【Objective】 This study was aimed to identify the genetic differentiation between IAS Grassland duck and Cherry Valley duck by genome resequencing technology, and investigate the genomic variation between the two breeds under different artificial selection to clarify the genetic basis for the formation of excellent traits.【Method】 In this study, 16 Cherry Valley ducks and 16 IAS Grassland ducks were adopted for genome resequencing.After filtering out SNP markers with high missing rates and low allele frequencies, high-quality SNPs were obtained for subsequent analysis.Then the genotypic data of the two breeds were analyzed by principal component analysis (PCA) to identify their genetic differentiation.The selected signals of IAS Grassland duck and Cherry Valley duck were comprehensively screened by population genetic differentiation index (Fst) and nucleic acid diversity ratio (Pi).【Result】 PCA analysis illustrated that IAS Grassland duck and Cherry Valley duck were significantly differentiated.The analysis values of Fst and Pi were calculated in 10 kb window and 5 kb step respectively, and the Top 1% of Fst and Pi values was taken as the threshold (Fst>0.177, Pi>0.885).A total of 410 kb candidate regions were screened in the signal overlap region of the two analysis methods, and a total of 21 candidate genes were annotated.GO and KEGG enrichment analysis showed that 12 candidate genes were enriched in two categories, including cellular component and biological process (P<0.05), and 2 genes were significantly enriched in the metabolic pathway, signal transduction, reproduction, immune response pathway, etc.(P<0.05).Among the significantly enriched genes, those related to lipid metabolism, amino acid metabolism, and immune modulation, including PDE3A, PRKAR2B, SEMA5A, SHANK2, STXBP6, and LOC101803508 (GOLGB1), were strongly selected.【Conclusion】 The results indicated that although both Cherry Valley ducks and IAS Grassland ducks were derived from Pekin ducks, they differentiated obviously after continuous artificial selection with different selection pressure and directions, IAS Grassland ducks had higher genetic diversity than Cherry Valley ducks.A series of candidate genes for genetic differentiation between the two duck breeds were predicted, and the relevant genes involved in the regulation of flavor and meat quality of White-feather meat ducks were mainly excavated, which provided a reference for the follow-up study of the characteristics of different White-feather meat ducks and the screening of variety specific molecular markers.

【Objective】 The purpose of this study was to reveal the role and regulatory mechanism of miR-145-4 in follicular development for egg duck.【Method】 The egg duck follicular granulosa cells were isolated and cultured.After miR-145-4 mimic and inhibitor were transfected, the expression of cell proliferation and apoptosis related genes were detected by Real-time quantitative PCR.RNAhybrid and Targetscan programs were used to predict the target gene and predict the potential binding sites of miR-145-4 to the 3'-UTR of phosphatidylinositol-3-kinase catalytic subunit α (PIK3CA) gene.The wild type and mutant type dual-luciferase reporter vector of PIK3CA gene 3'-UTR were constructed and co-transfeacted with miR-145-4 mimic and mimic-NC into duck granulose cells.The binding relationship between miR-145-4 and target gene PIK3CA was verified by double luciferase reporting system.And the effect of miR-145-4 on the expression of PIK3CA in granulosa cells of egg ducks was detected by Real-time quantitative PCR.【Result】 The results of Real-time quantitative PCR showed that after over expression of miR-145-4, compared with control group, the expression of CyclinB2 gene was extremely significantly decreased (P<0.01), and the expression of BCL2 gene was extremely significantly increased (P<0.01).After inhibiting miR-145-4, compared with control group, the expression of CyclinB2 gene was extremely significantly increased (P<0.01), and the expression of BCL2 was extremely significantly decreased (P<0.01).The predict results showed that miR-145-4 could be binding to PIK3CA gene 3'-UTR.PCR amplification and sequencing results indicated that the wild type and mutant type vectors of PIK3CA gene 3'-UTR were successfully constructed.The results of double luciferase assay showed that the double luciferase activity of wild-type group was significantly lower than that of mutant and empty vector co-transfection groups (P<0.05), indicating that miR-145-4 could bind to PIK3CA gene 3'-UTR.The results of Real-time quantitative PCR showed that the expression of PIK3CA gene decreased significantly after overexpression of miR-145-4 in egg duck follicular granulosa cells (P<0.05), while the expression of PIK3CA gene increased extremely significantly after inhibition of miR-145-4 (P<0.01).【Conclusion】 miR-145-4 promoted the apoptosis of follicle granulosa cells by positively regulating the target gene PIK3CA, and then regulated the follicular development of egg ducks.

【Objective】 The aim of this study was to clone and prediect its structure and function of growth differentiation factor 9 (GDF9) gene of Muscovy duck (Cairina moschata), and to detect its expression differences in Muscovy duck tissues at the nest stage and laying stage.【Method】 The ovary tissue cDNA in Muscovy duck was used as a template, the GDF9 gene was amplified and cloned by rapid-amplification of cDNA ends (RACE).Using biological information software, the similarity of the coding sequence of GDF9 gene was analyzed and evolutionary tree was constructed, and its basic physical and chemical properties and the structural characteristics of the coding amino acid sequence were analyzed.Real-time quantitative PCR was used to detect the expression of GDF9 gene in Muscovy duck of broody period and laying period.【Result】 The total CDS region sequence of GDF9 gene was 1 380 bp, which edcoded 459 amino acids.The similarity of GDF9 gene sequence of Muscovy duck with that of Aythya fuligula, Anas platyrhynchos, Cygnus atratus, Oxyura jamaicensis, Anser cygnodies domesticus, Aptenodytes forsteri, Gallus gallus, Bos taurus, Ovis aries, Sus scrofa and Mus musculus published in GenBank was 98.8%, 97.5%, 96.4%, 95.8%, 94.4%, 90.2%, 87.5%, 64.5%, 64.2%, 63.9% and 60.6%, respectively.The phylogenetic tree showed that Muscovy duck was closely related to Aythya fuligula and Anas platyrhynchos, but far from Mus musculus.The molecular weight of GDF9 protein was 52.81 ku.It was an unstable alkaline hydrophilic membrane protein.It had one signal peptide, 45 phosphorylation sites, 8 O-glycosylation sites, 4 N-glycosylation sites and a transforming growth factor-β (TGF-β) domain.The secondary structure of protein consists of random coil, alpha helix, T-turn and beta sheet composition, accounting for 58.61%, 22.22%, 16.56% and 2.61% respectively.The predicted results of the tertiary structure of GDF9 protein were consistent with the secondary structure.GDF9 protein might interact with BMP15, BMPR1B, BMPR2, TGFBR1, ACVR1B, POF1B, ZP2, ZAR1 and MOS proteins.The results of Real-time quantitative PCR showed that GDF9 gene was expressed in all tissues of Muscovy ducks at nesting and laying stages, the expression of genes in ovary were the highest in two stages, and they were extremely significantly higher than that in other tissues at the same time (P<0.01).Compared with different periods of the same tissue, the expression of GDF9 gene in spleen, heart and kidney in nesting Muscovy duck was significantly or extremely significantly lower than that in laying period (P<0.05 or P<0.01), and the expression in ovary was extremely significantly higher than that in laying period (P<0.01).There was no significant difference in the expression of GDF9 gene in other tissues between the two periods (P>0.05).【Conclusion】 In this study, the GDF9 gene of Muscovy duck was successfully cloned.GDF9 gene was expressed in all tissues of Muscovy duck.GDF9 gene was due to the differentially expressed genes in ovary, heart, kidney and spleen of Muscovy duck during egg laying and nesting.This study provided a theoretical basis for further exploring the function of GDF9 gene in Muscovy duck, and also laid a foundation for studying the biological function and molecular mechanism of GDF9 gene in ovarian development.

【Objective】 The purpose of the experiment was to study the heritability of the degree of striped eggs produced by ducks and the genetic correlation with other traits related to egg quality.【Method】 600 female ducks of the A line were selected and the eggs produced were collected.The degree of striped eggs was divided into 0, 1, 2, 3, and 4 grades according to the coverage area of the striped on the surface of the duck egg.The grade of striped eggs and other egg quality-related traits were observed and determined, the restricted maximum likelihood estimation method and linear mixed model were used to estimate heritability and genetic parameters of related traits.【Result】 During the experiment, 418 female ducks lay eggs among 600 female ducks and 1 171 breeding eggs were collected.Among them, there were 742, 135, 161, 93, and 40 striped eggs at grades 0, 1, 2, 3 and 4, which account for 63%, 12%, 14%, 8% and 3% of the total.The number of laying ducks which produced grades 0, 1, 2, 3 and 4 were 228, 55, 69, 46 and 20, respectively.The heritability of striped egg grade, average egg weight, average egg type index, and birth weight were 0.30, 0.29, 0.52, and 0.72, respectively, which belonged to high heritability.The heritability of the total number of eggs was 0.15, which was medium heritability.The striped egg grade had a higher genetic positive correlation (0.34) and a lower phenotypic positive correlation (0.12) with the total number of eggs.【Conclusion】 The striped egg grade, average egg weight, average egg type index and birth weight all had high heritability.The selection and elimination of some female ducks that produced higher grades of striped eggs could reduce the proportion of striped eggs produced by ducks.In this study, the grades of striped duck eggs were divided for the first time, which could accurately distinguish the differences between striped eggs, provided classification standards for subsequent related studies, and provided a basis and reference for genetic improvement of egg production performance.

【Objective】 The study was aimed to clarify the basic characteristics of circ-ZNF326 and its effect on the proliferation of small intestinal epithelial cells in ducks.【Method】 Taking the Mallard duck as the research object, the duck embryo intestine was collected and cultured for small intestinal epithelial cells, RNA was extracted and reverse transcribed into cDNA, primers were designed with reference to the full-length sequence of circ-ZNF326, partial sequences of circ-ZNF326 were obtained by PCR reaction amplification, sequencing was performed to verify its loop formation mode using reverse splice sites.The nuclear and cytoplasmic RNAs were extracted using the PARISTM Kit to detect their nucleoplasmic distribution in the cells.The full-length sequence of circ-ZNF326 was synthesised and ligated onto the circ-ZNF326 eukaryotic expression vector using double digestion and T₄ ligase, and then transformed into E.coli DH5α competent cells for expansion.The recombinant plasmid was detected by double enzyme digestion and agarose gel electrophoresis.The circ-ZNF326 overexpression vector was transfected into the small intestinal epithelial cells.The expression efficiency of circ-ZNF326 over expression was detected by Real-time quantitative PCR.The effect of over expression of circ-ZNF326 on the proliferation of small intestinal epithelial cells was detected by CCK-8.【Result】 circ-ZNF326 was formed by reverse splicing of exons 10, 11 and 12 of ZNF326 gene, and mainly distributed in the cytoplasm.circ-ZNF326 overexpression vector was successfully constructed and its expression efficiency was extremely significantly higher than that of no-load group (P<0.01).After transfection of circ-ZNF326 overexpression vector into small intestinal epithelial cells, the cell viability in circ-ZNF326 overload group was extremely significantly or significantly higher than that in no-load group within 24-72 h (P<0.01 or P<0.05).【Conclusion】 This experiment successfully verified the reverse splicing and annular structure of circ-ZNF326, proved that circ-ZNF326 was mainly enriched in the cytoplasm, constructed circ-ZNF326 overexpression vector, and confirmed that overexpression of circ-ZNF326 could improve the activity of small intestinal epithelial cells of laying ducks.The results provided a theoretical basis for further study on the biological functions of circ-ZNF326 and its regulation mechanism on the proliferation of small intestinal epithelial cells in laying ducks.

