【Objective】 The aim of this study was to clone porcine Microrchidia family CW-type zinc finger 2 (MORC2) gene,analyze the sequence characteristics using bioinformatics,and detect the expression of MORC2 gene in different tissues and the localization of MORC2 gene in ovary of pigs.【Method】 The complete CDS region of MORC2 gene was amplified and cloned with porcine ovary cDNA as template,and the similarity comparison and phylogenetic tree construction were carried out.The sequence of porcine MORC2 protein were predicted by bioinformatics software.The expression of MORC2 gene in different tissues of pigs were detected by Real-time quantitative PCR.The location of MORC2 protein in porcine ovary was detected by immunohistochemical method.【Result】 The length of MORC2 gene CDS was 3 102 bp,encoding 1 033 amino acids.The amino acid sequence of porcine MORC2 protein had 94.8%,94.6%,94.9%,91.9%,93.8%,93.9%,80.8% and 64.3% similarity with Human sapiens, Pan troglodytes, Macaca mulatta,Mus musculus,Bos taurus,Ovis aries,Gallus gallus and Danio rerio,respectively.Phylogenetic trees revealed that pig was most closely related to primates,ruminants and rodents were next,and the farthest related was Danio rerio.The molecular weight of porcine MORC2 protein was 117.44 ku,the isoelectric point was 8.16,the half-life was 30 h,and there were no transmembrane structure and signal peptides,it was an unstable hydrophilic protein.There were 174 phosphorylation modification sites and 81 glycosylation modification sites of MORC2 protein,it was mainly located in cell nuclear,less in cytoplasm and mitochondria.MORC2 protein contained the classical MORC protein family structure (GHKL-ATPase,zf-CW and CC domain).Tissue expression profile results showed that MORC2 gene was widely expressed in 11 tissues of pigs,the expression was the highest in liver,which was significant higher than that in other tissues (P＜0.05),and the expression were the lowest in heart,lung and muscle,which was significant lower than that in other tissues (P＜0.05).Immunohistochemical results showed that MORC2 protein was expressed in both granulosa cells and membrane cells of healthy pigs,and the expression was higher,but less in atretic granulosa cells and membrane cells.【Conclusion】 The CDS of porcine MORC2 gene was obtained,it was widely expressed in various tissues.MORC2 protein was expressed in both granulosa cells and membrane cells of healthy pigs.The results provided a theoretical basis for further research on the molecular mechanism of MORC2 protein regulating porcine ovarian development and follicular atresia.

【Objective】 This study was aimed to screen the core promoter region of melanocortin 1 receptor (MC1R) gene of silver fox,and provide a theoretical basis for exploring the expression regulatory mechanism of MC1R gene.【Method】 Using genomic DNA of silver fox as template,the sequence of the 5'-UTR region of MC1R gene was obtained by PCR amplification.The active region of the MC1R gene promoter of silver fox was predicted comprehensively using 3 online biology softwares.The deletion fragments of different lengths in 5'-UTR region of MC1R gene were amplified by PCR,which were cloned into pMD19-T vector,and the recombinant plasmid was transfected into melanin B16 cells.The luciferase activities of 10 missing fragments were detected by dual luciferase detection technique.【Result】 The 5'-UTR region sequence of MC1R gene in silver fox was successfully obtained,and ―596/+73 bp might be the active region of the promoter using biology softwares prediction.The detection of dual luciferase activity revealed that the constructed luciferase expression vectors of 10 different deletion fragments all had promoter activity.Among them,pGL3-MC1RP8 (―520/+73 bp) had strong activity,indicating that it was the core promoter region,which was basically consistent with the software prediction results.【Conclusion】 The core promoter region of MC1R gene in silver fox was determined to be ―520/+73 bp,which laid a theoretical foundation for the further study on coat color regulation mechanism of MC1R gene.

【Objective】 This study was aimed to predict and analyze the sequence conservation,transcription factors and target genes of bta-miR-34b/c and bta-miR-449a/b/c by bioinformatics,and explore the mechanism which affected spermatogenesis,so as to provide guidance and ideas for studying its biological function and mechanism of action.【Method】 The miRBase database was used to obtain and analyze the sequence characteristics of bta-miR-34b/c and bta-miR-449a/b/c in different species for similarity.TargetScan,miRWalk,miRDB and other methods were used to predict target genes of bta-miR-34b/c and bta-miR-449a/b/c,and the target genes were performed to GO functional and KEGG pathway enrichment analysis.The transcription factors that simultaneously regulated bta-miR-34b/c and bta-miR-449a/b/c were obtained by AnimalTFDB,and the TF-miRNA-mRNA interaction network was constructed.【Result】 bta-miR-34b/c and bta-miR-449a/b/c were highly conserved among different species.According to the prediction results,2 transcription factors and 49 candidate target genes were obtained.These target genes were mainly enriched in spermatogenesis,regulation of calcium-dependent exocytosis,regulation of insulin secretion and other GO items,as well as HIF-1 signaling pathway and thyroid hormone signaling pathway,Wnt signaling pathway and other KEGG pathways.The interaction network composed of bta-miR-34b/c,bta-miR-449a/b/c,upstream transcription factors NR2C2 and HIC1,and downstream target genes AXL,E2F3,DAAM1,etc affected spermatogenesis by regulating meiosis,cell cycle,apoptosis,sperm cell energy metabolism and other biological processes.【Conclusion】 The spermatogenesis were regulated by molecular interactive network of bta-miR-34b/c and bta-miR-449a/b/c with transcription factors and target genes.

【Objective】 This study was aimed to explore the effect of canine acidic (leucine-rich) nuclear phosphoprotein 32 ku (caANP32) on the RNA polymerase activity of Influenza A virus (IAV) from different species.【Method】 Real-time PCR method was used to analyze the tissue distribution of caANP32 family genes (caANP32A,caANP32B and caANP32E),then these genes were amplified and cloned to evaluate the supporting effect of caANP32 family proteins on the RNA polymerase activity of IAV in different species.【Result】 The results showed that the caANP32 family genes were similarly expressed in different tissues,and the expression abundance of caANP32B gene was significantly or extremely significantly higher than that of caANP32A and caANP32E genes in different tissues (P＜0.05;P＜0.01),the tissue abundance of caANP32E gene was extremely significantly or significantly higher than that of caANP32A gene in heart,cecum and brain (P＜0.01;P＜0.05),there was no significant difference in gene abundance between caANP32E and caANP32A genes in liver,lung and other tissues (P>0.05).Only one transcript of caANP32A gene was found in canine heart tissue and MDCK cells,however three different splice variants of caANP32B gene (caANP32B_X1,caANP32B_X2 and caANP32B_X3), and two different splice variants of the caANP32E gene (caANP32E_X1 and caANP32E_X2) were found in these samples.By analyzing the effects of ANP32 family members on the RNA polymerase activity of IAV from different species,it was found that caANP32A and caANP32B could support Canine influenza virus (H3N2_GD11) and Equine influenza virus (H3N8_JL89) RNA polymerase activity,but had lower activity to support Avian influenza virus (H9N2_ZJ12) RNA polymerase activity.The supporting ability of caANP32B_X2 to the RNA polymerase activity of IAV was significantly higher than that of caANP32B_X1 and caANP32B_X3,while caANP32E did not support the RNA polymerase activity of Avian influenza virus.【Conclusion】 This study analyzed the tissue distribution and sequence polymorphisms of the caANP32 family members,and clarified their role in supporting RNA polymerase activity of IAV from different species,providing a new basis for analyzing the molecular mechanism of MDCK as a cell line for influenza virus isolation and vaccine production.

