【Objective】 This study was aimed to screen the key genes, biological processes and signaling pathways that regulate feather follicle growth in the dorsal skin epithelium and mesenchyme during early development of chicken embryos, and provide a reference for understand the molecular mechanism of feather follicle growth during early development of chicken embryos.【Method】 The gene chip GSE62882 dataset was downloaded from the GEO database, and then the limma package of R language was used to conduct difference analysis on the dataset.At 7th and 9th days of embryonic stage, the differential genes of skin epithelium and mesenchyme, and co-upregulated and co-downregulated genes between shin epithelium and mesenchyme were screened.The STRING online database was used to analyze the protein-protein interaction network of differential genes, and co-upregulated and co-downregulated genes, visualized with Cytoscape 3.8.2 software, and the clusterProfiler package in R language was used to perform GO functional and KEGG pathway enrichment of differential genes, and co-upregulated and co-downregulated genes.【Result】 A total of 626 differential genes were screened, and 189 interaction relationships, 10 biological processes and 4 signaling pathways were obtained in skin epithelium on the 7th and 9th days of embryonic period.A total of 690 differential genes were screened, and 326 interaction relationships, 10 biological processes and 3 signaling pathways were obtained in skin mesenchyme on the 7th and 9th days of embryonic period.In skin epithelium and mesenchyme, 26 genes were co-upregulated and 48 genes were co-downregulated on the 7th and 9th days of embryonic period, and 34 interaction relationships of co-upregulated genes and co-downregulated genes were mainly enriched in Wnt signaling pathway.【Conclusion】 In this study, 26 co-upregulated genes and 48 co-downregulated genes were screened in skin epithelium and mesenchyme on the 7th and 9th days of embryonic period in chickens, which were mainly enriched in Wnt signaling pathway.The results provided a theoretical reference for the action mechanism of epithelium and mesenchyme in follicle growth and development.

【Objective】 The purpose of this study was to investigate the gene characteristics and expression patterns of DNA methyltransferase (DNMT) gene family in pig.【Method】 Bioinformatics methods were used to identify members of porcine DNMT gene family at the whole genome level, and the chromosomal localization, physicochemical properties, protein structure, subcellular localization, gene structure, conserved motif, phylogenetic relationship, collinear relationship and expression pattern of DNMT gene were analyzed.【Result】 A total of five porcine DNMT genes:PigDNMT1, PigDNMT2, PigDNMT3, PigDNMT4 and PigDNMT5, were identified, which were distributed on chromosomes 2, 3, 13, 14 and 17, respectively.Phylogenetic tree analysis of porcine DNMT gene family found that PigDNMT1 and PigDNMT4, as well as PigDNMT2 and PigDNMT5 displayed closer relationships.PigDNMT2 and PigDNMT5 were identical in number and order of motifs, but differ in gene structure.Phylogenetic tree analysis revealed that the DNMT gene family of pig, human, mouse and cattle were divided into four subfamilies, among which the DNMT gene family of pig and cattle displayed closer relationships.Gene collinearity analysis showed that there were four pairs of orthologous genes for DNMT in pig, human, mouse and cattle, indicating that there was a strong collinear relationship among the DNMT genes of the four species.Lengths of DNMT family protein in pig varied from 228 to 1 706 amino acids, and the molecular weight of DNMT proteins varied from 26 133.32 to 192 267.80 u, and the isoelectric points of DNMT protein varied from 6.00 to 9.06.Analysis of secondary structure prediction showed that DNMT proteins of pig were mainly composed of random coils.Subcellular localization analysis found that all DNMT genes of pig were located in the nucleus.Transcriptome analysis of sow gonadal axis found that PigDNMT1 showed different expression patterns in the hypothalamus, pituitary and ovary during the onset of puberty.【Conclusion】 In this study, a total of 5 DNMT genes were identified in pig, and it was found that PigDNMT1 in the gonadal axis showed different expression patterns during the initiation of puberty.This bioinformatics analysis provided some reference for analyzing the function of DNMT gene family in pigs.

【Objective】 This experiment was aimed to explore the long non-coding RNA (lncRNA) that played a regulatory role in bovine skeletal muscle development, and study its effect on the expression of bovine skeletal muscle satellite cells in proliferation and differentiation, so as to provide reference for the study of bovine muscle development related mechanisms.【Method】 A lncRNA obtained by high-throughput sequencing in the previous test was named lnc721. The biological information of lnc721 was analyzed using NCBI database, the coding ability and subcellular localization of lnc721 were determined by CPC website and nucleo-cytoplasmic separation experiment, respectively.The designed and synthesized lnc721 siRNA was transfected into bovine skeletal muscle satellite cells, and the bovine skeletal muscle satellite cells were cultured and differentiated through a mature in vitro myoblast induced differentiation model.The expression changes of lnc721 in different stages of cell development were analyzed in detail by EdU experiment, Real-time quantitative PCR and Western blotting, respectively.【Result】 lnc721 was located on chromosome 18 of the whole bovine genome, and its protein coding capacity was-1.33129, which was mainly located in cytoplasm.After interference with lnc721 expression, the EdU positive cellular rates was extremely significantly increased, and the mRNA expression of cell proliferation marker genes Pax7 and Ki-67 were extremely significantly up-regulated (P<0.01).Western blotting results further proved that interference with lnc721 extremely significantly promoted the expression of Pax7 protein (P<0.01).The mRNA and protein expression of the cell differentiation marker MyHC gene were extremely significantly down-regulated after interfering with the expression of lnc721 at the cell differentiation stage (P<0.01).【Conclusion】 Interference with lnc721 could promote the proliferation of bovine skeletal muscle satellite cells and inhibit the process of myoblast differentiation.

【Objective】 This study was aimed to analyze the interaction between bta-miR-377 and cocaine and amphetamine regulated transcript peptide(CART), a key regulatory factor of bovine follicle development, and to clarify the mechanism of bta-miR-377 on the expression of CART.【Method】 In this study, the binding sites of bta-miR-377 and bovine CART gene mRNA were predicted by bioinformatics, and the sequence conservation of miR-377 in different species was analyzed.The target relationship between bta-miR-377 and CART gene was verified by double luciferase reporter gene method.Three healthy Simmental cows were selected to collect their hypothalamic tissues and analyze the endogenous expression of bta-miR-377 and CART gene in the hypothalamus.PC12 and 293T cells were used as models for cell function verification.The abundance of CART gene mRNA in PC12 cells and CART protein in 293T cells transfected with bta-miR-377 mimics and NC mimics were detected by Real-time quantitative PCR and Western blotting, respectively.【Result】 bta-miR-377 had binding site to CART 3'-UTR and extremely significantly down-regulated the relative fluorescence activity of wild-type recombinant double fluorescent plasmid (P<0.01).The binding site sequences between the bta-miR-377 and CART 3'-UTR were highly conserved in different species.Lower sequence conservation in the miR-377 seed region occurred between cows and rats.Both of bta-miR-377 and CART were detected in bovine hypothalamus.Overexpressing bta-miR-377 mimics could significantly down-regulated the expression of CART gene mRNA and CART protein (P<0.01 or P<0.05).There was a negative correlation between bta-miR-377 and CART expression.【Conclusion】 Both bta-miR-377 and CART were expressed in bovine hypothalamic and bta-miR-377 inhibits CART expression at level of mRNA by specifically binding to CART 3'-UTR in PC12 cells.

【Objective】 This study was aimed to amplify porcine serum and glucocorticoid-inducible kinase (SGK) family genes and analysis their bioinformatics, as well as to explore their expression patterns in porcine adipose tissues and adipocytes.【Method】 SGK family genes were amplified by PCR using the cDNA of Tibetan pig adipocytes.The physicochemical properties and subcellular localization of the encoded proteins were predicted by online tools.The phylogenetic tree was constructed by Mega X software.The tissues from heart, liver, spleen, kidney, lung, back muscle, leg muscle, cervical fat, backfat, inguinal fat, and perirenal fat of 30-day-old Bama pigs and the inguinal adipose tissues of 7-day-old and 4-month-old Bama pigs were collected.The expressions of SGK family genes in these tissues were detected by Real-time quantitative PCR.The 30-day-old porcine inguinal adipose tissues were collected and stromal vascular fraction (SVF) cells were isolated and differentiated into white adipocytes.The expressions of SGK family genes and adipocyte differentiation marker genes CCAAT/enhance binding protein alpha (C/EBPα) and peroxisome proliferator activated receptor gama (PPARγ) in adipocytes were detected by Real-time quantitative PCR.【Result】 The sequence length of the CDS regions of SGK1, SGK2 and SGK3 genes were 1 296, 1 104 and 1 473 bp, encoding 431, 367 and 490 amino acids, respectively.The SGK1 and SGK2 were localized in cytoplasm, and the SGK3 was localized in nucleus.All three SGK proteins were hydrophilic proteins with the same structural domains and conserved motifs of their encoded proteins.Phylogenetic tree results indicated that pigs were most closely related to cattles.SGK1 and SGK3 genes were widely expressed in heart, liver, spleen, lung, kidney, various muscles and adipose tissues.SGK2 gene was highly expressed in adipose tissues of the cervical fat, backfat, inguinal fat and perirenal fat.The expression of SGK1 and SGK2 genes were extremely significantly higher in adipose tissues of 7-day-old than 4-month-old pigs (P<0.01), and there was no significant difference in the expression of SGK3 gene between 4-month-old and 7-day-old pigs (P>0.05).The expression of SGK3 gene was lower than that of SGK1 and SGK2 genes.Compared with undifferentiated adipocytes, the expression of SGK1 and SGK2 genes were extremely significantly up-regulated in differentiated adipocytes (P<0.01), and the expression of SGK1 gene was higher than that of SGK2 gene, while the expression of SGK3 gene did not change significantly (P>0.05).【Conclusion】 SGK family proteins had conserved structural domains and might play similar functions.SGK1 and SGK2 might be involved in regulating the differentiation of porcine adipocytes, which laid a certain theoretical foundation for exploring the molecular mechanism of porcine fat deposition.

【Objective】 The purpose of this study was to investigate the factors affecting the transcription of long chain noncoding RNA-bone morphogenetic protein 4(lncRNA-BMP4) promoters in chickens, and study the molecular mechanisms regulating the specific expression of lncRNA-BMP4.【Method】 Using the muscle genome in chickens as a template, the promoter region of lncRNA-BMP4 in chickens was amplified by PCR and cloned, the lncRNA-BMP4-EFGP vector was constructed, and the lncRNA-BMP4 promoter activity was qualitatively analyzed.The core region of the lncRNA-BMP4 promoter was screened by chromosome 5'-terminal deletion and double luciferase system detection.The potential transcriptional factors of the regulatory core region were predicted and analyzed by online tools, and the transcriptional factors that really affected lncRNA-BMP4 were screened by point mutation and dual luciferase system.The transcriptional regulation of DNA methylation and histone acetylation on lncRNA-BMP4 was verified by apparent modification.【Result】 The lncRNA-BMP4 promoter fragments(1 288 bp) were successfully amplified and ligated with a pEGFP-N1 vector.After transfection into chicken fibroblast cell line (DF-1), it showed fluorescent expression, indicating that lncRNA-BMP4 promoter had promotive activity.The results of chromosome 5'-terminal deletion and double luciferase system detection showed that the core promoter region was-832 to-651 bp.The transcription factors screened by Jaspar database analysis showed that the core regions were SOX17, CREB1 and STAT1.Double luciferase system showed that STAT1 could promote the activity of lncRNA-BMP4 core promoter region, DNA methylation inhibitor 5'-Azacd had no effect on lncRNA-BMP4 transcriptional activity, while histone acetylation inhibitor TSA extremely significantly increased its transcriptional activity (P<0.01).【Conclusion】 This study suggested that the transcriptional activity of lncRNA-BMP4 was positively regulated by STAT1 and histone acetylation, while DNA methylation did not affect its transcription.The results provided a theoretical basis for a detailed analysis of the function and molecular mechanism of lncRNA-BMP4.

