【Objective】 Differential codon usage bias are prevalent in genomes of almost all species.In order to provide a basis for Camelus ferus genome research and codon optimization,the codon usage bias and influencing factors of Camelus ferus genome were analyzed.【Method】 The codon usage characteristics and influence factors of protein-coding genes of Camelus ferus genome were analyzed by various methods such as bioinformatics analysis,ENC-plot,Neutrality-plot and PR2-plot analysis.Meanwhile,the codon preferences between Camelus ferus and other species were analyzed.【Result】 The GC content was higher than AT content in the coding region of Camelus ferus genome,and the codon terminal bases were preferred to G/C.There were 29 codons with a relative synonymous codon usage (RSCU) greater than 1,of which 14 ended in C and 9 ended in G.The results of ENC-plot,Neutrality-plot and PR2-plot analysis showed that the codon usage bias were influenced by both natural selection and mutation,and the main factor was natural selection.Fiftheen optimal codons mainly ended with C or G were identified,including AUC,GGC,CUG,GUG,GCC,UCC,CCC,AGA,ACC,UAC,CAC,AGC,UUG,CAG and CGG.The codon usage bias of Camelus ferus, Camelus dromedarius and Mus musculus was relatively close.【Conclusion】 The codon usage bias in Camelus ferus genome was influenced by both natural selection and mutation,with natural selection played a greater influence.The results would provide reference and guidance for the protection and utilization of Camelus ferus genetic germplasm resources,genetic engineering and efficient expression of heterologous genes.

【Objective】 This study was aimed to clone uncoupling protein-1 (UCP1) gene in Nubian goats and carried out bioinformatics analysis,and detect its expression difference of UCP1 gene in different tissues of Nubian goats,in order to provide data for studying its regulatory function in fat metabolism and analyzing the function of UCP1 gene in Nubian goats.【Method】 Using the cDNA from the subcutaneous fat tissue in Nubian goats as a template,the CDS region of UCP1 gene in Nubian goats was amplified by PCR and cloned,sequence similarity alignment and phylogentic tree contruction were carried out with other species,and the bioinformatics analysis of UCP1 protein was carried out.The expression of UCP1 gene in heart,liver,spleen,kidney,longissimus dorsi muscle,subcutaneous fat and abdominal fat of Nubian goats were detected by Real-time quantitative PCR.【Result】 The CDS sequence of UCP1 gene in Nubian goats was 918 bp and encoded 305 amino acids.The similarity of amino acid sequence of UCP1 gene between Nubian goats and Ovis aries, Bos indicus× Bos taurus, Bubalus bubalis, Oryx dammah, Cervus elaphus, Canelus ferus, Equus asinus, Aliuropoda melanoleuca and Homo sapiens were 98.1%,97.0%,96.5%,96.1%,95.8%,91.0%,87.0%,86.5% and 83.8%,respectively.The phylogenetic tree indicated that the closest was to Ovis aries and the furthest was to Homo sapiens.The results of bioinformatics analysis showed that the molecular mass of UCP1 protein in Nubian goats was 32.97 ku,with an isoelectric point of 9.29,which was a basic proteins.UCP1 protein lacked stability,an overall average hydrophilicity was 0.20,which possessed certain hydrophilicity.The lipid solubility coefficient was 91.70,the predicted value of the number of amino acid residues in transmembrane helices was 15.94,the predicted value of the number of transmembrane helices in the first 60 amino acids of the protein was 8.76,the overall probability of being located on the cytoplasmic side of the membrane was 29.03%,and it belonged to nonsecretory proteins.The secondary structure was composed of alpha helix (48.85%),random coil (27.54%),extended chain (16.39%),and beta turn (7.21%).UCP1 had a total of 28 phosphate sites,5 potential O-glycosylation sites and 2 N-glycosylation potential sites.Real-time quantitative PCR results showed that the expression of UCP1 gene in subcutaneous fat of Nubian goats was relatively high,which was significantly higher than other tissues (P＜0.05),while the expression of longissimus dorsi muscle in Nubian goats was the lowest.【Conclusion】The CDS sequence of UCP1 gene was successfully amplified,UCP1 was a basic protein lacking stability and hydrophilicity. UCP1 gene was widely expressed in various tissues of Nubian goats, which was mainly expressed in subcutaneous fat and spleen.The results provided a theoretical basis for subsequent research on the mechanism of UCP1 gene in the energy supply of fat thermogenesis.

【Objective】 To study the regulatory effect of fatty acid desaturases 1 (FADS1) gene on the metabolism of unsaturated fatty acids, and provide a reference for revealing its molecular mechanism. 【Method】 The FADS1 gene was overexpressed in the primary fetal fibroblasts isolated by vector pcDNA3.1-FLAG-FADS1, which was designed according to the CDS sequence of FADS1 gene. The constructed vector was transferred into the cells and screened by geneticin (G418) to obtain monoclonal cell lines. After transfection, cells were collected at 24 and 48 h respectively, and the expression of FADS1 gene was detected by Real-time PCR and western blotting, and the cell line with stable expression of FADS1 gene was obtained after G418 screening, named 1-4^#. These cells were used to detect the change of triglyceride content and analysis of unsaturated fatty acid composition through GC-MS.【Result】 The results showed that the overexpression vector pcDNA3.1-FLAG-FADS1 was successfully obtained, and the overexpression effect was detected at both the transcription level and the protein level. Then these cells were cultured in complete medium with G418 to obtain stable cell lines. Triglyceride assay showed that FADS1 gene overexpression could reduce the content of triglyceride in primary fetal fibroblasts (P＜0.05). The test results of unsaturated fatty acids showed that after overexpression of FADS1, the content of total fatty acids increased extremely significantly (P＜0.01) and increased by 1.8 times. In polyunsaturated fatty acids (PUFA), the contents of ω-3 PUFA and ω-6 PUFA were extremely significantly increased (P＜0.01), the relative contents of eicosapentaenoic acid(EPA), docosapentaenoic acid(DPA) and docosahexaenoic acid(DHA) were extremely increased (P＜0.01), and the ratio of ω-3/ω-6 was significantly increased (P＜0.05).【Conclusion】 In summary, the overexpression vector pcDNA 3.1-FLAG-FADS1 was successfully constructed, and a single clonal cell with stable hyperexpression of FADS1 gene was successfully screened in primary fetal fibroblasts, FADS1 gene could significantly increase the content of ω-3 PUFA and increase the ratio of ω-3/ω-6.

In recent years,with the popularity and cost reduction of RNA-sequencing (RNA-Seq) technology,it is increasingly used in animal science research.Several genes in blood are closely related to diseases and involved in immune,stress,metabolism and disease-related functions.For example,syntaxin 1A (STX1A) gene may be a molecular target for predicting ketosis.Blood RNA-Seq can predict blood marker related to the diagnosis of early pregnancy,such as preterm birth,recurrent fertilization failure and abortion (for example,lysophosphatidic acid receptor 3,LPAR3).The use of blood RNA-Seq to identify reliable predictors of feed efficiency is a strategy to reduce the collection of phenotypic data,which could help the field of nutritional regulatory genomics in livestock.There are markers related to growth traits and performance of livestock in blood.For example, CD48 gene is a key gene regulating the growth of beef cattle.Blood is an important line of defense of the immune system,which can transport various immune active substances and immune cells.The analysis of blood RNA-Seq provides an overview of systemic immunity. Interleukin 1 receptor type Ⅱ (IL1R2) is a major gene of immunomodulation in the wild state.Studies on the application of RNA-Seq in mammalian blood,especially in livestock,show that blood is rich in information about health status,which is expected to replace pathological tissue sampling to study the molecular characteristics of different physiological conditions and predict diseases.The genes expressed in the blood of animals with different dietary nutrition levels are different,which is helpful for the development of the field of nutritional regulatory genomics in livestock.In conclusion,the minimally invasive diagnosis of blood RNA-Seq is not only helpful to solve the molecular regulatory mechanism of different physiological states of animals,but also helpful to the application of molecular marker-assisted management of livestock in the future above these studies,which has the possibility of becoming a candidate practical biological sample.

【Objective】 The aim of this study was to study the mechanism of trypanosome invasion into host cells and to lay the foundation for the establishment of detection methods.【Method】 The isolated and preserved Trypanosoma evansi was used for culture propagation in Kunming White mice,and its paraflagellar fasciculus rod (PFR) gene was cloned and a phylogenetic tree was constructed.The physical and chemical properties,hydrophobicity,transmembrane region structure,secondary structure and tertiary structure of PFR protein were analyzed and predicted by bioinformatics methods.The prokaryotic expression vector pET28a-PFR was constructed,and the reactogenicity of PFR recombinant protein was detected by Western blotting.【Result】 The Yili strain of Trypanosoma evansi was successfully expanded in Kunming White mice,and the highest infection rate was reached on the 5th day.The PCR amplified fragment size of PFR gene was 834 bp,and the similarity with PFR gene of Trypanosoma brucei gambiense (XP_011775815.1) was 99.52%,and the phylogenetic tree based on the amino acid sequence of the PFR protein also showed that the strain had the closest genetic relationship with Trypanosoma brucei gambiense.The molecular formula of PFR protein was C₁₄₁₆H₂₂₈₆N₄₁₆O₄₄₂S₁₁,and the theoretical isoelectric point was 5.74.PFR protein was an alkaline,hydrophilic and unstable protein,without transmembrane region and signal peptide,and had 10 potential antigen epitopes,which were mainly located in cytoplasm.The secondary structure of PFR protein was mainly composed of alpha helix (92.34%),the tertiary structure was consistent with the secondary structure.The expression plasmid pET28a-PFR of PFR gene of Trypanosoma evansi was successfully constructed.After optimization of induction time,temperature and IPTG concentration,it was found that when IPTG concentration was 1 mmol/L and recombinant bacterial solution was induced at 37 ℃ for 6 hours,PFR protein expression was the highest and expressed in the form of inclusion body.Western blotting results showed that the recombinant protein reacted positively with Trypanosoma evansi positive serum.【Conclusion】 In this experiment, Trypanosoma evansi was successfully propagated in Kunming White mice,the PFR gene of Trypanosoma evansi was cloned,the prokaryotic expression vector of PFR was constructed,and the recombinant protein of PFR was induced to express.The protein had good reactivity,which provided a theoretical basis for the subsequent research on the function of the protein and the pathogenesis of Trypanosoma evansi.