【Objective】 Tripartite motif-containing 3 (TRIM3) gene of Large White pigs was cloned and analyzed by bioinformatics and tissue expression.【Method】 The full-length CDS sequence of TRIM3 gene in Large White pigs was amplified by PCR technology and cloned,pMD18-T vector was ligated and Escherichia coli DH5α competent cells were transformed,positive clones were screened by blue and white spots,sequenced after PCR identification of bacterial fluid,compared with TRIM3 gene sequences of different species and constructed phylogenetic tree,and bioinformatics analysis of its coding proteins was applied to a variety of online software.The relative expression of TRIM3 gene in different tissues of Large White pigs was detected by Real-time quantitative PCR.【Result】 The CDS sequence of TRIM3 gene in Large White pigs was 2 235 bp in total length and encoded 744 amino acids.Similarity and genetic evolution analysis results showed that the similarity between Large White pigs and Sus scrofa was the highest,up to 99.7%,and the similarity with Anas platyrhynchos was the lowest (75.1%).And the TRIM3 gene of Large White pigs was first clustered with Sus scrofa,and the relationship with Bos indicus and Capra hircus were closer.Bioinformatics analysis showed that the molecular mass of TRIM3 protein in Large White pigs was 80.58 ku,the theoretical isoelectric point (pI) was 8.32,and the instability coefficient was 40.85,which was a hydrophilic protein but not a secretory protein,and there were no glycosylation sites,which were predicted to have 60 phosphorylation sites,mainly in the cytoplasm;In the secondary structure of TRIM3 protein,random coil was the mainstay,accounting for 41.67%,and the prediction results of the tertiary structure model were consistent with the secondary structure.Tissue expression analysis showed that the TRIM3 gene in Large White pigs was distributed in heart,liver,spleen,lung,kidney,muscle,trachea and colon,and the expression in lung was the highest, and significantly higher than that in other tissues (P＜0.05).【Conclusion】 In this study,the full-length CDS sequence of TRIM3 gene in Large White pigs was successfully cloned and analyzed by bioinformatics and tissue expression,which provided a theoretical basis for the further study of the immunological function of TRIM3 protein in Large White pigs,and was of great significance to explore the molecular mechanism of TRIM3 gene in innate immunity and antiviral infection.

【Objective】 To analyze the bioinformatics function of cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and the regulation of genes related to reproductive performance in New Zealand White rabbit ovarian granulosa cells,in order to lay the foundation for exploring its regulatory function in follicular development.【Method】 According to the sequence of rabbit CYP11A1 gene in GenBank,specific primers were designed to clone CYP11A1 gene and construct pcDNA3.1-CYP11A1 overexpression recombinant vector from ovary tissue of New Zealand White rabbits.Phylogenetic tree was constructed by Mega 5.1 software. The amino acid composition,physicochemical properties,phosphorylation site,conserved domain,secondary structure,tertiary structure,subcellular location,protein cross-linking of CYP11A1 protein were predicted by bioinformatics softwares. Ovarian granulosa cells were further isolated from ovarian tissue of New Zealand White rabbits and identified by immunofluorescence using specific antibody follicle stimulating hormone receptor (FSHR).Three primers (siRNA-1,siRNA-2,siRNA-3) were designed to interfere the expression of CYP11A1 gene,and the one with the highest interference efficiency was screened,and the overexpressed recombinant vector pcDNA3.1-CYP11A1 and siRNA-CYP11A1 were transfected into ovarian granulosa cells.After RNA was extracted from granulosa cells,then Real-time quantitative PCR was used to analyze the effects of CYP11A1 overexpression and knockdown on the mRNA expression levels of reproductive genes such as HSD17B1,BMP15 and FSHR.【Result】 CYP11A1 gene was successfully cloned from New Zealand White rabbits.The full-length sequence of CDS of CYP11A1 gene of New Zealand White rabbits was 1 557 bp,encoding 518 amino acids,among which leucine (10.6%) was the highest,followed by arginine (7.6%),valine (7.4%) and alanine (7.2%).Evolutionary tree analysis showed that the protein sequence of rabbit CYP11A1 was closest to mice,humans and orangutans.Bioinformatics analysis showed that the theoretical isoelectric point of CYP11A1 protein was 7.4,the number of positive and negative charge residues was 50,the aliphatic amino acid index was 82.16,and the instability index was 39.54,among which the number of hydrophilic residues in amino acid sequence was more than that of hydrophobic ones.This protein had 40 potential phosphorylation sites,among which threonine,serine and tyrosine were the most abundant.The secondary structure of CYP11A1 protein consisted α-helix (48.31%),random coil (40.22%),extended chain (7.42%) and β-turn (4.04%),and the tertiary structure was predicted to be curved helix.Subcellular localization predicted that CYP11A1 protein was mainly distributed in mitochondria (52.2%),cytoplasm (17.4%) and nucleus (13.0%).It also had a domain of P450 family and interacted with multiple proteins related to reproductive performance (STAR,CYP21A2,CYP17A1 and FDX1).The expression of FSHR in isolated granulosa cells was positive,which could be used for subsequent experiments.After CYP11A1 gene was overexpressed in granulosa cells,the expression of HSD17B1 and FSHR genes were significantly upregulated (P＜0.05), the expression of BMP15 gene was extremely significantly upregulated (P＜0.01).After CYP11A1 gene was interfered,the expression of BMP15 and FSHR genes were extremely significantly down-regulated (P＜0.01),and the expression of HSD17B1 gene was significantly down-regulated (P＜0.05).【Conclusion】 CYP11A1 gene could regulate the expression of reproductive performance related genes HSD17B1,BMP15 and FSHR in ovarian granulosa cells,suggesting that CYP11A1 played a certain role in the reproductive process of female rabbits.

【Objective】 The extraction process of cichoric acid from Echinacea root was optimized,and the antioxidant effect of cichoric acid in intestinal cells was determined.【Method】 In the experiment,the economical organic solvent ethanol was selected as the extraction solvent of Echinacea root powder (80 mesh),and several factors such as extraction solvent concentration,extraction temperature,extraction time,extraction times,and the ratio of raw materials to solvent were set for analysis,and the results of each factor were obtained.The corresponding curve of acid yield and this factor,and then on the basis of single factor,select solvent concentration (30%,40%,50%),extraction temperature (60,70,80 ℃),extraction time (1,2,3 h),solid-liquid ratio (1∶12,1∶15,1∶18),four factors and three levels of orthogonal experiments were designed.With different concentrations of H₂O₂ (100,200,400,600,800,1 000 μmol/L) to treat human colon adenocarcinoma cells (Caco-2) and porcine small intestinal epithelial cells (IPEC-J2).The cells before and after oxidative stress were treated with chicory acid (0,100,200,400,600,800,1 000 μmol/L) for 24 h respectively.The protective and alleviating effects of chicory acid on oxidative stress of two intestinal cells were measured by MTT method.【Result】 The results of extraction process showed the optimal extraction conditions of chicoric acid were 50% ethanol at 70 ℃ with a solid-liquid ratio of 1∶18,ultrasonic-assisted extraction for 2 h.Finally,the yield of this process was 1.89 mg/g.The results of cellular antioxidant assay showed that chicoric acid had preventive and alleviating effects on both Caco-2 and IPEC-J2 cell lines which damaged by oxidative stress.The IC₅₀ values of its protective and alleviating effects were 172.6 and 207.5 μmol/L for Caco-2,133.1 and 196.5 μmol/L for IPEC-J2,respectively.【Conclusion】 The best extraction process of chicoric acid from Echinacea root powder was obtained,which provided a reference for industrial and efficient extraction of chicoric acid,and the cell antioxidant experiment showed the intestinal antioxidant ability of chicoric acid.The calculated IC₅₀value of its protective effect was smaller than that of the mitigation effect.It was further proved that chicoric acid had the potential as a future intestinal antioxidant drug.

【Objective】 The aim of this study was to explore the effect of bovine adipose-derived mesenchymal stem cells (bAD-MSCs) on the migration of bovine endometrial epithelial cells under oxidative stress.【Method】 Bovine endometrial epithelial cells were treated with 0,5,25,50,100,200 μmol/L H₂O₂ for 2,4,6,8 and 12 h,activity was detected by MTT assay and intracellular reactive oxygen species (ROS) level was detected by flow cytometry,in order to screen the best optimum condition of oxidative stress model of bovine endometrial epithelial cells.Six groups including control group,H₂O₂ group,bAD-MSCs co-culture group (1∶0.5),bAD-MSCs co-culture group (1∶1),MACT co-culture group (1∶0.5)and MACT co-culture group (1∶1) were established in the cell scratch test,and the migration ability of bovine endometrial epithelial cells was analyzed,the expression levels of extracellular regulated protein kinases (Erk) and phosphorylation extracellular regulated protein kinases (pErk) protein were detected by Western blotting.【Result】 The results of MTT assay showed that the cell survival rate of 4-12 h was 50%-80% when H₂O₂ concentration was 50 μmol/L,which was suitable for the establishment of oxidative stress model.Flow cytometry assay results showed that when bovine endometrial epithelial cells were stimulated with 50 μmol/L H₂O₂ for 2 h,the ROS level was significantly higher than that in control group and 4,6,8,12 h groups (P<0.05).The results of scratch test indicated that compared with control group,the migration ability of bovine endometrial epithelial cells in H₂O₂ group was significantly reduced (P＜0.05);While compared with H₂O₂ group,the migration ability of the bovine endometrial epithelial cells in bAD-MSCs co-culture groups was significantly increased (P＜0.05),and the cell migration ability in MACT co-culture groups was significantly decreased (P＜0.05).The results of Western blotting showed that compared with control group,the expression of pErk protein in bovine endometrial epithelial cells in H₂O₂ group was significantly decreased (P＜0.05);While compared with H₂O₂ group,the expression of pErk protein in bovine endometrial epithelial cells in bAD-MSCs co-culture groups was significantly increased (P＜0.05),and the expression of pErk protein in MACT co-culture groups was significantly decreased (P＜0.05).【Conclusion】 50 μmol/L H₂O₂ treatment for 2 h could successfully construct the oxidative stress model of bovine endometrial epithelial cells,and bAD-MSCs could promote the migration of bovine endometrial epithelial cells under oxidative stress and participate in regulating the expression of Erk protein.

