mutation was detected at position 631 947 bp of FGF14 gene,two alleles of A and G,and three genotypes of AA,AG,and GG.The results of χ² test showed that the distribution of genotype frequency and allele frequency of FGF14 gene in two Yanbian Yellow cattle populations of 16 and 24 months of age conformed to Hardy-Weinberg equilibrium (P>0.05).Polymorphic information content (PIC) analysis results showed that FGF14 gene was moderately polymorphic in both groups (0.25 < PIC < 0.5).The results of association analysis with growth traits showed that the different genotypes of FGF14 gene had significant influence on the traits of height at back and rump length of Yanbian Yellow cattle at 16 months of age (P<0.05),and had significant influence on the traits of body height,body weight and height at hip cross of Yanbian Yellow cattle at 24 months of age (P<0.05).In conclusion,it was speculated that FGF14 gene had a certain influence on the growth and development of Yanbian Yellow cattle,and provided reference for the subsequent modern molecular breeding and breeding of meat traits in Yanbian Yellow cattle.

The purpose of this study was to investigate the effects of resveratrol (RES) at different concentrations (0,0.5,2.0,5.0 μmol/L) on in vitro fertilization (IVF) and antioxidant capacity of ovine oocytes and the secretion of steroid hormones by cumulus cells.Sheep oocytes were fertilized in vitro after maturated in different concentrations of RES for 24 h,and the in vitro maturation (IVM) medium was collected for detecting the enzyme activity of superoxide dismutase (SOD),glutathione peroxidase (GSH-Px) and the content of monochrome display adapter (MDA).The ELISA method was used to detect the concentration of estradiol (E₂) and progesterone (P₄).The results showed that when compared to the control group,adding 0.5 μmol/L RES to the IVM medium significantly increased the cleavage rate (P<0.05),but had no significant effect on the fertilization rate and blastocyst rate (P>0.05);5.0 μmol/L RES significantly reduced the fertilization rate,cleavage rate and blastocyst rate (P<0.05),which had an inhibitory effect on embryonic development.Adding 0.5 μmol/L RES to IVM and IVC (in vitro culture,IVC) medium significantly increased the fertilization rate,cleavage rate and blastocyst rate (P<0.05).Compared to the control group,the addition of RES to IVM solution had a certain inhibitory effect on the secretion of E₂ by cumulus cells,5.0 μmol/L RES significantly reduced E₂ concentration (P<0.05);0.5 μmol/L RES significantly increased the P₄ secretion of cumulus cells (P<0.05).0.5 and 2.0 μmol/L RES increased the activity of SOD,GSH-Px and other enzymes,but there was no significant difference when compared with the control group (P>0.05),but significantly reduced MDA content (P<0.05),while 5.0 μmol/L RES significantly reduced the activity of antioxidant enzymes and increased the content of MDA (P>0.05).In conclusion,0.5 μmol/L RES simultaneously added to IVM and IVC medium could enhance the antioxidant capacity of oocytes and concentration of P₄,and reduced the content of MDA,thus improved the cleavage rate and blastocyst rate.

The purpose of this study was to explore the problems of low development efficiency of small amounts in vitro fertilization (IVF) embryos and limited development potential of zona pellucida-free embryos when cultured in vitro of mammals,to establish an efficient and stable in vitro production system for improving the developmental potential of living ovum-pick-up (OPU) and handmade clone (HMC) embryos in buffalos.The study first compared the effect of the number of fertilized eggs (1,3,5,10 and 20) co-cultured within a single microdroplet on the embryonic development effect.Then,OPU-IVF embryos were cultured using the well-of-the-well (WOW) system and the auxiliary co-culture system (adding agarosaccharide fragments of embedded in in vitro fertilization (IVF) fertilized egg in the cultivation of microdroplets).Furthermore,the embryos without zona pellucida were cloned and reconstructed by using the WOW system and compared with the traditional microdroplet system in vitro.The results showed that the blastocyst development rates of the 10 and 20 embryos groups were significantly higher than that of the 1,3 and 5 embryos groups.Compared with the microdroplet system,the assisted co-culture system and the WOW system significantly improved the blastocyst rate of OPU-IVF embryos(P<0.01).Moreover,the WOW culture system significantly promoted the cleavage rate and blastocyst rate of HMC reconstructed embryos(P<0.01).To sum up,embryo mass culture contributed to embryo development,and under the premise of ensuring a clear pedigree,the agar-sugar embedding assisted embryo co-culture system and the WOW system improved the in vitro development efficiency of OPU-IVF embryos,the WOW system would also be applied to the high-efficient culture of zona pellucida free embryos.