Single-cell RNA sequencing (scRNA-seq) technology is used to sequence the transcriptome at the single cell level,which can describe samples from multiple perspectives such as expression amount,cell size and cell composition,mainly including single cell isolation,mRNA reverse transcription,library construction,transcriptome sequencing and data analysis.Single cell isolation is the first step of this technology. Common cell isolation methods include serial dilution,micromanipulation,fluorescent activated cell sorting,laser capture microdissection and microfluidics.Many transcriptome sequencing methods based on different cell capture,cDNA amplification and library construction have been developed,such as CEL-seq2,Drop-seq,MARS-seq,SCRB-seq,Smart-seq,Smart-seq2,etc.Compared with the traditional sequencing method,scRNA-seq can identify the gene expression information of a single cell,record the spatial location of the cell,and track the trajectory of different cell lineages in the differentiation process,which brings great convenience for studying the molecular specificity of intramuscular adipocytes that are difficult to extract in large quantities and the occurrence and differentiation process.The author briefly introduced the key steps of scRNA-seq technology,compared and analyzed the advantages and disadvantages of single cell isolation and transcriptome sequencing methods,reviewed the origin of intramuscular adipocytes,and the application of scRNA-seq in the identification of subgroups and marker genes of intramuscular adipocytes,as well as the tracking of cell differentiation trajectory,in order to provide a reference for the further study of intramuscular adipocytes.

Neurodegenerative disease is a central nervous system disease based on the progressive degeneration and necrosis of neurons,which is commonly characterized by the accumulation of misfolded proteins and mitochondrial damage.As the center of cellular energy production,mitochondria are the main energy source of neurons and are essential for maintaining the structure and function of neurons.Damaged mitochondria lead to inadequate ATP supply and oxidative stress damage,and even resulte in cellular death.Mitophagy is a process by which cells selectively remove senescent or damaged mitochondria through the autophagy-lysosome pathway.It is an important part of mitochondrial quality control mechanism and plays an important role in maintaining cellular homeostasis.Many studies have suggested that mitophagy is closely related to the occurrence and development of neurodegenerative diseases.Activation of mitophagy or improvement of mitophagy abnormalities can alleviate neuronal damage caused by misfolded protein accumulation.In this paper,the author reviewed the mechanism of mitophagy,its regulation and its role in the occurrence and development of neurodegenerative diseases,which was in order to provide reference for the research and treatment of neurodegenerative diseases.

【Objective】 This experiment was conducted to study the effects of chicory on growth performance,slaughter traits and meat quality of Mahuang broilers with different sex.【Method】 Five hundred and seventy-six Mahuang broilers at 42-day-old were randomly divided into 8 groups with 6 replicates in each group and 12 broilers per replicate (half male and half female).A 2×4 (sex×chicory) two-factor design was used,the chicory addition levels were 0,3%,6% and 9%,respectively.The experiment lasted for 42 days.At the end of the test,the growth performance indexes were statisticed,and one broiler in each replicate was slaughtered for the measurement of relevant indexes of slaughter and immune organs,and samples of breast and leg muscles were taken for meat quality analysis.【Result】 ①Compared with no chicory,adding 6% and 9% chicory could significantly increase the F/G of Mahuang broilers (P＜0.05).The growth performance of male was significantly better than that of female (P＜0.05).②The Bursa of Fabricius index of female was significantly higher than that of male (P＜0.05).③The eviscerated rate of male was significantly higher than that of female (P＜0.05),while the abdominal fat rate was significantly lower than that of female (P＜0.05).④Compared with no chicory,adding 6% and 9% chicory could significantly improve the breast muscle b_24h^* of Mahuang broilers (P＜0.05).The breast muscle L_24h^* of female was significantly higher than that of the male (P＜0.05).⑤Sex and chicory level had significant interaction on the leg muscle shear force of Mahuang broilers (P＜0.05),and adding 6% and 9% chicory could significantly decrease the leg muscle shear force of male compared with no chicory (P＜0.05).Compared with the female,the a_45min^*,a_24h^* and b_45min^* of male leg muscle were significantly higher,while the L_45min^* and L_24h^* were significantly lower (P＜0.05).Sex and chicory level had no significant interaction on growth performance,immune organ index,slaughter performance and breast muscle quality of Mahuang broilers (P＞0.05).【Conclusion】 The growth performance,slaughter performance and muscle quality of male were better than those of female. Adding 6% and 9% chicory both could improve the meat quality of Mahuang broilers while have adverse effects on the F/G.According to the evaluation of various indicators,the optimum addition amount of chicory was 3%.

Resveratrol (RES) is a natural non-flavonoid polyphenol compound that exists widely in various plant tissues,such as grapes,tab wraps,and peanuts,it is an important substance that plants resist the invasion of pathogenic microorganisms and maintain their stability,so it is also called plant antitoxin.RES has good antioxidant,anti-inflammatory,and antibacterial characteristics,and plays an important role in improving animal resistance,improving immune capacity,and maintaining intestinal health.RES can promote the expression of nuclear factor E2-related factor 2 (Nrf2) in livestock and poultry by regulating Nrf2-Kelch-like oxide propylene-1 (keap 1),and improve antioxidant factor expression and antioxidizes activity,relieves oxidation stress,reduces the level of inflammatory factor expression and the inflammatory response by regulating nuclear factor-κB (NF-κB) signaling pathway.In terms of animal intestinal health,RES can promote the proliferation and differentiation of intestinal epithelial cells by regulating the Wnt/β-serial protein (Wnt/β-catenin) signaling pathway,improving intestinal mucosal barrier integrity,promoting intestinal microbial planting,and then improving the intestinal flora structure.As a natural supplementation,RES can improve the growth performance and growth potential of animals,and improve the quality of livestock and poultry products.At the same time,RES can enhance the immune function of animals,improve their ability to resist pathogens,and reduce the incidence of disease.At present,RES has been widely used in the production of livestock and poultry,but it still needs to be further understood by its specific mechanism.The authors introduced the specific mechanism of RES regulating Nrf2-Keap1,NF-κB and Wnt/β-catenin signaling pathways,and its application prospects in livestock and poultry production,in order to provide a theoretical basis for the in-depth application of RES in animal production.

【Objective】 This study was aimed to analyze the effect of Clostridium butyricum on the composition of plasma free fatty acid (FFA) in mice with abnormal lipid metabolism caused by high choline diet,so as to clarify the mechanism of Clostridium butyricum regulating lipid metabolism in high choline diet.【Method】 24 healthy male Kunming mice aged 4 weeks were randomly divided into 3 groups:Normal,model and Clostridium butyricum groups,with 8 mice in each group.After 8 weeks of high choline diet,7 d of administration and 12 h of fasting,the plasma,liver,epididymal fat pad and perirenal fat were collected.Weighing the fat and liver,the blood lipid was detected by biochemical analyzer.HE staining and oil red O staining were used to observe the changes of liver tissue structure and the deposition degree of lipid droplets.The content of trimethylamine oxide (TMAO) in plasma was detected by liquid mass spectrometry (LC-MS) technology.The composition of FFA in plasma was studied by gas chromatography-mass spectrometry (GC-MS) technology and multivariate statistical analysis.【Result】 Compared with model group,the body weight and fat growth in Clostridium butyricum group were effectively inhibited (P＜0.05).After Clostridium butyrate administration,the deposition of liver lipid in mice was decreased. Clostridium butyrate significantly or extremely significantly decreased the contents of plasma triglyceride (TG) and low density lipoprotein (LDL-C) (P＜0.05 or P＜0.01),and significantly increased the content of high density lipoprotein (HDL-C) in mice fed with high choline diet (P＜0.05).After administration of Clostridium butyricum,the plasma TMAO concentration in mice fed with high choline diet was significantly decreased (P＜0.05).In Clostridium butyrate group,the contents of saturated fatty acids (SFA) such as palmitic acid (C16∶0) and stearic acid (C18∶0) were significantly decreased,the contents of palmitoleic acid (C16∶1) and oleic acid (C18∶1 N9C) in monounsaturated fatty acid (MUFA) were significantly decreased,the content of n-3 type polyunsaturated fatty acids (PUFA) was significantly increased,the content of docosahexaenoic acid (DHA) was significantly increased,and the contents of linoleic acid (C18∶2 N6C) and gamma linolenic acid (C18∶3 N6) in n-6 type PUFA were significantly decreased (P＜0.05).In addition, Clostridium butyrate significantly decreased the contents of trans isooleic acid (C18∶1 N9t) and trans linoleic acid (C18∶2 N6t) in plasma (P＜0.05).【Conclusion】 Clostridium butyrate could regulate FFA composition and plasma TMAO content in mice fed with high choline diet,and improve lipid metabolism disorder.

【Objective】 This experiment was conducted to study the effects of Chinese herbal medicine feed additives on the nutritional composition,the residues of heavy metal and antibiotic,and the content of flavor substances of meat from different parts of Yanbian Yellow cattle.【Method】 A total of 100 healthy 27-month-old Yanbian Yellow cattle with similar body weight were randomly divided into two groups with 50 cattle in each group.A basal diet was served to control group,while a basal diet with 0.2% Chinese herbal medicine feed additives was served to experimental group.The pre-experimental period lasted for 10 days,and the experimental period lasted for 90 days.At the end of the experiment,10 cattle from each group were randomly selected for slaughter,the muscle samples were taken from five parts of beef,including brisket,ribeye,striploin,chuck and tenderloin,the conventional nutrients,amino acid,fatty acid,shear force,meat color,heavy metals,antibiotics,flavor substances and other indexes of beef were determined.【Result】 Compared with control group,the addition of Chinese herbal medicine feed additives in diet had no significant effect on the content of crude protein,crude fat,crude fiber,calcium and phosphorus of beef conventional nutrients (P＞0.05),but could increase the content of total amino acids and the total content of umami amino acids,sweet amino acids and bitter amino acids in beef,and increase the content of palmitoleic acid and linolenic acid in beef,but the difference were not significant (P＞0.05).Compared with control group,the shear force of brisket in Yanbian Yellow cattle in experiment group was significantly decreased (P＜0.05), the redness value of ribeye and tenderloin was significantly increased (P＜0.05),and the contents of nitrogen oxides and inorganic sulfides in striploin,ribeye and brisket were significantly reduced (P<0.05). The residues of heavy metals and antibiotic were not detected in beef of experiment and control groups.【Conclusion】 Chinese herbal medicine feed additives could improve the tenderness of brisket and the meat color of five parts of beef in Yanbian Yellow cattle,and increase the content of flavor substances in tenderloin and chuck.But there was no significant effect on the content of fatty acid and amino acid of beef.Additionally,the meat products won’t be harmed.The results provided a theoretical basis for the application of Chinese herbal medicine feed additives to improve the quality and safety of meat from Yanbian Yellow cattle.