【Objective】 This study was aimed to investigate the effect of high fat and high fructose diet on the morphological changes of retina in elder mice.【Method】 A total of twelve 4-week-old SPF male C57BL/6J mice were fed to 12 months old and then divided into normal diet group (ND) and high fat and high fructose diet group (HFHFD),with 6 mice in each group.The normal diet group was fed with ordinary diet,and the high fat and high fructose diet group was fed with high fat and high fructose diet.The eyeballs were taken at the age of 18 months,the left eyeballs were fixed with eyeball fixation solution,and the right eyeballs were frozen for standby.The structural changes of mouse retina were observed by HE staining,the expression of phosphorylated acetyl CoA carboxylase(p-ACC) was detected by immunohistochemistry (IHC) method,the expression of sterol regulatory element binding protein-1 (SREBP1) was detected by immunofluorescence (IF) method,and the expression of p-ACC,p-AMPK and SREBP1 protein were detected by Western blotting.【Result】 HE staining results showed that compared with normal diet group,the retinal tissues in the high fat and high fructose diet group showed uneven thickness of rods and vertebral layers,local hyperplasia,extension into the outer granular layer,loose arrangement of cells and so on.Immunohistochemistry results showed that compared with normal diet group,the expression of p-ACC in the high fat and high fructose diet group was significantly decreased (P＜0.05).Immunofluorescence results showed that compared with normal diet group,the expression of SREBP1 protein in the high fat and high fructose diet group was significantly increased (P＜0.05).Western bloting results showed that compared with normal diet group,the expression of p-AMPK and p-ACC protein in the high fat and high fructose diet group were significantly decreased (P＜0.05),and SREBP1 protein was significantly increased (P＜0.05).【Conclusion】 Retinopathy in elder mice could be induced by high-fat and high fructose diet,which could inhibit the activation of AMPK,reduce the expression of p-ACC and increase the expression of SREBP1.The results could provide a reference for further exploring the mechanism of high-fat and high fructose diet affecting retinal structural changes.

【Objective】 The experiment was conducted to study the effects of complete pellet diet energy to nitrogen ratio (ME/N) and gender on growth performance, nutrient digestion and metabolism of Jianzhou Da'er goats in house feeding.【Method】 Using 2×4 two factors experimental design, 64 five-month-old healthy Jianzhou Da'er goats were selected,32 males (26.56 kg±2.96 kg) and 32 females (25.75 kg±2.46 kg) were randomly divided into 4 groups respectively, with 8 goats in each group, and they were fed complete pellet diets with the ME/N of 0.59, 0.51, 0.43 and 0.35,respectively. After 5 days transition and 10 days adaption, 30 days feeding trial were carried out, after that, 4 goats were selected in each group according to the average weight for a 11-day digestion and metabolism trial, the total collection of feces and urine was conducted for 4 days after a 7-day adaptation.【Results】 ①The ADG and DMI of the test goats were affected by ME/N significantly (P＜0.05), and ADG was the hightest (259 g/d±58 g/d) when ME/N was 0.43. ADG and DMI in test rams were significantly higher than those in ewes (P＜0.05). There was a significant interaction between dietary ME/N level and gender on DMI (P＜0.05). ②UE was increased significantly (P＜0.05), GEI, FE, DE and apparent digestibility of GE had no significant difference (P＞0.05) with the decrease of ME/N. GEI,FE and DE in test rams were significantly higher than those in ewes (P＜0.05).③NI and N apparent digestibility were significantly increased with the decrease of dietary ME/N (P＜0.05). NI,NR,NPU and BV in test rams were significantly higher than those in ewes (P＜0.05). ④The apparent digestibility of EE and ADF increased first and then decreased (P＜0.05), while the others had no significant change (P＞0.05) with the decrease of ME/N. The apparent digestibility of DM, P, ADF and NDF in test rams were higher than that in ewes (P＜0.05). There was no significant interaction between dietary ME/N level and gender on nutrient apparent digestibility (P＞0.05).【Conclusion】 When the dietary energy concentrations were the same, reducing ME/N (increasing N concentration) appropriately could improve the growth performance of Jianzhou Da'er goats during growing-fattening period, but continuously reducing ME/N could not improve the growth performance, nutrient digestion and metabolism of the goats. Under this experimental conditions, the complete pellet diet with an ME/N of 0.43 was more suitable. Rams had better digestion and metabolism of nutrients and growth performance than ewes during the growth and fattening period.

【Objective】 This study was aimed to evaluate 6 kind of machine learning (ML) algorithms which were used to establish a dairy cow disease prediction model, and the importance of predictors. 【Method】 The production information,behavior information and disease information of a total of 944 lactating cows from December 2020 to November 2021 were selected as predictors to train and validated the models.Daily milk production,rumination,activity,parity,and lactation days were used as input variables,machine learning algorithms were used to establish a dairy cow disease prediction model,6 machine learning algorithms including Decision Tree (DT) C5.0,CHAID algorithm,Artificial Neural Network (ANN),Random Forests (RF),Bayesian Networks (BN) and Logistic Regression (LR) were evaluated,the importance of predictors and the improvement of model performance by including parity and lactation days were assessed as predictors.Sensitivity and specificity were used to evaluate the performance of the models,and the importance of input variables for models predictions was evaluated according to the weight ranking.【Result】 The sensitivity of DT C5.0 algorithm was greater than 85%,and the specificity was greater than 90%,which was the model with the best performance.The total sensitivity of RF was 56.8%,and the prediction performance for various types of coe was relatively stable.ANN,BN and DT CHAID had better prediction performance for diseases with a large sample size,up to 74.4%.The correct identification rate of LR for sick cow was less than 40.0%,and most of them were identified as healthy cattle.The sum of daily milk production was the most important predictor of RF,ANN,and LR,and the number of days of lactation was the most important predictor of DT C5.0,CHAID and BN.After adding parity and lactation days,the sensitivity of the model's prediction was significantly improved.【Conclusion】 Using machine learning algorithms to predict dairy cow diseases has shown potential,and among them,DT C5.0 was a more suitable model.What's more,milk production and lactation days were relatively important variables in disease prediction models.In addition,including parity and lactation days as predictors could improve the accuracy of model prediction.