To investigate the influencing factors of locomotion score (LS) and its impacts on production performance and blood routine index in Holstein,body condition score (BCS),LS and DHI (dairy herd improvement) of 1 486 lactating cows from two dairy farms,and the blood routine indexes of 101 lactating cows from one farm in Beijing were measured.The fixed model was used to analyze the impacts of herd,parity and lactation stage on LS,and the impacts of LS and BCS on daily milk yield,somatic cell score,milk composition and blood routine indexes in dairy cows.The results showed that both parity and lactation stage had significant impacts on LS.The LS increased with the increase of parit,the LS increased by 0.71 from first lactation to fifth and above lactation.The LS of cow in lactation stage Ⅲ(100-199 d) and Ⅵ (after 350 d) were significantly higher than cow in stage Ⅱ (45-99 d)(P<0.05),and both were higher than 2.The LS had no significant impacts on daily milk yield,milk composition and somatic cell score (P>0.05).However,the milk yield of the cow with LS≥4 was the lowest,and a decrease of 2.29 kg was found compared with the cow with LS=1.Among the various blood routine indexes,LS had significant impacts on red blood cell count,hemoglobin content,mean corpuscular hemoglobin concentration and lymphocyte count (P<0.05),and had no significant impacts on white blood cell count (P>0.05).With the increase of LS,the red blood cell count,hemoglobin content,mean corpuscular hemoglobin concentration and lymphocyte count of cows decreased with different extents.There were some problems on the body immunity and hematopoiesis of cows with poor LS.In dairy farm,regular evaluation of LS could be used to find the management problems of feet and legs,which had a great significance to reduce the loss of production performance and early culling caused by hoof disease.

The purpose of this experiment was to investigate the effects of different intensities of monochromatic green light on hatching,embryo development,and early growth and development of chickens during incubation.810 New Pudong chicken eggs with similar size were randomly divided into 3 groups (n=270).The eggs were hatched in dark,low illumination (20~50 lux),or high illumination (200~300 lux) LED monochromatic green light (λ=525 nm) respectively during 1~18.5 days of incubation period,with 24 h light per day.From 18.5 days of incubation,the eggs of 3 groups were transferred to the same hatcher without light.Incubation temperature and humidity conditions were consistent among groups.The hatchability of fertile eggs,percentage of early dead embryo,body weight (BW) at birth and 10 weeks of age,serum hormones (thyroid hormone,calcitonin,growth hormone,melatonin and osteocalcin),and blood physiological and biochemical indicators (alkaline phosphatase,insulin-like growth factor and calcium of one-day-old and 3-week-old chicks) were examined.The results showed that 3 groups had similar early dead embryo number and percentage.At the 20.5 d after incubation,the hatchability of fertile eggs in the low illumination group and the high illumination group was about 2.3 times and 5.2 times of that in the dark group.At the 21.5 d after incubation,the hatchability of fertile eggs in the dark group was 82.8%.This parameter of the low illumination group and high illumination group was 6.5% and 1.9% higher than that of the dark group.The percentage of healthy chicks of dark group was 98.6%,this parameter of the low illumination group was 0.4% lower than that of dark group,while high illumination group and dark group was almost the same.The birth BW of males of the high illumination group was lower than that of the other two groups (P<0.05),but there was no difference between the low illumination group and the dark group (P>0.05).The trend of birth BW of females was basically consistent with that of males.The birth BW of female chicks in the high illumination group was lower than those of other two groups (P<0.05),and there was no difference between other two groups (P>0.05).At 10 weeks of age,there was no difference in BW between males or females of 3 groups (P<0.05).For the day-old chicks,there was no difference in blood and hormones indicators among groups (P>0.05).For the 3-week-old New Pudong roosters,the serum melatonin level(P<0.05)in the high and low illumination groups were higher than that in the dark group,and there was no difference in other indicators (P>0.05).In conclusion,compared to the dark condition,providing proper intensity of monochromatic green light during the incubation could promote the development of chicken embryo,and advance the hatching process without impairing chicks’health and following growth performance.