Animal intestinal tract has a complex and dynamic microbial ecosystem, which plays a vital role in maintaining body health by promoting nutrient intake, host defense, immune regulation and so on.The structure and composition of colonies are different due to the changes of maternal body, diet, environment, physiological state and the interaction between different species.Waterfowls (including ducks and geese) are oviposition animals.Compared with mammals, its intestinal microbial system is special.In this paper, the establishment of intestinal microorganisms, the characteristics of microbial community structure and developmental changes in different parts of the intestine in waterfowl were introduced.The main functions of intestinal microorganisms in waterfowl were expounded from three aspects:The influence of intestinal microorganisms on the growth performance of waterfowl, the influence on nutrient digestion and absorption, and the relationship with the immune system.At the same time, the multiple factors affecting the intestinal microorganisms of waterfowl were analyzed from the four aspects of diet composition, physiological state of animals, external environmental factors, microbial factors and interactions, along with the prospect of future research ideas and development direction of gut microbes in waterfowl.In order to provide a theoretical basis for the design of feed formula, improving intestinal health and production efficiency in aquaculture, so as to accurately regulate the nutrition, immunity and growth process of waterfowl from the new target, intestinal microorganism, and support further in-depth study of intestinal microorganism of waterfowl.

【Objective】 By constructing the tissue expression profile and bioinformatics function of alpha-s1 casein(CSN1S1)gene in Guanzhong dairy goats, the function of CSN1S1 gene in milk composition synthesis of Guanzhong dairy goats was preliminarily investigated.【Method】 Guanzhong dairy goats were taken as the research object, two mutant forms of CSN1S1 were cloned which were named CSNIS1-1 and CSN1S1-2. The protein structure, physicochemical property and phosphorylation sites of CSN1S1 gene and its mutant form were analyzed using a variety of biological information softwares and online tools such as ProtParam, NetPhos, SingalP 4.1 Server, NPS and Phyre2. The relative expression of CSNIS1-1 and CSN1S1-2 in liver, spleen, mammary gland, kidney, uterus and fallopian tube of Guanzhong dairy goats were detected by Real-time quantitative PCR.【Result】 The results showed that two mutant forms CSN1S1-1 and CSN1S1-2 were found in mammary epithelial cells of Guanzhong dairy goats. Sequencing results showed that compared with CSN1S1 sequence, 3 bases mutations were found in CSN1S1-1 and CSN1S1-2, in addition, there were total 6 bases deletions in two positions of CSN1S1-2. The similarity was 99.07% between CSN1S1-1 and CSN1S1, and 97.20% between CSN1S1-2 and CSN1S1.The analysis of protein physicochemical properties showed that the mutation of bases in CSN1S1-1 resulted in the change of leucine at position 31 to proline and glutamine at position 92 to glutamic acid. In addition to the above changes, CSN1S1-2 changed from serine to cysteine at position 83, and changed from glutamic acid to glutamine at position 84. In addition, there were deletions of two serines at positions 81 and 82. Real-time quantitative PCR results showed that the relative expression trends of CSN1S1-1 and CSN1S1-2 in Guanzhong dairy goat tissues were basically the same, and the relative expression levels were higher in mammary gland, uterus and fallopian tube, and basically not expressed in liver, spleen and kidney. The expression of CSN1S1-2 in uterus was significantly higher than that of CSN1S1-1 (P<0.05).【Conclusion】 Two mutant forms CSN1S1-1 and CSN1S1-2 were found in the mammary epithelial cells of Guanzhong dairy goats, and the protein similarity between the two mutant forms and CSN1S1 reached more than 97%. CSN1S1-1 was obviously different from CSN1S1 in protein structure, and the deletion of 6 bases in CSN1S1-2 might be the direct cause of amino acid change, deletion and displacement of phosphorylation site. The expression of CSN1S1-2 in uterus was significantly higher than that of CSN1S1-1, and its potential function in uterus remains to be further explored.

【Objective】 This study was aimed to clone insulin-like growth factor 2 (IGF2) gene of Kunming dogs and obtain its sequence characteristics and spatio-temporal expression, to provide basic data for the study of body size and growth characteristics of working dogs.【Method】 The IGF2 gene CDS region of Kunming dogs was amplified by PCR technique, the structure and function of IGF2 protein in Kunming dogs were analyzed and predicted by bioinformatics method.The expression levels of IGF2 gene in heart, liver, spleen, lung, kindey and medial thigh muscle and liver at different growth stage in Kunming dogs were detected by Real-time quantitative PCR.【Result】 The CDS region of IGF2 gene in Kunming dogs was 717 bp which encoded 238 amino acids.The genetic tree analysis showed that the genetic distance of IGF2 gene from Kunming was closest to that of Red fox, and the genetic distance was farthest from that of chicken.The theoretical molecular weight and isoelectric point of IGF2 protein was 26.46 ku and 9.61, respectively.It had no signal peptide and transmembrane structure and belonged to hydrophilic non-secretory protein.The results of subcellular localization showed that IGF2 protein was mainly located in the nucleus (47.8%) and mitochondria (17.4%), and had 23 phosphorylation sites.The secondary structure of the protein was mainly composed of alpha helix and random coil.The prediction of tertiary structure was consistent with the results of secondary structure.The mRNA expression level of IGF2 gene in liver of 2.5 months old Kunming dogs was the highest, and was extremely significantly higher than that in kidney, lung and muscle (P<0.01).The expression of IGF2 gene in the liver of puppies (2.5-month-old) was extremely significantly higher than that of puppies (6-month-old), youth (1-year-old) and adults (1.7 and 2.5-year-old) (P<0.01).【Conclusion】 Kunming dog IGF2 gene was successfully cloned, and it was revealed that IGF2 was widely expressed in many tissues, which was highest in liver.The results could provide a basis for further study on the regulation mechanism of IGF2 gene in the growth and development of Kunming dogs.

【Objective】 The aim of this study was to clone and prokaryotic express the keratin associated protein 11.1(KAP11.1) gene of hair follicles, compare the expression of KAP11.1 gene in skin follicles of different sheep breeds, and explore the expression difference of KAP11.1 gene in local sheep breeds in Southern Xinjiang and its effect on wool quality.【Method】 The skin follicles on the body side of plain-type Hetian sheep, mountain-type Hetian sheep and Karakul sheep were used as the research materials, and the sequence of KAP11.1 gene in sheep (accession No.:HQ595347.1) in GenBank was used as the reference to design primers.PCR amplification of KAP11.1 gene was carried out to construct pMD19-T-KAP11.1 clone plasmid, and pET-28a(+)-KAP11.1 prokaryotic recombinant expression plasmid was constructed after double-enzyme digestion identification.Sequence analysis was carried out after PCR and double-enzyme digestion identification.The recombinant plasmid was transformed into E.coli BL21(DE3).SDS-PAGE and Western blotting were used for detection.The expression of KAP11.1 gene in skin follicles of different sheep was detected by Real-time quantitative PCR.【Result】 The CDS sequence of KAP11.1 gene in 3 sheep breeds was 480 bp, encoding 159 amino acids, which was an unstable hydrophobic protein.The similarity alignment results showed that compared with the reference gene, the two types of Hetian sheep was 99.79%, and mutations occurred at 423 bp, from C to T.The sequence homology of Karakul sheep was 99.38%, from G to T at 69 bp, from C to T at 93 bp, and from C to T at 423 bp.Phylogenetic tree analysis showed that 3 sheep breeds had the closest genetic relationship with Capra hircus, and the farthest genetic relationship with Bos indicus.The secondary structure of KAP11.1 was mainly composed of random coil.The prokaryotic recombinant expression plasmid pET-28a(+)-KAP11.1 was successfully constructed, and KAP11.1 protein (19 ku) was purified.The expression of KAP11.1 gene in skin follicles of mountain-type Hetian sheep and Karakul sheep were significantly higher than that of plain-type Hetian sheep (P<0.05), and there was no significant difference between mountain-type Hetian sheep and Karakul sheep (P>0.05).【Conclusion】 The 480 bp CDS sequence of KAP11.1 gene in sheep was cloned, the prokaryotic recombinant expression plasmid pET-28a(+)-KAP11.1 was successfully constructed, 19 ku KAP11.1 protein was obtained, and KAP11.1 gene was expressed in skin follicles of 3 sheep breeds.

【Objective】 The aim of this study was to clone Jiaxian Red cattle DNA polymerase beta (POLB) gene and analyze its bioinformatics.【Method】 The complete CDS region of cattle POLB gene was cloned by PCR with the cDNA of muscle tissue in Jiaxian Red cattle as the template.Genetic distance analysis and phylogenetic tree were constructed by online softwares to analyze the physical and chemical properties, hydrophobicity, phosphorylation sites, transmembrane structure, signal peptide, secondary structure, tertiary structure and interaction proteins of POLB protein.【Result】 The sequence of CDS region of Jiaxian Red cattle POLB gene was 1 008 bp, encoding 335 amino acids.Phylogenetic tree analysis showed that the amino acid sequence of Jiaxian Red cattle POLB gene was closest related to sheep and other ruminants, followed by other mammals and poultry, and was farthest related to zebrafish (fish).The molecular weight of Jiaxian Red cattle POLB protein was 38.26 ku, the theoretical isoelectric point (pI) was 9.10, the half-life was 30 h, the instability coefficient was 31.06, and the total average hydrophilic coefficient was -0.659, indicating that it was a stable hydrophilic protein.Predictive analysis of phosphorylation sites showed that Jiaxian Red cattle POLB protein contained 17 serine phosphorylation, 12 threonine phosphorylation and 4 tyrosine phosphorylation sites.The prediction results of transmembrane region and signal peptide showed that Jiaxian Red cattle POLB protein did not belong to transmembrane protein and secretory protein.The prediction of secondary structure and tertiary structure showed that the structure mainly consisted of alpha helix, beta turn, extended strand and random coil.POLB protein was predicted to interact with XRCC1, FEN1, PARP1 and LIG3 by online softwares.【Conclusion】 The CDS sequence of Jiaxian Red cattle POLB gene was successfully cloned in this study, and it was closest to sheep.POLB protein was a hydrophilic protein without transmembrane structure and signal peptide, and had the highest degree of interaction with XRCC1 protein.The results could provide references for further exploring the biological function of POLB gene.