【Objective】 The aim of this study was to investigate the effect of transport stress on the intestinal flora of horses,and to explore the preventive effect of Lactobacillus acidophilus on transport stress in horses.【Method】 For the experiment,ten 24-month-old Ili horses were chosen and randomly divided into test group and control group.After all horses had been acclimatized for one week,the test group was formally fed Lactobacillus acidophilus capsules in their diets,while the control group was fed the diets without any adjustment.After 15 d of feeding,the test group and the control group were transported at the same time on day 16 over a distance of about 400 km.The jugular blood of the control group was collected before and after the transportation for physiological indicators detection,and the horse feces were collected before feeding,15 days after feeding and 8 hours after transportation.The sample before feeding the bacteria in the test group was set as group A1,the sample after feeding the bacteria for 15 d was set as group A2,the sample after transportation for 8 h was set as group A3,and the sample after transportation for 8 h in the control group was set as group T3.Analyzing the variety of equine intestinal flora involved high-throughput sequencing on the Illumina MiSeq platform.【Result】 Blood physiological indices before and after transport in the control group showed that the intermediate cell ratio and hemoglobin were significant decreased (P＜0.05),and the hemoglobin concentration was extremely significantly decreased (P＜0.01) after 8 h of transport.Supplemental feeding of Lactobacillus acidophilus and transportation could cause differences in the composition of intestinal flora in horses.Supplemental feeding of Lactobacillus acidophilus increased the population of helpful bacteria,including Rikenellaceae_RC9_gut_group and Lachnospiraceae_AC2044_group,while transport increased the quantity of harmful bacteria,including Bacteroidetes and Proteobacteria.【Conclusion】 Transportation could cause stress reaction of horses.Adding Lactobacillus acidophilus to the diet could restore the intestinal flora disorder of horses caused by transportation stress,and increase a small amount of beneficial bacteria,so as to alleviate the flora disorder caused by transportation and had a beneficial impact on the body.

【Objective】 The effects of different doses of Zanthoxylum bungeanum essential oil (EOZB) on the production performance and gastrointestinal tissue structure of Small-tail Han sheep (STH) were measured and analyzed in this study,in order to provide a theoretical reference for the application of EOZB in ruminants.【Method】 Twenty 3-month-old STH with similar initial body weight were randomly divided into four groups:control group (CON) and experimental groups Ⅰ,Ⅱ and Ⅲ.The control group was fed the basal diet,while experimental groups Ⅰ,Ⅱ and Ⅲ were supplemented with 5,10 and 15 mL/kg of EOZB in the basal diet.The test period was 52 d.At the end of the test,all test sheep were slaughtered and gastrointestinal tissue samples were collected from 10 sites (rumen,reticulum,omasum,abomasum,duodenum,jejunum,ileum,cecum,colon and rectum).The effect of EOZB on the gastrointestinal tract organization of STH was systematically evaluated by production performance and gastrointestinal papilla length,papilla width,submucosal thickness,muscle thickness,intrinsic membrane thickness,stratum corneum thickness,villus length,villus width,crypt depth and other indicators.【Result】 ①EOZB could improve the productive performance of STH. Compared with the control group, the average daily gain, dry matter intake and F/G of EOZB10 group were significantly increased, and significantly higher than the other two test groups (P＜0.05). ②EOZB could promote the development of stomach on STH. Compared with the control group, the thickness of ruminal submucosa and muscularis increased significantly (P＜0.05), the thickness of corneum decreased significantly (P＜0.05), the length and width of reticulum and omasum papillae increased significantly (P＜0.05), the thickness of omasum submucosa, muscularis and lamina propria increased significantly (P＜0.05), and the thickness of abomasum mucosa and submucosa increased significantly (P＜0.05). In the three test groups, the above indexes in EOZB10 group were also higher than those in the other two groups (except for the thickness of rumen muscle layer, the width of reticulum papilla and the thickness of abomasum submucosa), and some indexes reached significant levels (P＜0.05). ③EOZB could promote small intestine development in STH. Compared with the control group, duodenal villus length, muscular layer thickness, jejunal villus width, recess depth and muscular layer thickness in EOZB10 group increased significantly (P＜0.05), and ileal villus length, villus width and villus length/recess depth increased significantly (P＜0.05). In the three test groups, the length and width of ileal villus in EOZB10 group were significantly higher than those in the other two groups (P＜0.05). ④EOZB could promote the development of large intestine in STH. Compared with the control group, the rectum muscular layer thickness and colon mucosal layer thickness in EOZB10 group were significantly increased (P＜0.05). In the three test groups, the rectum muscular layer thickness in EOZB10 group was significantly lower than that in EOZB15 group, and the colon muscular layer thickness and mucosal layer thickness were significantly lower than those in EOZB15 group, but significantly higher than those in EOZB5 group (P＜0.05). 【Conclusion】 Adding EOZB to the diet could improve the production performance and promote the development of gastrointestinal tissue structure in STH,under the conditions of this test,10 mL/kg of EOZB was the most effective.

【Objective】 This study was designed to explore the application effects of biotin in diet of laying ducks and its effects on ovarian development and tibia characteristics.【Method】 A single factor completely randomized experimental design was adopted.A total of 432 healthy Fujian Longyan ducks in peak laying period were randomly divided into 6 groups with 6 replicates in each group and 12 ducks in each replicate.A wheat-soybean meal based diet was adopted,and the dietary biotin supplemental levels were 0,0.05,0.10,0.15,0.20 and 0.25 mg/kg,respectively.The experiment lasted for 12 weeks. At the 12th week, 4 eggs and 2 test ducks were randomly selected from each repetition for blood sampling and slaughter sampling to detect egg quality, blood biochemical indicators, ovarian development and tibia traits.【Result】 ①Dietary biotin supplemental level had no effect on laying performance (P＞0.05).②When dietary supplemental biotin level was 0.10 mg/kg,the yolk weight was significantly lower than that of 0.05 and 0.20 mg/kg groups (P＜0.05). With the increase of dietary supplemental biotin level, the yolk cholesterol (CHO) content was on the rise (P＜0.05).Compared with the control group,the malondialdehyde(MDA) content in yolk was significantly decreased at 0.25 mg/kg biotin supplemental level (P＜0.05).③Dietary biotin supplemental level had no significant effects on plasma biochemical indexes,ovarian development and tibia characteristics of laying ducks (P＞0.05).【Conclusion】 Dietary biotin supplement had no effect on production performance,ovary development and tibia characteristics of laying ducks in peak period.When biotin supplemental level was 0.25 mg/kg,the degree of lipid oxidation in yolk was reduced,while cholesterol deposition in egg yolk was increased when it was 0.10 to 0.25 mg/kg.

【Objective】 The feasibility of fermented navel orange pomace for Muscovy duck breeding was explored by investigating the effects of fermented navel orange pomace on intestinal morphology,intestinal mucosal immune function and microbial diversity in Muscovy ducks.【Method】 A total of 180 Muscovy ducks (16-day-old) weighing (267.33±9.50) g were randomly divided into two groups (half male and half female in each group), 6 replicates each group,15 ducks each repetition.The control group was fed the basal diet,and the experimental group was supplemented with 100 g/kg fermented navel orange pomace in the basal diet.The prefeeding period was 7 days,and the formal experiment lasted for 48 days.At the end of the experimental period,the duodenal tissues and cecal contents were collected from Muscovy ducks.The duodenal tissue samples were fixed with 4% paraformaldehyde.The intestinal morphological indexes and intestinal mucosal immune function index were detected by HE staining and immunohistochemistry,respectively.High-throughput sequencing technology was used to sequence 16S rDNA of cecal contents from experimental Muscovy ducks to analyze the changes of cecal microbial diversity.【Results】 ①Compared to control group,100 g/kg fermented navel orange pomace could extremely significantly increased the villi height (P＜0.01),as significantly elevated surface area of villi and positive rate of intraepithelial lymphocyte (P＜0.05) in duodenum of Muscovy duck.②Differential analysis between two groups showed that 100 g/kg fermented navel orange pomace extremely significantly inhibited the abundance of harmful bacteria such as Deferribacterota,Anaerovoracaceae and Acholeplasma (P＜0.01),and extremely significantly increased the abundance of beneficial bacteria such as Flavonifractor, Helicobacter and Megamonas (P＜0.01) compared to control group.Results of function prediction showed that 100 g/kg fermented navel orange pomace clearly depressed metabolism of terpenoids and polyketides of KEGG pathways in microbes of the cecum(P＜0.05) compared to control group. 【Conclusion】 Fermented navel orange pomace could improve intestinal morphology and structure,enhance the mucosal immune function of intestine and regulate the intestinal microbial diversity and metabolism.

With the improvement of people’s living standard and the increase of dietary consumption demand,the livestock and poultry breeding industry has been able to develop rapidly,which resulted in the increasing shortage of domestic feed resources,especially protein resources.With the high production and low price of foreign protein raw materials,China’s feed industry is overly dependent on imports for production.In order to promote the balanced development of domestic and foreign feed trade,as well as to reduce feed costs and optimise feed production structure,finding and developing new protein feed resources is one of the difficult tasks that need to be solved urgently in the breeding industry.Insects are a kind of productive resource with rich protein content,which can replace some protein feed resources.Nowadays,insect feed resources are gradually becoming a research hotspot.As a kind of resource insects with wide application prospects, Hermetia illucens L.has the characteristics of fast reproduction,mixed feeding and strong resistance,rich in protein and fat,and contains active substances such as antibacterial peptides,chitin and lauric acid,which are often used in breeding production.The use of Hermetia illucens L.as a protein substitute in the diet can improve the production performance of poultry,product quality,intestinal health and immune function,and also have the role of resource utilization of poultry manure.The author reviewed the biological and nutritional characteristics of Hermetia illucens L.and its impact on poultry production and application,so as to provide a reference basis for further resource exploitation and research on the mechanism of action and standardised application of Hermetia illucens L.in poultry production.