This study was designed to evaluate the synergistic effect of VA5 immunopotentiator on pigeon ND4416 inactivated vaccine.160 healthy pigeons were randomly divided into four groups,including ND4416 strain inactivated vaccine group (NDV group),ND4416 strain inactivated vaccine and immunopotentiator (VA5) mixed group (NDV+VA5 group),La Sota inactivated vaccine group (La Sota group) and normal saline as the control group (C group),to assess their vaccine efficacy against virulent pigeon NDV by serological analysis and animal testing.Pigeons sera were collected at different time points after immunization and measured the HI antibody titer of each group.The results showed that VA5 immunopotentiator significantly improved the serum antibody level of pigeons immunized with pigeon Newcastle disease inactivated vaccine (P<0.05).In addition,comparative test of spleen lymphocyte transformation was conducted at various time points after immunization.The results indicated that VA5 effectively stimulated the lymphocyte transformation of immune pigeon.Pigeons in each groups were challenged with ND4416 strain at the 30th,90th and 180th d after immunization.The results presented that the NDV+VA5 group had 100% protection rate and higher than La Sota group.The duration of immunization test showed that the antibody titer of NDV+VA5 group reached peak 11.20log₂ at the 21st d,remained 7.50log₂ at 180th d,and the protection rate remained 100% at 180th d.It indicated that VA5 immunopotentiator sustained the immune duration of pigeon NDV vaccine up to 180 d.Moreover,the in vitro detoxification test results suggested that VA5 immunopotentiator reduced the in vitro detoxification cycle of pigeons after challenge.Overall,this study suggested VA5 immunopotentiator could significantly improve the immune efficacy of pigeon Newcastle disease inactivated vaccine,which provided a basis for further enhancing the efficacy of Newcastle disease vaccine,as well increased experimental data for the application of immunopotentiators.

The purpose of this study was to investigate the growth promoting effect and the safety of the vaccine in crossbred buffalo calves immunized with somatostatin and corticostatin co-expression DNA vaccine.The identified plasmid of pVGS/2SS-2A-S/CST-asd was electroporated into attenuated Salmonella Choleraesuis C500 to construct the co-expression live vector vaccine of somatostatin and corticostatin,and evaluate the effect on the growth performance of the crossbreds buffalo after nasal immunization.Twenty four 2-6 months of age crossbred buffalo calves with similar body weight were randomly divided into 4 groups immunized with the vaccine in low dosage group (TL),middle dosage group (TM),high dosage group (TH) and negative control group (NC).Immunization was performed once a day for 3 consecutive days,and a booster was given 2 weeks late.The results showed that the antibody of SS and CST could be produced on 2 weeks after the primary immunization,and TL group was the best,showing higher antibody level and positive rate.At 7 weeks,the positive rate of experimental group showed a downward trend.The daily gain of each experimental group was higher than that of control group at 2 and 7 weeks after the primary immunization,but there was no significant difference between experimental groups (P>0.05),while the average daily gain of antibody positive group (P) was significantly higher than that of negative group (N) at 2 weeks (P<0.05).The results of related hormones showed that the levels of growth hormone (GH) and insulin growth factor 1 (IGF-1) were higher in experimental groups at 2 and 7 weeks after the primary immunization.In each experimental group,TL group showed the best performance,both extremely significantly higher than that of control group except GH level at 7 weeks after the primary immunization (P<0.01),antibody positive group was also significantly higher than negative group (P<0.05).Cytokine test results showed that the interleukin 4 (IL-4) level in experimental groups at 2 weeks after the primary immunization was higher than that of control group,and the IL-4 level in TL and TM groups was extremely significantly higher than that of control group (P<0.01),especially in TL group.The IL-4 level in TL and TH groups at 7 weeks after the primary immunization was significantly higher than that of control group (P<0.05).At 2 weeks after the primary immunization,the level of immune interferon γ (INF-γ) in control group was extremely significantly higher than that of experimental groups (P<0.01),but at 7 weeks after immunization was significantly lower than that of TL and TH groups (P<0.05).The levels of IL-4 and INF-γ in antibody positive group were significantly higher than that of negative group at 2 and 7 weeks after the primary immunization (P<0.05),respectively,and there was no significant difference among other groups (P>0.05).The levels of blood glucose (GLU),total protein (TP),total cholesterol (CH) and urea nitrogen (BUN) in serum showed that except that the level of total protein in TL group at 2 weeks after the primary immunization was significantly higher than that of control group (P<0.05),there was no significant difference of other indexes among each group (P>0.05).In order to confirm the safety of the vaccine,the genes of water,soil and fecal samples after immunization were amplified,which were not detected in PCR sensitivity range.The results indicated that the vaccine could achieve a good immune effect,make the body produce a significant immune response,and the effect of the low dosage group was the best.The vaccine did not cause adverse effects on the environment.