【Objective】 The purpose of this study was to construct the expression system of feline serum albumin (FSA) in Pichia pastoris, and to explore its optimal basic expression conditions, so as to provide a new method for the production of FSA.【Method】 The FSA base sequence was obtained from GenBank, and the codon was optimized and synthesized.The optimized codon was constructed into vector pPIC9K.After PCR and double enzyme digestion verification, the recombinant plasmid FSA-pPIC9K was transformed into Pichia pastoris GS115 by electric shock transformation.The positive strains were obtained by colony PCR after screening by MD plate and G418.The positive strains were randomly selected and induced by methanol for 96 h, the expression supernatant was taken for Western blotting verification.The strains with high expression were selected to induce expression for 96 h at different temperatures (24, 26, 28 and 30 ℃), pH (4.0, 5.0, 6.0, 7.0 and 8.0) and methanol induction (0.5% was added every 24 h in one group and 0.5%, 1.0%, 1.5% and 2.0% were added every 12 h in the other four groups).The expression supernatant was taken for Western blotting verification.【Results】 The colony PCR results showed that two bands with the size of about 2.3 and 2.2 kb of target gene and Pichia pastoris AOX1 gene were obtained.After 96 h of induced expression, the expression supernatant was taken for Western blotting verification, and the specific bands appeared at about 70 ku.Through the optimization of basic conditions, it was found that the protein was highly expressed at 28 ℃, pH 6.0 and 0.5% methanol was added every 12 h.【Conclusion】 Pichia pastoris GS115 could stably express FSA.

【Objective】 This experiment was conducted to study the effects of early weaning transfer on piglet behavior, growth performance and blood indexes, and to explore the appropriate weaning transfer mode, so as to provide reference for improving the welfare level of piglets.【Method】 216 (half of castrated male and female) Duroc×Large White×Yorkshire piglets with good health and similar weight (6.98 kg±0.63 kg) were randomly divided into three groups (there were 6 litters in each group and 12 heads in each litter).Group Ⅰ was control group, piglets did not wean or transfer into herds.Group Ⅱ was weaned transfer group, piglets did wean and transfer into herds.Group Ⅲ was weaned non-transfer group, piglets did wean but not transfer into herds.Early weaning was carried out at 21 days of age, and the experimental period was 14 days.During the experiment, the behavior performance, skin injury, mortality and diarrhea rate of piglets were observed and recorded, the blood of piglets was collected, and the serum cortisol and immune indexes were measured.【Result】 Compared with the control group, the feeding, resting and playing behaviors of piglets in groups Ⅱ and Ⅲ decreased significantly (P<0.01), and inquiry, solitude, wailing, huddling, aggression and phobia behaviors were extremely significantly increased (P<0.01).3-5 days after treatment, the degree of skin damage of piglets was extremely significantly higher than that of control group (P<0.01).The piglets in group Ⅱ had significantly lower body weight and average daily gain at 35 days of age than control group (P<0.05), and extremely significantly higher diarrhea rate than control group and group Ⅲ (P<0.01).The order of piglet mortality among groups was group Ⅰ(0)P<0.01), but the cortisol concentration in group Ⅲ was extremely higher than that in control group and extremely lower than that in group Ⅱ at 3 and 5 days after weaning and transfer (P<0.01).The concentration of immunoglobulin A (IgA) and immunoglobulin G (IgG) in groups Ⅱ and Ⅲ were extremely significantly lower than that in control group on the 7th day (P<0.01).The concentrations of interleukin-12 (IL-12) and interferon-γ (IFN-γ) were not significantly different among three groups (P>0.05).【Conclusion】 Early weaning not only affected the behavior, the concentration of cortisol and immunity of piglets, but also leaded to the decline of growth performance, the increase of skin damage, mortality and diarrhea rate.However, the stress of early weaning without transfer on piglets was significantly lower, which could reduce the adverse effects on piglets.

【Objective】 The purpose of this experiment was to explore the effects of dietary methionine on performance, antioxidant and stress responses of broilers under high-stocking density.【Method】 396 1-day-old AA broilers were selected and fed uniformly at the age of 1-21 days.At the end of 21 days, broilers with similar body weights were selected and divided into 5 groups, with 6 replicates in each group.The feeding density of broilers in control group was 14 birds/m², and the methionine level was 0.40%;the test groups were set at 20 birds/m² stocking density, with methionine level at 0.35%, 0.40%, 0.45% and 0.50% in the diet, respectively.At the end of the experiment (42 d), blood and liver samples were collected to measure the stress level and antioxidant capacity.【Result】 ① Compared with normal-density feeding group, high-density rearing significantly reduced the final body weight, average daily feed intake and the average daily body weight gain of broilers during 22-42 days (P<0.05), and significantly increased broiler mortality rate (P<0.05).In the case of high-density feeding, the weight gain ratio of feed consumption was the highest when the methionine level was 0.35%, and it decreased linearly with the increase of the methionine level of the diet (P<0.05).The mortality rate could be reduced by 0.45% and 0.50% methionine diets, compared with 0.35% methionine level (P<0.05).② Compared with normal-density feeding group, T-AOC of high-density feeding group was significantly reduced (P<0.05), while SOD activity and PCO level were significantly increased (P<0.05).In high-density feeding groups, blood PCO level was significantly decreased as methionine level was increasing (P<0.05).③Compared with normal-density feeding group, high-density rearing significantly reduced TP concentration (P<0.05) and increased ALT activity (P<0.05) in plasma.In the case of high-density feeding, the plasma TP concentration and AST activity was significantly higher at 0.50% methionine level than that of the 0.35% and 0.40% methionine groups (P<0.05).The ALT activities of 0.40% and 0.45% methionine groups were significantly lower than that of 0.35% methionine group (P<0.05).④ Compared with normal-density feeding group, high-density rearing significantly increased HSP70 mRNA expression in liver (P<0.05).In the high-density feeding groups, HSP70 was significantly higher in 0.35% and 0.50% methionine groups than 0.45% level group (P<0.05).Compared with normal density feeding group, HMGCR mRNA expression was increased significantly when methionine level was 0.35% and 0.50% (P<0.05).【Conclusion】 The stocking density and the level of methionine addition had a certain impact on broiler's growth performance, stress level, and antioxidant capacity.High-density breeding could cause broiler stress and cause damage to liver function.Under high-density feeding, methionine restriction could increase mortality rate of broilers, and relatively higher methionine also could impair liver health.When methionine level was 0.45%, it had better anti-stress and antioxidant effects, which was beneficial to the growth of broilers.

【Objective】 To study the relationship between the growth rate of growing finishing pigs and fecal microbiota, and find the microbial flora related to the growth characteristics of pigs.【Method】 50 piglets with similar birth weights were selected and carried out feeding and management in the same feeding environment, and transfered sick pigs to the experimental group in time.At the age of 125 days, 4 pigs with the highest and lowest body weight were divided into high body weight (51.30 kg±2.57 kg, HW) and low body weight (36.90 kg±2.50 kg, LW) groups.The fecal microflora diversity, colony composition and correlation with growth rate of 125-day-old growing finishing pigs were analyzed by 16S rRNA sequencing.【Result】 Alpha diversity analysis showed that there was no significant difference between HW and LW groups (P>0.05).PCA results showed that it could clearly distinguish the flora structure in HW and LW groups.The analysis of the structure and composition of fecal microbiota showed that the dominant phyla in HW and LW groups were Firmicutes, Bacteroidetes and Spirochaetes, and the dominant genus were Treponema_2, Lactobacillus, Streptococcus and uncultured_bacterium_f_Muribaculaceae.The abundance of Bacteroides and Fibrobacteres in LW group was extremely significantly or significantly higher than that in HW group (P<0.01 or P<0.05), and Patescibacteria was significantly lower than that in HW group (P<0.05).The difference analysis of the fecal microbial structure and composition showed that 16 unique bacteria phyla were detected in HW group, 1 unique phyla and 24 unique genera were detected in LW group.The resuts of the correlation between intestinal flora and growth rate showed that 10 bacterial phyla and 18 genera were positively correlated with body weight (BW) and average daily gain (ADG), 7 bacteria phyla and 12 genera were negatively correlated with BW and ADG.【Conclusion】 There were significant differences in the flora of pig fecal microbiota with different growth rates at the same age, and the different flora might have a certain regulatory effect on the growth rate of pigs.This results could provide a reference for the development of probiotics and the improvement of the growth rate of growing pigs.

【Objective】 This experiment was conducted to investigate the effects of dietary berberine (BBR) on organ indexes, intestinal antioxidant capacity and intestinal immunity in Yellow-feathered broilers.【Method】 A total of 360 one-day-old Yellow-feathered chickens were randomly divided into 3 groups with 6 replicates in each group and 20 chickens in each replicate.The control group (NC) was fed with basal diet, the antibiotic group (PC) was fed with basal diet +200 mg/kg oxytetracycline calcium +250 mg/kg nasitide, the berberine group (BBR) was fed basal diet +250 mg/kg BBR.At 21, 42 and 63 d, one chick with the similar average body weight from each replicate was randomly selected and sacrificed.The heart, liver, spleen and bursa of Fabricius were collected and weighed respectively.The weight was recorded and the organ index was calculated.The jejunal mucosa was scraped to determine the activities of superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC), and glutathione peroxidase (GSH-Px), as well as the maldialaldehyde (MDA) content.The total RNA was extracted from jejunal mucosa and synthesized for cDNA to determine the mRNA expression of the tight junction proteins and the inflammatory cytokines.【Result】 There was no significant difference in the indexes of organs among groups (P>0.05).Compared with NC group, the total antioxidant capacity (T-AOC) was significantly increased and the content of malondialdehyde (MDA) was significantly decreased by dietary BBR supplementation in jejunal mucosa of Yellow-feathered broilers at 63 d (P<0.05).The mRNA expression of Occludin and zonula occludens-1 (ZO-1) in jejunal mucosa was significantly higher in PC group compared with those in NC group and BBR groups at 21 d (P<0.05).Compared to NC and PC groups, the mRNA expression of Claudin-1 in the jejunal mucosa of Yellow-feathered broilers at 63 d was significantly higher in BBR group (P<0.05), and the mRNA expression of nucleotide-binding oligomerization domain-containing protein 1 (NOD1) at 21 and 42 d was significantly down-regulated in BBR groups (P<0.05).Moreover, the mRNA expression of interleukin-1β (IL-1β) and IL-8 (IL-8) of Yellow-feather broilers was decreased in BBR and PC groups when compared with PC at 42 d (P<0.05).Compared to NC group, the mRNA expression of tumor necrosis factor-α (TNF-α), IL-1β, and IL-8 was decreased in BBR group at 63 d (P<0.05).【Conclusion】 The addition of 250 mg/kg berberine to the diet could enhance the intestinal antioxidant capacity, improve the intestinal barrier and reduce the expression of intestinal inflammatory factors in Yellow-feathered broilers at 63 d, so as to improve the intestinal immune function.