Intestinal microorganisms,which is called the 'hidden immune organ’ of animals and is crucial to maintain body health,can not only participate in host metabolism,but also affect the host’s immune system.The author mainly introduced the development history of culturomics and its significance to the study of animal intestinal microorganisms,and the advantages and disadvantages of traditional microbial culture methods and molecular biology methods in the study of microorganisms. The culturomics is a new method for the isolation and identification of microorganisms which is based on traditional microbial culture methods and simultaneously uses multiple culture conditions to culture microorganisms,supplemented by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA (16S ribosomal RNA) gene sequencing technology to isolate and identify microorganisms.This method combines the advantages of traditional microbial culture technology and molecular biology technology.This method has the advantage of discovering,finding,and obtaining in the study of excavating 'new microorganisms’.In microbial studies,target strains can be customized for verification and the gut microbiome can be clearly understood through rich annotations.In addition,the author analyzes the research and application of culturomics in the intestines of poultry,pigs,ruminants and other animals,and puts forward the impact of environmental conditions on intestinal microorganisms,such as the impact of human contact on intestinal flora,and the differences between intestinal flora of the same species and different genders,hoping to provide reference for the research and application of culturomics in animal intestinal microorganisms.

【Objective】 The aim of this study was to evaluate the effects of fetal bovine serum (FBS) supplementation during postwarming culture on vitrified porcine parthenogenetic blastocysts.【Method】 The porcine parthenogenetic blastocysts on the 5th day of in vitro culture were used in this experiment,and the fresh and vitrified blastocysts were cultured in the embryo culture medium containing 10% FBS (V/V) for 48 h.This experiment was divided into four groups:Fresh,Fresh+FBS,Vitrified and Vitrified+FBS groups.The blastocysts expansion and hatching rates,the percentages of membrane damage and apoptosis and total cell number were determined.Moreover,the intracellular reactive oxygen species (ROS) levels,mitochondrial activity and expression levels of genes related embryo development in the blastocysts were detected.【Result】 Compared with the Fresh and Vitrified groups, the complete expansion rate, hatching rate and total number of blastocyst cells in the Fresh+FBS and Vitrified+FBS groups were significantly increased (P<0.05), while the cell membrane damage rate and apoptotic cell rate were significantly decreased (P<0.05). Compared with the Fresh group, ROS levels were significantly increased in the Vitrified group, while ROS levels were significantly decreased in both the Fresh+FBS and Vitrified+FBS groups (P<0.05). The mitochondrial activity of Vitrified+FBS group was significantly higher than that of Vitrified+FBS group and significantly lower than that of Fresh+FBS group (P<0.05), but there was no significant difference between Vitrified+FBS group and Fresh group (P>0.05). Compared with the Fresh group, POU class 5 homeobox 1(POU5F1) gene expression level in Vitrified group was significantly increased (P<0.05) and catalase (CAT)gene expression level was significantly decreased (P<0.05). There were no significant differences in the expression levels of proliferating cell nuclear antigen (PCNA), DNA methyltransferase 3A (DNMT3A), superoxide dismutase 1 (SOD1) and BCL2-associated X protein (BAX):BCL2L1 between the Fresh and Vitrified groups (P>0.05). The expression levels of PCNA, SOD1 and CAT genes in Fresh+FBS and Vitrified+FBS groups were significantly higher than those in Fresh and Vitrified+FBS groups (P<0.05).【Conclusion】 This study found that fresh and vitrified parthenogenes blastocysts were cultured in 10% FBS on the 5th day of in vitro culture, and their development ability and quality of embryo were significant improved.

Quantitative trait is an important trait in sheep breeding,which is controlled by micro-effect polygenes and has low heritability.However,traditional breeding methods are difficult to improve sheep breeding efficiency.Improving the efficiency of animal breeding is very important for breeding selection and economic production benefits.With the continuous innovation and development of new breeding technologies,genomic selection (GS) has become a powerful tool in breeding technology,and it has been successfully used in species with greater individual economic value.It has the advantages of shortening generation interval,improving breeding accuracy,reducing production cost and improving economic benefits of livestock and poultry.In recent years,as a result of the continuous maturity of genome technology and the upgrading and optimization of various statistical models,and lower high-density SNP chip prices,report about genome selective breeding of empirical and simulation studies emerge in endlessly,and the genome choose technology has been gradually develop in sheep breeding,especially in important trait of sheep have been many reports.However,due to the large number of sheep breeds,large differences in local traits and slightly low individual economic value,although the new technology of genome breeding has been very mature,it is still not widely used in sheep breeding.In order to have a more comprehensive understanding of the research status of this technology in sheep breeding,and based on the important position of selection and matching,the author reviews the research progress on genomic selection in sheep breeding,mainly summarized the application and current situation of genomic selection in sheep important traits from phenotyping,genotyping and different models,discussed its advantages and challenges,and looked forward to the future development direction of genomic selection.

Cryopreservation of oocytes and embryos can be combined with reproduction techniques such as superovulation and embryo transfer to break the limitations of physiology,region and time on the reproductive potential of female animals,which is of great significance for international circulation of germplasm,propagation of excellent domestic animals and conservation of germplasm of endangered animals.However,oocytes or embryos are prone to membrane damage,endoplasmic reticulum and mitochondrial damage and lipid peroxidation during cryopreserved process due to the abundant lipid content,which reduces their developmental ability after thawing and greatly limits the application of cryopreserved oocytes and embryos.Many researches have shown that reducing the lipid content of oocytes or embryos before cryopreservation is beneficial for improving the survival rate,blastocyst rate,and pregnancy rate of them after thawing.At present,there are three popular methods to reduce lipid content such as removing lipid droplets by micromanipulation after polarization by centrifugation,promoting lipid metabolism by adding chemicals,and regulating the expression of genes related to lipid metabolism.In this review,the mechanisms of damages caused by high lipid content in oocyte and embryo were briefly expounded,the principles,effects and limitations of methods that reduce the content of lipids by centrifugation or chemicals were summarized,and the possibility of lipid removal by regulating the expression of genes related to lipid metabolism was discussed.This review could provide some reference for the development of more stable cryopreservation technology of oocyte and embryo.

China has a long history of goat breeding and rich variety resources,which play an important role in the national economy.Wool can be divided into homogeneous fleece and heterogeneous fleece according to fiber type.The characteristic of homogeneous fleece is that the coat is composed of the same type of wool fibers,and its characteristics are that the fiber diameter,length,curl,and other appearance characteristics of wool are basically the same.The characteristic of heterogeneous fleece is that there are different types of wool fibers in the coat,such as down fleeces,medullary fleeces,and non-curly and oligo-curly fleeces.The chemical,physical and mechanical properties of wool fibers of heterogeneous fleece are different from those of homogeneous fleece. Hair follicle is an important organ with periodic development and lifelong regeneration,which controls the development and natural shedding of the hair.The hair follicles can be divided into primary follicles and secondary follicles according to their formation time and structure.The primary follicles develop subsequently developed coarse hairs,while secondary hair follicles produce unmyelinated villi.Being relatively thicker,longer,and less curved,the medullated wool is mainly used to process coarse textiles,blankets,carpets,and felt products.In contrast,non-medullated wool is an excellent raw material for the wool spinning industry because it is generally not more than 40 μm in diameter and has much curvature.Hair follicle development is affected by different genes and signaling pathways,such as Wnt signaling pathway,bone morphogenetic protein (BMP) signaling pathway,fibroblast growth factor (FGF) signaling pathway,sonic hedgehog(SHH) signaling pathway,Notch signaling pathway,keratin family genes, BMPs family genes,homologous box genes (Hox),etc.The author reviewes the literature on the development and regulation of hair follicles in the fetal skin of sheep,intending to provide a reference for the selection and breeding of sheep whose main breeding objectives are to improve coat yield and coat quality.

【Objective】 The purpose of this study was to prepare a safe and stable quantitative Real-time PCR positive control for African swine fever virus (ASFV).【Method】 The ASFV nucleotide sequence containing P72, CD2 v and MGF360-12 L gene fragments was synthesized,and the three gene segments were inserted in series into the Adenovirus vector PacAd5.The recombinant Adenovirus plasmid and Adenovirus skeleton plasmid were co transfected into 293A cells,and the expression of green fluorescent protein (EGFP) was observed by inverted fluorescent microscope.After 10 days of culture,the pseudovirus packaging was detected by PCR.The prepared pseudovirus particles were added with freeze-dried protective agent to prepare a positive control,and the homogeneity and stability tests were carried out,and the prepared positive control was subjected to the absolute quantification of the number of nucleic acid copies.【Result】 The 293A cells co-transfected with recombinant Adenovirus plasmid and Adenovirus skeleton plasmid showed spot fluorescence 24 h later,the cells showed a tendency to form island like infection area 7 days after transfection,and the cells showed obvious cytopathic effects such as pyknosis and abscission 10 days after transfection.The harvested pseudovirus liquid was detected by PCR and sequenced,and the result showed that it could amplify about 2 400 bp positive band,and the sequencing sequence was consistent with the inserted fragment.The homogeneity test showed that the coefficient of variation of Ct value of the positive control was less than 1%,indicating good homogeneity.Accelerated thermal stability test showed that the prepared positive control sample remained stable after being placed at 4 ℃ for 7 days,treated at room temperature (25 ℃) and 37 ℃ for 24 h respectively.The positive control still had good stability after DNase Ⅰ treated for 1 h.The storage period test showed that the prepared positive control could be stored stably for 6 months at －20 ℃.The prepared positive control was detected by ddPCR,and the result showed that the nucleic acid copy number of the prepared positive control pseudovirus solution was (2.26±0.09)×10⁴ copies/μL.【Conclusion】 The prepared ASFV pseudovirus positive control containing P72, CD2 v and MGF360-12 L gene fragments by using lentiviral packaging technology in this study were constructed successfully,which could be used for DNA extraction and molecular detection.

【Objective】 This study was aimed to monitor the current Porcine circovirus type 2 (PCV2) strains circulating in Hunan province and its capsid protein (Cap) variation,predict the cytotoxic T lymphocyte (CTL) epitopes,and provide some insights for the development of PCV2 novel vaccines and virus clearance.【Method】 In this study,PCV2 complete genome amplification,sequencing and phylogenetic analysis were performed on 17 PCV2 positive tissue samples collected from 6 regions in Hunan province from 2019 to 2021,and bioinformatic methods were used to analyze the amino acid variation of Cap and its CTL epitopes were predicted.【Result】 The phylogenetic tree showed that 1 strain of PCV2a,7 strains of PCV2b and 9 strains of PCV2d were obtained from the 17 PCV2 genome sequences.Multiple sequence alignments of Cap protein revealed that there were 16 mutated amino acid residues located on the capsid surface,and 11 residues were involved in the formation of conformational epitopes.Furthermore,among the predicted 9 potential CTL epitopes,epitopes 16－24,28－36,136－144 and 179－187 amino acids were highly conserved in 1 610 PCV2 genotypes from GenBank.The four conserved epitopes were further analyzed by the 3D structure of the TCR-pMHC complex,the results showed that all the four peptides could form stable TCR-pMHC complexes with MHC Ⅰ molecule and TCR.【Conclusion】 This study showed that PCV2b and PCV2d became the predominant genotypes in Hunan province with highly variability.These predicted CTL epitopes could be used as candidate epitopes.