【Objective】 This study was conducted to investigate the effects of Bacillus licheniformis and Bacillus amyloliquefaciens supplementation on the growth performance,blood routine,serum biochemical indicators and antioxidant and inflammation indexes of weaned piglets.【Method】 A total of 108 28-day-old weaned piglets (Duroc×Landrace×Yorkshire) were selected and divided into 3 groups according to body weight,with 6 replicates per group and 6 piglets per replicate.The piglets in control group were fed with basal diet,and the other two groups were supplemented with 1 g/kg Bacillus licheniformis or 1 g/kg Bacillus amyloliquefaciens in the basal diet.The experiment lasted for 28 days.At the 14th and 28th days,fasting piglets were weighed and piglets intake were calculated,two piglets were selected from each replication,and the plasma samples were collected from the anterior vena cava,the blood parameters were detected by hematology analyzer and kit.【Result】 There were no significant difference in average daily gain (ADG),average daily feed intake (ADFI) and feed/gain ratio (F/G) among three experiment groups (P>0.05).Compared with control group,the diarrhea rate of weaned piglets in Bacillus amyloliquefaciens group was significantly decreased (P＜0.05).At the 14th day,the percentage of blood lymphocytes percentage of weaned piglets in Bacillus licheniformis and Bacillus amyloliquefaciens groups were significantly increased,while the contents of interleukin-2 (IL-2) and malondialdehyde (MDA),and the activities of lactate dehydrogenase and aspartate aminotransferase were significantly decreased (P＜0.05).At the 14th day,the serum alkaline phosphatase activity of weaned piglets in Bacillus amyloliquefaciens group was significantly decreased,while the hemoglobin was significantly increased (P＜0.05).At the 28th day,the content of IL-2 of weaned piglets in Bacillus amyloliquefaciens group was significantly decreased,while the contents of total protein and globulin were significantly increased (P＜0.05).【Conclusion】 The supplement of Bacillus licheniformis or Bacillus amyloliquefaciens had no outstanding effects on the growth performance,but it could improve the blood biochemical indicators and protect the liver of weaned piglets,and the supplement of Bacillus amyloliquefaciens could also improve the metabolism of piglets and decrease the diarrhea rate of piglets.

Gut microbiota plays an important role in regulating host physiology,metabolism and immune function,and is one of the important factors affecting pig health and economic traits.In recent years,the researches and understanding of porcine gut microbiota have become more and more in-depth.Understanding the composition of porcine gut microbiota can provide the references for improving pig health and production performance from the perspective of gut microbiota.The author firstly reviewed the composition of gut core bacteria in different developmental stages and gut locations of commercial and Chinese indigenous pig breeds.Then,the effects of host genetic background,diets,sex,environment,and the use of antibiotics,probiotics and feed additives on the gut microbial composition were systematically summarized.Finally,the main roles of porcine gut microbiota on feed efficiency,fat deposition,host behavior and immune inflammation response were summarized.

【Objective】 The effects of citrus extract on protein metabolites in the hindgut digesta of finishing pigs were studied in this experiment,in order to provide basis for the regulation of citrus extract on the generation of odor from the source.【Method】 One hundred and eight 56-day-old Duroc×Landrace×Large White pigs weighted (19.73±0.39) kg were randomly assigned to 3 groups with 6 pans per group and 6 pigs per pan (same male and female).Pigs in the control group were fed basal diets,pigs in the antibiotic group were fed the basal diets supplemented with 75 g/t chlortetracycline,and pigs in the citrus extract group were fed the basal diets supplemented with citrus extract (250 mL/t during 56 to 112 days of age and 200 mL/t during 113 to 194 days of age).The cecal and colonic digesta was collected from finishing pigs at 194 days of age,and the contents of free amino acids,phenols,indoles,and biogenic amines in the digesta were determined.【Result】 ①In cecal digesta,compared to control group,adding citrus extract and antibiotic significantly decreased the levels of taurine,aspartic acid,alanine,valine,leucine,lysine,histidine,arginine,indole,and methylamine (P＜0.05).Adding citrus extract tended to decrease the levels of threonine,serine,glutamic acid,glycine,citrulline,isoleucine,proline,and skatole (0.05≤P＜0.10),significantly decreased the levels of phenol,putrescine,tyramine,and spermine (P＜0.05).Compared to antibiotic group,dietary with citrus extract decreased the levels of putrescine and tyramine (P＜0.05).②In colonic digesta,compared to control group,dietary with citrus extract and antibiotic significantly decreased the levels of aspartic acid,serine,isoleucine,cystathionine,leucine,γ-aminobutyric acid,total amino acids,phenol,p-cresol,skatole,and methylamine (P＜0.05),and tended to decrease the levels of proline and indole (0.05≤P＜0.10).The levels of serine,isoleucine,cystathionine,leucine,total amino acids, and p-cresol in the citrus extract group were significantly lower than those in antibiotic group (P＜0.05).Compared to control group,dietary with citrus extract decreased the levels of threonine,glutamic acid,glycine,ornithine,and tyramine (P＜0.05),and tended to decreased the level of spermidine (0.05≤P＜0.10).The levels of phenylalanine,tryptophan,and lysine in the citrus extract group were significantly lower than those in control and antibiotic groups (P＜0.05).【Conclusion】 Dietary with citrus extract decreased the protein metabolite levels in the hindgut digesta of finishing pigs,reduced the production of odor.

【Objective】 The metabolizable energy values of different sources of rapeseed meals and the utilization of their nutrients by geese were evaluated using set algorithm.【Method】 Thirty healthy,200-day-old adult male geese were selected and randomly divided into five groups (groups Ⅰ,Ⅱ,Ⅲ,Ⅳ,and Ⅴ),with six replicates in each group and one goose in each replicate.4 types of rapeseed meal were extracted from rapeseed imported from Canada and rapeseed locally produced in Suzhou,Suqian and Yancheng,Jiangsu province,respectively.Metabolic test was conducted by forced feeding method with a feeding amount of 80 g.Four types of rapeseed meals were used to replace 30% of the basal diet in groups Ⅰ,Ⅱ,Ⅲ,and Ⅳ,respectively, the basal diet was forced fed in group Ⅴ,and endogenous feces were collected for endogenous correction.【Result】 ① Gross energy (GE),dry matter (DM),crude protein (CP),crude fat (EE),crude ash (Ash),acid detergent fiber (ADF),neutral detergent fiber (NDF),calcium (Ca),and total phosphorus (TP) contents in four kinds of rapeseed meals were 17.48 MJ/kg,88.95%,42.22%,2.47%,7.93%,22.42%,37.81%,0.83%,and 1.09%,respectively.② The mean values of apparent metabolic energy (AME) and true metabolic energy (TME) of four rapeseed meals in geese were 12.27 and 13.05 MJ/kg,respectively,and there were significant differences among groups in AME and TME (P＜0.05).The mean CP true utilization of four rapeseed meals by geese was 44.84%.The mean apparent utilization of DM,EE,Ash,ADF,NDF,Ca,and TP were 70.71%,76.48%,38.07%,43.31%,51.67%,46.09%,and 48.99%,respectively, there were significant differences between groups in utilization rates of CP,DM,EE and NDF (P＜0.05).【Conclusion】 Rapeseed meal was rich in nutrients,and its metabolic energy was similar to that of soybean meal and wheat bran. Geese could utilize its EE,NDF,Ca,and TP at a higher level,but CP utlization was at a lower level.The metabolic energy,ADF,NDF,and TP utilization of rapeseed meals by geese in different regions differed significantly,with AME and TME in group Ⅰ being higher than the other three groups and CP utilization in test group Ⅳ being the highest.

【Objective】 The effects of plant essential oils (13.5% thymol and 4.5% cinnamaldehyde) on immunity function,antioxidant capacity and gut microbiota of Shan Partridge ducks were studied.【Method】 A total of 200 forty-week-old healthy ducks with similar laying performance and body weight were divided into 4 treatments with 5 replicates per group and 10 Shan Partridge ducks per replicate,and the ducks were fed the basal diet supplemented with 0,50,150,200 mg/kg plant essential oil,the experiment lasted 30 days.On the 28th to 30th day of the experiment,eggs (close to the average egg weight) were randomly selected from each group for analysis of egg quality.At the end of the experiment,one duck was selected for each replicate,blood was collected to detect serum immunoglobulin (Ig),complement protein,antioxidant capacity,and after slaughter the contents of duck cecum were collected for the detection of intestinal gut microbiota.【Result】 Compared with the control group, the egg weight, eggshell weight, albumen weight, yolk weight and transverse diameter were significantly increased (P＜0.05), and the serum malondialdehyde content significantly decreased in the group of plant essential oil (P＜0.05),but there was no significant difference in the proportion of egg components among the groups (P＞0.05); The serum immunoglobulin A, immunoglobulin G, immunoglobulin M contents and the serum glutathione peroxidase content of ducks were significantly increased (P＜0.05), the relative abundance of Bacteroidetes, Parabacteroides and Prevotellaceae NK3B31 group were increased,and the relative abundance of Ralstonia in cecum of shan partridge duck were decreased (P＜0.05) in the 150 mg/kg and 200 mg/kg plant essential oil groups.【Conclusion】 The dietary supplementation with plant essential oil could increase egg weight,immune function and antioxidant capacity of ducks,and improve caecal microbiota structure of Shan Partridge ducks.Overall consideration,the appropriate amount of plant essential oil to be added was 150 mg/kg.