The study was aimed to establish a simple in vitro culture system for piglet small intestinal epithelial cells,and to provide materials for researches on porcine epidemic diarrhea virus (PEDV).In this study,newborn piglets that did not eat colostrum were used as the initial donors,and the primary cells were separated in vitro by scraping the intestinal mucosa from the intestinal lumen and mechanical separation and dispersion.The piglet intestinal epithelial cells were purified using 0.1% trypsin differential digestion method.Compared the effects of newborn weak piglets and normal piglets as donors on the activity of primary intestinal epithelial cells.MTT method was used to compare the proliferation activity of primary cells of different generations.Immunofluorescence and Real-time RT-PCR were used to detect the infection and proliferation of PEDV strain CV777 in primary intestinal epithelial cells.The results showed that the isolation and culture method in vitro used in this study could obtain primary intestinal epithelial cells with good proliferation activity,with obvious S-type cell proliferation curves.The cells with high purity and single morphology could be obtained by differential digestion,and had good proliferation activity after five consecutive passages.The proliferative activity of intestinal epithelial cells isolated from weak piglets and normal piglets had no obvious difference,and provided new way for reducing the cost of primary cell culture.The results of immunofluorescence and Real-time RT-PCR showed that PEDV could infect the primary intestinal epithelial cells,and replicated and proliferated in them.In this study,we established a simple,practical and low-cost method for in vitro culture of piglet primary small intestinal epithelial cells.The primary cells cultured by this method could be used as basic materials for the isolation and culture of PEDV and related research.

Canine coronavirus (CCoV) and feline coronavirus (FCoV) belong to α-genus coronavirus of coronavirus family,porcine transmissible gastroenteritis virus (TGEV),porcine epidemic diarrhea virus (PEDV) also belong to the same genus.Genetic evolution analysis showed that different genotype of the virus could produce new variant strains through gene recombination,which caused great obstacles to the diagnosis and control of the disease.β-genus coronaviruses include bovine coronavirus (BCoV),canine respiratory coronavirus (CRCoV) and severe acute respiratory syndrome coronavirus (SARS-CoV).Among them,CRCoV has the highest homology with BCoV,but there are great differences in genomic structure,pathogenic mechanism and infection symptoms between this kind of coronavirus and α-coronavirus.CCoV and FCoV are widely spreading around the world,characterized by high morbidity and low mortality.Due to the characteristics of RNA virus and the influence of environmental selection pressure,the viruses continue to mutate and evolve,and new virulent strains appear one after another.The virulence of feline infectious peritonitis virus (FIPV) is greatly enhanced,some specific point mutations in the virus genome change the cellular tropism against the host.The pathogenesis of the virus mainly depends on the antibody-dependent enhancement (ADE) induced by virus infection.The epidemiological investigation and prevention and control of CCoV and FCoV should not only rely on the single factor of vaccine immunity,but also comprehensively consider the virulence of the virus,environmental conditions,pet self-immune resistance, and so on.The identification of CCoV and FCoV should be based on clinical symptoms,combined with routine hematological examination,serum biochemical examination and laboratory diagnosis techniques to prevent false positive and false negative results.

The aim of this investigation was to find out the prevalence of Francesella tularensis in some provinces of China. Using the general and subspecies PCR detection method for Francesella tularensis established in our laboratory, PCR detection of Francesella tularensis in rabbits and ticks was carried out in provinces with high-density rabbits feeding and some provinces and cities with sheep and cattle stock. The results showed that DNA detection of Francesella tularensis from rabbit tissues and ticks carried by cattle and sheep was positive. 12 out of 218 rabbit samples were positive (5.5% positive rate), and 15 tick samples were positive (3.1%) of 490 tick samples. In terms of geographical distribution, most of the positive rabbit samples were from Shandong, Henan and Sichuan province, however, ticks collected from Yunnan and Shandong province showed a higher positive rate. PCR detection showed that Francesella tularensis subspecies in this investigation was F.h, which was a subspecies with strong toxicity. This investigation revealed that Francesella tularensis presented in rabbits and ticks, public health and safety risks of Francesella tularensis existed in Shandong and Yunnan province, which should be paid more attention by the relevant scientific research institutions, medical institutions and government departments.