【Objective】 The purpose of this study was to explore the proportion of compound microecological preparation in piglet feed and the effect on piglets after replacing antibiotics.【Method】 A total of 80 healthy weaned piglets (Duroc×Landrace×Yorkshire, weaned at (28±2) days, weighted (9.31±0.52) kg) were randomly divided into five groups, 4 replicates per group and 4 piglets per replicates (half male and half female), respectively.The diet in control group was basal diet.The diet in low dose group (LOW) was basal diet supplemented with 0.1% compound microecological preparation (1×10¹¹ CFU/kg).The diet in medium dose group (MED) was basal diet supplemented with 0.2% compound microecological preparation (2×10¹¹ CFU/kg).The diet in high dose group(HIG) was basal diet supplemented with 0.3% compound microecological preparation (3×10¹¹ CFU/kg).The diet in antibiotic group (ANT) was basal diet supplemented with 75 mg/kg chlortetracycline.The experiment was lasted for 28 days.The initial and final body weights, diarrhea of piglets, and feed intake of repeats were accurately recorded.Blood samples were collected on 28 days for the measurement of serum immune parameters and biochemical indices.The contents of cecum were collected, and the changes of intestinal microbiota were analyzed by 16S rRNA sequencing.【Result】 Compared with control group, ①The average daily gain (ADG) and average daily feed intake (ADFI) of MED group were significantly increased (P<0.05), and there was no significant difference between MED and ANT groups (P>0.05).The feed/gain ratio (F/G) of MED and HIG groups were decreased significantly (P<0.05), and had no significant difference compared to ANT group (P>0.05).The diarrhea rate of piglets decreased significantly in LOW and MED groups (P<0.05), and had no significant difference compared to ANT group (P>0.05).②Thymus index of MED and HIG groups, and spleen index of MED group were significantly increased (P<0.05).The thymus and spleen index of MED group were significantly higher than ANT group (P<0.05).③ The high-density lipoprotein (HDL) of the LOW, MED and HIG groups were increased significantly (P<0.05), and the serum albumin (ALB) contents of MED and ANT groups were significantly increased (P<0.05).The activity of aspartate transaminase (AST) were significantly decreased in LOW, MED and ANT groups (P<0.05).④ The serum immunoglobulin G(IgG) levels in LOW, MED and ANT groups were significantly increased (P<0.05).The immunoglobulin A(IgA) and immunoglobulin M(IgM) levels in MED and ANT groups were significantly increased (P<0.05), and there was no significant difference between MED and ANT groups (P>0.05).⑤ The relative abundance of Firmicutes were increased significantly (P<0.05), in LOW, MED and ANT groups, and the relative abundance of Prevotella_1 decreased significantly (P<0.05) in LOW and ANT groups.The relative abundance of Proteobacteria in HIG and ANT groups were significantly reduced (P<0.05).The relative abundance of Rikenellaceae and Campylobacter were decreased significantly (P<0.05), while the relative abundance of Lactobacillaceae and Lactobacillus were increased significantly (P<0.05) in LOW, MED, HIG and ANT groups.The relative abundance of Alloprevotella in HIG group was increased significantly (P<0.05).The relative abundance of Lachnospira in MED, HIG and ANT groups were decreased significantly (P<0.05).The relative abundance of Faecalibacterium in LOW, HIG and ANT groups were increased significantly (P<0.05).Compared with the control group and ANT group, the relative abundance of Pseudomonadaceae in LOW and HIG groups were decreased significantly (P<0.05), and the relative abundance of Ruminococcaceae in LOW group was increased significantly (P<0.05).【Conclusion】 Adding compound microecological preparations to the diet could improve the growth performance and immune function of weaned piglets, improve the abundance of cecal microbes, reduce the abundance of intestinal pathogenic bacteria, and reduce the rate of diarrhea.The effect of adding 0.2% compound probiotics (the number of viable bacteria was 2×10¹¹ CFU/kg) was better, and was similar to that of antibiotics.

【Objective】 The effects of adding different proportions of alfalfa meal on the growth performance, serum biochemical indicators, antioxidant properties and immune indicators of fattening pigs were studied in this experiment, in order to find out the optimal addition ratio of alfalfa meal in the diets of fattening pigs.【Method】 120 Du×Long×Large pigs with an average weight of (75.5±1.03) kg were selected and randomly divided into 4 groups, with 6 replicates in each group, and 1 pen per replicate.0 (control group), 5%, 10%, and 15% alfalfa meal were added to the basal diets for feeding experiments.The pre-test period was 7 days and the official period was 45 days.The initial weight, final weight of each pig and daily feed intake per pen were determined.After the test, 1 pig was randomly selected from each pen, 10 mL of blood was collected from anterior vena cava to determine the serum biochemical indexes, antioxidant indexes and immunoglobulin content.【Results】 Compared with control group, the average daily gain of each experimental group was significantly reduced (P<0.05).The feed-to-gain ratio (F/G)of 5% and 10% alfalfa groups had no significant difference (P>0.05), while it was significantly increased in 15% alfalfa group (P<0.05).The activities of antioxidant dismutase (SOD) were significantly increased in 3 experimental groups (P<0.05).The triglyceride (TG) content was significantly increased (P<0.05), and the content of malondialdehyde (MDA) was significantly reduced (P<0.05) in 5% alfalfa group.The total antioxidant capacity (T-AOC) and catalase (CAT) activity were significantly increased (P<0.05), while the content of MDA was significantly reduced (P<0.05) in 10% alfalfa group.【Conclusion】 Adding 15% alfalfa meal would significantly increase F/G of fattening pigs, and the addition of 5% and 10% alfalfa could increase the antioxidant capacity.It was recommended that 5%-10% alfalfa meal should be added to the diet of finishing pigs.

【Objective】 This study was aimed to analyze the polymorphism of MHC-DRB3 gene in Ovis ammon polii, determine its allele number, nucleotide and amino acid polymorphic loci, as well as genetic relationship and evolutionary relationship with domestic sheep.【Method】 PCR-SSCP method was used to detect the alleles of MHC-DRB3 gene exon 2 in 39 Ovis ammon polii, 159 Taxkorgan sheep, 72 Tibetan sheep, 76 Ningxia Tan sheep, 36 Kirgiz sheep and 26 Duolang sheep.The differential alleles of different sheep breeds were cloned and sequenced, and the haplotype sequences of alleles were analyzed.The physical and chemical properties, structure and function of MHC-DRB3 gene exon 2 coded protein in Ovis ammon polii were analyzed by bioinformatics online softwares.【Result】 This study obtained 66 MHC-DRB3 gene alleles, including 23 alleles for Ovis ammon polii, 14 alleles for Taxkorgan sheep, 12 alleles for Tibetan sheep, 12 alleles for Ningxia Tan sheep, 4 alleles for Kirgiz sheep, and 1 allele for Duolang sheep.The number of nucleotide polymorphic loci of allele sequence of MHC-DRB3 gene exon 2 in Ovis ammon polii, Taxkorgan sheep, Tibetan sheep, Ningxia Tan sheep and Kirgiz sheep were 74, 76, 45, 49 and 25, respectively.It showed that Ovis ammon polii and Taxkorgan sheep had higher polymorphisms than Tibetan sheep, Ningxia Tan sheep and Kirgiz sheep.The phylogenetic analysis results showed that the MHC-DRB3 gene of Ovis ammon polii and Taxkorgan sheep both diverged into two branches.The MHC-DRB3 gene haplotypes of Ovis ammon polii, Taxkorgan sheep and Tibetan sheep had high similarity.The product encoded by MHC-DRB3 gene exon 2 of Ovis ammon polii was an unstable hydrophilic protein.The secondary structure mainly composed of random coil and alpha helix, and its biological effects were mainly reflected in cytoplasm.【Conclusion】 In this study, the number of alleles of MHC-DRB3 gene exon 2 and the polymorphic loci of nucleotide and amino acid of MHC-DRB3 gene in Ovis ammon polii and five species of domestic sheep were determined, it provided materials for further exploring the regulatory mechanism and evolutionary significance of MHC-DRB3 gene in sheep.

【Objective】 The purpose of this study was to study the relationship between differentially expressed gene and sperm freezing injury of Yunnan Semi-fine Wool sheep before and after sperm freezing.【Method】 The semen of three Yunnan Semi-fine Wool sheep were collected by electrical stimulation and divided into two parts.One was directly used to extract sperm RNA, and the other was cryopreserved for 7 days before sperm RNA extraction.Using the obtained sperm RNA, the mRNA transcriptomes of fresh and frozen-thawed sperm were examined by single-cell whole transcriptome sequencing, and mRNA transcriptome libraries were constructed and sequenced with Hiseq2500 sequencer, and the sequencing results were further filtered, and the differentially expressed genes were analyzed by GO function annotation and KEGG enrichment analysis.【Result】 6 semen samples had a total of 623 399 754 original sequencing sequences.After filtration, 514 313 802 (82.50%) high-quality Clean reads were obtained.The bioinformatics analysis results showed that there were 1 213 genes with significant difference before and after the freezing and thawing process.Among them, the abundance of 1 208 genes were down-regulated, and 5 genes were up-regulated after freezing and thawing.Through GO annotation and KEGG enrichment analysis, the differential genes were classified into 56 GO terms and were participate in 40 signal pathways.Their biological functions were closely related to sperm functions mainly including cellular process, metabolic process, response to stimulation, reproductive process, reproduction, membrane and antioxidant.Before and after sperm freezing, the main genes involved in inflammation, cytokine-cytokine receptor interaction and apoptosis signal pathway.【Conclusion】 The results showed that the cryoinjuries of sperm of Yunnan Semi-fine Wool sheep might be related to the biological process that the obtained differentially expressed genes were involved.In addition, the obtained differential genes might also be used as potential molecular markers to evaluate the quality of sheep frozen semen.

【Objective】 The purpose of this study was to investigate the effects of different concentrations of rutin and different freezing rates on the cryopreservation of Duroc boar sperm and their interaction, in order to provide basis for guiding production practice and improving utilization rate of excellent breeding pigs.【Method】 The experiment was divided into 6 groups:Blank control group (frozen diluent solution Ⅰ), and experimental groups (0.2, 0.4, 0.6, 0.8 and 1.0 mmol/L rutin were added to the frozen diluent solution Ⅰ, respectively).Semen were frozen at 1 cm (fast freezing) and 3 cm (slow freezing) above the liquid nitrogen surface, respectively.After being stored in liquid nitrogen for 30 days, sperm motility parameters, membrane integrity rate (MI), acrosome integrity rate (AI), mitochondrial membrane potential(MMP), DNA integrity rate, ROS level, MDA and ATP content, and the activities of SOD, CAT and GSH-Px were measured to evaluate the frozen-thawed semen quality.【Result】 In terms of freezing rate, in the same concentration of rutin freezing solution, the slow freezing effect was better than the fast freezing effect, and the difference was significant (P<0.05).In terms of rutin concentration, the effect of adding 0.6 mmol/L rutin was the best (P<0.05), followed by 0.8 mmol/L.In terms of their interaction, there was an interaction between freezing rate and rutin concentration, especially sperm motility parameters, membrane integrity, acrosome integrity, DNA integrity, mitochondrial membrane potential and ATP content of slow freezing×0.6 mmol/L rutin combination were significantly higher than those of other combination experimental groups (P<0.05), and the level of ROS was significantly lower than that of other combination experimental groups(P<0.05).The activities of SOD, CAT and GSH-Px were significantly increased compared with other groups.【Conclusion】 There were interaction effects between different rutin concentrations and different freezing rates, and adding 0.6 mmol/L rutin into the slow freezing diluent had the best effect.

With the rapid development of molecular biology and animal genetics and breeding technology, the research on fine traits of livestock and poultry has also moved from phenotypic breeding to molecular breeding of genes associated with single or multiple important traits.For example, the research on the meat quality trait of pigs has obtained many key genes in affecting the backfat thickness, intramuscular fat content, ham weight and so on.In recent years, African swine fever has caused a great blow to the pig industry, and the research on pig disease has once again become a hot spot.In the study of disease resistance traits in pigs, many key genes, such as CD163, are directly involved in the invasion mechanism of Porcine respiratory and reproductive syndrome virus.In addition to the influence of disease, the reproductive traits of pigs also play a vital role in the development of pig industry.With the in-depth study of important traits, a large number of multidimensional data of important breeding value genes have been accumulated, and a variety of gene screening techniques have been developed, especially the discovery of CRISPR-Cas9 gene editing technology and its successful application as a genome-wide screening tool, as well as the combined application of functional genomic screening and multi-omics in the post-genomic era, the application and development of these technologies have further promoted the development of the field of genetic breeding.The author systematically summarized important breeding value genes related to meat, disease resistance and reproductive traits and their mining technology in pigs, in orded to provide guiding suggestions and references for the genetic improvement of pigs.