【Objective】 The purpose of this experiment was to find out the effects of Jianpi Xiaodao Chinese herbal compound on the growth performance,rumen fermentation parameters and serum biochemical indexes of 6-month-old Simmental cattle×local yellow cattle (Simmental crossbred cattle).【Method】 28 Simmental crossbred cattles weighing 193 kg±20 kg were selected and randomly divided into control group and experimental group respectively.The control group was fed with basal diet,and the experimental group was fed with basal diet supplemented with 0.5% Jianpi Xiaodao Chinese herbal compound.After 7 d of pretrial period,cattles were fed for 90 d.Rumen fluid and blood were collected before morning feeding on 91 d,and rumen short-chain fatty acids and blood physiological and biochemical indexes were measured.【Result】 Compared with control group,in the experimental group,the average daily gain weight (ADG) was significantly increased (P＜0.05),the feed-to-gain ratio (F/G) was decreased,there were no significant differences in rumen fluid pH and contents of acetic acid,propionic acid,butyric acid,total short-chain fatty acid and ratio of acetic acid to propionic acid (P＞0.05),the content of serum total cholesterol (CHO) was significantly higher,the contents of total bilirubin (TBIL),direct bilirubin (DBIL), alanine aminotransferase (ALT) and malondialdehyde (MDA) were significantly decreased (P＜0.05),the contents of immunoglobulin A (IgA),immunoglobulin G (IgG),gastrin (GAS),triiodothyronine (T₃) and reduced glutathione (GSH) were significantly increased (P＜0.05). The contents of motilin (MTL) and growth hormone (GH) were increased but there was no significant difference (P＞0.05).【Conclusion】 The supplementation of Jianpi Xiaodao Chinese herbal compound in the diet of 6-month-old Simmental crossbred cattle could regulate hormone secretion,promote forage digestion and absorption,enhance antioxidant capacity,improve body immunity,and then improve the growth performance.

【Objective】 The objective of this study was to evaluate the genetic diversity and genetic structure of Sus scrofa in Guizhou,so as to provide theoretical basis for further conservation genetics research and population ecological management,and then lay a foundation of the research of Sus scrofa conservation biology and its adaptation to the environment.【Method】 The mitochondrial Cytb gene of Sus scrofa was amplified by PCR and sequenced.Combined with 55 mitochondrial Cytb gene sequences of Sus scrofa downloaded from GenBank in other 9 regions in China,Mega 7.0 software was used for base composition and genetic distance analysis,and DNAsp6 software was used for genetic diversity analysis.【Result】 The ratio of Cytb gene (1 140 bp) of Sus scrofa in Guizhou was similar with other region,and AT content was higher than GC content.The 11 sequences had 6 variation sites and 4 haplotypes.The haplotype diversity was 0.600,and the nucleotide diversity was 0.00118.Haplotype diversity and nucleotide diversity were only higher than those of Sus scrofa populations in Jiangxi and Northeastern region.The genetic distance within species was small,and the genetic relationship was close.The neutral test Tajima's D and Fu's Fs values were negative,and the difference was not significant.The mismatch distribution analysis showed a unimodal distribution,it showed that Sus scrofa population in Guizhou was relatwely stable in history.The haplotype diagram was constructed using Network,and the analysis of haplotype phylogenetic tree constructed by Mega 7.0,which showed the haplotypes of Sus scrofa in Guizhou were shared with populations reported in other region (Hap_1 and Hap_10).【Conclusion】 This study concluded that Sus scrofa population in Guizhou was experiencing an expansion phase,but the population genetic diversity was lower than the populations in other region in China,the genetic distance of intrapopulation was small,and moderate comparing with the populations from other region in China.Therefore,the conservation management on genetic biodiversity of Sus scrofa in Guizhou should be paid more attention.

【Objective】 This study was aimed to investigate the effect of myocyte enhancer factor 2C (MEF2C) gene polymorphism on the body weight and body size traits in Qiandongnan Xiaoxiang chickens.【Method】 251 300-day-old Qiandongnan Xiaoxiang chickens were used as the research object,the polymorphism of all (15) exons of MEF2C gene were analyzed by PCR amplification and direct sequencing technology,and the single nucleotide polymorphism (SNP) was screened.The correlation between the genotypes corresponding to different SNPs and body weight or body size traits in Qiandongnan Xiaoxiang chickens were analyzed using SPSS 18.0 statistical analysis software.【Result】 Only 3 SNPs (g.1819 T＞C,g.2426 T＞C and g.2543 C＞T) were found in exon 12 of MEF2C gene in Qiandongnan Xiaoxiang chickens,and no SNP was found in other exons.χ² test results showed that 3 SNPs deviated from Hardy-Weinberg equilibrium in Qiandongnan Xiaoxiang chickens (P＞0.05).The results of association analysis showed that the body oblique length,chest depth,and tibia circumference of g.1819 T＞C TC genotype were extremely significantly higher than those of TT and CC genotypes,and the body weight was extremely significantly higher than that of TT genotype (P＜0.01);The tibial length of TC genotype was significantly higher than that of TT and CC genotypes,and the keel length was significantly higher than that of TT genotype (P＜0.05).g.2426 T＞C and g.2543 C＞T were linkage balance sites,which had extremely significant effect on the tibia circumference (P＜0.01).【Conclusion】 The exon of MEF2C gene in Qiandongnan Xiaoxiang chickens were highly conserved,and g.1819 T＞C could be used as a reference site for molecular marker-assisted breeding of Qiandongnan Xiaoxiang chickens.

【Objective】 This experiment was conducted to investigate the effects of exogenous RF-amides related peptide 3 (RFRP-3) and melatonin (MLT) on the growth and reproductive performance of male Kunming mice at the beginning of estrus,and the relationship between them.【Method】 Male Kunming mice at the beginning of estrus were given normal saline,exogenous RFRP-3,MLT,and a mixture of RFRP-3 and MLT,respectively.The effects on average weight gain,average feed intake,feed gain ratio (F/G),testicular development,the secretion levels of serum growth hormone (GH) and luteinizing hormone (LH),and the relative expression of growth and reproduction-related genes in mice were examined.【Result】 Exogenous RFRP-3 inhibited the expression of GH gene,suppressed body weight gain,and improved F/G;While exogenous MLT promoted the expression of GH gene,resulting in faster body weight growth and lower F/G.Under the combined treatment of RFRP-3 and MLT,the expression of GH gene increased,and the body weight gain was similar to that of MLT group.RFRP-3 inhibited the expression of melatonin receptor 1A (Mtnr1A) and LH genes and the secretion of testosterone,and the number of spermatogonia at all levels and leydig cells in the testes of mice decreased;MLT promoted the expression of Mtnr1A and LH genes and the secretion of testosterone,and the leydig cell density extremely significantly increased (P＜0.01).The MLT and RFRP-3 cotreatment decreased the number of spermatogonia and leydig cells,and significantly decreased the secretion of testosterone (P＜0.05).【Conclusion】 Exogenous RFRP-3 inhibited the growth and reproductive performance of male mice at the puberty,while exogenous MLT promoted the growth and reproductive performance of male mice.