【Objective】 The purpose of this study was to explore the optimal extraction conditions of cordycepin from Cordyceps militaris residual medium by the hydrothermal reflux method.【Method】 The single factor design and the orthogonal experiment design were conducted according to four factors including the liquid to material ratio, extraction repetitions, extraction temperature and extraction time, and the cordycepin content was measured by high-performance liquid chromatography (HPLC) method, which were used to decide the optimal extraction parameters of cordycepin from Cordyceps militaris residual medium by the hydrothermal reflux method.【Result】 The results of methodology showed that the relative standard deviation (RSD) of precision, repeatability, and stability for HPLC detection of cordycepin content in the extract of Cordyceps militaris residual medium were less than 2%, the RSD of accuracy was 2.64%, and the standard curve had high linear correlation.The results of the orthogonal experiment showed that the order of factors affecting the extraction efficacy of the hydrothermal reflux method were extraction time, extraction repetitions, liquid to material ratio and extraction temperature, and extraction time and extraction repetitions had significant effects on cordycepin content in extract (P<0.05), while extraction temperature and liquid to material ratio had not obvious effects (P>0.05).The results of the single factors and the orthogonal experiment design showed that the optimal extraction process factors of cordycepin from Cordyceps militaris residual medium were the ratio of liquid to material 25:1, repetitions 5 times, extraction temperature 70 ℃, and extraction time 60 min, respectively.Under these extraction conditions, the cordycepin content of Cordyceps militaris residual medium was 1.48 mg/g.【Conclusion】 The optimal extraction parameters of the hydrothermal reflux method in this study could effectively extract the active components from Cordyceps militaris residual medium, the operation process was easy and practical, and the yield of the active components of the extract was stable.The results provided reference for the development of product from Cordyceps militaris residual medium.

【Objective】 The study was aimed to investigate the alleviating effect of nuclear factor erythroid 2-related factor 2 (Nrf2) on oxidative damage induced by heat stress of broiler cardiomyocytes.【Method】 Primary broiler cardiomyocytes were purified through different-speed adherence method and chemical purification method.The cardiomyocytes were randomly divided into control group, heat stress model group (HS group) and Nrf2 activator pre-treatment heat stress group (TBHQ+HS group).The cardiomyocytes of the control group were cultured without any treatment, the cardiomyocytes of the HS group were treated in a high-temperature (43 ℃) incubator for 2 h, the cardiomyocytes of the TBHQ+HS group were pretreated with 50 μmol/L tert-butylhydroquinone (TBHQ) for 12 h and then heat stress treatment was carried out.The relative viability of the cells was measured by MTT method.The activity of superoxide dismutase (SOD), the content of malondialdehyde (MDA) and the activity of lactate dehydrogenase (LDH) in cell culture medium were detected by colorimetry method.Real-time PCR was used to detect the mRNA expression levels of Nrf2, heme oxygenase-1 (HO-1), glutamate cysteine ligase (GCLC) and NAD(P)H:quinone oxidoreductase 1 (NQO1).The expressions of Nrf2 total protein and HO-1 protein were detected by Western blotting.【Result】 Compared with the control group, the morphology of primary cardiomyocytes was changed and vacuolar reticular structure was shown in heat stress group, the relative activity and SOD activity of cardiomyocytes were decreased extremely significantly (P<0.01), the content of MDA was increased extremely significantly (P<0.01), and the activity of LDH in cell culture medium was increased extremely significantly (P<0.01).The difference of NQO1 and GCLC mRNA expression in the downstream of Nrf2/antioxidant response element (ARE) signal pathway was not significant (P>0.05), the expression of Nrf2 and HO-1 mRNA was remarkably increased (P<0.05), but there was no difference in Nrf2 total protein and HO-1 protein expression (P>0.05). In TBHQ pre-treatment heat stress group, compared with the heat stress group, the vacuolar reticular structure reduced in cardiomyocytes and the relative activity of cardiomyocytes was raised significantly (P<0.05), the activity of SOD was increased extremely significantly (P<0.01), the content of MDA was diminished meaningly (P<0.05), the activity of LDH enzyme in cell culture medium was decreased extremely significantly (P<0.01).The expression of NQO1 and GCLC mRNA in the downstream of Nrf2/ARE signal pathway was increased significantly(P<0.05), the expression of Nrf2 mRNA and total protein was increased significantly (P<0.05), besides, the expression of HO-1 mRNA and protein were all raised extremely significantly (P<0.01).【Conclusion】 Activation of Nrf2 could up-regulate the transcription of antioxidant genes and the expression of protein in the downstream of Nrf2 signaling pathway, improve the antioxidant capacity of heat stressed broiler cardiomyocytes, and alleviate the oxidative damage induced by heat stress.It suggested that Nrf2 might be an effective molecular target for broiler cardiomyocytes to resist oxidative damage induced by heat stress.

【Objective】 The failure of passive transfer (FPT) of newborn calves will lead to significant increase in incidence rate and mortality rate of calves, and affect later growth and development.This study was aimed to measure the production and quality of colostrum of Angus cows imported from abroad, and evaluate the passive immunity effect of newborn calves, in order to provide a scientific basis for the formulation of colostrum management measures for newborn calves.【Method】 15 healthy Angus primiparous cows (24 months of age) and 15 Angus multiparous cows (36 to 48 months of age) were randomly selected, which were milked immediately after calving to determine the colostrum yield, nutritional component and immunoglobulin concentration.15 Angus primiparous and multiparous newborn calves were selected, respectively, the birth weight were measured, the blood samples were collected after birth without colostrum (0 h) and after colostrum (24 to 36 h) to determine the content of serum total protein and physiological and biochemical indicators.【Result】 The average colostrum yield of primiparous cows was extremely significantly lower than that of multiparous cows, but the contents of milk protein, non-fat milk solids, lactose, ash, and density and electrical conductivity of colostrum in primiparous cows were extremely significantly higher than that in multiparous cows (P<0.01).The immunoglobulin concentration of colostrum in primiparous and multiparous cows were 28.72% and 26.24%, the pass rate were 93.33% and 91.67%, respectively, there were no significant difference (P>0.05).The passive immunization failure rate of of primiparous and multiparous newborn calves were 33.3% and 25.0%, respectively.The average body weight of primiparous newborn calves was extremely significantly lower than that of multiparous newborn calves (P<0.01).The average birth weight and globulin content of passive immunity success calves were extremely significantly higher than that of passive immunity failure calves (P<0.01).【Conclusion】 The main cause of FPT in Xinjiang Angus calves might be closely related to the insufficient colostrum intake of newborn calves due to low colostrum production of cows.Calves with lower birth weight were at higher risk for FPT.This study provided a scientific basis for formulating a feasible colostrum supplementary feeding plan for newborn Angus calves.

【Objective】 The objectives of current study were to reveal the population characteristics of tympanic temperature in lactating cows and its change with temperature-humidity index (THI).Further, the influence factors of tympanic temperature and its correlation with rectal temperature under moderate heat stress were investigated.【Method】 The records of tympanic temperature, rectal temperature, as well as environmental temperature and humidity were measured from 638 Holstein dairy cows from Beijing in August, 2021.Descriptive statistics of tympanic temperature, rectal temperature and THI were summarized.Then the correlations between 6 h tympanic temperature and rectal temperature in 161 Holstein dairy cows were analyzed.Finally, a linear mixed model was employed to test the significances of effects, such as collecting time, parity, lactation stage, and reproductive status on tympanic temperature and rectal temperature.【Result】 In Beijing, the average tympanic temperature of Holstein cows in summer was 39.01 ℃±0.40 ℃ under moderate heat stress (THI was 76.11 to 83.18) during the measurement time.Under this circumstance, tympanic temperature was differed between day and night, and when THI changed, it showed a slight delay.To be specific, tympanic temperature was lowest in the forenoon and stay heated in the afternoon and nightfall, with 2 abnormal peak values in the morning and afternoon.The tympanic temperature and rectal temperature were significantly correlated (P<0.05, r=0.35).In addition, tympanic temperature and rectal temperature rose gradually with THI increasing, and the regression coefficients of tympanic temperature and rectal temperature on THI were 0.09 (P<0.05, R²=0.09) and 0.10 (P<0.05, R²=0.08), respectively.Collecting time and parity had significant effects on tympanic temperature, while parity and lactation stage significantly affected rectal temperature (P<0.05).THI had significant regression on both tympanic temperature and rectal temperature of lactating cows (P<0.05).【Conclusion】 The results of current study revealed the rhythmic changes and influencing factors of cow body temperature.The tympanic temperature could effectively reflect the real-time body temperature of lactating cows, which provided a theoretical basis for the fine management of farm.

【Objective】 The aim of this study was to explore the biological characteristics of limbal stem cells (LSCs) isolation, culture, identification and multidirectional differentiation potential of Beijing You chickens.【Method】 Limbus cornea of 10-day-old healthy Beijing You chicken embryos was isolated by dispase Ⅱ combined with trypsin two-step method.The cells were cultured in vitro and the growth curve was drawn.The expression of LSCs specific markers cytokeratin 3 (CK3), CK12, cell cycle regulatory protein p63, ATP binding box transporter (ABCG2) and cytokeratin 19 (CK19) were identified by immunofluorescence and RT-PCR.Multidirectional differentiation potential was detected by osteogenic and chondrogenic induction.【Result】 The cells of Beijing You chickens showed strong refraction, polygonal shape, typical S shaped growth curve, and population doubling time (PDT) was decreased with the increase of generations.Immunofluorescence assay showed that ABCG2, p63 and CK19 were positive, while CK3 and CK12 were negative.The results of RT-PCR showed that the isolated cells expressed p63, CK19 and ABCG2, but did not express CD45, indicating that the isolated cells were LSCs.After osteogenic induction and differentiation of LCSs, mineralized calcium nodules stained with alizarin red could be seen.RT-PCR results showed that osteopontin (OPN) and collagen type Ⅰ(Col-Ⅰ) which were key osteogenic genes were expressed in LCSs.After induction and differentiation of chondroblasts, the chondroblasts were stained with alicin blue.The results of RT-PCR showed that the expression of key chondroblast gene transcription factors Y-frame 9 (SOX-9) and type Ⅱ collagen (Col-Ⅱ) were detected.【Conclusion】 In this study, LSCs were successfully isolated from limbus cornea of 10-day-old Beijing You chicken embryos, and they had good proliferative activity and osteogenic and chondrogenic abilities.The results could provide references for the conservation of poultry LSCs resources.