【Objective】 The purpose of this study was to study the virulence characteristics of a strain of Infectious bursal disease virus (IBDV) isolated from Guangxi and its relationship with the sequence characteristics of VP2 gene, so as to provide reference for the epidemiological research and epidemic prevention and control of IBDV.【Method】 The virus was isolated and identified by PCR, chicken embryo subculture, adaptive culture of DF-1 cells and agar diffusion test, and its VP2 gene was amplified.The nucleotide and amino acid sequences were compared with other IBDV strains with different virulence by Mega 7.0 and MegAlign softwares, and the pathogenicity to SPF chicken was tested.The changes of IBDV copy number were determined by Real-time PCR.【Result】 One strain of IBDV was successfully isolated, and named as GX20210126 strain.SPF chicken embryos inoculated with this strain could cause bleeding, dwarfing, liver yellowing, bleeding and acitoid necrosis.Moreover, GX20210126 had good cell adaptability to DF-1 and could cause significant cytopathic changes.The agar diffusion test showed that the strain had good antigenicity, and a white precipitation line could be formed between the virus liquid and the IBDV-positive serum.VP2 gene evolution analysis showed that the GX20210126 strain was on the same branch with the international standard super virulent strain, and the amino acid similarity was between 86.8% and 99.6%.It had the highest similarity with the UK661 strain, and the amino acid similarity was 99.6%.Only two amino acid positions changed, which were D279N and I272T.The 8-day-old SPF chickens were inoculated with EID₅₀ 10^-3.8/0.1 mL, 0.5 mL per chicken.After inoculation, the chickens showed fluffiness, droopiness, white and green water feces, and other clinical symptoms.Necropsy showed muscle and liver hemorrhage, kidney urate deposition, and bursa atrophy.What was more, IBDV copy number peaked on the 5th day.【Conclusion】 The IBDV epidemic strain GX20210126 in Guangxi had the genetic sequence characteristics of a super virulent strain.Artificial infection of chickens could lead to typical clinical symptoms and pathological changes of IBDV.

【Objective】 This study was aimed to investigate the genotypes and evolutionary characteristics of Porcine circovirus type 2 (PCV2) isolated from Guangdong province, and provide reference for PCV2 prevention and control and vaccine strain screening in Guangdong province.【Method】 PCV2 whole genome sequences from 4 positive samples were amplified by PCR, and then sequenced and phylogenetically analyzed.MegAlign software was used to analyze the key variation of ORF2 amino acid sequences of 4 Guangdong isolates of PCV2 and reference strains at home and abroad.The capsid protein encoded by ORF2 gene of 4 Guangdong isolates of PCV2 and 4 vaccine strains were predicted by Jameson-Wolf method of DNAStar Protean software.【Result】 The sequence length of 4 Guangdong isolates of PCV2 was 1 767 bp.Phylogenetic tree analysis showed that all the 4 isolates belonged to PCV2d genotype with nucleotide homology of 97.7%-99.1%, 91.7%-99.8% with 54 reference strains at home and abroad, PhuTho/G40312/2018 (Viet Nam, accession No.:LC602996), QZ1410 (Jiangsu, accession No.:MG732832) and GXBB1501211 (Guangxi, accession No.:MH756609) had similar genetic relationships.Analysis of key amino acid variation showed that there were 8 amino acid specific mutation sites were found in Cap protein, which were Y3C, F8Y, T56S, R116K, V123I, K164E, R169G and T216A.Antigen analysis showed that the Cap protein antigenic index of Guangdong isolates was significantly different from that of the four vaccine strains, mainly in the amino acid regions of 7-12, 47-53, 80-90, 160-170 and 205-213.【Conclusion】 In this study, 4 strains of PCV2 isolated from parts of Guangdong province were all 2d subtypes, and there were many mutation sites in the amino acid sequence of Cap protein, and the antigenic index was also significantly different from that of the vaccine strain, suggesting that the epidemic trend of PCV2 in Guangdong province was gradually dominated by 2d subtypes.

【Objective】 The aim of this study was to express the nucleocapsid protein of Porcine reproductive and respiratory syndrome virus (PRRSV) by Adenovirus AdMax system expression vector, and to study its immunogenicity.【Method】 Referring to the PRRSV N gene sequence published in GenBank (accession No.:KT945017.1), the PRRSV N gene was synthesized and connected to the Adenovirus shuttle vector pDC316-mCMV-EGFP, transformed into E.coli Top10 competent cells, and the recombinant shuttle plasmid pDC316-N was constructed.The recombinant shuttle plasmid pDC316-N and the framework plasmid PBHGLOX(delta)E1, 3Cre of AdMax Adenovirus system were co-transfected into 293A cells to obtain the recombinant Adenovirus rAd-N.The obtained recombinant Adenovirus solution was identified by PCR and sequencing.The 293A cells were infected with the identified correct rAd-N virus solution.The recombinant Adenovirus was expanded for culture and the TCID₅₀of the virus was detected, RT-PCR and Western blotting were used to detect the expression and reactogenicity of recombinant Adenovirus.Mice were immunized with recombinant Adenovirus, the serum was collected, the antibody level was detected with PRRSV antibody detection kit, and the immune effect on mice was preliminarily evaluated.【Result】 A 400 bp PRRSV N gene band was amplified by PCR.The sequencing results were correct, indicating that the recombinant Adenovirus was successfully constructed.After concentration, it was determined that its TCID₅₀ was 10^-10.239.The results of RT-PCR and Western blotting confirmed that the target gene was correctly expressed at the gene and protein levels, and the molecular weight of the protein was about 14 ku.The detection of mouse specific antibody showed that mice immunized with recombinant Adenovirus rAd-N could quickly produce specific antibody against PRRSV, which was significantly different from the control group (P<0.05).The effect of recombinant Adenovirus combined with Gel adjuvant was the best, which was up to 7.84 U/L.【Conclusion】 The recombinant Adenovirus expressing PRRSV N protein was successfully constructed, which had good immunogenicity, and laid a foundation for the establishment of indirect ELISA for PRRSV antibody and the further development of PRRSV antibody detection kit.

【Object】 The aim of the experiment was to explore the pathogenicity of subgroup Ⅰ Fowl adenovirus (FAdV) isolates GDDL-4(FAdV-4), GDB3-8a(FAdV-8a), GDT08-8b(FAdV-8b) and GDWYT-11(FAdV-11) to SPF chicks, and develop more effective prevention measures.【Method】 The challenge (GDDL-4, GDB3-8a, GDT08-8b and GDWYT-11) and control groups were established, the challenge groups were injected with 0.2 mL 10⁵ TCID₅₀virus into leg muscles, and the control group was injected with equal volume of PBS.The clinical symptoms, autopsy lesions, histopathology of SPF chicks were observed, the detoxification and viral load of SPF chicks were detected.【Result】 The results showed that 4 target strains could all cause depressed, emaciated and clustered.Pathologic autopsy showed that severe hepatitis-hydropericardium syndrome, especially GDDL-4, with mortality rate of 85%, the overall mortality rate was 0 to 85%.The results of detoxification analysis showed that GDDL-4 and GDB3-8a groups had high titer detoxification (≥ 10⁴ copies/μL), while GDT08-8b and GDWYT-11 groups could only detect low titer detoxification (≤ 10³ copies/μL).The organ index showed that there were no significant difference between GDT08-8b and control groups, but the other challenge groups could cause organ swelling or atrophy to varying degrees.The histopathology sections showed that the structures of liver, kidney and bursa of Fabricius were destroyed, it showed that a large number of lymphocytes infiltrated into liver cells, the renal interstitial congestion and hemorrhage, and the lymphocyte necrosis of bursa of Fabricius.The results of viral load analysis showed that GDDL-4 and GDWYT-11 groups had the highest viral titers in liver, while GDB3-8a and GDT08-8b groups had the highest viral titers in duodenum.【Conclusion】 The results indicated that the mortality of subgroup Ⅰ FAdV was high.4 strains of isolates could cause the pathological changes of inclusion body hepatitis in liver of chicks, and could be repeatedly detoxified.Moreover, the isolates had different affinity to different tissues and organs, with the highest viral load in liver and duodenum.

【Objective】 The aim of this study was to investigate the effect of Porcine circovirus (PCV3) on the expression of growth-related hormones and functional genes in piglets, and explore the mechanism of PCV3 causing weight loss in piglets.【Method】 Eight 23 day-old non-infected piglets were randomly divided into PCV3 infected group and DMEM group.Piglets of PCV3 infected and DMEM groups were intramuscular injected with 3 mL of PCV3 virus (10⁹ copies/mL) and 3 mL of DMEM in the neck respectively.The piglets were weighed before injection (0 d) and 3, 7, 14 and 21 d after injection, the clinical manifestations were observed, and serum, oral swab, nasal swab and anal swab were collected.Real-time quantitative PCR method was used to detect the viral load of PCV3, ELISA was performed to determine the content of triiodothyronine (T₃) and thyroxine (T₄) in the serum after infected 3, 7, 14 and 21 d.Real-time quantitative PCR was utilized to determine the relative expression of growth hormone receptor (GHR), insulin-like growth factor type 1 (IGF-1), insulin-like growth factor type 1 receptor (IGF-1R) genes in longissiums dorsi, spleen and liver.【Result】 The final weight and average daily gain of the piglets in infected group were extremely significantly lower than those in DMEM group (P<0.01).The viral load of serum, oral swab, nasal swab and anal swab in infected group reached the highest at 14 d.The content of T₃ in the infected group was extremely significantly higher than that in DMEM group on the 14th and 21st day of infection (P<0.01), and there was no significant difference in the content of T₄ (P>0.05).Compared with DMEM group, the relative expression of IGF-1 gene in longissimus dorsi was extremely significantly decreased (P<0.01), there was no significant difference in the relative expression of GHR, IGF-1 and IGF-1R genes in leg muscle and spleen (P>0.05), the relative expression of GHR gene in liver was extremely significantly increased (P<0.01), and the relative expression of IGF-1R gene was increased significantly (P<0.05).【Conclusion】 PCV3 infection in piglets could cause abnormal metabolism, reduce the growth-promoting and mitogenic effects of IGF-1 on the back muscles, leading to weight loss in piglets.