【Objective】 The aim of this study was to investigate the function and mechanism of miR-140-5p in the differentiation of preadipocytes 3T3-L1.【Method】 When the convergence degree of 3T3-L1 cells reached 100%,the adipogenic differentiation was induced.The cells of differentiation day ―1 (the day before differentiation induction),days 0,1,2,3,5 and 7 were collected,and the expression of miR-140-5p was detected by Real-time quantitative PCR.miR-140-5p mimics and NC were transfected into 3T3-L1 cells to induce adipogenic differentiation.Oil red O staining was used to observe the formation of lipid droplets.Real-time quantitative PCR was used to detect the relative expressions of adipogenic marker genes CAAT enhancer binding protein β (C/EBPβ),CAAT enhancer binding protein δ (C/EBPδ) and peroxisome proliferator-activated receptor γ (PPARγ).The target gene of miR-140-5p was predicted by miRandn and TargetScan online websites,the sequence conservation of the binding site sequence of P300/CBP-related factor (PCAF) 3'-UTR sequence and miR-140-5p in different species such as mice and humans was analyzed by comparing the sequence differences.miR-140-5p mimics,inhibitor and NC were transfected into 3T3-L1 cells and the cells were induced to differentiate into adipocytes.The relative expression of miR-140-5p and PCAF was detected by Real-time quantitative PCR,and the protein level of PCAF was detected by Western blotting.PEGFP-N1-PCAF and PEGFP-N1 were transfected into 3T3-L1 cells.Oil red O staining was used to observe the formation of lipid droplets.Real-time quantitative PCR was used to detect the relative expression of PCAF,C/EBPδ and PPARγ genes.Western blotting was used to detect the expression levels of C/EBPβ,C/EBPδ and PPARγ proteins.Three PCAF siRNAs (siRNA1,siRNA2 and siRNA3) and siRNA NC were transfected into 3T3-L1 cells.The protein level of PCAF was detected by Western blotting to screen the best PCAF siRNA.Optimal PCAF siRNA and siRNA NC were transfected into 3T3-L1 cells,and the formation of lipid droplets was observed by oil red O staining.The relative expression of PCAF,C/EBPβ,C/EBPδ and PPARγ genes were detected by Real-time quantitative PCR,and the expression of C/EBPβ and PPARγ protein were detected by Western blotting.miR-140-5p mimics,NC,PGL0-PCAF 3'-UTR vector and PGLO empty vector were co-transfected into 293T cells,respectively.The targeting relationship between miR-140-5p and PCAF gene was detected by double luciferase report test.【Result】 In the process of inducing adipogenic differentiation of 3T3-L1 cells,compared with the day before inducing differentiation,the relative expression of miR-140-5p was extremely significantly increased on the day 1 and day 2 of adipogenic differentiation (P＜0.01) and was significantly increased on the day 3 day of differentiation (P＜0.05).Compared with NC group,the number of lipid droplets in miR-140-5p mimics group was increased significantly,and the relative expression of lipid marker genes C/EBPδ and PPARγ in miR-140-5p mimics group were extremely increased significantly (P＜0.01).The result of target gene prediction showed that miR-140-5p had the expected binding site with PCAF.The results of conservation analysis showed that the binding site sequence of target gene PCAF was highly conserved among different species.Compared with NC group,the relative expressions of miR-140-5p and PCAF gene in mimics group were extremely significantly increased (P＜0.01),while those in inhibitor group were extremely significantly decreased (P＜0.01),PCAF was significantly decreased (P＜0.05),and the expression of PCAF protein was significantly increased (P＜0.05).Compared with PEGFP-N1 group,the number of lipid droplets in PEGFP-N1-PCAF group was increased,and the relative expression levels of PCAF and PPARγ genes and the levels of C/EBPβ,C/EBPδ protein were extremely significantly increased (P＜0.01),PPARγ protein was significantly increased (P＜0.05).PCAF siRNA1,siRNA2 and siRNA3 all extremely significantly inhibited the expression of PCAF protein (P＜0.01),and siRNA3 had the most significant effect,so siRNA3 was selected for the follow-up test.Compared with NC group,the number of lipid droplets in 3T3-L1 cells in PCAF siRNA3 group was less,the expression of PCAF,C/EBPβ,C/EBPδ and PPARγ genes and the level of C/EBPβ protein were extremely decreased significantly (P＜0.01).The results of double luciferase report test showed that there was no target relationship between miR-140-5p and PCAF gene.【Conclusion】 The experiment proved that the expression of endogenous miR-140-5p was increased during the differentiation of 3T3-L1 cells.miR-140-5p might promote the adipogenic differentiation of 3T3-L1 cells by indirectly up-regulating the expression of PCAF gene.

【Objective】 This study was aimed to explore the changes of testis and epididymal tissues in different days old Hyla rabbits before sexual maturity to provide histological basis for the determination of their puberty and sexual maturity.【Method】 45 male Hyla rabbits were randomly divided into 9 groups,with 5 in each group,the testis and epididymis of one group of rabbits were collected every 15 days from 30 days of age,and their growth and development rules were examined and analyzed using gross morphology and histoanatomy.【Result】 With the increase of daily age,testicular index,testicular weight,testicular long diameter,testicular short diameter,and testicular thickness were gradually increased,and all the indexes of each group at 150 days of age were significantly higher than those in other groups (P＜0.05).30,45 and 60 days old testis with endophyte sperm tubules were sparsely arranged.The stromal component of the testis of 75,90 and 105 days old were gradually increased and the lumen was gradually formed,the plasmic cells of 120,135 and 150 days old were further proliferated and distributed in groups,round and long-shaped unformed sperm appeared from 90 days old,a small amount of formed sperm appeared in the lumen of the epididymis at the 120 days old testis,and a large number of sperm was densely distributed in the testicular lumen at 150 days old.Moreover,the rabbit sperm tubule area,epithelial cell thickness and number of supporting cells were significantly greater at 120,135 and 150 days old than those in the other groups (P＜0.05).The number of interstitial cells did not differ significantly between the groups above 30 days old (P＞0.05),but all were significantly greater than 30 days old.The diameter of rabbit epididymal head,body and tail at 120,135 and 150 days old were significantly higher than those in the other groups (P＜0.05),the columnar epithelial thickness and cilia length of the epididymal head were significantly higher from 105 days old than in the 30,40,60,75 and 90 days old groups (P＜0.05).【Conclusion】 The testis and epididymis of Hyla rabbits developed synchronously with the increase of age,reaching the initial estrus at 120 days old,and the testis and epididymis basically reached sexual maturity at 150 days old.

【Objective】 This study was aimed to investigate the effect of compound additive composed of melatonin and dinoprostatin on sex controlled frozen semen quality and conception rate after artificial insemination.【Method】 Thirty sex controlled frozen sperm of dairy cows was divided into experimental and control groups,250 μL compound semen additive composed of 24 ng/mL melatonin,1.5 mg/mL dinoprostatin and semen dilution was added to experimental group,and 250 μL semen dilution was added to control group.The experimental and control groups were mixed with thawed sex controlled frozen semen in dairy cows according to the volume of 1∶1, and incubated at room temperature for 0, 2 and 4 h. The viability rate, acrosome integrity rate and high-energy mitochondrial ratio were analyzed by immunofluorometric assay, and the conception rate of 216 heifers and 82 first lactating dairy cows were determined by 28 day early pregnancy test after artificial insemination. 【Result】 Compared with control group,there was no significant difference in the viability rate,acrosome integrity rate and high-energy mitochondria ratio after treatment with compound additives for 0 h (P＞0.05);The viability rate, acrosome integrity rate and high-energy mitochondria ratio were significantly increased after treatment with compound additives for 2 h (P＜0.05);There was no significant difference in the viability rate and acrosome integrity rate after treatment with compound additives for 4 h (P＞0.05),but the high-energy mitochondria ratio was significantly increased (P＜0.05).In the insemination test,the conception rates of heifers and first lactating dairy cows in experimental group were 65.22% and 48.21%,which were significantly higher than control group (P＜0.05).【Conclusion】 The compound additive composed of melatonin and dinoprostatin could promote the thawed semen quality of dairy cows and the conception rate of heifers and first lactating dairy cows after insemination.

【Objective】 The study was to investigate the effect of miR-449b on early embryo development in sheep.【Method】 Purified sheep sperm and mature oocytes were obtained by Percoll density gradient centrifugation and in vitro maturation of cumulus-oocyte complexes (COCs) for 16-18 h,respectively.And fibroblasts were isolated from the ear tissues of one-month-old Suffolk sheep.Firstly,the relative expression of miR-449b in sperms,mature oocytes and fibroblasts were compared by Real-time quantitative PCR.Mature oocytes were injected with 0.9% normal saline (Con group),NC mimic (NC mimic group) and miR-449b mimic (miR-449b mimic group) by microinjection,respectively.Then the embryonic cleavage rate and blastocyst rate were counted on the 2nd and 7th day after parthenogenetic activation. Mature oocytes were fertilized in vitro (IVF), 2-cell embryos in IVF, Con, NC mimic and miR-449b mimic groups were collected, and the changes of histone H3K9me3 were compared by immunofluorescence method. 【Result】 The expression of miR-449b in sperm was significantly higher than that in mature oocytes and fibroblasts (P＜0.05). Compared with Con group, the cleavage rates of embryos in miR-449b mimic and NC mimic groups were significantly decreased (P＜0.05), and the cleavage rate of embryos in NC mimic group was significantly lower than that of miR-449b mimic group (P＜0.05). The blastocyst rate of embryos in miR-449b mimic group was increased, but the difference was not significant (P＞0.05). Histone H3K9me3 was expressed in 2-cell embryos in IVF, Con, NC mimic and miR-449b mimic groups, and was expressed in the nucleus. Compared with Con group, the expression level of histone H3K9me3 in 2-cell embryos in NC mimic group was significantly increased (P＜0.05), and was significantly decreased in miR-449b mimic group (P＜0.05). The expression level of histone H3K9me3 in 2-cell embryos in NC mimic group was significantly higher than that in miR-449b mimic group (P＜0.05), but there was no significant difference between miR-449b mimic and IVF groups (P＞0.05). 【Conclusion】 miR-449b could significantly increase the development rate of early sheep embryos and reduce the expression of H3K9me3 in early embryos.