【Objective】 This study aimed to investigate the effects of supplementation with two different organic zinc sources on the growth performance and zinc metabolism of newborn dairy heifers.【Method】 Thirty newborn Holstein heifers with similar body weight (BW 39.4 kg±0.8 kg) were selected and randomly divided into 3 groups, with 10 heifers in each group.Heifers in the control group (CON) was fed basal diet without organic zinc supplementation, and heifers in organic zinc groups were fed with 522.82 mg/d of zinc proteinate (Zn-Pro) or 467.88 mg of zinc methionine (Zn-Met) (equivalent to 80 mg Zn).The experiment lasted for 14 d.Before morning feeding on day 1 and 14, the body weight of each calf was measured to calculate the average daily gain.Milk intake was recorded daily and used to calculate average daily feed intake and F/G.The feces score was assessed by a specialized person before morning feeding every day, and the incidence of diarrhea was calculated.Before morning feeding on day 14, the serum samples were collected for the determination of calcium, phosphorus, zinc, copper and iron concentrations, activities of alkaline phosphatase and superoxide dismutase, metallothionein concentration, malondialdehyde concentration and total antioxidant capacity.【Result】 Compared with the CON group, supplementation with Zn-Met significantly increased the average daily gain of dairy heifers (P<0.05), supplementation with Zn-Pro and Zn-Met decreased the incidence of diarrhea and increased the serum zinc concentration of heifers (P<0.05), and the serum zinc concentration of heifers in Zn-Met group was significantly higher than that in Zn-Pro group (P<0.05).Compared with the CON group, supplementation with Zn-Met significantly decreased the serum copper concentration of heifers (P<0.05), but there was no significant difference between the Zn-Pro group and the control group(P>0.05).Compared with the CON group, supplementation with Zn-Pro and Zn-Met both significantly increased the serum concentration of metallothionein (P<0.05), but only Zn-Met increased serum activity of alkaline phosphatase (P<0.05).Superoxide dismutase activity and total antioxidant capacity in serum of heifers were increased by Zn-Met supplementation (P<0.05), but supplementation with Zn-Pro had no significant effect (P>0.05).Supplementation with Zn-Pro and Zn-Met reduced the concentration of malondialdehyde in serum of dairy heifers (P<0.05).【Conclusion】 Supplementation with Zn-Met in the diet improved the growth performance, reduced the incidence of diarrhea, and improved the zinc metabolism of dairy heifers, whose effects were better than that of Zn-Pro.Therefore, it was suggested to add 80 mg Zn/d as Zn-Met to the diet of dairy heifers.

【Objective】 The objective of this trial was to investigate the protective effect of quercetin on the intestinal tract of Porcine epidemic diarrhea virus(PEDV)-infected piglets by studying the effect of quercetin on the intestinal morphology, intestinal antioxidant function and relative expression of genes related to jejunal lipid metabolism in pigs infected with PEDV.【Method】 Eighteen healthy 7-day-old weaned piglets were randomly divided into 3 groups:, control group, PEDV group, and quercetin+PEDV group, with 6 replicates per group and one pig per replicate.The trial lasted for 8 days.On the 0-7th days of the trial, the piglets in the quercetin+PEDV group were orally administered with quercetin at the dosage of 10 mg/kg BW, the rest of the groups were orally administered with equal volume of artificial milk.On the 5th day of the trial, piglets in the PEDV group and PEDV+quercetin group were administered with PEDV (10^4.5TCID₅₀), and piglets in control group were given the same volume of PBS solution.On 8th day of the trail, all piglets were sacrificed, duodenum, jejunum, ileum and colon samples were taken for testing and small intestinal tissue was sliced.【Result】 Compared with the control group, in the PEDV group, the villous height and the ratio of villi height to crypt depth of the jejun and ileum were reduced (P<0.01), the activities of glutathione peroxidase (GSH-Px) and total superoxide dismutase (T-SOD) were decreased in jejunum, ileum, and colon (P<0.05), the viabilities of catalase (CAT) was decreased in duodenum, jejunum, ileum and colon (P<0.05), and the contents of malondialdehyde (MDA) was increased in ileum and colon (P<0.05), the relative expression of apolipoprotein A1(APOA1), apolipoprotein B(APOB), apolipoprotein C2 (APOC2), fatty acid binding protein 2 (FABP2), acyl-coenzyme A synthase long-chain family member 3 (ACSL-3) and fatty acid synthase (FASN) genes were downregulated in jejunum (P<0.01).Compared with the PEDV group, in the PEDV+quercetin group, the villus height and villus height to crypt depth ratio were increased in the jejunum and ileum of piglets (P<0.01), the activities of GSH-Px and T-SOD were increased in the jejunum, ileum and colon (P<0.05), the viabilities of CAT was increased in duodenum, jejunum and colon (P<0.05), the contents of MDA was decreased in the ileum and colon (P<0.05), the relative expression of APOB, FABP2, and FASN genes were upregulated in the jejunum(P<0.01).【Conclusion】 Dietary quercetin supplementation could alleviates the intestinal injury of piglets caused by PEDV infection, the intestinal antioxidant capacity and jejunal lipid metabolism of piglets were improved.

【Objective】 The influences of different weaning strategies on growth, serum biochemical indexes and antioxidant capacity of yak calves were investigated in the present study, with the purpose of exploring scientific early fostering pattern in yak calves.【Method】 24 healthy newborn yak calves with similar body weight were selected and randomly divided into 3 treatment groups.8 calves (half males and half females) were assigned to each group.The calves in control group (GF) were breastfed with dam on grazing pasture, while the calves in early weaning group (EW) were breastfed for 15 days after birth and then separated from the dam.These calves were sebsequently fed with milk replacer by gradual transition, and starter concentrate and natural grass were provided for ad libitum intake.Milk replacer feeding was stopped when the starter concentrate intake of calves reached 0.5 kg/d.At 90 days of age, the calves were switched to grazing feeding on natural grassland without concentrate.Calves in early weaning with probiotics group (EWP) followed the same weaning protocol as EW group, and probiotics compound (Lactobacillus, Bacillus and yeast, ≥ 9.98×10¹¹ CFU/g) was supplemented into the milk replacer and starter concentrate.The body weight and size indexes were measured at 30, 60, 90 and 150 days of age.Blood samples were collected from jugular vein of calves before morning feeding at 30, 60 and 90 days of age.Serum samples were separated for the analysis of metabolites, hormones, immunoglobulins and antioxidant capacity.【Result】 At 30 days of age, the body weight, body size, serum glucose (GLU), triglycerides (TG), cholesterol (CHO), growth hormone (GH), insulin-like growth factor 1 (IGF-1), and immunoglobulin A, G (IgA, IgG) concentrations of calves in EW and EWP groups were significantly lower than those in GF group (P<0.05), and the cortisol level of EW group was significantly higher than that of GF group(P<0.05).There was no significant difference in body weight and body size among treatments from 60 to 150 days of age (P>0.05).At 60 days of age, the serum IGF-1, glutathione peroxidase (GSH) levels were significantly increased in EW and EWP groups compared to GF group (P<0.05).At 90 days of age, the serum GLU, blood urea nitrogen (BUN), IGF-1, thyroxine (T₄) and IgA concentrations of calves in EW and EWP groups were significantly greater than those in GF group (P<0.05).Moreover, calves in EWP group had significantly higher TG, GH concentrations and catalase (CAT) enzyme activity compared to GF group (P<0.05).【Conclusion】 In comparison with the breastfeeding model with grazing dam, both of the two early weaning strategies had a negative impact on yak calf in the early stage (30 days of age).However, the supplementations of milk replacer and starter concentrate after early weaning (60-90 days of age)were beneficial to improve the later growth, nutritional metabolism, immunity and antioxidant capacity of calves.Besides the beneficial effect of early weaning strategy, probiotics supplementation in early weaning further relieved the weaning stress, and promoted the growth and antioxidant capacity of yak calves.

【Objective】 The study was to investigate the role of quercetin in the prevention and treatment of Porcine epidemic diarrhea virus (PEDV) infection in piglets.【Method】 Eighteen healthy weaned piglets (Ducoc×Landrace×Yorkshire, 7-day-old neonatal) with similar body weight were selected and randomly divided into 3 groups (control, PEDV and PEDV+ quercetin groups), with 6 replicates in each group and one piglet per replicate, the trial lasted for 11 d.After 3 d acclimation period, piglets in the PEDV+ quercetin group were orally administered with 10 mg/kg BW of quercetin during the days 4-10 of the experiment, the other groups of piglets were given the same volume of artificial milk.On the day 8 of the experiment, the piglets in PEDV and PEDV+ quercetin groups were orally administered with 1×10^4.5 TCID₅₀ PEDV, and the control group was given with the same volume of PBS.On the day 11 of the experiment, all piglets were weighed and orally administered with D-xylose (0.1 g/kg BW) on an empty stomach.One hour later, jugular vein blood samples were harvested to detect blood biochemical indexes, plasma diamine oxidase (DAO) activity and D-xylose content, and then all piglets were slaughtered and jejunal mucosa was taken to detect the relative expression of intestinal barrier function related genes, including villin, claudin-1, occludin and intestinal fatty acid binding protein (iFABP).【Result】 Compared with the control group, in PEDV group the average daily gain of piglets was significantly decreased (P<0.05), the fecal score was significantly increased (P<0.05);The content of HDL in blood was decreased significantly (P<0.05), and the contents of creatinine and urea nitrogen were increased significantly (P<0.05);The content of D-xylose in plasma was decreased significantly (P<0.05);The relative expressions of claudin-1 and iFABP genes in jejunum were significantly down-regulated(P<0.05).Compared with PEDV group, in PEDV +quercetin group the average daily gain of piglets and the content of D-xylose in plasma were significantly increased (P<0.05);The contents of creatinine and urea nitrogen in blood were significantly decreased (P<0.05);The relative expressions of claudin-1, villin and iFABP genes in jejunum were significantly up-regulated (P<0.05).【Conclusion】 10 mg/kg BW quercetin could alleviate the growth inhibition and renal function injury of piglets caused by PEDV infection to a certain extent, and improve the intestinal absorption and barrier function.