【Objective】 The experiment was aimed to study the acute brain injury caused by African swine fever virus (ASFV) and the related pathological mechanisms of NF-κB mediated brain edema under experimental conditions.【Method】 Eighteen healthy Landrace pigs were randomly divided into two groups:Challenge group (n=13) and control group (n=5).The challenge group was injected intramuscularly with the ASFV strain Pig/HLJ/18 with a dose of 10² HAD₅₀/mL (HAD₅₀:half of the red blood cell adsorption), the control group was injected with the same volume of normal saline, and the experiment lasted for 15 days.During the experiment, the clinical symptoms were observed, the dead pigs were dissected and the brain lesions were observed.Virus localization was performed by in situ PCR, HE staining and toluidine blue staining were used to observe the histological changes of brain.Immunohistochemical staining was used to detect the expression of TNF-α, IL-1β, IFN-γ, NF-κB, MMP-9, and AQP-4 in brain tissue.【Result】 9 pigs in the challenge group showed clinical neurological symptoms, and the main brain lesions were meningeal congestion and different degrees of brain parenchymal edema.ASFV was mainly located in cerebral microvessels.The early stage of the lesion was mainly cerebral edema, loose structure of cerebral cortex, widening of perivascular space, swelling and rounding of neurons and light staining.In the middle and late stage, in addition to the characteristics of cerebral edema, cerebral vascular congestion, a large amount of micro thrombosis and lymphocyte infiltration were also seen.Immunohistochemical results showed that TNF-α, IL-1β, IFN-γ, NF-κB, and MMP-9 were mainly expressed in neurons and glial cells in the challenge group, and the positive cells of AQP-4 were mainly astrocytes around capillaries.Compared with the control group, the positive expression rates of the above 6 factors in the challenge group were extremely significantly higher (P<0.01).【Conclusion】 In summary, the main manifestations of acute African swine fever brain injury were meningeal vascular congestion, hemorrhage and brain parenchymal edema, and histological lesions showed viral encephalitis.ASFV infected the brain and promoted abnormally high expression of inflammatory factors.By activating the NF-κB signaling pathway, it regulated the transcription of TNF-α, IL-1β and IFN-γ, which increased the production and release of these inflammatory factors, expanded the inflammatory response, and affected the permeability of the blood-brain barrier.It could also up-regulate the expression of MMP-9 and AQP-4, destroy the blood-brain barrier, and promote the occurrence and development of cerebral edema.

【Objective】 The study was aimed to design a multi-epitope vaccine against Swine acute diarrhea syndrome coronavirus (SADS-CoV) S, M and E proteins.【Method】 IEDB server was used to predict the MHC class Ⅰ molecular binding epitopes of SADS-CoV S protein, M protein and E proteins of T lymphocytes.T lymphocyte MHC class Ⅱ molecule binding epitopes were predicted by NetMHCIIpan 4.0 Serve.Immunomedicine Group and IEDB server were used to predicted B lymphocyte epitopes.The overlapping epitope regions were selected as the dominant epitopes from the prediction results of each server, and the highly conservative and non-sensitizing dominant epitope regions were selected by using IEDB and AllerTOP v 2.0 servers, which were connected in series into a multi epitope vaccine through a flexible linker.The antigenicity, physicochemical properties, N-glycosylation sites, secondary and tertiary structures of the constructed multi-epitope vaccine were evaluated.The binding capacity of multi-epitope vaccines to immune receptors was evaluated by molecular docking and silico cloning was carried out.【Result】 The results showed that the multi-epitope vaccine constructed with highly conservative and non-sensitizing dominant epitopes had a relative molecular weight of 35.30 ku.It was a stable hydrophilic protein with good antigenicity.There was one N-glycosylation site, and the alpha helix, beta sheet, random coil and beta turn were 22.11%, 20.35%, 50.88% and 6.67% in the secondary structure, respectively.Ramachandran mapping of tertiary structure found that the residue base in the dominant region accounted for 90.00%.After refinement, the residue base in the dominant region increased to 91.92%.Mapping of prominent epitopes of tertiary structure also proved that the multi-epitope vaccine had good immunogenicity, and molecular docking showed that the multi epitope vaccine had high affinity with TLR3.Finally, codon optimization, reverse translation and silico cloning ensure the efficient and stable expression of the designed multi epitope vaccine in E.coli K12 expression system.【Conclusion】 The constructed multi-epitope vaccine might effectively express and induce strong T-cell and B-cell immune responses.This study provided a new method for the design of SADS-CoV multi-epitope vaccine, and provided theoretical basis and data support for the research and development of SADS-CoV multi-epitope vaccine.

【Objective】 This study was aimed to understand the genetic evolution and pathogenicity of Fowl adenovirus (FAdV) in Jiangsu area, and provide reference for the epidemiological study of FAdV and disease prevention and control.【Method】 Serological typing of the virus was determined by chick embryo passage culture, PCR identification, electron microscope observation, whole genome sequencing, sequence alignment and similarity analysis, and the isolated strain were injected into the chest muscle with a dose of 5×10^5.33 EID₅₀ to infect 10-day-old SPF chicks for animal regression experiments.【Result】 PCR results showed that the isolated strain was positive for group Ⅰ FAdV, and the spherical, membraneless, icosahedral structure of adenovirus was observed under transmission electron microscope.The whole genome and Hexon gene sequence analysis showed that the isolated strain was serotype 11 of FAdV D, named JSNT-1 strain.The mortality rate of SPF chicks infected with this strain was 10%(1/10).Autopsy of the dead chickens showed that the liver was discolored and yellow, bleeding and swelling, and the edge of liver was blunt and round, kidney was swollen, bleeding and pale.Detoxification could be detected in oral cavity and cloaca by Real-time PCR test, and the virus was distributed in many tissues and organs in the chicken body.The histopathological results showed that the liver cells of the dead chickens were degenerated and necrotic, with basophilic intranuclear inclusion bodies, glomerular epithelial cells were degenerated and necrotic.A large number of inflammatory cell infiltrations were seen in the myocardium.【Conclusion】 The isolated strain was serotype 11 of FAdV, and the clinical incidence was not obvious after infection with SPF chickens, and the fatality rate was low.The sick and dead chickens could produce characteristic inclusion body hepatitis lesions, and the virus could replicate in and outside the chickens.

【Objective】 This study was aimed to study the histopathological characteristics of mirror carps infected with Carp edema virus (CEV) and the distribution of CEV in various tissues, in order to provide references for the diagnosis, prevention and control of Carp edema virus disease (CEVD).【Method】 The pathological changes of heart, liver, skin, spleen, kidney, brain and gill tissues of infected mirror carps were observed by HE staining.The immunohistochemistry was used to detect the distribution and protein expression of CEV in the tissues of mirror carps.Three probes were designed in the conserved region of CEV P4a gene and labeled at the 5'end FAM for in situ hybridization to detect the nucleic acid distribution of CEV in the tissues of mirror carps.Real-time quantitative PCR was used to detect the viral load in each tissue of diseased mirror carps.【Result】 HE staining analysis showed that the gills of the diseased mirror carps were swollen and hyperemic, and there were red blood cells and inflammatory cells between the gills.The liver cells were deeply stained and atrophied, and the bile duct was bleeding.There was obvious space and hemorrhage in spleen tissue.The renal tissue hyperemia was obvious and the renal tubules were obviously closed.The results of immunohistochemistry showed that the CEV P4a protein was highly expressed in gill and liver tissues of the diseased fish, and less expressed in other tissues.The results of in situ hybridization showed that CEV nucleic acids were abundant in gill tissue, and a small amount of positive signals were found in heart, spleen and kidney.Real-time quantitative PCR assay indicated that the viral load of gill was extremly significantly higher than that in myocardium, liver, skin, spleen, kidney and brain tissues (P<0.01).【Conclusion】 After infection with CEV, visceral tissues of mirror carps showed varying degrees of congestion and bleeding, the virus was mainly distributed in gill, indicating that gill was the main target organ for CEV proliferation.

【Objective】 This experiment was conducted to investigate the effects of interleukin-10 (IL-10) on the replication of Porcine circovirus type 2 (PCV2), screen high infectious PCV2 cell lines, and improve the virus titer of PCV2, and lay a foundation for the subsequent vaccine research and development and the study of the role of IL-10 in PCV2 infection.【Method】 Porcine IL-10 gene was amplified by PCR, the target gene was linked to lentivirus expression vector (pCDH-CMV-MCS-EF1-GFP+Puro), and the recombinant plasmid pCDH-CMV-IL-10 was obtained.It was co-transfected with package plasmids psPAX2 and pMD2.G into 293T cells for lentivirus packaging.PK-15 cells were infected with the collected lentivirus supernatant and screened by puromycin to obtain PK-15-IL-10 cell lines.The control cells were named PK-15-pCDH and PK-15, respectively.After PCV2 was infecting PK-15-IL-10, PK-15-pCDH and PK-15 cell lines, cell fluid was collected at 24, 48 and 72 h, respectively, and cell viability was detected by CCK-8.The expression of IL-10 gene and the replication of PCV2 were detected by Real-time quantitative PCR and Western blotting.Indirect immunofluorescence assay (IFA) was used to observe the replication of PCV2 in cells and determine the virus titer of PCV2 (TCID₅₀).【Result】 The recombinant plasmid pCDH-CMV-IL-10 was successfully constructed.293T cells were co-transfected with the packaged plasmids psPAX2 and pMD2.G, and the cell status was the best and the fluorescence was the strongest at 48 h.PK-15 cells were co-transfected with lentivirus supernatant for 48 and 72 h, respectively.The fluorescence of pCDH-CMV-IL-10 group was the strongest, and they were cultured in complete medium with puromycin concentration of 2.5 μg/mL to obtain stable cell lines with green fluorescence.Real-time quantitative PCR and Western blotting results showed that the expression of IL-10 gene in pCDH-IL-10 cell lines was significantly higher than that in control group PK-15-pCDH and PK-15.The copy number of PCV2 was increased by four times, and its replication capacity was enhanced.After the virus was diluted and passed for 3 generations, PCV2 in PK-15-IL-10 cells was extremely significantly higher than that in PK-15 cells (P<0.01).Cell proliferation assay showed that overexpression of porcine IL-10 gene had no significant effect on cell viability.IFA results showed that fluorescence in PK-15-IL-10 cells was stronger than that in PK-15 cells.TCID₅₀ of PCV2 in PK-15-IL-10 cells was extremely significantly higher than that of PK-15 cells at 48 h after infection (P<0.01).【Conclusion】 The lentiviral expression vector of pCDH-CMV-IL-10 was successfully constructed and used to infect PK-15 cells.After further culture, PK-15-IL-10 cell line expressing IL-10 was screened, and infection with PCV2 could promote the replication of PCV2 in PK-15 cells.These results provided reference for later vaccine research, and laid a foundation for further study on the effect of IL-10 on the replication of PCV2 in PK-15 cells.

【Objective】 This study was aimed to clarify the main bacterial pathogens and their biological characteristics that cause respiratory symptoms of piglets in a large-scale pig farm in Luohe, Henan province.【Method】 Oral and nasal swabs of infected piglets and tissues and organs such as pleural effusion, lung, liver and spleen of dead pigs were collected aseptically for PCR detection of pathogens.The biological characteristics of the isolate was analyzed by isolation and culture of bacteria, morphological observation, satellite test, PCR amplification of 16S rDNA gene, construction of genetic evolution tree, multiple PCR identification of serotypes of isolated strains, pathogenicity test of mice and drug sensitivity test.【Result】 Round, smooth, moist, colorless and transparent microcolonies grew on the BHI solidplate containing V factor, but did not grow on the medium without V factor.The isolates were stained with Gram staining, and Gram-negative bacteria were seen under the microscope, mostly in the shape of a single ball club and long rod.Atypical 'satellite colony' was formed around the culture of Staphylococcus aureus.16S rDNA sequence analysis showed that the isolate was in the same branch as Haemophilus parasuis (Hps) with more than 97% similarity.The isolate was identified as Hps serotype 4.When the isolated Hps was injected into mice intraperitoneally, bleeding in various organs of the infected mice was induced, especially in lung.High doses of infection of Hps serotype 4 could cause death in mice.Drug sensitivity test showed that the strain had strong resistance to tetracycline, ciprofloxacin and co-trimoxazole compound sulfamethoxazole.After rational administration, a good therapeutic effect was achieved.【Conclusion】 A strain of Hps serotype 4 was successfully isolated, which could cause bleeding in different degrees in multiple organs of infected mice, and was resistant to tetracycline, ciprofloxacin and co-trimoxazole.This results provided a theoretical basis for the clinical diagnosis and treatment of Hps.