【Objective】 The purpose of this study was to investigate the biological characteristics and antibacterial activity in vitro of a Lactobacillus plantarum GX20200417-1,and discuss the possibility of its application in production practice.【Method】 The GX20200417-1 strain was inoculated into PBS with different pH and different concentrations of bile salt,and artificial gastrointestinal juice,its tolerance to acid,bile salt and artificial gastrointestinal juice were analyzed by colony count. The antibacterial activities of Lactobacillus plantarum GX20200417-1 strain against Escherichia coli, Staphylococcus aureus and Salmonella were detected by Oxford cup method.The degradation ability of Lactobacillus plantarum to mycotoxin was studied by ELISA method after co-culture with mycotoxin.Finally,the mice were fed for safety test.【Result】 Lactobacillus plantarum GX20200417-1 strain could tolerate pH 2.0 and 0.3% bile salt,and had at least 1×10⁴CFU/mL viable bacteria in artificial gastrointestinal juice.The supernatant of Lactobacillus plantarum culture had obvious inhibitory effect on Escherichia coli, Salmonella and Staphylococcus aureus,it could degradation gibberellin and aflatoxin,but it has no effect on the degradation of vomiting toxin.The mice were safe without side effects after Lactobacillus plantarum GX20200417-1 strain gavage.【Conclusion】 Lactobacillus plantarum GX20200417-1 strain had excellent probiotic properties,and had certain development potential in livestock and poultry production.

【Objective】 The purpose of this experiment was to construct the PA-LF1 fusion gene expression vector of Bacillus anthracis (B. anthracis) attenuated C40-202 strain and to detect its prokaryotic expression and regenicity,so as to provide the basis for the preparation of anthrax subunit vaccine,the diagnosis of anthrax and the detection of vaccine immune effect.【Method】 Two pairs of primers were designed based on the sequences of B. anthracis PA and LF genes in GenBank,and the PA and LF1 gene fragments were amplified from B. anthracis weakly virulent strain C40-202 by PCR.After digestion by Xho Ⅰ restriction endonuclease,the PA-LF1 fusion gene fragment was obtained by connecting the sticky ends.SWISS-MODEL analysis software was used to predict the tertiary structures of PA,LF1 and PA-LF1 fusion proteins respectively.The fusion fragments were cloned into pET32a(＋) plasmid to construct recombinant plasmid pET32a-PA-LF1,and the recombinant plasmid pET32a-PA-LF1 was transferred into E.coli BL21(DE3) competent cells for induced expression.SDS-PAGE was used to analyze the expression of the recombinant protein,and Western blotting was used to identify the fusion protein and analyze its reactogenicity.【Result】 PA, LF1 and PA-LF1 fusion genes were successfully amplified from B. anthracis weakly virulent strain C40-202.The B. anthracis PA-LF1 fusion gene expression plasmid pET32a-PA-LF1 was successfully constructed,and the tertiary structure of the fusion protein was predicted to fold to the correct spatial conformation.The recombinant plasmid was induced,purified and detected by SDS-PAGE,and the fusion protein with molecular weight of 96 ku was obtained.Western blotting showed that the purified recombinant protein had specific binding with the sheep serum immunized with anthrax spore vaccine Ⅱ,indicating that it had good reactivity.【Conclusion】 The PA-LF1 fusion protein had good reactogenicity and could provide a theoretical basis for anthrax diagnosis and vaccine immunity testing,and also could be used as a candidate component for an anthrax subunit vaccine.

Avian influenza virus (AIV) has a huge impact on the development of animal husbandry and the safety of public health because of its characteristics such as strong variability,sorts of subtypes,diversity of infection hosts and so on.At present,although traditional inactivated vaccines play an important role in the prevention of avian influenza,there are still disadvantages such as immune failure,multiple vaccinations,easy occurrence of adverse reactions and so on.Therefore,it is very necessary to develop new vaccines to make up for the shortcomings of traditional vaccines.Nanoparticle vaccines have the advantages such as good encapsulation,stable structure,high targeting,strong immunogenicity and so on,which can be used as candidates for novel influenza virus vaccines.This paper first introduced the reasons for the difficulty in preventing and controlling avian influenza and the characteristics of nano-vaccine,and then reviewed the toxicity mechanisms of virus-like particle vaccines,self-assembled protein vaccines,polymer nanoparticle vaccines,inorganic nanoparticle vaccines and nanoparticles.Besides,it also summarized the research progress of AIV nanoparticle vaccine in recent years,and briefly described the advantages and disadvantages of using different antigens,applying different nanomaterials,and using different drug methods.Last but not least,it combined current studies on nano-vaccine to predict a new approach that nanoparticle vaccines could be used for influenza virus prevention and control in the future,and analyzed and prospected the application prospect of avian influenza nano-vaccine in veterinary medicine.

【Objective】 This study was aimed to investigate the immunomodulatory effects of prepared Radix Rehmanniae polysaccharide (PRRP) on cyclophosphamide (CTX)-induced immunesuppression in mice,and provide scientific evidence for the clinical use of PRRP on immune decrease diseases and development of dietary supplement.【Method】 Thirty six Kunming mice were randomly grouped into six groups:Blank control group,model group, Astragalus polysaccharide (APS) group,and three PRRP groups,which oral administer 50,100,200 mg/kg PRRP as PRRPL,PRRPM and PRRPH groups,respectively,six mice each group.Except normal control group,mice in other groups were given 80 mg/kg CTX via peritoneal injection to induce immunosuppression situation.After model was prepared,the mice were given drugs,once a day,for continually 15 days.The body weight,phagocytosis of peritoneal macrophage,immune organ index,tissue damage of thymus and spleen,splenic lymphocyte proliferation and cell cycle were measured,productions of cytokines in serum were determined,and Peyer’s patches (PPs) in intestinal were also counted.【Result】 Compared with blank control group,the index of spleen in model group was extremely significantly improved (P＜0.01),red and white pulp divided unclear,the number of splenic lymphocyte was decreased;The index of thymus was reduced,structure was destroyed with unclear cortex and medulla;The splenic lymphocyte proliferation,phagocytosis of peritoneal macrophage and interleukin-1 β (IL-1β),IL-6 and γ-interferon (INF-γ) in serum were significantly or extremely significantly decreased (P＜0.05; P＜0.01);The splenic lymphocytes were arrested in G0/G1 stage.Compared with model group,the spleen index in mice was extremely significantly decreased by PRRP (P＜0.01),the thymus and spleen structures were destroyed by CTX,the splenic lymphocyte proliferation was extremely significantly induced by advanced and lipopolysaccharide (LPS) (P＜0.01);The levels of IL-1β,IL-6,tumor necrosis factor α (TNF-α) and INF-γ in serum were significantly or extremely significantly improved (P＜0.05; P＜0.01);The splenic lymphocyte arrest was adjusted,and the phagocytosis of peritoneal macrophage in PRRPH group was significantly improved (P＜0.05).There were no significant changes of PRRP and before and after modeling on the number of PPs in intestinal (P＞0.05).【Conclusion】 PRRP could accelerate recovery of CTX-induced immunosuppression in mice,and could be used as drugs or dietary supplement for the treatment of immune decrease diseases.

【Objective】 In order to reveal the anti-inflammatory and antioxidation effects of Euphorbiae humifusae (EH) in swine intestinal epithelial cells (IPEC-J2).【Method】 Different concentrations of EH aqueous extract (0,5,10,50,125,200 μg/mL) were used to treat IPEC-J2 cells for 12 hours.The viability of IPEC-J2 cells was detected by CCK-8 method,determining the optimal concentration of EH treatment.IPEC-J2 cells were randomly divided into control group (CT),lipopolysaccharide group (LPS),EH+LPS group (ELP),with three replicates in each group.The cells in CT group were cultured in DMEM medium containing 10 % fetal bovine serum.The cells in LPS group were treated with 5 μg/mL LPS.The cells in ELP group were co-cultured with 5 μg/mL LPS and EH,cells in each group was treated for 12 hours,collecting the cells and supernatant.The expression of interleukin-6 (IL-6),tumor necrosis factor α (TNF-α),kelch like epichlorohydrin related protein 1 (Keap1),nuclear factor E2 related factor 2 (Nrf2),heme oxygenase-1 (HO-1) mRNA were detected by real time fluorescent quantitative PCR,and the content of malondialdehyde (MDA),superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the supernatant were detected by ELISA,and the reactive oxygen species (ROS) level in the cells was detected by chemical fluorescence method.【Result】 Compared with EH untreated cells,5,50 μg/mL EH increased significantly the viability of IPEC-J2 cells (P＜0.05).EH concentration was at 10 μg/mL,the increased cell viability was very significant (P＜0.01).When EH concentration was at 125 μg/mL,IPEC-J2 cell viability decreased,there was no statistical significance (P＞0.05).EH concentration is at 200 μg/mL,the decreased cell viability was extremely significant (P＜0.01).The concentration of EH treatment was 10 μg/mL in subsequent experiment.Compared with the CT group,in LPS group,the IL-6 and TNF-α genes mRNA expression in IPEC-J2 cells were extremely significantly increased (P＜0.01),and the content of MDA in the supernatant and the fluorescence intensity of cells ROS were extremely significantly increased (P＜0.01).The expression of Keap1 ,Nrf2 and HO-1 genes mRNA were extremely significantly reduced (P＜0.01),the content of antioxidant enzymes SOD and GSH-Px were extremely significantly decreased (P＜0.01).Compared with LPS group,in ELP group,the mRNA expression of IL-6 gene was extremely significantly reduced (P＜0.01),the mRNA expression of TNF-α gene was significantly decreased (P＜0.05).The content of MDA in the supernatant and the fluorescence intensity of cells ROS were extremely significant decreased (P＜0.01).The mRNA expression of Keap1 ,Nrf2 and HO-1 genes were significantly up-regulated (P＜0.01),the content of GSH-Px was increased extremely significant (P＜0.01).The content of SOD was also significantly increased (P＜0.05).【Conclusion】 EH aqueous extract played an anti-inflammatory and antioxidant role in IPEC-J2 cells by activating the Keap1/Nrf2 signal,increasing the contents of antioxidant enzymes SOD and GSH-Px, activating the expression of downstream target genes HO-1,and 10 μg/mL EH water extract had the best effect.