【Objective】 This experiment was conducted to investigate the effect of glycyrrhizic acid(GA) on the immune function of Yellow-feathered broilers.【Method】 A total of 300 one-day-old healthy Yellow-feathered broiler chickens with similar body weight were selected and randomly divided into 6 groups, with 5 replicates in each group, 10 in each replicate, namely the normal saline group (NS), lipopolysaccharide (LPS) group, astragalus polysaccharide group(APS), high-dose glycyrrhizic acid group(GA_H), middle-dose glycyrrhizic acid group(GA_M) and low-dose glycyrrhizic acid group(GA_L).The NS group was intraperitoneally injected with 2 mL of 0.9% sterile saline in the morning of 14, 16, 18 and 20 days old, and the other groups were injected with an equal volume of LPS solution (1.5 mg/kg BW) at the same time to establish immune stress model in vivo. NS group and LPS group were fed with the basal diet, while APS group, GA_H, GA_M and GA_L experimental groups were supplemented with 400 mg/kg astragalus polysaccharides (APS) and 120, 100, 80 mg/kg of GA on the basis of the basal diet at the stage of establishment of the stress model and 3 days before and after the stage (11-23 days).The experimental period was 49 days.At 21, 28, 35, 42 and 49 days of age, 2 chickens were randomly selected in each replicate to collect wing vein blood, and the contents of CD4, CD8 and immunological active material IgA, IgG and IgM in serum were determined by enzyme-linked immunosorbent assay (ELISA).【Result】 Compared with NS group, at the age of 21-49 days, the serum CD4 molecule content of the other 5 groups of broilers was extremely significantly decreased (P<0.01), while the serum CD8 molecule, IgA, IgG, IgM (except GA_H group and APS group) were increased.Compared with LPS group, the serum CD4 molecular contents of broilers in APS group and three GA groups were increased, while the serum CD8 molecular contents were extremely significantly or significantly decreased (P<0.01;P<0.05).The serum IgA, IgG and IgM contents were extremely significantly decreased (P<0.01).In addition, among the three GA groups, the serum CD4 content of broilers was increased with the increase of GA supplementation, and the increase in the GA_H group was the closest to that in the APS group, especially at 42 days of age, there was no significant difference between GA_H group and APS group (P>0.05), and all were extremely significantly or significantly higher than those in the LPS group (P<0.01;P<0.05)。However, the serum CD8, IgA, IgG, and IgM contents of broilers showed a decreasing trend with the increase of GA supplementation, and the decrease in GA_H group was the closest to that in APS group.There was no significant difference in CD8 molecular content between the two groups (P>0.05), and at 35 and 49 days of age, there was no significant difference in CD8 molecular content between GA_H group and NS group (P>0.05).In terms of IgA content, there was no significant difference between GA_H group and APS group except at 35 days of age (P>0.05), and at 21 and 49 days of age, there was no significant difference between GA_H group and NS group (P>0.05).In terms of IgG content, there was no significant difference between GA_H group and APS group except at the age of 28 days (P>0.05), and at the age of 35 days, there was no significant difference between GA_H group and NS group (P>0.05).In terms of IgM content, except for 42 days, there was no significant difference between GA_H group and APS group at other days (P>0.05).At 21 days, there was no significant difference between GA_H group and NS group (P>0.05).【Conclusion】 GA could effectively relieve the immune stress response of Yellow feather broiler chickens induced by LPS stimulation, and enhance the immune regulation ability.

【Objective】 The aim of this study was to investigate the effects of genetic variation of myosin heavy chain 3 (MYH3) and myosin heavy chain 13 (MYH13) on meat quality traits in pigs.【Method】 The meat quality traits (intramuscular fat (IMF), moisture, drip loss, pH, meat color (L^*, a^* and b^* values)) of longissimus dorsi muscle samples in Beijing Black pigs were measured.The promoter and CDS regions of MYH3 and MYH13 genes were genotyped by PCR and Sanger sequencing.The SNPs associated with meat quality traits were determined by covariance analysis using sex, field and age as covariates, the expression of MYH3 and MYH13 genes were detected by Real-time quantitative PCR.【Result】 A total of nine SNPs were screened in this study.There was a missense mutation (c.5782 G>C) in CDS region of MYH3 gene, which was significantly correlated with IMF content (P<0.05), the expression of MYH3 gene was positively correlated with IMF content.There were four SNPs in CDS region of MYH13 gene, of which two SNPs (c.1923 G>A and c.1308 G>A) were significantly correlated with meat color a^*value, one SNP (c.963 G>A) was significantly correlated with drip loss, one SNP (c.237 G>T) was significantly associated with meat color L^* value (P<0.05).There were four SNPs in promoter region of MYH13 gene, two SNPs (rs699287502 and rs318639161) were significantly associated with meat color L^* value, two SNPs (rs321315318 and rs330770991) were significantly related to drip loss (P<0.05), and the expression of MYH13 gene was negatively correlated with drip loss.【Conclusion】 One SNP of MYH3 gene was significantly associated with IMF, and its gene expression was positively correlated with IMF content.Three SNPs of MYH13 gene were significantly associated with drip loss, the expression of two SNPs of MYH13 gene promoter region was negatively correlated with drip loss.Three SNPs of MYH13 gene were significantly associated with meat color L^* value, two SNPs of MYH13 gene were significantly associated with meat color a^* value.These SNPs could be used as candidate gene functional loci for meat quality traits in Beijing Black pigs.

【Objective】 The aim of this study was to investigate the relationships between the phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit beta (PIK3CB) gene polymorphism and reproductive and milk production traits in Chinese Holstein cattle.【Method】 The single nucleotide polymorphisms (SNP) site of PIK3CB gene in Chinese Holstein cattle was screened using hybrid pool sequencing.SNP genotyping and population genetic analysis were performed in 1 160 Holstein dairy cows based on Kompetitive Allele Specific PCR (KASP) method.The linear model was employed to perform the association between SNP and 11 reproductive and milk production traits based on unit point and haplotype combinations.【Results】 A total of 17 SNPs were detected in PIK3CB gene, and 7 SNPs were screened for subsequent analysis.The association analysis revealed that a total of 7 SNPs were significantly or extremely significantly associated with various target traits (P<0.05 or P<0.01).The individuals with AA genotype at g.130433743 A>G in exon and GG genotype at g.130448069 G>A in variable splice region had the lowest mating interval, milk yield, milk protein and milk fat, and the highest somatic cell score, which suggested the individuals with the above genotypes had shorter mating intervals between the first and last mating and relatively poor milk production performance.The individuals with AA genotype at g.130387717 G>A had the lowest mating age and the lowest first to last mating interval of young cattle, and the highest milk yield, milk protein yield and milk fat.The above mentioned 3 SNPs could be considered as candidate SNPs for reproductive and milk production traits in Chinese Holstein cattle.Haplotype analysis revealed that 6 SNPs of g.130387717 G>A, g.130430832 A>-, g.130433743 A>G, g.130433982 C>T, g.130446073 C>T and g.130448069 G>A were closely linked to form a haplotype block, and were significantly or extremely significantly associated with various target traits (P<0.05 or P<0.01).Among the various haplotypes, H2H3 and H2H4 haplotype combinations had better reproductive and milk-producing performance, and were the advantageous haplotype combination.【Conclusion】 Polymorphism of PIK3CB gene had abundant variants which was associated with reproductive and milk production traits in Chinese Holstein cattle. g.130433743 A>G, g.130448069 G>A and g.130387717 G>A could be considered as potential molecular markers to provide the theoretical basis for balanced breeding of Chinese Holstein cattle.

【Objective】 The purpose of this study was to explore the metabolic mechanism of testis development in Guanzhong dairy goats from the perspective of metabolomics.【Method】 Six 1 month old weaned male and six 24 months old physically mature male of Guanzhong dairy goats were selected to collect testis tissue, and the testis tissues were chemically detected by liquid chromatography mass spectrometry (LC-MS) technology.The chemical detection, comparison and identification of different metabolites in two groups were carried out through the combination of multi-dimensional and single dimensional analysis.The potential key pathways in developmental metabolism of Guanzhong dairy goats were screened out using the enrichment differential metabolite analysis.【Result】 A total of 334 differential metabolites were screened out in testis of Guanzhong dairy goats at 1 and 24 months old, of which 137 were significantly upregulated and 197 were significantly downregulated.The top 36 differential metabolites were compared and identified, which were classified into 4 categories:Lipids and lipid-like molecules, organic acids and their derivatives, unclassified compounds, and phenylpropanes and polyketides.The correlation analysis showed that during testis development of Guanzhong dairy goats from 1 and 24 months old, lipids and lipid-like molecules had the largest positive correlation with glycerophospholipids, and carboxyl groups and their derivatives had the largest negative correlation with glycerophospholipids.The enrichment analysis of different metabolites resulted in 7 potentially important metabolic pathways, which were choline metabolism, arginine and proline metabolism, central carbon metabolism in tumor, ferroptosis, glutathione metabolism, aminoacyl-tRNA biocomposition, and taurine and hypotaurine metabolism.【Conclusion】 The results of this experiment revealed that the differential metabolites and metabolic mechanisms of testis in Guanzhong dairy goats at 1 and 24 months old from the perspective of metabolomics, and provided abasis for future research on the development of mammalian testis.

【Objective】 This study was aimed to investigate the sequence characteristics, polymorphisms of plemorphic adenoma gene 1 (PLAG1) gene and its effects on birth weight and body size in goats.【Method】 Boer goats were selected as the object, the PLAG1 gene were cloned and sequenced, the expression pattern of PLAG1 gene in various tissues of Boer goats at different ages and birth weight was identified by Real-time quantitative PCR, the PLAG1 protein level of leg muscle in lambs with different birth weight was detected by Western blotting.The SNPs in CDS and 3'-UTR of PLAG1 gene in Boer goats were further screened, and its correlation between different genotypes and birth weight and body size in Boer goats were analyzed.【Result】 The total length of PLAG1 gene CDS in Boer goats was 1 500 bp, encoding 499 amino acids.PLAG1 protein was relatively conservative.The tissue expression profile showed that the expression of PLAG1 gene in heart, small intestine and leg muscle in lambs with large birth weight were significant or extremely significantly higher than those with small birth weight, the expression of different tissues in fetal Boer goats was significantly or extremely significantly higher than that in adult ewes (P<0.05 or P<0.01). The expression of PLAG1 protein in leg muscles of lambs with large birth weight was extremely significantly higher than those with small birth weight (P<0.01).6 SNPs of PLAG1 gene 3'-UTR in Boer goats were screened, which were 2664 A>G, 2712 A>G, 2721 T>C, 2879 T>A, 2990 A>T and 3270 A>G, respectively.Correlation analysis showed that the birth tube circumference of AG genotype individuals at 2664 A>G was significantly longer than that of GG genotype (P<0.05);The birth length of TT genotype individuals at 2721 T>C was significantly longer than that of TC genotype (P<0.05);The birth tube circumference of TA genotype individuals at 2879 T>A was significantly longer than that of AA genotype (P<0.05);The birth weight of AA genotype individuals at 2990 A>T was significantly higher than that of AT genotype (P<0.05);The birth length of GG genotype individuals at 3270 A>G was significantly longer than that of AA genotype, and the birth chest circumference of AG genotype individuals at 3270 A>G was significantly larger than that of AA genotype (P<0.05).【Conclusion】 The expression of PLAG1 gene in different tissues of fetal goats were higher than those of adult ewes, the expression during early development was related to the birth weight.PLAG1 gene could be used as a molecular marker for early growth and breeding of Boer goats.

【Objective】 The purpose of this study was to explore the population characteristics of milking speed indicators of Holstein cows and the influence of non genetic factors on them.【Method】 In this study, a total of 906 748 milking records from 8 757 dairy cows in a large-scale dairy farm in East China were collected.Six milking speed indicators were defined, including single milking yield (SMY), milk yield in first two minutes (MIN2), milking time (DUR), the proportion of MIN2 to SMY(PMY2), average flow rate (AMF) and the peak of flow rate (PMF).Subsequently, descriptive statistics of the above mentioned six indicators were obtained, and the Pearson correlations among these indicators were calculated.Furthermore, the impacts of season, parity, lactation stage, first calving age and milking shift on milking speed indicators were analyzed using a fixed model.【Result】 Among the six milking speed indicators, except for PMY2, the frequency distribution of SMY, MIN2, DUR, AMF and PMF were approximately normal distribution, and each indicator had a relatively large variation with the variation coefficient of about 30%.There were significant correlations among six milking speed indicators, and the coefficients ranged from -0.84 to 0.89.The milking speed was moderately or strongly correlated with milk yield, weakly negatively correlated with milking time, and weakly positively correlated with PMY2.It was found that season, parity, lactation stage, age of first calving and milking shift had highly significant impacted on milking speed indicators(P<0.01).Among different seasons, SMY and DUR in winter were the largest, AMF in summer and PMF in autumn were the highest.Among different lactation stages, SMY, DUR, AMF and PMF in the second lactation stage(45-99 d) were the largest.Among different milking shifts, SMY, DUR, AMF and PMF at night shifts (00:00-06:00) of dairy cows were the highest.【Conclusion】 The milking speed indicator with large variation was sensitive to physiological and environmental changes, which could provide useful information for selection of milk production performance.