In this study,191 strains of avian pathogenic Escherichia coli (APEC) were isolated from duck farms in and around Jiangsu province.The serotype,virulence gene distribution and drug resistance of 21 strains (one from each farm) were detected,and the correlation between serotype,virulence gene distribution and drug resistance was analyzed,in order to provide reference for the prevention and control of APEC.The serotypes of 21 APEC strains showed that there were 12 strains of O65,accounting for 57.14% of all strains.The results of virulence gene detection showed that 5 virulence genes had a high distribution rate,among which the positive rate of fimA gene was 100%,and the positive rates of ECs3737,ECs3703,tsh and irp2 genes were 90.5%,85.7%,57.1% and 42.9%,respectively.There were 6 strains (28.57%) with five virulence genes.The results of drug sensitivity test showed that 21 APEC strains had multiple drug resistance,and 100% strains were resistant to enrofloxacin,doxycycline,vancomycin and erythromycin.Among all the strains,85.71% and 14.29% were resistant to more than 10 and 21 kinds of drugs,respectively.The relationship among serotypes,virulence gene distribution and drug resistance showed that there were 13 strains with more than 4 virulence genes,9 of which were O65 serotypes.Among the 13 strains with more than 4 virulence genes,9 strains (69.23%) were resistant to more than 15 drugs,and 3 strains (23.08%) were resistant to more than 20 drugs.The results showed that the serotypes of Escherichia coli isolated from ducks in Jiangsu province and its surrounding areas were complex,carrying a variety of virulence genes,and the drug resistance was serious.

In order to understand the drug resistance of Riemerella anatipestifer from Muscovy duck,this study carried out bacterial isolation and culture,Gram staining microscopy,pathogen detection,biochemical test,16S rRNA sequence analysis,PCR identification,drug sensitivity test and drug resistance gene detection for Muscovy duck suspected of Riemerella anatipestifer infection.The results of bacterial isolation showed that on the blood agar medium,the isolated bacteria grew creamy needle tip size colonies with smooth surface,neat edge,luster and translucency.The Gram-negative bacillus brevis was detected by Gram-negative staining microscopy,and it was named GZQN201907.In the biochemical test of GZQN201907,urea reaction was positive,but glucose,maltose,lactose and other biochemical reactions were negative.The 16S rRNA phylogenetic tree was in the same branch as Riemerella anatipestifer.And the OmpA gene of PCR identification results of the isolated strain were positive.The drug sensitivity test was sensitive to 18 antibiotics including cefuroxime,erythromycin and ceftazidime,moderately sensitive to carboxypicillin and ciprofloxacin,and sensitive to neomycin and cotrimoxazole.And the resistance genes could detect the β-lactam resistance genes VIM and TEM,tetracycline resistance genes tetB,macrolide resistance genes ermB and ermF.The results of drug sensitivity test and drug resistance gene detection indicated that GZQN201907 showed the same resistance phenotype and gene detection results for β-lactam,tetracycline and macrolide.In the animal regression test,all the ducklings inoculated with GZQN201907 died within 72 h,while the control group showed no symptoms,indicating that GZQN201907 was virulent to the ducklings.One strain of Riemerella anatipestifer from Muscovy duck was successfully isolated,which laid a foundation for the prevention and treatment of Riemerella anatipestifer from muscovich.

The aim of this study was to search for novel non-ribosomal peptide antimicrobial substances based on the screening of bacterial secondary metabolites.The bacteria isolated from soil,sea water and common marine organisms in Yantai coastal area were isolated and purified.E.coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were selected as indicator bacteria,and E.coli B2 (bla_NDM-5+mcr-1) and methicillin-resistant Staphylococcus aureus (MRSA) T144 were used as indicator for secondary screening.The genome was extracted and the PCR products were sequenced to determine the active species.The secondary metabolites of bacteria were extracted by organic extraction,purified by gel chromatography and preparative liquid chromatography,and purity was detected by analytical liquid chromatography.The results of sequencing showed that the active strain belonged to Bacillus amyloliquefaciens sp.,and was named as Bacillus amyloliquefaciens 9-14 (active bacterium 9-14).The results of antibacterial test showed that the metabolites of active bacterium 9-14 had high inhibitory effect on Staphylococcus aureus ATCC 29213,MRSA T144,E.coli ATCC 25922 and E.coli B2.The metabolites of active bacterium 9-14 were cyclic lipopeptides composed of amino acid chains,which belonged to the derivatives of ibuprofen.The biological characteristics and antibacterial spectrum of the metabolites of active bacterium 9-14 were studied.The results showed that the metabolites of active bacterium 9-14 had good thermal stability and acid-base stability.And the antibacterial activity of the antibacterial substance treated with trypsin,pepsin,protease K and papain was not significantly weakened and had good stability.The antibacterial substance also had inhibitory effect on Staphylococcus aureus and E.coli,but had no activity against Pseudomonas aeruginosa,Klebsiella pneumoniae,Enterococcus faecalis and Bacillus cereus.In this study,a new antibacterial substance was obtained,which could be used as the precursor of antibacterial drugs,and could provide certain reference for food safety and disease control.