【Objective】 The study was aimed to forecast the material basis, action target and pathway of Bailongsan in treating chicken diarrhea by the novel method of network pharmacology.【Method】 All the active ingredients of Pulsatillae Radix, Coptidis Rhizoma and Gentianae Radix et Rhizoma in Bailongsan, and related potential drug targets were collected by TCMSP.The disease targets related to chicken diarrhea were retrieved from GeneCards and OMIM databases.The drug targets were mapped to disease targets by Venny platform, and the intersection targets were uploaded to the STRING database for analysis.PPI networks were constructed and the key targets were obtained, and GO function and KEGG pathway enrichment analysis were performed for the key targets.【Result】 A total of 27 active ingredients, 57 drug targets and 4 207 disease targets were screened.There were 45 intersection targets.The protein interaction network found that 45 proteins including MYC, ESR1, IL-6 and PTGS2 might be the key targets of Bailongsan in treatment of chicken diarrhea.The GO enrichment analysis identified 112 GO items.The enrichment analysis of KEGG pathway identified 22 related signal pathways.【Conclusion】 The Bailongsan might act on the key targets such as JUN, MYC, ESR1 and IL-6 to treat chicken diarrhea by quercetin, kaempferol, and isorhamnetin, etc.And the pathways were involved in cytokine-cytokine receptor interaction, p53 signaling pathway, MAPK signaling pathway and other pathways.

【Objective】 The purpose of this experiment was to investigate the effects of miR-145a-3p on Brucella induced autophagy of macrophages (RAW264.7) and its effect on the intracellular survival of Brucella.【Method】 First, miR-145a-3p mimics, miR-145a-3p inhibitor and NC mimics of autophagy related miRNA miR-145a-3p were synthesized and transfected into GFP-RFP-RAW264.7 macrophages.Then, Brucella infected the cells for 24 h, and the effect of miR-145a-3p on autophagy was observed by laser confocal microscopy.Bioinformatics softwares such as TargetScan and miRBase were used to predict the target protein of mir-145a-3p.The recombinant plasmids PmirGLO-ATG14-3'UTR and PmirGLO-ATG14-3'UTR-mutation were constructed, and were identified by double enzyme digestion with SacⅠ and KpnⅠ, and the targeting relationship between miR-145a-3p and autophagy related gene 14 (ATG14) was verified by double luciferase reporting system.When RAW264.7 cells were cultured to 60% confluence, miR-145a-3p mimics, miR-145a-3p inhibitor and NC mimics were transfected respectively, after transfection for 7 h, they were infected with Brucella, and PBS was added as the uninfected control.The cells were collected after 24 h of culture, and the regulatory effects of miR-145a-3p on ATG14 mRNA and protein expression were detected by Real-time quantitative PCR and Western blotting.Finally, the macrophages transfected with miR-145a-3p were infected by Brucella, and the bacterial colony was counted to verify the effect of miR-145a-3p on the intracellular survival of Brucella.【Result】 miR-145a-3p mimics promoted Brucella induced autophagy, while miR-145a-3p inhibitor inhibited Brucella induced autophagy.The results of software prediction showed that the target of miR-145a-3p was due to the autophagy related protein ATG14-3'UTR.The results of double enzyme digestion showed that the recombinant plasmids PmirGLO-ATG14-3'UTR and PmirGLO-ATG14-3'UTR-mutation were successfully constructed.When the interaction between miR-145a-3p mimics and ATG14-3'UTR was verified by double luciferase reporter gene system, the fluorescence value of miR-145a-3p mimics group was decreased significantly compared with NC mimics group (P<0.01).Compared with the NC mimics group, when not infected with Brucella, in miR-145a-3p mimics group, the level of ATG14 mRNA was extremely significantly decreased (P<0.01) and the level of ATG14 protein was significantly decreased (P<0.05), whlie the level of ATG14 mRNA was significantly increased in miR-145a-3p inhibitor group (P<0.01);After Brucella infection, the level of ATG14 mRNA in miR-145a-3p mimics+Bru group was extremely significantly increased (P<0.01), and the level of ATG14 mRNA in miR-145a-3p mimics+Bru group was significantly higher than that of NC mimics+Bru group (P<0.05).miR-145a-3p mimics promoted the expression level of ATG14 protein.The overexpression of miR-145a-3p decreased the number of intracellular survival of Brucella (P<0.05).【Conclusion】 miR-145a-3p was highly expressed after Brucella infection, and promoted autophagy by targeting ATG14 and inhibited Brucella replication.

【Objective】 This study was aimed to study the proportion of prophages carried by Escherichia coli (E.coli) and the virulence genes as well as drug resistance genes carried by prophages, understand the effect of prophages in pathogenic E.coli on drug resistance and virulence of strains, ensure the biosafety of phage-producing bacteria, and provide a reference for the basic research and application of E.coli phages.【Method】 In this study, the whole genome information of E.coli was downloaded from NCBI, the number of prophages carried in E.coli through the online website was predicted, the genomic characteristics such as GC content, genome size, and proportion of bacterial genome of prophages and the type of intact prophages were analyzed, a preliminary classification of prophages was made, and the intact prophages carrying drug resistance genes, the number of drug resistance genes, drug resistance phenotypes, drug resistance mechanisms and virulence genes, and the distribution of virulence gene families were also statistically analyzed.【Result】 112 strains of E.coli carried 1 024 intact prophages, 287 questionable prophages, and 505 incomplete prophages.Most of the 1 024 intact prophages were similar to long tailed phages, accounting for 70.21% (719/1 024).There were 63 phage types in 1 024 complete type prophages, among which the phage BP4795 (accession No.:NC_004813.1) type accounted for the highest proportion, reaching 19.24%(197/1 024), followed by the phage DE3(accession No.:NC_042057.1), accounting for 11.72%(120/1 024).The GC content of E.coli carrying prophage was 38%-57%, E.coli prophage genomes vary in size was 3.1-152.6 kb, prophages within 80 kb accounted for 97.03%(1 762/1 816).65 of 1 024 complete prophages carried resistance genes, and a total of 253 carried resistance genes, which were from 11 antibiotic resistance gene families, respectively, and the complete prophage resistance gene carriage rate was 6.35%;Out of 1 024 prophages, 438 of the prophages carrying one or more virulence genes, with a high carriage rate of 42.78%, carried a total of 1 274 virulence genes, and on average each complete type prophage carried 2.9 virulence genes, from 9 different virulence factor families, respectively.【Conclusion】 E.coli usually carried prophages, and parts of the prophages carried drug resistance genes and virulence genes.The risk should be fully evaluated in practical application, attention should be paid to prevent the transfer of drug resistance genes and virulence genes caused by prophages between the strains.

【Objective】 The study was aimed to explore the pathogens, pathogenicity and drug resistance of Pseudosciaena crocea in a farm in Zhejiang province.【Method】 The diseased Pseudosciaena crocea was dissected under sterile conditions, the liver and kidney tissues were taken, and the bacteria were isolated and cultured by scribing.The species characteristics of the isolated bacteria were identified by morphological observation, physiological and biochemical test and 16S rRNA gene sequence analysis.The pathogenicity of the isolated bacteria to Pseudosciaena crocea was evaluated by artificial infection and tissue section.Finally, the drug resistance of the isolated bacteria was detected by drug sensitive paper method.【Result】 The appearance of the isolated bacteria on the culture medium was yellow round colonies with convex, smooth surface and neat edges.After Gram staining, there were single scattered or paired distribution of Corynebacterium red under an optical microscope.The isolated strain could grow on MacConkey agar and produce acid by glucose.16S rRNA gene sequence similarity analysis and phylogenetic tree showed that the isolated strain had more than 97.0% similarity with Chryseobacterium sp. and clustered into one class.The results of animal regression test showed that the isolated bacteria could cause obvious lesions in the liver, kidney and spleen of Pseudosciaena crocea, and the median lethal dose was 6.32×10⁴ CFU/mL, indicating strong pathogenicity to Pseudosciaena crocea.The results of drug sensitivity showed that the isolated bacteria were sensitive to amikacin and levofloxacin, moderately sensitive to gentamicin, neomycin and erythromycin, and resistant to cefixime, ampicillin, kanamycin and furazolidone.【Conclusion】 In this study, the pathogenic strain of Chryseobacterium isolated from diseased Pseudosciaena crocea was reported, and its pathogenicity and drug resistance were studied to provide references for the prevention, control and treatment of aquatic animal diseases caused by Chryseobacterium sp..

【Objective】 This study was aimed to explore the effect of selenized garlic polysaccharide on the function of mouse peritoneal macrophages, in order to provide a basis for the mining and clinical application of selenium garlic polysaccharide (sGPS).【Method】 The garlic polysaccharide(GPS) was obtained by isolation and purification, and modified by HNO₃-Na₂SeO₃ to obtain sGPS₃, GPS₅ and sGPS₆.Taking the mouse peritoneal macrophages as the research object, the mouse peritoneal macrophages were treated with sGPS₃, GPS₅, sGPS₆ at five concentrations (6.25, 12.5, 25, 50 and 100 μg/mL)and 10 μg/mL lipopolysaccharide (LPS group) for 48 h, at the same time, the cells without drugs were set as control group. The neutral red method was used to determine the phagocytic function of mouse peritoneal macrophages, and the CCK-8 method was used to determine the proliferation, in order to screen out sGPS with the best activity. The ELISA method was used to determine the effect of sGPS with the best activity on the contents of NO, TNF-α, IFN-γ, IL-1β, IL-6, and IL-12 in the supernatant of macrophages. The mouse peritoneal macrophages swallow chicken red blood cells and mouse carbon clearance experiments were used to detemine the phagocytic indexes and phagocytic percentage of blank control group (normal saline), high (2 mg/mL), medium (1 mg/mL) and low (0.5 mg/mL) doses of sGPS with the best activity.【Result】 Except 6.25 μg/mL sGPS₃, the A_{490 nm} values of 6.25, 12.5, 25, 50 and 100 μg/mL peritoneal macrophages in sGPS₃, sGPS₅ and sGPS₆ groups were significantly higher than those in control group (P<0.05).Among them the A_{490 nm} values 50 and 100 μg/mL of sGPS₆ groups were significantly higher than that of the LPS group (P<0.05).Therefore, sGPS₆ was selected for follow-up test.The contents of NO, TNF-α, IL-1β and IL-6 in the supernatant of mouse peritoneal macrophages of sGPS₆ at the concentrations of 12.5, 25, 50 and 100 μg/mL were significantly higher than those in control group (P<0.05).The contents of IFN-γ, IL-12 in the supernatant of mouse peritoneal macrophages of sGPS₆ at the concentrations of 25, 50 and 100 μg/mL were significantly higher than those in control group (P<0.05).Among them, the contents of NO, TNF-α and IL-12 of mouse peritoneal macrophage supernatant at 100 μg/mL of sGPS₆ were significantly higher than the LPS group (P<0.05).The phagocytic index and phagocytic rate at 2 and 1 mg/mL sGPS6 were significantly higher than those of the blank control group (P<0.05).【Conclusion】 Selenium garlic polysaccharide could enhance the phagocytic function and proliferation ability of the mouse peritoneal macrophages, and promote the secretion of cytokines TNF-α, IL-1β and IL-6.