【Objective】 The aim of this study was to investigate the effect of adding different concentrations of taurine to Modena diluent under negative pressure on the quality and antioxidant capacity of Landrace boar sperm stored at room temperature, in order to provide reference for the improvement of pig semen preservation system.【Method】 Taurine was added to Modena diluent at the concentration of 0(control group), 1, 5 and 10 mmol/L, respectively, and the semen of each group were stored under the condition of-0.04 Mpa for 11 days.On 1st, 3rd, 5th, 7th, 9th and 11th days, sperm motility, viability, malformation rate, average path velocity (VAP), curvilinear velocity (VCL) and beatcross frequency (BCF) were detected by computer-aided sperm analysis system (CASA).The total antioxidant capacity (T-AOC) and H₂O₂ content of semen were determined by kits.【Result】 The motility and viability of boar sperm in the three taurine groups were significantly higher than those in control group during the storage period of 1-11 days (P<0.05).On the 11th day, the malformation rate of boar sperm in 5 mmol/L taurine group was significantly lower than that in other groups (P<0.05).From the 3rd day, the VAP of boar sperm in 1, 5 and 10 mmol/L taurine groups were significantly higher than those in control group (P<0.05), and the 5 mmol/L taurine group had the best effect.On the 11th day, the VCL and BCF of boar sperm in 5 mmol/L taurine group were significantly better than those in other groups (P<0.05).Taurine could significantly increase the T-AOC of boar sperm (P<0.05), but the T-AOC was almost undetectable on the 11th day.From 1 to 3 days, there was no significant difference in the H₂O₂ content of boar sperm among all test groups, from 5 to 11 days, the H₂O₂ content of the three taurine groups was significantly lower than that of the control group (P<0.05).【Conclusion】 Under the condition of -0.04 Mpa, adding taurine to Modena diluent could significantly improve the quality parameters and antioxidant properties of boar semen stored at room temperature, and the best concentration was 5 mmol/L, and the storage time was less than 9 days.

【Objective】 The aim of this study was to test the quality of long-term frozen embryos and forzen semen of Hu sheep in national livestock gene banks, and to evaluate the effect of cryopreservation.【Method】 The frozen embryos (preserved for 20 years) and frozen semen (preserved for 20 and 30 years) produced in 1988 and 1998 and stored in the national livestock gene bank were resuscitated.After corresponding identification, embryo transfer and artificial insemination were carried out respectively.At the same time, 0-year frozen semen and fresh semen were used as controls to determine the conception rate and the lambskin performance, growth performance and breeding performance of offspring, and test the preservation effect.【Result】 A total of 36 embryos were thawed in this experiment, including 22 class-A embryos, 7 class-B embryos, 5 class-C embryos and 2 class-D embryos.The recovery rate of frozen embryos reached 94.44% (34/36), the rate of class A embryos reached 61.11% (22/36).34 embryos were transplanted and 17 embryos were lambed.The lambing rate of frozen embryos was 50.00%.The average sperm motility of 30-year frozen semen was 35%, the conception rate was 58.57%, and the average number of lambs was 1.90.The average sperm motility of 20-year frozen semen was 31%, the conception rate was 53.66%, and the average number of lambs was 1.95.The body shape and appearance of embryo resuscitation transplanted sheep, 30-year and 20-year frozen semen progenies were consistent with the characteristics of pure Hu sheep, and there were no abnormal individuals.The first grade lambskin percentage of the lambs were 31.25%, 42.03% and 23.81%, respectively, which maintained the lambskin characteristics of the Hu sheep in that year.The growth, development and reproductive performance were basically consistent with those of the existing Hu sheep population.【Conclusion】 The method of long-term preservation of frozen embryos and semen of Hu sheep by cryopreservation was feasible, which was of great significance to the conservation of local breeds.

Wild-type p53-induced phosphatase 1(Wip1) is a member of protein phosphatase 2C (PP2C) family, which regulates a number of critical signal molecules, such as p53, MAPK and Chk1/Chk2, and so on, it plays an important role in physiological and pathological processes of cell cycle, proliferation, differentiation, apoptosis, senescence, autophagy and DNA damage repair in animal.With the rapid development of molecular biology, many studies have reported that the deletion of Wip1 gene will imbalance the level of reproductive hormones in mice, and it affects spermatogenesis by regulating signal pathways such as ATM, Wnt, apoptosis and inflammation, resulting in the decrease of animal fecundity.In addition, Wip1 gene also regulates DNA damage response and dephosphorylation through dynamic balance to affect oocyte and embryonic development, so as to regulate the reproduction of female animals.The immune system is an important system in the body to perform immune response and immune function, and it is closely related to tumorigenesis.Knock-out Wip1 gene will also increase the sensitivity to pathogens, affect the migration and apoptosis of T cells, B cells and neutrophils, and lead to inflammation.As a proto-oncogene, Wip1 gene also participates in tumorigenesis by regulating various signal molecules, affecting DNA damage repair, cell cycle progression and apoptosis.Therefore, Wip1 gene plays an important role in biological processes such as animal reproductive regulation and immune inflammation.At present, Wip1 gene has been attracting more and more attention, especially its mechanism of regulating the occurrence and development of animal diseases has become a research hotspot.This study mainly reviewed the regulatory effect of Wip1 gene on animal reproduction and immune, in order to provide new ideas for livestock breeding, disease prevention and targeted therapy

【Objective】 This study was aimed to establish a rapid and accurate indirect ELISA method for detecting Streptococcus suis autolysin (atl) antibody.【Method】 A pair of primers was designed according to the Streptococcus suis atl gene sequence registered in GenBank, and the atl gene sequence was obtained from Streptococcus suis by PCR.The recombinant plasmids pET-32a-atl and pGEX-4T-1-atl were constructed, and then were transferred into BL21 competent cells for induction expression.The purified atl-His fusion protein was used as immunogen to immunize New Zealand White rabbits to prepare polyclonal antibody.The reactivity of atl-GST fusion protein was detected by Western blotting, using the purified atl-GST fusion protein as the coating antigen and the positive serum of Streptococcus suis infection as the standard serum, an indirect ELISA method for the detection of Streptococcus suis autolysin antibody was established.184 serum samples were detected by the ELISA method established in this test and the commercial Streptococcus suis type 2 ELISA antibody detection kit at the same time, and the clinical samples were detected by the method established in this test.【Result】 atl gene was successfully amplified from Streptococcus suis.The constructed recombinant plasmids was induced, expressed and purified to obtain 40 ku atl-His and 48 ku atl-GST fusion proteins.Western blotting result showed that rabbit anti atl-His antiserum had good reactivity with atl-GST fusion protein.The results of indirect ELISA showed that the critical value of negative serum and positive serum was 0.318, and the positive serum was still positive when diluted to 1:1280.The intra-batch and inter-batch coefficients of variation were both less than 10%, indicating that the method had good reproducibility and sensitivity. 96 positive samples were detected by the ELISA method and 66 positive samples were detected by the commercial Streptococcus suis type 2 ELISA antibody test kit, with a compliance rate of 71.7%.A total of 458 pig serum samples from different feeding stages of some pig farms in Jiangxi were detected by the established method, of which 277 positive samples were detected, and the positive rate was 60.4%.【Conclusion】 The indirect ELISA method for detecting Streptococcus suis autolysin antibody infection was successfully established and preliminarily applied, which laid a foundation for the seroepidemiological investigation of Streptococcus suis.

【Objective】 The aim of this study was to understand and master the epidemiological characteristics and biological characteristics of Novel duck reovirus (NDRV), and to provide theoretical basis and technical support for the prevention and treatment of NDRV.【Method】 The tissues suspected of Duck reovirus infection was collected from a duck farm in Hebei, and after aseptic treatment, it was inoculated with SPF chicken embryos to isolate the virus, and the third-generation allantoic fluid was harvested for hemagglutination test and in vitro cell culture.The virus was identified by observation and indirect immunofluorescence assay (IFA).The isolated virus was subjected to animal regression test, and its σC gene was analyzed by Mega 7.0.【Result】 The isolated virus couldn't agglutinate chicken erythrocytes, and could kill chicken embryos.The dead chicken embryos were hemorrhagic and congested.The virus was NDRV positive by PCR identification, and other pathogens (Avian reovirus, Duck hepatitis virus, Duck Tembusu virus, Duck plague virus and Fowl adenovirus 4) were all negative, and the isolated virus was named BD/CHN/2020 strain.Spherical virus particles with a diameter of 60-80 nm were observed by electron microscopy and no envelope could be seen after viruses were purificated.The strain could be proliferated stably on BHK cells and LMH cells and produced a cytopathic effect (CPE) of cell fusion.IFA results showed that specific green fluorescence could be observed under a laser confocal microscope after BD/CHN/2020 strain was inoculated in BHK cells.After the BD/CHN/2020 strain was subcutaneously inoculated, the diseased ducks showed clinical symptoms such as depression and white loose stools.The autopsy showed pathological changes such as spleen hemorrhage, enlargement, and white necrotic foci.Sequence alignment showed that the BD/CHN/2020 strain and NDRV (TH11, 091 and other strains) were in the same branch, and had the highest similarity of nucleotide and amino acid sequences to the NDRV SY strain, which were both 99.7%, and belonged to the NDRV.Compared with the inactivated vaccine TH11 strain (KC493571.1) and the attenuated vaccine JS01-105P strain (CCTCC No.:V202168), the amino acid site mutations of BD/CHN/2020 strain had amino acid site mutations at sites 93, 120, 132, 158, 253 and 298.【Conclusion】 One NDRV BD/CHN/2020 strain was successfully isolated.The isolated strain had strong pathogenicity to Peking duck.Compared with the domestic vaccine strain, the σC protein of the BD/CHN/2020 strain had already undergone amino acid changes.The results laid the foundation for the epidemiology and vaccine development of NDRV disease.