Avian pathogenic Escherichia coli(APEC) is an important pathogen that causes localized and systemic infection in avian species of all ages.It is also an important reservoir or source of virulence genes of human extraintestinal pathogenic Escherichia coli (ExPEC).In order to understand deeply the infection progresses,pathogenesis,host immune responses and genetic resistance mechanism of APEC,and evaluate the efficacy of drugs and vaccines,several experimental infection models were established to evaluate the virulence of APEC through different approaches.According to the different systems involved,it can be divided into respiratory system,vascular system,musculoskeletal system,dermatological system,reproductive system,gastrointestinal system and chicken embryo system.In addition,there are infection experiments in mice and rats,tissue culture cells and explants infection experiments in vitro.The author highlights the establishment,pathogenesis,host responses and application of the different APEC experimental infection models.

In order to study the tolerance of Enterococcus spp.from companion animals to common antibiotics in Beijing,320 samples were collected from dogs and cats in a pet hospital in Beijing from May 2015 to January 2016.The strains were isolated by selective culture medium and identified by VITEK-2 Compact 60.The sensitivity test of 11 kinds of common antibiotics in 8 categories was carried out for the isolated and identified strains.At the same time,the high-level gentamicin and high-level streptomycin resistance test were carried out to analyze the drug resistance and multiple drug resistance of Enterococcus.A total of 318 strains of non repetitive enterococci with different colony morphology and biochemical characteristics were obtained.The isolation rates of Enterococcus faecalis (49.06%) and Enterococcus faecium (29.87%) were higher.The resistance rates of Enterococcus to high-level gentamicin and high-level streptomycin were 39.94% and 43.40%,respectively.The resistance rates to tetracycline (78.62%),erythromycin (67.30%) and quinoproptin dapfoptin (43.71%) were very high.All of them were sensitive to tigecycline,and the non-resistant rate to vancomycin and nitrofurantoin was also high (98.74%).Enterococcus tested showed different degrees of resistance to class 1 to class 7 antibiotics,and the multidrug resistance was common,and the multidrug resistance rate was as high as 57.23%.The number of resistant antibiotics was the highest (n=65).A total of 44 different drug resistance profiles were obtained.Among them,erythromycin-quinoptin-daptoptin-tetracycline-high level aminoglycoside resistant spectrum accounted for the highest proportion (45/318,14.15%).This study confirmed that Enterococcus spp.isolated from companion animals in Beijing was highly resistant to common antibiotics,and multidrug resistance was prevalent.Therefore,it was necessary to strengthen the monitoring of drug resistance.

The purpose of this study was to understand the prevalence of arboviruses in Jinghong city,Yunnan province.Three sentinel cattle were set up in Menghan township of Jinghong city in 2019,blood samples were collected regularly for isolation and identification of arboviruses,a total of 7 virus isolates were obtained.Viral nucleic acid were identification by RT-PCR,and the results showed that two strains of epidemic hemorrhagic fever virus (EHDV) with serotypes of 6 and 7,two strains of bluetongue virus (BTV) with serotypes 4 and 5,D’Aguilar virus (DAV) serotype in one strain of PALV and two unidentified circoviruses were isolated,respectively.The ORF region of virus Seg-2 and Seg-3 sequences was compared and analyzed,all the 7 regional strains of the virus were Eastern,which was most closely related to strains in Japan,Australia and India.The blood and serum of three sentinel animals were tested by viral nucleic acid and serum neutralization test,which proved that all three animals were infected with the corresponding virus.When animals were infected with the virus,specific antibodies in the serum rise rapidly,it reached peaks 3 to 4 weeks later and could remain at this level for a long time.However,the content of viral nucleic acid in the blood decreased rapidly after reaching the peak from 2 to 4 weeks.The isolation,sequence characteristics and infection characteristics of Jinghong arboviruses in animals were reported in this study,the results provided data support for further understanding of local bovine arboviruses.At the same time,7 strains of virus were isolated from 3 cattle,suggesting that there might be more arboviruses in the area.