【Objective】 This study was aimed to investigate the regulatory role of microRNA-188-5p (miR-188) on the proliferation, migration and apoptosis of breast cancer cells, in order to provide a theoretical basis for the development of breast cancer related treatment drugs.【Method】 The mouse breast cancer cells (4T1) was cultured to establish 4T1 mouse xenograft model.The tumour tissues and adjacent tissues were then separated, and the expression of miR-188 was detected by Real-time quantitative PCR.The oligos miR-188 mimics, mimics-NC, miR-188 inhibitor and inhibitor-NC were transferred into 4T1 cell lines, and the non transfected cells was blank control (Con), the expression level of miRNA-188 was detected by Real-time quantitative PCR, cell proliferation was detected by CCK-8 method, cell migration was detected by cell scratch test, the apoptosis rate was detected by flow cytometry, the expression of apoptosis related proteins was detected by Western blotting.【Result】 The expression of miR-188 in peritumoral tissues was significantly higher than that of tumour tissues (P<0.05).The results of cell transfection showed that compared with Con group, the relative expression of miR-188 in miR-188 mimics group was significantly up-regulated (P<0.05), and in miR-188 inhibitor group was significantly down regulated (P<0.05).There was no significant difference between mimics-NC and inhibitor-NC groups.Therefore, the follow-up tests were analyzed with mimics-NC and inhibitor-NC as the control of miR-188 mimics and miR-188 inhibitor, respectively.Cell proliferation test and cell scratch test showed that compared with mimics-NC group miR-188 significantly reduced the proliferation rate and migration rate of 4T1 cells (P<0.05).Cell apoptosis test results showed that compared with mimics-NC group miR-188 mimics significantly promoted 4T1 apoptosis (P<0.05);Compared with inhibitor-NC group, miR-188 inhibitor significantly inhibited apoptosis (P<0.05).Western blotting results showed that miR-188 mimics significantly decreased the expression of matrix metalloproteinase-9 (MMP-9) protein (P<0.05), significantly increased the expression of adenosine diphosphate ribose polymerase (PARP1), cysteine protease-3 (caspase-3) and Bcl-2-associated X protein (Bax), and significantly inhibited the expression of anti-apoptotic protein B lymphoma-2 (Bcl-2) (P<0.05).【Conclusion】 miR-188 could significantly inhibit the proliferation and migration of 4T1 cells and promote the apoptosis of 4T1 cells, indicating that miR-188 could be used as a tumor regulator to inhibit the progression and metastasis potential of breast cancer.

【Objective】 The purpose of the experiment was to screen the best kinds of carbon source, nitrogen source and inorganic salt of Qingfei granule solid-state fermentation medium, optimize the composition of Qingfei fermentation medium and improve the content of epigoitrin in the formula.【Method】 A high performance liquid chromatography (HPLC) method was established for the determination of epigoitrin in fermented products.The carbon source (glucose, sucrose, mannitol, maltose, corn flour, soluble starch and herb), nitrogen source (peptone, soybean powder, bran, yeast extract, ammonium sulfate, ammonium chloride and urea) and inorganic salt(calcium carbonate, potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate and sodium chloride) components of Qingfei granule solid-state fermentation medium were screened by single factor test.The content of epigoitrin in each gram of solid-state fermentation medium was determined by HPLC.Four factors (carbon source, nitrogen source, inorganic salt and water) and three levels response surface analysis was used to optimize the proportion of each medium component.The selected optimal medium component was used to replace the corresponding substances in the basic medium.The fermentation bacterium Bacillus amyloliquefacines was inoculated and cultured at 37 ℃ and 130 r/min for 22 h.Then the content of each component of the medium was analyzed by response surface analysis with the content of epigoitrin in each gram of solid fermentation culture as the evaluation index.【Result】 The optimum solid-state fermentation medium for Qingfei granules was traditional Chinese medicine powder, inorganic salt was calcium carbonate, and nitrogen source was soybean powder.The content of each medium optimized by response surface analysis was 30% traditional Chinese medicine powder, 10% of soybean powder, 0.2% of CaCO₃ and 59.8% of water.At this time, the content of epigoitrin in solid-state fermentation was 0.0184 mg/g.However, the content of epigoitrin in the prescription of Qingfei granules before optimization was 0.0150 mg/g, with extremely significant difference (P<0.01), it indicated that the fermentation process of Bacillus amyloliquefaciens was more conducive to the release of effective components in the prescription of Qingfei granules.【Conclusion】 The optimized solid-state fermentation substrate was easy for the growth of Bacillus amyloiquefaciens.

【Objective】 The effects of silver nanoparticles (AgNPs) on the in vitro antibacterial activity and biofilm formation of multidrug resistant Streptococcus suis were studied to provide reference for solving the drug resistance problem of Streptococcus suis.【Method】 Five clinically isolated multi-drug resistant Streptococcus suis (S1018, S894, S786, S815 and S844) were used as the research objects.The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of AgNPs against Streptococcus suis were determined by micro broth dilution method.The antibacterial activity of AgNPs was evaluated by measuring the growth rate of Streptococcus suis under the action of AgNPs and the inhibition rate of different concentrations of AgNPs on Streptococcus suis.Furthermore, the biofilm formation ability of each strain and the effect of AgNPs with concentrations of 6.25, 12.5, 25 and 50 μg/mL on biofilm formation of 5 multi-drug resistant Streptococcus suis strains were evaluated by crystal violet staining.【Result】 The MIC and MBC of AgNPs against S1018 strain were both 12.5 μg/mL, and the MIC and MBC of AgNPs against the other four multi-drug resistant Streptococcus suis strains were all 25.0 μg/mL.AgNPs had good antibacterial activity against multi-drug resistant Streptococcus suis.The inhibition rate of AgNPs on 5 strains of Streptococcus suis could reach 96.60%-99.70% at the concentration of 25-100 μg/mL, and the growth rate of bacteria was significantly inhibited under the action of 1/2MIC, 1MIC and 2MIC AgNPs.5 strains of multi-drug resistant Streptococcus suis could form biofilms, and the inhibition rates of AgNPs at concentrations of 6.25, 12.5, 25, 50 μg/mL on their biofilm formation were 17.98%-45.58%, respectively.【Conclusion】 AgNPs inhibited the activity and biofilm formation of multi-drug resistant Streptococcus suis.

【Objective】 The aim of this experiment was to study the antibacterial activity of licochalcone A against Pseudomonas aeruginosa isolated from mink, and to provide experimental basis for the rational use of licochalcone A on the infective treatment clinically.【Method】 The minimal inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of licochalcone A against Pseudomonas aeruginosa were determined by microbroth dilution method and plate counting method, respectively.The germicidal efficacy of 1MIC, 2MIC, 4MIC and 8MIC of licochalcone A against Pseudomonas aeruginosa was determined by plate counting method.The effect of 1/16MIC, 1/8MIC, 1/4MIC and 1/2MIC of licochalcone A on the growth of Pseudomonas aeruginosa was determined by absorbance method.The crystal violet staining was used for the determination of the biofilm inhibition with 1/8MIC, 1/4MIC, 1/2MIC, and 1MIC of licochalcone A and the biofilm clearance with 1/4MIC, 1/2MIC, 1MIC and 1MBC of licochalcone A.The model of skin wound and skin abscess were established in mice to evaluate the therapeutic effect of licochalcone A on Pseudomonas aeruginosa infected mice.【Result】 Licochalcone A showed a good antibacterial activity against Pseudomonas aeruginosa from mink.The MIC and MBC values were 3.125 and 12.5 μg/mL, respectively.The results of time-bactericidal curve showed that the number of Pseudomonas aeruginosa was decreased gradually in 2MIC and 1MIC groups within 6 hours, and then remained at about 1×10⁵ CFU/mL, while in 8MIC and 4MIC groups the number of Pseudomonas aeruginosa was in a downward trend, and no colonies were formed after 14 and 16 hours, respectively.Licochalcone A with 1/16MIC had little effect on the growth of Pseudomonas aeruginosa, but 1/8MIC, 1/4MIC and 1/2MIC could inhibit the growth of Pseudomonas aeruginosa.The inhibitory rate of licochalcone A with 1MIC on biofilm formation of Pseudomonas aeruginosa was about 70%, and 1MBC could clear about 50% of preformed biofilm.The results of in vivo experiments showed that licochalcone A could promote wound healing and reduce the formation of skin abscess in mice infected with Pseudomonas aeruginosa.Compared with the control group, the number of bacteria in abscess in licochalcone A and ampicillin groups was extremely significantly decreased (P<0.01).【Conclusion】 Licochalcone A had good antibacterial activity against Pseudomonas aeruginosa isolated from mink in vitro and in vivo, and could be further used in the development of the preparation for clinical treatment of Pseudomonas aeruginosa infection.

Pruritus is one of the typical clinical symptoms of dermatosis in canine, which is usually accompanied by other skin symptoms such as alopecia and erythema.The pruritus not only troubles the animal itself, but also affects the life quality of the pet-owner.The mechanism of itching is complex.At present, there have been a large number of clinical and basic researches on pruritus diseases in humans and dogs at home and abroad, revealing the relationship between pruritus and the neuroimmune system, and introducing the concept of "pruritus-scratching" cycle, and indicating that the independent effects of the immune system, the skin barrier and the nervous system and the interaction between them are the key factors in pruritus, and any problem in them can start the "pruritus-scratching" cycle.The clinical etiology of pruritic skin diseases in canine is complex.Refering to the the classification of pruritus which was proposed by International Forum for the Study of Itch in 2007, the diseases that cause pruritus are divided into six categories correspondingly according to the etiology, and the diseases which can cause dermatologic pruritus are divided infectious diseases, allergic diseases, neoplastic diseases, and the most important in clinic is canine allergic pruritus.The diagnostic methods and differential diagnosis of canine pruritic skin diseases are diverse, and the information collected from various aspects of the sick dogs are needed.The differential diagnosis are carried out step by step in a certain sequence.There are still problems in the treatment of canine pruritic skin diseases, such as long treatment cycles and easy recurrence of the disease.At present, the commonly used western medicine drugs have disadvantages such as large side effects, single target and high price, and the Chinese medicine prescription which has complex components has many effective components, so it can treat pruritus with complex mechanism through multiple channels and multiple approaches, which has advantages and broad prospects in the treatment of canine pruritic skin disease.This article reviewed the latest pruritus mechanism, classification and diagnosis of canine pruritic skin diseases, and Chinese and Western medicine treatment ideas, so as to provide a reference for the diagnosis and treatment of clinical pruritus-related diseases in small animals.