【Objective】 The purpose of the experiment was to identify the etiology and genotype of calves suffering from diarrhea in a large-scale cattle farm in Xinjiang.【Method】 15 fecal samples were randomly collected from calves suffering from diarrheal diseases in a cattle farm in Xinjiang using the antigen diagnostic kit method.The virus was isolated and passaged in Marc-145 cells.Indirect immunofluorescence assay (IFA) and negative-staining direct electron microscopy were performed on the isolated strains to further identify the pathogen.The VP6 and VP7 genes of the isolates were amplified and sequenced by PCR and genetic evolution analysis.【Result】 The results of the antigen diagnostic kit showed that 3 stool samples were positive for Bovine rotavirus (BRV) antigen.Positive fecal samples were inoculated into Marc-145 cells, and only 1 sample showed obvious cytopathic effect (CPE) in the ninth passage.The IFA results showed that the Marc-145 cells inoculated with this isolate had bright green fluorescence, but no fluorescence was seen in the control group.Circular virus particles of about 65 nm were observed by electron microscope, and were named as XJ-2022 strain.The target bands of VP6 and VP7 genes were obtained by PCR amplification with lengths of 1 356 and 342 bp, respectively.Gene similarity comparison and genetic evolution analysis showed that VP6 gene had the highest similarity and the closest genetic evolutionary relationship with the human group A Rotavirus reference strain DB2015-066 (LC367318.1), and VP7 gene had the highest similarity and genetic evolutionary relationship with bovine G10 Rotavirus reference strain XJX2(MN937506.1), the isolate was identified as group A G10 Rotavirus.【Conclusion】 The genotype of group A G10 BRV XJ-2022 strain was successfully isolated, which was the first multi-host gene reassortment virus discovered in Xinjiang.

【Objective】 The aim of the experiment was to establish a canine nanobody (Nb) library against Canine parvovirus(CPV) and screen out the Nb with neutralizing activity.【Method】 Heavy chain variable region (VH) amplification was performed using cDNA from spleen tissue of high free Beagle dogs, then connected VH to pBSD vector to establish pBSD-Nb library.The high-affinity nanobodies were screened out from pBSD-Nb library by bacterial display technology combined with flow cytometry detection, and then positive clones were obtained and sequenced.The upstream and downstream primers of Nb genes were designed by Primer Premier 5.0 software, then Nb gene fragments were amplified by PCR.The target protein was expressed by pET-27b and purified by denaturation and renaturation.The purified protein were analyzed by SDS-PAGE.Nb was coated with 96 well plate, and the affinity of Nb to CPV was detected by ELISA method.The inhibition of Nb on CPV infection of F81 cells was detected by in vitro virus neutralization test.【Result】 PCR amplification results showed that a obvious band at 384 bp was consistent with the expected size, which proved that the pBSD-Nb library was successfully constructed.After the bacteria were shown, the results of flow cytometry showed that 9 clones of Nb with high positive peak deviation were screened and named Nb1, Nb2, Nb3, Nb4, Nb5, Nb6, Nb7, Nb8 and Nb9, respectively.9 clones of Nb gene fragments were amplified by PCR, and a band of 384 bp was obtained.SDS-PAGE results showed that there was a band at about 14 ku, indicating that the purified protein was successfully obtained.ELISA results showed that 6 of the 9 Nb strains had strong affinity for CPV.In vitro virus neutralization test showed that 1 strain of Nb could neutralize the virus and inhibit F81 cytopathic changes at an effective concentration of 0.05 mg/mL.【Conclusion】 In this study, the pBSD-Nb library was successfully established, 6 clones of Nb with high affinity for CPV were screened, and 1 clone of Nb with neutralizing activity was successfully obtained, providing a new solution for the development of CPV the rapeutic antibody.

【Objective】 The aim of this study was to explore the pathogenicity of Bovine viral diarrhea virus (BVDV) and the immune effect of BVDV E2 recombinant protein in New Zealand White rabbits.【Method】 The BVDV virus was purified and its titer was determined by Reed-Muench method. In the pathogenicity analysis test, 10 New Zealand White rabbits were randomly divided into infection group and control group, with 5 rabbits in each group. The infected group was challenged with 1 mL purified BVDV virus (nasal drip 500 μL, ear edge intravenous injection 500 μL), and the control group was treated with equal volume of normal saline, once a day for 3 days. The clinical symptoms and body temperature were observed every day. Blood samples were collected from auricular vein on 6, 9, 12, 15 and 17 days after inoculation. On the 17th day of infection, nasal swabs were collected for RT-PCR identification. After collection, trachea, lung, spleen and small intestine tissues were dissected and pathological sections were prepared to observe the pathological changes. In the immune effect evaluation experiment, 10 New Zealand White rabbits were randomly divided into the immune group and the control group, with 5 rabbits in each group. New Zealand White rabbits were immunized with E2 recombinant protein (1 mg/rabbit) mixed with adjuvant by intramuscular multi-point injection, and the control group was immunized with equal volume of normal saline, two immunizations were given at an interval of 14 days.Serum samples were collected on 0, 7, 14, 21 and 28 d after the first immunization, and the levels of specific antibodies against recombinant protein were detected by indirect ELISA. On the 28th day after the first immunization, nose swabs were collected for RT-PCR identification, and trachea, lung, spleen and small intestine tissues were collected for pathological sections to observe the pathological changes and immunohistochemistry detection.【Result】 The titer of the purified virus was 4.16×10⁶ TCID₅₀/mL. Compared with control group, some New Zealand White rabbits in the infected group had reduced activities and slightly reduced food intake within 6 days, and gradually returned to normal after 6 days. Diarrhea symptoms appeared on the 13th day of infection, and the body temperature increased slightly on the 5th day, but all fluctuated within the normal range. Compared with control group, white blood cells and platelets in infection group were significantly and extremely significantly decreased on the 6th and 9th day of challenge (P<0.05 or P<0.01). On the 12th, 15th and 17th days of challenge, white blood cells, platelets and lymphocytes in the infection group were extremely significantly decreased (P<0.01). RT-PCR test of nasal swabs was positive, and histopathological changes of trachea, lung, spleen and small intestine were mild to severe. The results of indirect ELISA showed that the titer of serum antibody was 1:16 to 1:32 on the 7th day after the first immunization. The nasal swabs of New Zealand White rabbits in the immune challenge group were negative by RT-PCR. Histopathological observation showed slight histopathological changes in trachea and lung in the immune challenge group. On the 28th day after the first immunization, the titer of serum antibody was 1:256 to 1:512. Immunohistochemical test results showed that the results were negative in the immune group and positive in the control group.【Conclusion】 A model of disease in New Zealand White rabbits was established by intranasal and auricular vein injection of BVDV, BVDV E2 subunit vaccine could stimulate the body to produce specific antibodies and play the role of immune defense.

The reproductive capacity is an important factor to determine the production efficiency of pigs, and the abortion caused by virus infection seriously affects the reproductive efficiency of pigs.Understanding the pathogenic mechanism of porcine abortion-related viruses is helpful to improve the reproductive efficiency of pigs.However, a large number of studies have focused on the protein or genomic DNA of viruses and hosts.In recent years, long noncoding RNA (lncRNA) has revealed the interaction between viruses and hosts from a new perspective, it provides a new way to study the interaction between them.lncRNA is a transcription with a length of more than 200 nt, its function takes mainly through interaction with DNA, RNA, chromatin and protein, and is specifically expressed in certain developmental stages, tissues and disease states, and participates in various regulation of body.lncRNA is a key factor regulating the interaction between virus and host.After virus infects host, lncRNA is differentially expressed, and the corresponding target genes are enriched in inflammatory and immune-related signaling pathways, participating in inflammatory, immune and antiviral responses of body.In-depth understanding of the regulatory role of lncRNA between porcine abortion-related virus and host is of great significance for the prevention and treatment of porcine abortion caused by virus infection.In this paper, lncRNA and its relationship with porcine abortion-related viruses, host lncRNA and virus interaction and regulatory pathways were reviewed, and the existing problems and application prospects were introduced, in order to provide theoretical basis for porcine disease resistance breeding, development and design of porcine abortion-related drugs, and target treatment of abortion-related diseases.

Parasitic diseases seriously affect the health and safety of humans and animals around the world, and the economic losses are huge.Therefore, it is necessary to develop targeted vaccines for parasitic diseases to block the transmission of parasitic diseases between animals, and between animals and humans.Parasites have a variety of immune evasion mechanism, the developed subunit vaccine, live attenuated vaccine, inactivated vaccine have not achieved the desired preventive effect, and commercialized vaccines are mostly injectable vaccines, ordinary injection vaccines are cumbersome operations, high transportation and storage requirements and easy to cause animal stress, directly affecting economic costs, so in actual production we need new vaccines with more simple operation, easy storage, lower immunization cost to effectively prevent and control parasitic diseases.Many studies have shown that oral vaccines are easy to administer and only need to be vaccinated by oral administration and the host has a high level of antibody titer, which is expected to become an effective means of preventing parasitic diseases.Oral vaccine is a new type of vaccine, relying on the host's mucosal immune system to produce an effect, that is, through the body's mucosal surface inoculation can induce the body to produce lasting mucosal immunity, humoral immunity and cellular immunity, providing efficient immune protection for the host.Compared with traditional vaccines, the most significant advantages of oral vaccines are that they are easy to administer and have little stress, and they can also form a strong mucosal immune barrier.However, factors such as the environment of the gastrointestinal tract and the susceptibility to the formation of immune tolerances have also created great challenges for the development of oral vaccines.The author reviewed the mechanism of the mucosal immune system and oral vaccines, as well as the research progress of parasite oral vaccines in recent years and advantages and challenges of oral vaccines in order to provide a theoretical basis for the development of parasite oral vaccines.

【Objective】 The study was aimed to determine the inhibitory effect of artemisinin on Bovine viral diarrhea virus (BVDV) replication using molecular docking as well as in vitro tests, and provide new ideas for antiviral drug and formulation development.【Method】 BVDV-NS5B protein was used as the target, and the three-dimensional crystal structure of BVDV-NS5B protein was retrieved through the PDB database, and the structure was appropriately modified.The structure of artemisinin from TCMSP database was retrieved, and Autodock software was applied to perform molecular docking and score the binding energy.The maximum safe concentration of artemisinin for MDBK cells was subsequently determined using a CCK-8 kit.The selected concentrations of artemisinin were subjected to gradient dilution, and three different ways of spiking drugs, including adding virus before adding drug, adding drug before adding virus, and the simultaneous action of traditional Chinese medicine and virus, were used for drug antiviral test to determine the optimal drug inhibitory concentration, prophylactic concentration and killing concentration against virus.Real-time quantitative PCR was used to measure the copy number of BVDV under the three modes of drug action to further define the effect of drugs on BVDV replication.【Result】 Molecular docking data indicated that artemisinin interacted with BVDV-NS5B with a binding free energy of -28.6748 kJ/mol.The optimal drug safety concentration of artemisinin on MDBK cells was 100 μmol/L.The three modes of action could effectively affect the replication of BVDV, and artemisinin had the most obvious inhibitory effect on BVDV.【Conclusion】 Artemisinin could interact with the NS5B protein target of BVDV, and could effectively inhibit the replication of BVDV in MDBK cells.This study laid a foundation for the screening of anti BVDV traditional Chinese medicine.