Liquid chip technology is a novel biomolecular detection technology which integrates laser technology,flow cytometry,digital signal processing and traditional chemical technology.It is widely used in various immunological analysis and nucleic acid detection.Single and mutiplex analysis are supported,with protein and nucleic acid targets detected in a variety of detection methods in high throughput manner.It has advantages of high throughput,easy operation,wide range of application,good repeatability,high specificity,less sample needed,high sensitivity and stablility,and low cost.Therefore,they are gradually replacing the traditional detection and quantitative pathogen methods,such as Real-time fluorescent quantitative nucleic acid amplification detection system (qPCR),enzyme-linked immunosorbent assay (ELISA) and other detection methods.Animal infectious diseases seriously endanger the development of the breeding industry,futhermore,some zoonoses such as highly pathogenic avian influenza also pose a serious threat to human health.An efficient and sensitive diagnostic system will help to screen a large number of samples during the outbreak of infectious diseases and prevent the spread of infection.The development of liquid chip technology provides a new platform for high-throughput detection and disease prevention.In this review,the principle and advantages of liquid chip technology are briefly described.The research progress of liquid chip technology in the detection of animal infectious diseases,including pigs diseases,poultry diseases,rabbit diseases,dog diseases,rodent diseases and other animal infectious diseases are summarized.We believe that in the future,this technology will become an important analytical and testing tool in clinical diagnosis,basic research,new drug development,judicial identification,food health supervision,biological weapons prevention and other fields.The development of this technology will greatly promote the research and development of life science.

The purpose of the experiment was to study the effect of Momordica grosvenori saponins on improving the effect of high-sucrose/high-fat diet combined with streptozotocin induced type 2 diabetes in rats.The type 2 diabetes rat model was established with a high-sucrose and high-fat diet combined with streptozotocin.Successfully modeled rats were given the total saponins of Momordica grosvenori (100,200,400 mg/kg) or metformin (268 mg/kg) by gavage,once a day for 4 weeks.Recording the "three more and one less"index (24 h internal urine output,water intake and feed intake,body weight);Detecting of biochemical indicators,analysis of fasting blood glucose (FBG),serum total cholesterol (TC),triglycerides (TG),low-density lipoprotein cholesterol (LDL-C),high-density lipoprotein cholesterol (HDL-C),insulin (INS),catalase (CAT),superoxide dismutase (SOD),malondialdehyde (MDA),alanine aminotransferase (AST),aspartate aminotransferase (ALT),blood urea nitrogen (BUN) and creatinine (Cr) levels.Hematoxylin-eosin staining (HE) was used to observe the pathological changes of the pancreas.Compared with the model group,the feed intake,water intake,urine output,blood indicators FBG,INS,MDA,TC,TG,BUN and Cr levels,AST and ALT activities were significantly or extremely significantly decreased in the total saponins of Momordica grosvenori each dose group and metformin group (P<0.05;P<0.01),while CAT,SOD activities and HDL-C level were significantly or extremely significantly increased (P<0.05;P<0.01).In pancreatic tissue,the number of pancreatic cells was increased significantly,and the pathological damage of the pancreas was significantly reduced. Momordica grosvenori saponins and metformin had obvious effects on improving glucose metabolism disorders,and at the same time could significantly reduce insulin resistance,enhance the body’s ability to resist oxidative stress and improve liver and kidney functions.

In order to explore the mechanism of chlorogenic acid on the inhibition and elimination of drug resistance of antibiotic resistant Escherichia coli from chicken,the third generation of avian Escherichia coli drug-resistant reverse strains,cultivated in the LB broth whose chlorogenic acid concentration was 1.25 mg/mL (1/2 MIC),was separated through photolithography,the minimal inhibitory concentration (MIC) of levofloxacin was determined through microplate method,and the molecular mechanism of chlorogenic acid eliminating drug resistance of Escherichia coli was further analyzed through transcriptome sequencing method.The results showed that the MIC of drug-resistant reverse strains to levofloxacin decreased from 16 μg/mL to 4-8 μg/mL,indicating that to a certain extent,chlorogenic acid of 1.25 mg/mL could eliminate the resistance of Escherichia coli to levofloxacin.To further analyze the molecular mechanism of chlorogenic acid on the elimination of drug resistance of Escherichia coli from chicken,transcriptome sequencing was used to compare the gene expression of this kind of Escherichia coli before and after the elimination of drug resistance.And it was found that the expression levels of 12 genes were significantly down-regulated after chlorogenic acid treatment,which the results of fluorescence quantitative PCR were basically consistent with.After GO functional enrichment analysis and KEGG metabolic pathway enrichment analysis,it was found that the differentially expressed genes were functionally concentrated in membrane composition and DNA recombination,and in metabolic pathway,the differentially expressed genes were related to metabolism.bhsA and cysP in the differentially expressed genes were Coli-HB chromatin transcribed RNA (GenBank No.:CP020933).oqxA,oqxR,emaA,pinR,xerD,folA,repA,repC,adhP,IS26 were Coli-HB plasmid 1 transcribed RNA (GenBank No.:CP020934).It could be seen from the distribution of differentially expressed genes that chlorogenic acid could weaken the resistance of bacteria,inhibit the activity of drug-resistant genes,and inhibit the recombination of bacterial DNA,etc.To some extent,it could eliminate the drug-resistance of Escherichia coli to levofloxacin and play a role in inhibiting the bacteria and eliminating the drug-resistance.