【Objective】 To investigate the O antigen,virulence gene and drug resistance of Escherichia coli (E.coli) isolates from ducks in Rongchang,Dazu and Longchang regions.【Method】 107 strains of bacteria isolated from duck diseased materials from 2014 to 2021 were inoculated in MacConkey medium under sterile conditions for culture and purification,identified by 16S rDNA gene amplification sequencing and biochemical test,detected O antigen and 16 virulence genes by PCR technology,and conducted drug sensitivity test by Kirby-Bauer disk diffusion method.【Result】 107 isolates identified as E.coli.O antigen identification tests identified nine O antigens,of which the dominant antigens were O78 (37.00%),O7 (25.00%),O121 and O145 (both 15.00%),and five O78+O145 and O7+O145 fusion strains were detected.The virulence gene detection test detected 11 virulence factors,of which the strongly pathogenic virulence genes were Tsh gene with a detection rate of 25.23%, fyuA gene with a detection rate of 31.78%, estB gene with a detection rate of 31.78%, Vat gene with a detection rate of 2.80% and iucA gene with a detection rate of 44.56%.3 virulence genes, ompA, yijP and ibeB,had the highest carriage rates of 100.00%,96.26% and 85.98%,respectively.The drug sensitivity test results showed that the isolates were most sensitive to aminoglycosides,minocycline and polymyxin,and resistant to macrolides and clindamycin.All isolates were multi-drug resistant,with 30.00% of isolates showing 7-fold resistance.【Conclusion】 In this study,107 strains of pathogenic E.coli from ducks were identified,which were highly pathogenic and drug resistant,and virulence gene carrier and drug resistance detection found that the virulence of E.coli had a certain relationship with its drug resistance.O antigen detection found five new O antigen fusion strains,the dominant O antigen was constantly changing,indicating that the genetic structure of E.coli in Southwest China was changing,and there might be new bacterial antigens.The results of this study provided a basis for the prevention and control of duck colibacillosis and vaccine preparation in Southwest China.

【Objective】 This study was aimed to explore the effective active ingredients and the molecular mechanism of Gui Qi Yimu oral liquid in the treatment of Sus scrofa Qi and blood two deficiency syndrome using network pharmacology and molecular docking technology.【Method】 The main active ingredients and targets of Gui Qi Yimu oral liquid were obtained from the Traditional Chinese Medicine Systematic Pharmacology Database and Analysis Platform (TCMSP) and Traditional Chinese Medicine Integrated Database (TCMID),and the targets of Sus scrofa Qi and blood two deficiency syndrome were acquired from GeneCards,OMIM and Malacards databases.The drug-ingredient-target network was constructed by using Cytoscape 3.9.0 software,and the interaction relationship between target proteins was constructed by using the STRING database and Cytoscape 3.9.0 software. DAVID database was used to analyze the Go function and KEGG signal pathway enrichment of co-acting targets.The core ingredients and targets were docked by Autodock Tools1.5.7 software.【Result】 The main active ingredients of Gui Qi Yimu oral liquid included quercetin,kaempferol and isorhamnetin.It corresponded to 76 key targets,6 335 disease targets and 41 intersection targets.Tumor necrosis factor (TNF),IL6 and IL10 were shown as key targets in PPI network. A total of 12 items were selected by GO functional enrichment analysis, among which 7 items were biological processes, including immune response, positive regulation of STAT protein tyrosine phosphorylation, response to glucocorticoid, etc. 2 items were cell component, including extracellular component and extracellular void. 3 items were molecular function, including cytokine, growth factor activity, and IL2 receptor binding. KEGG signaling pathway enrichment analysis showed that Guiqi Yimu oral liquid played its role mainly through cytokine receptor interaction, JAK-STAT signaling pathway and PI3K-Akt signaling pathway. The molecular docking results showed that the binding energies of quercetin,kaempferol,isorhamnetin and other core ingredients with key targets of Sus scrofa Qi and blood two deficiency syndrome were all less than 0 kJ/mol.【Conclusion】 Gui Qi Yimu oral liquid might act on the targets of RPN1,RNASE1 and RNH1 through the main active ingredients,such as quercetin,kaempferol and isorhamnetin.The treatment of pigs with Sus scrofa Qi and blood two deficiency syndrome was carried out by participating in cytokine receptor interactions,JAK-STAT signaling pathway,PI3K-Akt signaling pathway and other pathways.

【Objective】 The purpose of this study was to screen the natural products with anti-SVA activity from natural products library and to discover the mechanism of their antagonism.【Method】 Incorporating the use of the luciferase report technology,the high throughput platform for in vitro screening of anti-SVA natural products was established.In this platform,recombinant Luciferase reporter virus (rSVA-NLuc) system and BHK-21 cell were used to rapidly screen natural products against SVA from natural products library.The natural products with inhibitory effect on luciferase activity at the concentration of 10 μmol/L were screened from the natural product library,and their inhibitory activity was further verified by quantitative Real-time RT-PCR,at the same time,maximal nontoxic concentrations were determined by cytotoxicity test.The anti-viral mechanisms,which covering 4 main processes of the virus infection cycle including adsorption,entry,replication,assembly and release,were further studied with quantitative Real-time RT-PCR and 50% tissue culture infective dose(TCID₅₀).【Result】 16 natural products against SVA were screened from a library containing 560 natural products.Four safe and effective molecules were identified by quantitative Real-time RT-PCR and cytotoxicity test,namely (20S)-protopanaxatriol,paxilline,fangchinoline and hypocrellin B.Among them,(20S)-protopanaxatriol could inhibit the adsorption,replication of SVA.Paxilline could inhibit the adsorption,entry,replication,assembly and release of SVA.Fangchinoline mainly inhibited the entry and replication of SVA.Hypocrellin B could inhibit the adsorption,entry,replication,assembly and release of SVA.【Conclusion】 A total of 4 natural products against SVA were screened out from the natural products library.These compounds had good antiviral activity and different anti-viral mechanism.This study provided an important reference for the further development of anti SVA drugs.

【Objective】 This study was aimed to understand the infection,pathogenicity and drug resistance of Streptococcus from yak in Lhasa,Tibet,and provide new data for the study of Streptococcus from yak.【Method】 Bacterial isolation and culture,colony morphology observation,microscopic examination with Gram staining,biochemical identification and 16S rRNA sequence PCR amplification were performed on 55 yak nose swabs to identify the species characteristics of the isolates.The pathogenicity of the isolated bacteria was evaluated by the artificial infection test in mice.Finally,the drug resistance of the isolated bacteria was detected by disk diffusion method.【Result】 The isolated bacteria grew gray-white,smooth α-hemolytic round colonies on the blood plate,and Gram staining microscopic examination showed chain-like positive cocci. The isolated bacteria could ferment glucose, lactose, trehalose and sorbitol, but could not ferment maltose and glycerol. The nitrate reduction test, indigo substrate test, V-P test and MR test were negative. 16S rRNA gene sequence alignment analysis showed that,25 strains of Streptococcus (Tibet-1 to Tibet-25) were identified,and the detection rate was 45.46% (25/55),including 17 strains of Streptococcus pluranimalium and 8 strains of Streptococcus equi.The similarity between the 16S rRNA gene sequence of isolated bacteria and the Streptococci published on NCBI was 97.04%-99.77%.Among them,11 strains of Streptococcus pluranimalium from yaks,including Tibet-1,Tibet-2,Tibet-3,Tibet-5,Tibet-9,Tibet-10,Tibet-11,Tibet-14,Tibet15,Tibet-16 and Tibet-17,had more than 99% similarity with KM981770.1.The isolated Streptococcus from yak could cause diarrhea in mice. Streptococcus could be isolated from the organs of infected mice by enrichment culture.The results of drug sensitivity test showed that the isolated strains were highly sensitive to 11 antibiotics,including doxycycline,erythromycin,penicillin,and so on.It was highly resistant to compound sulfamethoxazole.【Conclusion】 This study isolated Streptococcus pluranimalium and Streptococcus equi from yak,and analyzed their pathogenicity and drug resistance,which provided a reference basis for the prevention,control and treatment of the disease.

【Objective】 The purpose of the test was to identify the primary pathogen causing recurrent pneumonia in intensive cattle farms in Shihezi region of Xinjiang,and in vitro drug sensitivity analysis of this pathogen was performed simultaneously.【Method】 Nasal swabs from 10 cattle with suspected symptoms and 1 lung tissue from dead cattle were collected and tested by PCR using primers specific for Mycoplasma bovis (M.bovis),and the positive samples were cultured and purified.Morphological observation,Dienes staining,biochemical test,16S rRNA sequencing and evolutionary analysis were carried out on the colony of the purified isolate.The growth curve of the isolate was determined by measuring the color change unit (CCU),and drug sensitivity test was carried out on the isolate.【Result】 The PCR results indicated that 7 M.bovis positive samples were detected in the 10 nasal swabs,and 1 of the lung of dead cattle collected was also positive.Needle like colonies grew on PPLO solid medium coated with lung tissue grinding fluid culture medium,after purification,the colony morphology of the isolates was typical fried egg like.A distinct dark blue central umbilicus was visible with Danies’ staining.Biochemical experiments showed that the isolate did not hydrolyze gelatin,arginine,esculin,nonfermentable lactose,glucose and mannitol,did not break down urea,and could reduce triphenyltetrazolium chloride.16S rRNA sequencing showed that the isolate shared 99.7% identity with the M.bovis international standard strain PG45 and 99.9% identity with the domestic M.bovis endemic strains XBY01,Ningxia-1,NM2012 and Tibet-10.The results of growth curve measurement showed that the isolate entered the logarithmic growth period after 6 h in the medium,reached the peak at 54 h and entered the stable period,and entered the decline period after 72 h.Susceptibility test revealed that the isolate was sensitive to nitrofurantoin,tetracycline and doxycycline,gentamycin and kanamycin,and moderately sensitive to norfloxacin and ofloxacin,whereas resistance developed to ciprofloxacin,lomefloxacin,azithromycin and erythromycin.【Conclusion】 In this study,a strain of M.bovis isolate was successfully identified from lung tissue,and 54 h was the optimal harvest time for this isolate,which was less different from most of the endemic strains of M.bovis in China,the overall genetic evolution was relatively stable,and there was certain drug resistance.The result of this study provided a reference for local prevention and control of M.bovis,and also laid a foundation for the research on the pathogenic mechanism of M.bovis and the development of vaccines.