【Objective】 The study was aimed to investigate the effects of endoplasmic reticulum IP3 receptor (IP3R) inhibitor 2-aminoethyl diphenylborinate (2-APB) on the developmental capacity of frozen-thawed bovine MⅡoocytes.【Method】 Oocytes matured in vitro for 24 h were digested with 0.1% hyaluronidase to remove cumulus cells,and were randomly divided into control group and 2-APB group,the open pulled straw (OPS) method was used for vitrification.The control group was vitrified directly,2-APB group was pretreated with 10 μmol/L 2-APB for 10 min before vitrification.Oocytes were thawed after 2 d,the survival rate of oocytes after thawed (0 h) and warmed for 2 h were counted.After recovering for 2 h, cortical granules (CG)were labeled with FITC-PNA fluorescent probe to detect distribution,intracellular reactive oxygen species (ROS) and glutathione (GSH) contents were deteced by 2',7'- DCHFDA and Cell Tracker Blue CMF2HC respectively.The non-vitrified oocytes (Fresh group)matured in vitro for 24 h were parthenogenetic activated as well as those of the control and 2-APB groups,the cleavage and blastocyst rates were counted at 2 and 7 d,respectively.【Result】 Compared with control group,the survival rate of frozen-thawed oocytes at 0 and 2 h in 2-APB group was significantly increased (P＜0.05),the proportion of CG distributed in cortical area in 2-APB group was significantly increased (P＜0.05),the proportion of CG injury type was significantly decreased (P＜0.05),the content of GSH was significantly increased (P＜0.05),and the content of ROS was significantly decreased (P＜0.05),the cleavage and blastocyst rates of parthenogenetic activated embryos in 2-APB group were significantly increased (P＜0.05),and there was no significant difference with the Fresh group (P＞0.05).【Conclusion】 Pretreatment of bovine MⅡ oocytes with 2-APB before vitrification could increase GSH content,decrease the proportion of CG injury type and ROS content,improve the frozen-thawed oocyte quality and early embryo developmental capacity.

【Objective】 This study was aimed to enrich the long non-coding RNA (lncRNA) expression profile of pig peripheral blood lymphocytes after African swine fever virus (ASFV) infection,and further explore the regulatory network affecting the Toll-like receptor signaling pathway.【Method】 Experimental animals were infected with ASFV, and peripheral blood was collected on the 7th day and isolated to obtain peripheral blood lymphocytes.The lncRNAs in peripheral blood lymphocytes were sequenced by Illumina high-throughput genomic sequencing.The raw data were processed and screened to obtain differentially expressed lncRNA,and target gene prediction was performed.GO function and KEGG signaling pathway enrichment analysis were performed using bioinformatics methods,and preliminary mapping of the lncRNA-mRNA regulatory networks related to the Toll-like receptor signaling pathway and 4 lncRNAs including lncRNA-ENSSSCG00000041959 were verified by Real-time quantitative RT-PCR.【Result】 A total of 73 differentially expressed lncRNAs were screened,including 38 upregulated and 35 downregulated lncRNAs.GO function analysis showed that target genes were significantly enriched in regulating immune system process,defense response,biological response,viral response and innate immunity.KEGG signaling enrichment analysis showed that most target genes were related to cell circulation,disease and immune response,including immune related signaling were Toll-like receptor signaling pathway,TNF signaling pathway,and intestinal immune network producing IgA.lncRNA-ENSSSCG00000041959-RIPK1 and lncRNA-ENSSSCG00000041959-IRAK1 might be important regulatory networks affecting Toll-like receptor signaling pathway.The result of Real-time quantitative RT-PCR was consistent with those of sequencing.【Conclusion】In this study,lncRNA-ENSSSCG00000041959-RIPK1 and lncRNA-ENSSSCG00000041959-IRAK1 were initially identified as the lncRNA-mRNA regulatory network affecting the Toll-like receptor signaling pathway,which laid a theoretical foundation for further exploration of lncRNA regulation of the immune response of ASFV infection.

【Objective】 This study was aimed to establish an ELISA method for detecting antibodies against African swine fever virus (ASFV).【Method】 In this study,recombinant truncated p72 (p72s) protein was used as the detection antigen,and the optimal working concentration of antigen,serum to be detected and enzyme-labeled antibody were determined by square titration.The reaction conditions of ELISA were optimized,such as antigen coating,ELISA plate sealing,serum/enzyme-labeled antibody reaction and substrate color developing.The threshold evaluation criteria of receiver operating characteristic (ROC) curve were used to determine the negative and positive critical values of the method.The specificity,sensitivity and intra- and inter-batch reproducibility of the method were detected.Finally,a total of 124 serum samples were detected by the established ELISA method and commercial kits respectively.The positive detection rates were compared by the ELISA and the kit,and the ELISA results were verified by Western blotting.【Result】 The optimal antigen coating concentration of ELISA was 0.5 μg/mL,the best dilution ratios of serum and HRP-conjugated antibody were 1∶100 and 1∶5 000,respectively.The reaction conditions were as follows:The antigen was incubated at 37 ℃ for 1 h and then coated at 4 ℃ overnight;The ELISA plate was blocked at 37 ℃ for 1 h or at room temperature for 2 h;The reaction time of serum or the enzyme-labeled antibody were at 37 ℃ for 60 or 45 min,respectively;And the substrate was kept away from light for color developing at room temperature for 20 min.The determination criteria of negative and positive serum were as follows:When the D_{450 nm} value of the serum to be tested was ≥0.365,it was determined as positive;When that was ＜ 0.365,it was judged as negative.This method only specifically binds to anti-ASFV positive serum,but did not cross react with antiserums against Classical swine fever virus, Pseudorabies virus and Porcine reproductive and respiratory syndrome virus.The minimum amount of total protein detected in ASFV antiserum was 0.091 to 0.153 mg/mL,which was lower than that of commercial kit (0.110 to 0.554 mg/mL).The mean coefficients of variation for intra-batch and inter-batch repeatability tests were 4.70% and 5.125%,respectively.The positive rates of serum samples detected by the established method were 75.81% (94/124) and those did by commercial kit were 32.26% (40/124).Further verification showed that the ELISA results were consistent with those of Western blotting.【Conclusion】 An ELISA antibody detection method based on ASFV-p72s protein was established.This method was specific,sensitive and reproducible.It could be used for clinical diagnosis and epidemiological investigation of ASF,which had great potential for development and application.

【Objective】 This study was aimed to express and purify the nucleocapsid (N) protein of Porcine deltacoronavirus (PDCoV) and then prepare its polyclonal antibody (PcAb).【Method】 The full-length coding sequence of N gene of PDCoV was amplified by RT-PCR using the genomic RNA of the PDCoV CHN-HN-1601 strain as the template.The resulting amplicon was cloned into the prokaryotic expression vector pET-28a(+) to construct a recombinant plasmid pET-28a-PDCoV-N.After verification by enzyme digestion and sequencing,the recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells,which were induced by 0.5 mmol/L IPTG at 37 ℃ for 12 h.The recombinant PDCoV-N protein which carried both an N-terminal and a C-terminal 6×His tag was purified from the supernatant of the lysate of pET-28a-PDCoV-N-transformed E.coli BL21 (DE3) using Ni-NTA agarose under native conditions.The purified N protein was used to immunize New Zealand White rabbits to prepare antisera,from which the PcAb was purified by Protein A/G agarose affinity chromatography.The purified PcAb was identified by Western blotting and indirect immunofluorescence assays,and the titer of PcAb was determined by indirect enzyme-linked-immunosorbent assay (ELISA).【Result】 The results showed that the recombinant N protein was expressed in both soluble and inclusion body forms with a molecular weight of about 42 ku.The purity of the purified recombinant N protein reached 90% and the protein concentration was 0.45 mg/mL.The purified polyclonal antibody has high purity,the titer of the purified PcAb was 1∶6 400,and that the PcAb was able to specifically recognize the recombinant N protein and PDCoV,and had no cross-reaction with other important porcine enteric coronaviruses causing diarrhea in pigs,such as Porcine epidemic diarrhea virus (PEDV),porcine Transmissible gastroenteritis virus (TGEV) and Porcine rotavirus (PRoV).【Conclusion】 The successful preparation of recombinant PDCoV-N protein and its PcAb provided valuable biomaterials for the further study of the function of N protein,the development of serological detection methods,the preparation of subsequent immunochromatographic strip and the basic research of PDCoV.