【Objective】 This research was aimed to obtain antimicrobial peptides with low molecular weight, low hemolysis and good stability.【Method】 One peptide (YKK-18) was selected from the amino acid sequence of bovine hemoglobin β-subunit with the guidance of the structure-activity theory of antimicrobial peptides, and three modified peptides (LJ-1, LJ-2 and DLK-3) with 18 amino acid residues were designed using YKK-18 as a template.The antimicrobial activities of four peptides were reflected by minimum inhibitory concentration (MIC), the thermal stability, acid-base stability, salt ion stability and repeated freeze-thaw stability of peptides with antimicrobial activities were analyzed by agarose diffusion method, and the hemolysis of peptides with antimicrobial activities was determined by spectrophotometry.【Result】 The four designed peptides were all positively charged hydrophilic peptides, and the similarity of amino acid sequence were less than 50% compared with antimicrobial peptides in APD3 database.Secondary structure of four designed peptides had high content of alpha helix.The amphiphilic degree of modified peptides were high than template peptide.The template peptide YKK-18 had no antimicrobial activity against all tested strains, while three modified peptides had different degrees of antimicrobial activity against Escherichia coli, Salmonella, Pseudomonas aeruginosa, Staphylococcus and Candida albicans. DLK-3 peptide and LJ-1 peptide had better antimicrobial activity than LJ-2. But four designed peptides had no antimicrobial activity against Proteus mirabilis.Under the conditions of high temperature of 100 ℃, pH 4.0 to 10.0, physiological concentration of salt ions (Na⁺, K⁺, Ca²⁺, Mg²⁺, Fe³⁺, Cu²⁺) and 14 times of repeated freezing and thawing, three modified peptides had good antimicrobial activity.The hemolysis of DLK-3 was dose-dependent, and at 200 μg/mL the hemolytic rate was over 50%.However, the hemolytic rates of LJ-1 and LJ-2 were still lower than 5% at high concentration (200 μg/mL).【Conclusion】 These findings would provide a reference for molecular design and modification of antimicrobial peptides, and the obtained antimicrobial peptides LJ-1 and LJ-2 would have the potential to be used as substitutes for antibiotics.

【Objective】 This study was aimed to explore the effect of long non-coding RNA(lncRNA) Gm35082-20 on pyrolysis during Brucella infection, which laid a foundation for further research on the involvement of lncRNA in regulating immune response induced by Brucella.【Method】 The chromosome location and exon information of Gm35082-202 were searched and analyzed by Ensemble database, the secondary structure of Gm35082-202 was predicted though RNAfold online software, KEGG and GO enrichment analysis were used to investigate the function of Gm35082-202.RAW264.7 cells infected by Brucella at different time (0, 4, 8, 12, 24, 36 and 48 h) with MOI 0.01 were collected, and the expression of Gm35082-202 detected by Real-time quantitative PCR.The subcellular localization of Gm35082-202 was predicted by LncLocator website, and the expression of Gm35082-202 was detected by Real-time quantitative PCR in the nuclear and cytoplasmic.3 pieces siRNAs of Gm35082-202(siRNA1, siRNA2 and siRNA3) were designed and transfected to RAW264.7 cells, then the expression of Gm35082-202 was detected by Real-time quantitative PCR to screen siRNA with the highest interference efficiency for subsequent tests.After RAW264.7 cells were transinfected with Gm35082-202-siRNA and Gm35082-202-NC for 24 h, and then infected with Brucella.PBS was used as blank control group (PBS), and only Brucella infected cells were used as infection control group (Bru).Cells in PBS, Bru, Gm35082-202-siRNA (Bru-siRNA) and Gm35082-202-NC (Bru-NC) groups were collected, the mRNA and/or protein expressions of NLRP3, Caspase-1, Caspase-11, IL-1 and IL-18 were detected by Real-time quantitative PCR and Western blotting, respectively.The secretion levels of IL-1β and IL-18 in supernatant of each group were detect by ELISA.The intracellular viability of Brucella in each group was compared by colony count.【Result】 Gene structure analysis showed that Gm35082-202 was a lncRNA of 525 nt (Trianscript ID:ENSMUST00000208164.2), contained 2 exons, located in chromosome 7 of mouse(Chr7:100 324 295-100 326 967), its secondary structure contained multiple stem loops and multi-branched internal loop structures.KEGG signaling pathway enrichment analysis showed that Gm35082-202 mainly involved in regulating NOD-like receptor, calcium regulation and cytokine signaling pathway.GO function analysis showed that the function of Gm35082-202 mainly involved in DNA transcription, mitochondrial components and metal ion binding.After Brucella infected RAW264.7 cells, compared with 0 h group, the expression of Gm35082-202 was significantly increased at 4 and 8 h (P<0.05), and extremely significantly increased at 12, 24, 36 and 48 h (P<0.01).The prediction results of subcellular localization showed that Gm35082-202 was mainly distributed in nucleus (58%), followed by cytoplasm (35%).When RAW264.7 cells were infected by Brucella for 0 h, the distribution of Gm35082-202 in the nucleus was 63.4%, while the distribution of Gm35082-202 decreased to 24.5%, 29.6% and 30.4% at 4, 24 and 36 h, respectively.The expression of Gm35082-202 was extremely significantly reduced by Gm35082-202-siRNA1 and Gm35082-202-siRNA3 (P<0.01), and Gm35082-202-siRNA1 had the highest interference efficiency.Compared with Bru group, in Bru-siRNA group, the expressions of NLRP3, Caspase-1, Caspase-11, IL-1β and IL-10 genes were extremely significantly reduced(P<0.01), the expressions of NLRP3 and Caspase-11 protein were significantly decreased(P<0.05), whlie the expression of Caspase-1 protein was extremely significantly decreased(P<0.01), and the secretion of IL-1β and IL-18 in supernatant was extremely significantly decreased (P<0.01), and the intracellular Brucella count was increased significantly (P<0.05).【Conclusion】 Brucella promoted pyroapoptosis of macrophages by up-regulating Gm35082-202 expression, which could provide references for further exploring the molecular mechanism of Gm35082-202 in regulating pyroapoptosis of host cells during Brucella infection.

【Objective】 This experiment was aimed to study the clinical effect of Lianhuachaiqin soluble powder on fever, cough, anorexia, thirst and drinking in chickens caused by wind-heat invading lung.【Method】 The broiler model of wind-heat invading lung syndrome was established by artificial challenge of chicken Infectious bronchitis virus.150 chickens with typical clinical symptoms of wind-heat invading lung syndrome were included in the experiment, and were randomly divided into 5 groups:Lianhuachaiqin soluble powder high, medium, low dose groups (Lianhuachaiqin soluble powder concentrations were 4, 2, 1 g/L, respectively), Ma Xing Shigan granule control group (1 g/L), and model control group, 30 chickens in each group.Another 30 chickens were used as healthy control group.Except for the healthy control group and the model group, the experimental groups were treated with drinking water continuously for 5 days.The changes of mental state, body temperature, respiratory symptoms, intake and drinking water of chickens before and after administration were recorded, and the indexes of immune organs and immune cell subsets in peripheral blood of chickens in each group were detected to comprehensively investigate the clinical effect of Lianhuachaiqin soluble powder.【Result】 The total effective rates of high and medium dose groups of Lianhuachaiqin soluble powder on fever and cough caused by wind-heat invading lung were 90.00% and 86.66% respectively, which were significantly higher than those in the model control group (P<0.05).And it had a good alleviating effect on the decrease of feed intake and the increase of drinking water in diseased chickens (P<0.05).On the other hand, the Lianhuachaiqin soluble powder could increase the immune organ index and promote the proliferation of CD3⁺, CD4⁺ and CD8⁺ T lymphocytes in the peripheral blood of diseased chickens, and the high and medium doses were significantly higher than those in the model control group (P<0.05).【Conclusion】 The results showed that Lianhuachaiqin soluble powder had good relieving and therapeutic effect on chicken fever, cough, loss of appetite and thirst caused by wind-heat invading lung. Combined with drug dose-effect relationship and economic cost, it was recommended that the clinical dosage of Lianhuachaiqin soluble powder was 2 g/L, given in drinking water for 5 days.

【Objective】 The aim of this study was to explore the anti-inflammatory mechanism of the main active components of Sophora japonica L.(S.japonica L.) based on network pharmacology.【Method】 The effective components and targets of S.japonica L.were screened from the TCMSP database and constructed an active component-target network diagram using the Uniprot database and the Cytoscape 3.6.1 software.Combined with the NCBI, GeneCards and OMIM databases to search the anti-inflammatory related targets of S.japonica L., the core target genes were uploaded to the STRING platform to build a protein-protein interaction network (PPI). DAVID database was used to analyze key targets by GO function and KEGG pathway enrichment.【Result】 The results showed that there were six active components in S.japonica L.including quercetin, isorhamnetin, kaempferol, beta-sitosterol, N-[6-(9-acridinylamino) hexyl] benzamide and quercetin-3-methyl ether.It could act on 152 anti-inflammatory targets including IL10, NFKBIA, ICAM1, MMP2, BCL2L1, STAT1, VCAM1, IFNG, MAPK14 and IL2.There were 16 items related to biological processes, including positive regulation of transcription from RNA polymerase Ⅱ promoter, transcription DNA-templated and gene expression, negative regulation of apoptotic process, inflammatory response and cellular response to lipopolysaccharide (LPS), etc.;6 items related to molecular function, including enzyme binding, transcription factor binding, protein binding, etc.;2 items related to cellular components, including extracellular space and cytosol. And 21 related signaling pathways, including TNF signaling pathway, Toll-like receptor signaling pathway, T cell receptor signaling pathway, NOD-like receptor signaling pathway, cancer pathway, hepatitis B, HTLV-Ⅰ infection and influenza A, etc.【Conclusion】 The results showed S.japonica L. might act on key targets such as quercetin, isorhamnetin, kaoneferol, beta-sitosterol and other active ingredients, played an anti-inflammatory role mainly through TNF, Toll-like receptor, T-cell receptor and NOD like receptor signaling pathways acting on RB1, CDKN1A, IKBKB, CHU, IL2, CXCL10 and other key targets.The results indicated that the anti-inflammatory mechanism of S.japonica L. might involve in multiple components, targets and pathways.

【Objective】 The aim of this study was to investigate the alleviating effect of diosgenin (Dio) on kidney injury induced by cisplatin (CP) in rats.【Method】 Twenty-four healthy male Wistar rats were randomly divided into control group (C), Dio group (Dio), model group (CP) and Dio intervention group (Dio+CP).C and CP groups were given a gavage of 6 mL 0.5% CMC-Na, Dio and Dio+CP groups were given a gavage of 60 mg/kg for 10 days.On day 7 of the experiment, CP and Dio+CP groups were injected with CP (6 mg/kg) through tail vein, and then Dio was gavaged continuously every day.After 72 h of CP administration, the rats in each group were sacrificed, and the renal tissues were separated aseptically, and the renal pathological changes were observed by HE staining.Total antioxidant capacity (T-AOC), catalase (CAT) activity and malondialdehyde (MDA) content were detected by kit.Renal tumor necrosis factor-α (TNF-α) and nuclear factor κB p65 (NFκB p65), cyclooxygenase-2(COX-2), interleukin-1 β (IL-1β) and IL-10 mRNA and protein expressions were detected by Real-time quantitative PCR and Western blotting respectively.The expressions of kidney receptor interacting protein kinase-1 (RIPK1) and RIPK3 protein were detected by immunochemistry.【Result】 HE staining results showed that the renal structure was normal in control and Dio groups.In CP group, the epithelial cells of the renal tubules were exfoliated and vacuolated, and the renal structure was destroyed.The damage of the renal tubules in Dio+CP group was significantly reduced.Compared with control group, the content of MDA was extremely significantly increased (P<0.01), the activities of T-AOC and CAT in CP and Dio+CP groups were extremely significantly decreased(P<0.01).Compared with CP group, the activities of T-AOC and CAT in kidney of Dio+CP group were extremely significantly increased (P<0.01), while the content of MDA was extremely significantly decreased (P<0.01).The results of Real-time quantitative PCR and Western blotting showed that compared with control group, the mRNA and protein expressions of TNF-α, COX-2, NFκB p65 and IL-1β, in the kidney of CP group were extremely significantly increased (P<0.01).The expressions of IL-10 mRNA and protein were extremely significantly decreased (P<0.01).Compared with CP group, the mRNA and protein expressions of TNF-α, COX-2, NFκB p65 and IL-1β in Dio+CP group were significantly or extremely significantly decreased (P<0.05; P<0.01), the mRNA and protein expression levels of IL-10 were extremely significantly increased (P<0.01).Immunohistochemical results showed that compared with control group, the expressions of RIPK1 and RIPK3 proteins in kidney of CP group were extremely significantly increased (P<0.01).Compared with CP group, the protein expressions of RIPK1 and RIPK3 protein in kidney of Dio+CP group were extremely significantly decreased (P<0.01) and significantly(P<0.05) decreased, respectively.【Conclusion】 Dio could alleviate CP-induced renal injury in rats by inhibiting renal oxidative stress, inflammatory response and programmed necrosis of cells.