This experiment was aimed to study the nutritional content and value of the muscles of two mallards (White feather mallard and mallard),and provide basic data and theoretical basis for the development and utilization of mallard meat products.60 White feathered mallards and mallards (30 for each breed,half of male and female) were chosen,and the composition and content of pectoral muscle crude protein,crude fat,cholesterol,amino acids,fatty acids,trace elements and vitamins were determined according to national standards and the nutritional values of muscle were evaluated.The results showed that the content of crude protein in muscle of White feather mallard was significantly lower than that of mallard (P<0.05).17 kinds of amino acids with contents higher than 0.01% were detected in the muscles of the two kinds of mallards,among which the contents of threonine,histidine,serine and proline in the muscles of the White feather mallard were significantly higher than those of mallard (P<0.05).The contents of lysine,glutamate and arginine were significantly lower than those of mallard (P<0.05).13 kinds of fatty acids with contents higher than 0.01% were detected,and the contents of stearic acid and oleic acid were significantly lower than those of the mallard.9 mineral elements (sodium,magnesium,potassium,calcium,manganese,iron,copper,zinc and selenium) in two kinds of mallards were detected,and there was no significant difference between the two kinds of mallards (P>0.05).8 kinds of vitamins were detected,and the content of vitamin B₁ in muscle of White feather mallard was significantly higher than that of mallard (P<0.05),but the contents of vitamin D and vitamin E were significantly lower than that of mallard (P<0.05).The ratio of amino acids in muscle of two kinds of mallards was close to the ideal model recommended by WHO,which was rich in mineral elements and vitamins for human body,and had a broad prospect of development and utilization.

In order to investigate the pollution status of DOX in soil and reduce the detection cost,a detection method of DOX in three soils was established by solid phase extraction and high performance liquid chromatography (HPLC-UV) in this study.The mixture of acetonitrile and Na₂EDTA-Mcllvaine (1:1,V/V) was used as extraction reagent for soil samples.HLB solid phase extraction column was used for enrichment and purification.Agilent ZORBAX SB-C18 (4.6 mm×250 mm×5 μm) column was used for separation.0.01 mol/L oxalic acid (A),acetonitrile (B) and methanol (C) were used as mobile phase system for HPLC detection at 355 nm.The limit of detection (LOD) was determined by 3 times S/N and the limit of quantitation (LOQ) was determined by 10 times S/N.The results showed that DOX peaked in 10 min.In the range of 0.1-10 μg/mL,the linear relationship between the peak area of DOX and the concentration was good.The regression equation was y=22 747x+3.3256,and the correlation coefficient was 0.9999.The LOD and LOQ of DOX in this method were 0.05 and 0.1 mg/kg,respectively.When the added concentration of DOX was 0.1 μg/g in soil,the recoveries of DOX in loam,sandy loam and sandy were 58.96%,75.84% and 83.06%,respectively,and the coefficient of variation(CV) was 1.00%-9.54%.The recoveries of DOX in loam,sandy loam and sandy were 63.89%,70.48% and 81.07%,respectively,and the CV 2.40%-8.83% when added 0.2 μg/g.The recoveries of DOX in loam,sandy loam and sandy soil was 59.02%,76.16% and 81.29% respectively,and the CV was 1.52%-5.89% when added 0.4 μg/g.This method was efficient,stable and specific.It could be used for the detection and quantification of DOX in different types of soil,which was helpful for the establishment of the detection method of DOX in the environment.