【Objective】 The aim of this study was to predict the potential target and mechanism of Mahuang Gancao decoction in the treatment of pulmonary edema,and to verify it through animal experiments in vivo,so as to provide a reference for further research on the pharmacological effects of Mahuang Gancao decoction.【Method】 The potential active ingredients and related targets of Mahuang Gancao decoction were screened by Traditional Chinese Medicine Systems Pharmacology Database (TCMSP).The related targets of pulmonary edema were obtained from the GeneCards database.And then,the intersection of the active ingredient targets and disease targets was selected.The network diagram of ingredient-target was established with Cytoscape 3.6.GO fuction and KEGG pathway enrichment analysis were carried out in DAVID database.The active ingredients with the highest values and the key core targets were docked and verified by AutoDdock Vina to support the prediction results of network pharmacology.Finally,BALB/c mice were used for experimental verification.2% ammonium chloride was used to construct the pulmonary edema model.Mice were randomly divided into blank group,ammonium chloride model group and Mahuang Gancao decoction treatment group.The blank group was intraperitoneally injected with 200 μL normal saline every morning,and the ammonium chloride model group and the Mahuang Gancao decoction treatment group were intraperitoneally injected with 200 μL 2% ammonium chloride solution,the ammonium chloride model group and the blank group were intragastrically administered with 200 μL normal saline every night,and the Mahuang Gancao decoction treatment group was intragastrically administered with 200 μL 1 g/mL Mahuang Gancao decoction.After 5 days of continuous treatment,the mRNA expression levels of interleukin-6 (IL6),inducible nitric oxide synthase (NOS2),mitogen-activated protein kinase 3 (MAPK3) and matrix metalloproteinase 9 (MMP9) genes were detected by Real-time quantitative PCR.【Result】 There were 23 and 92 potential pharmaceutical active ingredients in ephedra and licorice,respectively,and 239 targets of the two components.5 196 targets of pulmonary edema were identified.There were 213 intersecting targets of drugs and diseases.The core targets were RAC-α serine/threonine protein kinase (AKT1),cell tumor antigen p53 (TP53), IL6,vascular endothelial growth factor A (VEGFA),estrogen receptor 1 (ESR1),epidermal growth factor receptor (EGFR),MAPK3,peroxidase synthase 2 (PTGS2),signal transducer and activator of transcription 3 (STAT3),MMP9,NOS2,etc.954 items were obtained by GO function enrichment analysis,including 710 items of biological process,involving metabolic process,gene transcription process,biological regulation process,drug stimulation process,etc.Cell component 92 items,mainly included cell membrane,cytoplasm,nucleus,organelles,etc.152 items of molecular function mainly included enzyme catalysis,protein binding,transcriptional regulation,etc.The results of the KEGG pathway enrichment analysis were involved in 170 signaling pathways,mainly related to tumor necrosis factor (TNF),PI3K-Akt,IL17 and other signaling pathways.Results of molecular docking showed that the key active ingredients had a good binding ability with the core targets.Real-time PCR results showed that compared with the blank group,the mRNA expression of IL6, MAPK3 and MMP9 genes in ammonium chloride model group were significantly or extremely significantly up-regulated (P＜0.05 or P<0.01),and the mRNA expression of NOS2 gene was extremely significantly down-regulated (P＜0.01).Compared with the ammonium chloride model group,the mRNA expression of IL6, MAPK3 and MMP9 genes in Mahuang Gancao decoction treatment group were significantly or extremely significantly down-regulated (P＜0.05 or P<0.01),and the mRNA expression of NOS2 gene was extremely significantly up-regulated (P＜0.01).【Conclusion】 Mahuang Gancao decoction could treat pulmonary edema through multiple components,multiple targets and multiple pathways.Quercetin,kaempferol,naringenin,luteolin and so on,which were active components,might exert inflammatory control and immunomodulatory effects on TNF,PI3K-Akt,IL17 signaling pathways through acting on IL6,MAPK3,MMP9,NOS2 and other targets.

【Objective】 Our previous study had shown that traditional Chinese medicine (TCM) combined with probiotics had obvious protective effect on diarrhea caused by pathogenic E.coli in mice.This study was aimed to further explore the protective mechanism of TCM combined with probiotics on diarrhea caused by pathogenic E.coli in mice.【Method】 Sixty mice were randomly divided into five groups: Blank group,model group,TCM group,probiotics group and TCM combined probiotics group.The diarrhea model of mice was established by intraperitoneal injection with 1×10⁷CFU/kg E.coli isolate suspension,TCM group was gavaged with TCM (2.5 g/kg dandelion extract,0.5 g/kg Astragalus total flavone and 0.5 g/kg Astragalus polysaccharide),and probiotics group was gavaged with Bacillus subtilis suspension (2×10⁸CFU/kg),TCM combined with probiotics group was gavaged with the corresponding dose of TCM and Bacillus subtilis (5×10⁷CFU/kg) mixture,once a day for 7 days.The pathological changes of small intestine tissue were observed by HE staining,the ultrastructural changes were observed by scanning electron microscope.The contents of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in intestine homogenate and serum of mice were determined by ELISA,the expression of TNF-α and IL-6 mRNA in intestine tissue and blood were determined by RT-PCR.The expression of nuclear factor-κB (NF-κB) p65 protein in small intestine was determined by immunohistochemical staining.【Result】 TCM combined with probiotics could improve the pathological and histological changes of small intestine,repair the phenomenon of high dehydration and atrophy of small intestinal villi and obvious shedding of microvilli.At the same time,TCM combined with probiotics could significantly reduce the contents of TNF-α and IL-6 in small intestine homogenate and serum (P＜0.05 or P＜0.01),significantly inhibit the expression of TNF-α and IL-6 mRNA in small intestine tissue and blood (P＜0.05 or P＜0.01).Furthermore,TCM combined with probiotics could significantly decrease the high expression of NF-κB p65 protein in small intestine.【Conclusion】 TCM combined with probiotics regulatesd NF-κB signaling pathway,inhibited the excessive expression of TNF-α and IL-6 mRNA,reduced the contents of TNF-α and IL-6 in intestine homogenate and blood,improved intestinal gland degeneration and mucosal inflammatory cell infiltration,as well as small intestinal villi and microvilli damage,thus playing a protective role against diarrhea caused by E.coli in mice.

【Objective】 The purpose of the experiment was to investigate the prevalence and drug resistance of Staphylococcus aureus in raw milk in some areas of Xinjiang,and provide a theoretical basis for monitoring Staphylococcus aureus in raw milk in this area and ensure the quality and safety of raw milk.【Method】 A total of 110 raw milk samples were randomly collected from large-scale dairy farms in Northern and Southern Xinjiang. Staphylococcus aureus was isolated and identified from milk samples by enrichment culture,isolation and purification,Gram staining,biochemical identification and PCR.The phenotypic resistance and drug resistance genes of Staphylococcus aureus to 16 antimicrobial agents were analyzed by broth microdilution and PCR.【Result】 18 strains of Staphylococcus aureus were isolated from 110 milk samples.The isolated strains showed light yellow,smooth and convex round colonies.Gram staining microscopy showed purple,short chain arrangement of Gram-positive bacteria.The biochemical test results conformed to the biochemical characteristics of Staphylococcus aureus.The isolated strains were identified as Staphylococcus aureus by 16S rDNA and nuc gene PCR amplification,with a separation rate of 16.36%(18/110).Drug susceptibility results showed that Staphylococcus aureus isolates had high resistance rates to ampicillin,penicillin and sulfaisoxazole,with resistance rates of 100%,83.33% and 77.78% respectively,while they were highly sensitive to vancomycin,compound minophen,oxacillin,cefalotin,ceftiofur,rifaximin and ciprofloxacin,with sensitivity rates of 100%,100%,94.44%,94.44%,94.44%, 94.44% and 94.44%,respectively.Among them,14 strains of Staphylococcus aureus were multidrug resistant,and the multidrug resistance rate was 77.78%.The detection results of drug resistance genes showed that the detection rate of macrolide resistance gene ermB (50.00%) was the highest,followed by β-lactam resistance gene mecA (27.28%) and Sul1 (22.22%).【Conclusion】 The prevalence and drug resistance rate of Staphylococcus aureus isolated from raw milk obtained from dairy farms in Xinjiang were still relatively serious,and the phenomenon of multi-drug resistance existed,which emphasized the importance of monitoring the drug resistance of Staphylococcus aureus in raw milk.

【Objective】 The aim of this study was to explore the protective mechanism of adipose-derived stem cells conditioned medium (ADSCs-CM) in alleviating pancreatic oxidative stress injury induced by sodium taurocholate and trypsin.【Method】 One Chinese garden dog was selected to isolate and culture adipose-derived stem cells (ADSCs) and prepare conditioned medium (CM). Nine dogs were randomly divided into control group (CON group),AP model group (AP group) and CM treatment group (CM group).The AP and CM groups were retrogradely injected with a mixture of 5% sodium taurocholate (0.1 mL/kg) and trypsin (3500 U/kg) through the pancreaticobiliary duct to establish a AP model,The CON group was injected with the same volume of normal saline.After surgery, CM group was intravenously injected with ADSCs-CM(0.1 mL/kg),the CON group and AP group were injected with the same volume of normal saline.Blood and pancreatic tissue samples were collected 24 h after surgery,histopathology,B-ultrasound,canine pancreas-specific lipase (cPL),blood routine,biochemical indexes were detected 24 hours after operation,and the levels of total nitric oxide synthase (T-NOS),inducible nitric oxide synthase (i-NOS),glutathione (GSH) and glutathione peroxidase (GPx) in pancreas and serum were detected by colorimetry.【Result】 B-ultrasound showed that the pancreas echo in AP group and CM group was uneven,the surrounding fat was hyperechoic. Pathological tissue sections showed that the pancreatic swelling and hemorrhage,peripheral steatosis,severe duodenal hemorrhage,interstitial edema,a large number of cell necrosis and inflammatory cell infiltration,and severe hemorrhage of pancreas were occurred in the AP group.The histopathological changes of pancreas and surrounding tissues in the CM group were milder than that of in the AP group. The cPL tests were all positive.Compared with the CON group,the contents of Cl^-,ALB,GLB and TP in the AP group decreased significantly (P＜0.05),while the activities of AMY,LIPA,plasma T-NOS,pancreatic i-NOS and T-CHO,GSH contents increased significantly (P＜0.05).In the CM group,the contents of Na⁺,Cl^-,GLB and TP decreased significantly (P＜0.05),while the activities of AMY,pancreatic GPx,and the contents of plasma GSH,pancreatic GSH increased significantly (P＜0.05).Compared with the AP group,the contents of TCO₂,Na⁺,Cl^-,ALB,TCHO and plasma T-NOS activity in the CM group decreased significantly (P＜0.05),pancreatic GPx activity and plasma GSH content increased significantly (P＜0.05).【Conclusion】 ADSCs-CM could reduce the degree of pancreatic injury in AP model dogs,and the protective mechanism was closely related to the decrease of plasma T-NOS activity and the increase of pancreatic GPx and plasma GSH levels.This study could provide a theoretical basis for the treatment of AP and the application of ADSCs-CM.