【Objective】 The experiment was aimed to study the effective components and corresponding gene targets of Salvia miltiorrhiza and Ligusticum chuanxiong in the treatment of ischemic stroke through network pharmacology, and to explore its mechanism of action.【Method】 Using Traditional Chinese Medicine System Pharmacology Enumeration Platform (TCMSP), GeneCards and OMIM database, the potential targets of Salvia miltiorrhiza and Ligusticum chuanxiong for the treatment of ischemic stroke were screened, and GO function and KEGG pathway enrichment analysis were carried out.The sham-operated group, the model group, and the low-dose, medium-dose and high-dose Salvia miltiorrhiza-Ligusticum chuanxiong groups were set up respectively, and the cerebral ischemia-reperfusion model rats prepared by suture were intervened by means of preventive and therapeutic administration.The 24-h blood reperfusion model rats were scored for neurological function by Zea-longa method, and hematoxylin-eosin (HE) staining was used to observe the pathological changes of brain tissue for preliminary verification.【Result】 In this study, a total of 72 main active ingredients of Salvia miltiorrhiza-Ligusticum chuanxiong pairs were screened, 7 from Ligusticum chuanxiong, 65 from Salvia miltiorrhiza, and 1 972 target genes for ischemic stroke.There were 94 common targets, including IL6, IL10, TNF, MMP9, VEGFA, CASP3, etc.The results of GO function and KEGG pathway enrichment suggested that the treatment of ischemic stroke by Salvia miltiorrhiza-Ligusticum chuanxiong was related to PI3K-Akt, cGMP-PKG, VEGF and other signal pathways.Animal experiments showed that Salvia miltiorrhiza-Ligusticum chuanxiong could reduce the pathological changes of brain tissue, reduce neurological deficits, and improve cerebral ischemia-reperfusion injury.【Conclusion】 Salvia miltiorrhiza-Ligusticum chuanxiong medicine pair might improve cerebral ischemia-reperfusion injury through PI3K/Akt signaling pathway, and played an anti-apoptotic effect.

【Objective】 The purpose of this study was to clarify the serotype, virulence gene carrier and drug resistance characteristics of clinical isolates of Streptococcus agalactiae (S.agalactiae) from tilapia in Zhangzhou, Fujian, and to provide new data for the study of S.agalactiae from fish.【Method】 Samples of diseased fish suspected of S.agalactiae infection were collected from some tilapia farms in Zhangzhou city, Fujian province.The isolates were identified by PCR identification and sequencing alignment through 16S rDNA-specific fragment primers and 16S rDNA universal primers.The serotypes of the isolates and the distribution of four major virulence genes (cylE, sodA, gapC and scpB genes) were detected by PCR.The susceptibility tests of 29 kinds of antibiotics in 11 categories was tested by disk diffusion method, and the multi drug resistance was analyzed.【Result】 The isolated colonies were white and round, with neat edges, smooth and wet surface, and showed β-hemolysis.Gram staining was positive, and the bacteria were arranged in chains of different lengths, single or in pairs.VP test and CAMP were positive, and enzyme contact test was negative, it could ferment trehalose, which accorded with the typical characteristics of S.agalactis.A total of 14 fish-derived S.agalactiae were isolated and identified.The serotypes of the 14 isolated strains were all type Ⅰa.The detection rates of virulence genes cylE, sodA and gapC were all 100%, but none of the virulence gene scpB was detected.The resistance rates of the isolated strains to sulfisoxazole, gentamicin and kanamycin were all 100%, and the resistance rates to streptomycin, neomycin and methotrexate were all more than 70%.The isolates were all multi-drug-resistant strains, and 78.57% of the strains were resistant to more than 6 kinds of drugs.【Conclusion】 This study clarified the serotype, virulence gene and drug resistance characteristics of tilapia S.agalactis in Zhangzhou, so as to provide reference for further study on the pathogenic mechanism and effective clinical prevention and control measures of S.agalactis in tilapia.

【Objective】 The aim of this study was to investigate the mycotoxin contamination of buffalo milk in different seasons under different feeding modes in Guangxi.【Method】 Two seasons from October to November 2020 (autumn) and April 2021 (spring) were selected, and eight samples of raw buffalo milk were randomly collected from three breeding modes(large-scale, breeding cooperatives or breeding communities, free-range breeding) in each season.A total of 48 samples were collected.Mycotoxin contamination of buffalo milk, such as aflatoxin M₁(AFM₁), ochratoxin A(OTA), HT-2 toxin(HT-2), T-2 toxin(T-2), α-zearalenol(α-ZEL), zearalenone(ZEN) and deoxynivalenol(DON) were determined by ELISA.【Result】 Among the 48 samples, 16 (33.33%) samples were detected AFM₁.The highest detection rate was 43.75% in farming cooperatives or farming communities, and the lowest was 18.75% in free-range farming.The content of AFM₁ in the containminated samples was lower than the national limit standard (0.5 μg/kg).Two samples (4.17%) were exceeded the European Union Standard (0.05 μg/kg).The adult provisional maximum tolerable daily intake (PMTDI) of HT-2, T-2, α-ZEL, ZEN and DON in raw buffalo milk were far lower than the set value of Joint FAO/WHO Expert Committee on Food Additives(JECFA).The adult provisional tolerable weekly intake (PTWI) of OTA in raw buffalo milk was also lower than the set value of JECFA.Moreover, the content of OTA was lower than the European limit standard (<2 ng/mL).In addition, compared with the breeding cooperatives or breeding communities mode, the content of HT-2 of raw buffalo milk produced by both free-range and large-scale modes were significantly reduced (P<0.05).The content of T-2 of raw buffalo milk produced by the large-scale mode was significantly lower than free-range mode (P<0.05).In raw buffalo milk, the average content of AFM₁ and the proportion of AFM₁ content exceeding the European limit standard in autumn were higher than those in spring, but the detection rate of AFM₁ in spring was higher than that in autumn.Among the 3 feeding modes, the detection rate of AFM₁ in the samples of free-range mode in spring and autumn was the lowest.The average contents of OTA and DON in raw buffalo milk produced by each breeding mode in autumn was higher than that in spring.【Conclusion】 The current quality of raw buffalo milk in Guangxi was safe(the content of AFM₁ was less than 0.5 μg/kg, the adult PMTDI of HT-2, T-2, α-ZEL, ZEN, DON and the adult PTWI of OTA were lower than the set value of JECFA), but a variety of mycotoxins had been detected in raw buffalo milk, and the risk of mycotoxin contamination might be a concern.

【Objective】 This study was aimed to find out the causes of morbidity and mortality of geese in a goose farm in Jiangsu province and reduce economic losses.【Method】 The dead geese were dissected and the pathogenic bacteria identified by bacterial isolation and purification, staining observation, biochemical test, MALDI-TOF MS identification and 16S rDNA determination.The characteristics of the isolated strains were explored by drug sensitivity test and mouse pathogenicity test.【Result】 A Gram-negative short rod-shaped bacterium was isolated from the liver of diseased geese.The results of biochemical tests showed that the strain could ferment glucose, maltose, citrate and urea.MALDI-TOF MS identification showed that the strain was Plesiomonas shigelloides.16S rDNA sequence analysis showed that the similarity between the isolated strain and Plesiomonas shigelloides was the highest, which was more than 98%.The results of drug sensitivity test showed that the isolated bacteria were sensitive to 10 kinds of antibiotics such as levofloxacin, meropenem, cefoxitin and gentamicin.The isolate was intermediary to tetracycline, cefepime, ceftazidime and cefazolin, and resistant to 6 kinds of antibiotics including azithromycin, compound sulfamethoxazole and ampicillin.The isolate was typical multidrug-resistant bacterium.The results of mouse pathogenicity test showed that the median lethal dose of the isolated bacteria to mice was 5.0×10^6.5 CFU.【Conclusion】 This experiment first reported the isolation of pathogenic Plesiomonas shigelloides from geese.Levofloxacin, meropenem, cefoxitin and other effective commonly used clinical antibiotics were screened through drug sensitivity test.The isolate was highly pathogenic to mice, suggesting that the potential risks brought by it should not be ignored.

【Objective】 This study was aimed to analyze the pathogenicity of Escherichia coli(E.coli) from dairy cows in Hebei province and the antibacterial effect of Chinese herbal medicine in vitro.【Method】 30 feces samples with typical clinical symptoms were collected from dairy cows in Hebei province.The collected samples were cultured and observed, and the biochemical identification, serotype identification, transmission electron microscope observation, PCR amplification and pathogenicity study were carried out.9 kinds of Chinese herbal medicines were extracted by traditional decoction and alcohol extraction.The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by 96 well plate double dilution plate method.Chinese herbal medicines with strong antibacterial effect were selected and combined in two, the inhibition zone diameter(IZD) was measured by Oxford cup method, MIC and MBC were measured by 96 well plate double dilution plate method, and the antibacterial effect of the drug was evaluated.【Result】 12 strains of E.coli isolated from dairy cows were pathogenic, with a detection rate of 40%.The dominant serotype was O55, with a detection rate of 58.3%.In the liquid extract of Chinese herbal medicine, Phus chinensis and Schisandra chinensis had the strongest antibacterial effect, MIC and MBC were both 7.80 mg/mL, followed by Pructus mume, Prunella vulgaris and pomegranate peel, with a better antibacterial effect, MIC and MBC were both 15.63 mg/mL.In the alcohol extract of Chinese herbal medicine, Phus chinensis had the strongest antibacterial effect, MIC and MBC were both 3.97 mg/mL, followed by Schisandra chinensis, MIC and MBC were both 7.80 mg/mL.In the combination of two drugs, the combination of Schisandra chinensis and Pructus mume showed synergistic effect, and the antibacterial effect was the strongest, IZD was 26.00 mm, MIC and MBC were both 3.97 mg/mL.Secondly, the antibacterial effect of Phus chinensis and Pructus mume were better, IZD was 23.66 mm, MIC and MBC were both 7.80 mg/mL.【Conclusion】 The pathogenic Escherichia coli was successfully isolated in this study, and the dominant serotype was O55.Single and compound Chinese herbal medicines with good antibacterial effect were screened, which provided a scientific basis for the clinical application of traditional Chinese medicine in the prevention and treatment of cow diarrhea caused by Escherichia coli.