【Objective】 This study was aim to reveal the anti-inflammatory and antioxidant effects of heme oxygenase-1 (HO-1) in macrophages.【Method】 Different concentrations of lipopolysaccharide (LPS,0,3,5,10,15,20,25 μg/mL),HO-1(0,0.02,0.04,0.06,0.08,0.10 μg/mL) and zinc protoporphyrin (ZPP;0,5,10,15,20,30 ng/mL) respectively were used to treat macrophages (RAW264.7) for 12 hours.The viability of RAW264.7 cells was detected by CCK8 method,and the optimal concentration of RAW264.7 cells treated with LPS,HO-1 and ZPP was calculated. RAW264.7 cells were randomly divided into control group (CT),LPS group (LPS),LPS+HO-1 group (LH),HO-1 group (HO-1),LPS+HO-1+ ZPP group (LHZ),with three replicates in each group.The cells in CT group were seeded in DMEM medium containing 10% fetal bovine serum.The cells in LPS group were treated with LPS.The cells in LH group were treated with LPS and HO-1.The cells in HO-1 group were treated with HO-1.The cells in LHZ group were co-cultured with LPS,HO-1 and ZPP.Cells in each group was treated for 12 hours,collecting the cells and supernatant.The contents of interleukin-6 (IL-6),IL-8,tumor necrosis factor-α (TNF-α), reactive oxygen sepecies (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the supernatant were detected by ELISA.The mRNA expression of IL-6,IL-8,TNF-α,nuclear factor-E2-related factor 2 (Nrf2) and HO-1 was measured by quantitative Real-time PCR.The expression of Nrf2,HO-1 protein was detected by Western blotting.【Result】 Compared with cells treated by 0 μg/mL LPS,the concentration of 20 and 25 μg/mL LPS extremely significantly decreased the viability of RAW264.7 cells (P＜0.01).Compared with cells treated by 0 μg/mL HO-1,the concentration of 0.08 and 0.10 μg/mL HO-1 extremely significantly increased cell viability (P＜0.01).Compared with cells treated by 0 ng/mL,ZPP with 15,20 and 30 ng/mL extremely significantly reduced cell viability (P＜0.01).The results showed that the concentration of LPS,HO-1 and ZPP treatment was 20 μg/mL,0.08 μg/mL,15 ng/mL in subsequent experiment,respectively.Compared with CT group,IL-6,IL-8 and TNF-α mRNA expression in LPS group were extremely significantly up-regulated (P＜0.01).The contents of IL-6,IL-8,TNF-α,MDA and ROS were extremely significantly increased (P＜0.01),and the contents of GPx and SOD were extremely significantly decreased (P＜0.01).Compared with LPS group,IL-6,IL-8 and TNF-α mRNA expression in LH group were extremely significantly decreased (P＜0.01).The contents of IL-6,IL-8,TNF-α,MDA and ROS were extremely significantly decreased (P＜0.01),and the contents of GPx and SOD were extremely significantly increased (P＜0.01).Compared with the LH group,IL-6,IL-8 and TNF-α mRNA expression in LHZ group were extremely significantly up-regulated (P＜0.01).The content of IL-6,IL-8,TNF-α,MDA and ROS were extremely significantly increased (P＜0.01),while the contents of GPx and SOD were extremely significantly decreased (P＜0.01).The results of Nrf2/HO-1 signal pathway molecules expression showed that the mRNA and protein expressions of Nrf2 and HO-1 in LPS group were extremely significantly up-regulated compared with CT group (P＜0.01).Compared with LPS group,Nrf2 and HO-1 mRNA expression in LH group were extremely significantly down-regulated (P＜0.01),Nrf2 protein expression was extremely significantly decreased (P<0.01),and HO-1 protein expression was extremely significantly increased (P＜0.01).Compared with LH group,Nrf2 and HO-1 mRNA expression in LHZ group was extremely significantly up-regulated (P＜0.01),Nrf2 protein expression was extremely significantly increased (P＜0.01),and HO-1 protein expression was extremely significantly decreased (P＜0.01).Compared with CT group,the indexes of HO-1 group were not significantly different (P>0.05).【Conclusion】 20 μg/mL LPS induced macrophages to produce inflammatory response and oxidative stress.0.08 μg/mL HO-1 had anti-inflammatory and antioxidant effects.HO-1 regulated Nrf2 signaling pathway to exert anti-inflammatory and antioxidant effects.

【Objective】 This study was aimed to investigate the species and drug resistance of pathogenic bacteria in raw milk samples and environmental samples of mastitis from some dairy farms in Inner Mongolia,so as to provide clinical medication guidance and reference for the prevention and control of mastitis.【Method】 Bacteria were isolated from 468 samples (199 samples of mastitis raw milk and 269 samples of mastitis cow breeding environment) from 4 large-scale farms in Inner Mongolia by plate streak method.The isolated bacteria were identified by morphological observation,Gram staining microscopy and 16S rDNA sequencing.Drug susceptibility testing was performed on the main pathogenic bacteria that cause mastitis.【Result】 There were small,white and yellow colonies on the high salt mannitol medium,56 strains were highly similar to Staphylococcus aureus by 16S rDNA sequencing and comparison,and the isolation rate was 11.97%.On eosin-methylene blue medium,there were black purple and metallic luster colonies.After 16S rDNA sequencing and comparison,44 strains were highly similar to Escherichia coli,with a separation rate of 9.40%.In 199 raw milk samples,the isolation rates of Staphylococcus aureus and Escherichia coli were the highest,it was 21.11% and 17.09%,respectively.In 269 environmental samples,the isolation rates of Staphylococcus aureus and Escherichia coli were 5.20% and 3.72%,respectively.The results of drug sensitivity test showed that Staphylococcus aureus was relatively resistant to penicillin,sulfamethoxazole,ampicillin,amoxicillin/clavulanic acid,and the drug resistance rates were 90.63%,78.13%,75.00% and 68.75%,respectively,and the sensitivity rate to gentamicin,ciprofloxacin and compound sulfamethoxazole was higher. Escherichia coli was significantly resistant to ampicillin,cefothiophene and sulfamethoxazole,and the drug resistance rates were 100%,94.29% and 45.71%,respectively,it was highly sensitive to streptomycin,kanamycin,gentamycin,polymyxin E,meropenem,amoxicillin/clavulanic acid and rifaximin.【Conclusion】 The main pathogenic bacteria causing dairy cow mastitis in Inner Mongolia were Staphylococcus aureus and Escherichia coli. Staphylococcus aureus had relatively high resistance to penicillin,sulfamethoxazole,ampicillin,amoxicillin/clavulanic acid, Escherichia coli had obvious drug resistance to ampicillin,cephalothiophene and sulfamethoxazole.

With the large-scale and intensive development of dairy farming in China,and the excessive pursuit of dairy production performance,it is easy for dairy cows to occur diseases.Subclinical ketosis,subclinical hypocalcemia,mastitis,foot-and-mouth disease,respiratory diseases and bovine viral diarrhea are mainly nutritional and infectious diseases,which threaten the health and welfare of dairy cows and bring about the decrease of milk quality and the increase of financial loss of farm.The timely and rapid detection of diseases is a key step to control the infection among livestock,reduce the damage of economy,and protect the food safety.Although cell culture methods,enzyme-linked immunoabsorbent assay (ELISA),polymerase chain reaction (PCR) and some other methods have been applied in the detection of diseases of livestock and poultry,it’s still difficult to carry on the detecting work due to the limitations of time consuming and high-cost and complicated procedures of present methods,and unsuitable for point-of-care testing of diseases of dairy cow.Biosensor depends on a transductor to convert the sign originating from specific biochemical interaction between the biomarker and the bioreceptor into a measurable signal,and on a signal amplification system to detect the biomarker qualitatively or quantitatively.There is great potential for biosensor in detecting metal ion,pathogenic bacteria with advantages of simple operation,high sensitivity and strong specificity.Currently,there are various kinds of biosensors designed for the detection of diseases of diary cow.In this review,we provided an overview of the corresponding electrochemical and optical biosensors for common diseases of dairy cows.The descriptions illustrated the detection strategy,detection range and limit and other information of each biosensor.In the last section,this paper prospected the development tendency of biosensor,in order to provide references for the healthy development of dairy cow.

Periodontal disease is a kind of disease in which plaque and calculus formed by bacteria on the surface of teeth gradually erode the periodontal tissue and cause inflammation of surrounding tissues.According to the development process of the disease,it can be divided into two stages:Gingivitis and periodontitis.Periodontal disease is particularly common in small animal clinical diseases.About 80% of mature dogs worldwide suffer from periodontal disease.Due to the weak awareness of pet owners on oral health care and the disease is highly seretive irreversible damage of periodontal supporting tissue often occurs when dogs are treated,and the risk of systemic diseases caused by bacteria and their virulence factors entering the whole body through blood circulation is greatly increased.However, the core pathogenic bacterium of canine periodontal disease,the cause of imbalance in the flora and the pathogenesis of the canine periodontal disease is unclear.This paper reviewed the research progress of the canine periodontal disease in the small animal clinical field at hoem and abroad by compiling the microbiota most relevant to canine periodontal disease based on high-throughput sequencing methods,integrating the latest clinical diagnostic classification criteria for canine periodontal disease,comparing and analyzing the advantages and disadvantages of Chinese and Western medicine for the prevention and treatment of canine periodontal disease,and analyzing the relationship between canine periodontal disease and systemic diseases.We hope to provide a theoretical reference for the clinical treatment of this disease,to promote veterinarians to carry out the treatment of this disease more efficiently in clinical practice,and to provide ideas for clinical research related to canine periodontal disease.