【Objective】 This study was aimed to express Seneca virus A (SVA) VP1 protein in Escherichia coli (E.coli), and establish the detection method of SVA antibody by liquid chip technology.【Method】 According to the SVA VP1 gene sequence (KY747514.1) from GenBank,the VP1 gene was synthesized based on the codon preference of E.coli,and cloned into pCold TF vector to construct the recombinant plasmid pCold TF-VP1.The pCold TF-VP1 was transformed into E.coli BL21 (DE3) competent cells and induced by IPTG.The inducing conditions were studied by optimizing the induction time and IPTG concentration.Recombinant VP1 protein was purified by nickel column.The purified SVA VP1 protein was coupled with fluorescent microspheres,and the microspheres were reacted with SVA negative and positive serum.Luminex 200 system was used to detect the background and the median fluorescence intensity (MFI) of the negative and positive serum.【Result】 The recombinant plasmid pCold TF-VP1 was successfully transformed into E.coli BL21 competent cells and expressed as soluble protein with the molecular weight of 82 ku.The VP1 protein was expressed at the highest yield when inducing at 16 ℃ for 3 h and the final concentration of IPTG was 1.2 mmol/L.The average MFI of negative control was 17 (＜100).The average MFI of the test microspheres was 2 339.5 (＞2 000),indicating that SVA VP1 protein was successfully coupled with the microspheres.The coupled fluorescent microspheres could be used to detect SVA antibody in porcine serum.【Conclusion】 VP1 protein of SVA was successfully expressed in E.coli.The expression conditions were optimized,the target protein was purified and could be used to develop a liquid chip analysis for detection of SVA antibody.

【Objective】 The inactivated Pasteurella multocida vaccine was prepared to effectively control the occurrence and prevalence of Pasteurella multocida in ducks in Guizhou province.【Method】 The local epidemic strain of A:L1 type Pasteurella multocida,isolated and preserved in the laboratory,was used as the strain.The growth curve of the strain was determined by the plating method and the median lethal dose (LD₅₀) was calculated by improved Karber's method.After the bacteria were cultured to a final concentration of 1×10¹⁰ CFU/mL,the oil adjuvant inactivated vaccine and carbomer adjuvant inactivated vaccine were prepared respectively,and then the immunized duckling test was conducted after the vaccine's quality was inspected.The protection rate of the two vaccines were evaluated and compared by the challenge protection test,and the histopathological observation was performed on the liver,lung and spleen of the ducks in the challenge test.【Result】 The vaccine strain of A:L1 type Pasteurella multocida reached to the higestaed after 18 h,the concentration of viable bacteria was reached to 1.0×10¹⁰ CFU/mL,and the LD₅₀ was reached to 5 CFU.The safety of the two vaccines was qualified.The challenge protection test results showed that after first immunization,the immune protection rate of the carbomer adjuvant inactivated vaccine could reached 62.5%,which was higher than that of the oil adjuvant inactivated vaccine (50.0%) and the commercial inactivated vaccine (50.0%).After the second immunization,carbomer adjuvant inactivated vaccine had obvious advantages,and the immune protection rate could reached 87.5%,which was higher than that of oil adjuvant inactivated vaccine (75.0%) and commercial inactivated vaccine (62.5%).The histopathological results showed that carbomer adjuvant inactivated vaccine could provide best protective effects on the lung after the first immunization,and in terms of the protective effects of liver,lung and spleen after the second immunization,the carbomer adjuvant inactivated vaccine group had obvious advantages.【Conclusion】 The carbomer adjuvant inactivated vaccine prepared by using the A:L1 strains prevalent in Guizhou had obvious immune effects,which could play an important role in the prevention and control of Pasteurellosis multocida in Guizhou.

【Objective】 This study was aimed to investigate the infection status of the equine intestinal parasites in Guangdong, offer guidance for the prevention and control of parasites infection in the region.【Method】 375 fresh horse fecal samples were collected from 24 farms in 7 cities in Guangdong province,which were processed by the saturated salt solution and saturated sucrose solution separately.Eggs/oocysts were observed with the microscope and identified based on literatures.The number of eggs/oocysts per gram of feces (EPG) was calculated by McGrady counting method.The infection rate and infection intensity were calculated.Fecal egg count reduction test (FECRT) was performed on positive samples with EPG＞500 to test whether anthelmintic resistance existed.In addition,the DNA of 5 young foals under 1-year-old was extracted from fecal samples for Cryptosporidium detecting using nested PCR method.【Result】 120 of 375 samples were tested positive for parasite eggs/oocysts,and the total infection rate was 32.00%.A total 4 intestinal parasites were identified,which were Strongylus app.,Parascaris equorum,Strongyloides westeri and Oxyuris equi,and the total infection rates were 30.40%,3.70%,0.80% and 0.30%,respectively.The total infection rate of horses aged from 1-4 years old was significantly higher than that of other ages (P＜0.05),but there was no significant difference in parasitic infection between different genders ((P＞0.05).The investigated horses were mainly with low EPG (＜200),accounting for 83.47% of the total number of horses.FECRT results showed that the commonly used anthelmintic drugs (mainly composed of ivermectin and albendazole) were effective in the equestrian club where the sample was located and there was no evidence of anthelmintics resistance.5 fecal samples of foals under 1-year-old were negative for Cryptosporidium.【Conclusion】 The parasite control situation of equestrian clubs in some areas of Guangdong province was generally good.There were differences in parasite control effects among equestrian clubs in the region.It was suggested that all equestrian clubs in the region should strengthen the monitoring of intestinal parasites of horses,and use drugs rationally according to the monitoring situation in the field,so as to avoid the prevalence of parasites in the field to develop resistance to the used deworming drugs.

【Objective】 This study was aimed to explore the biological characteristics and genome characteristics of broad-spectrum Escherichia coli O157∶H7 phage.【Method】 The broad-spectrum Escherichia coli O157∶H7 lytic phage was seperated from a swine farm sewage in Nanning of Guangxi using the double-layer agar culture method.The host range and the corresponding titers of phage were determined through the methods of spot tests and plaque tests.The methods of transmission electron microscope observation,determination of the best multiplicity of infection in the plural,one-step growth curve drawing,thermal sensitivity and pH stability evaluation,sterilization experiment and the whole genome sequencing were used to analyze the morphological features,biological characteristics and the whole genomic characteristics of phage.【Result】 A broad-spectrum lytic phage against Escherichia coli O157∶H7 was successfully isolated and purified which was named as vB_EcoM_GXBP08.Phage vB_EcoM_GXBP08 could lyse 14 Escherichia coli strains with high efficiency,and the phage titers could reach 10⁹ to 10¹⁰ PFU/mL.Using Escherichia coli O157∶H7 CVCC4050 as host bacteria,the optimal multiplicity of infection (MOI) of phage vB_EcoM_GXBP08 was 1.One-step growth curve indicated that the latent period of vB_EcoM_GXBP08 was 20 min,the outbreak period was 50 min,and the outbreak quantity was 154 PFU/cell. The tolerable temperature range of phage vB_EcoM_GXBP08 was 30 to 70 ℃,and it could maintain activity at pH 4.0 to pH 10.0.The results of bactericidal experiment against CVCC4050 showed that phage vB_EcoM_GXBP08 had good germicidal efficacy when MOI was 1.According to transmission electron microscope observation and the whole genome analysis,phage vB_EcoM_GXBP08 belonged to Caudovirales order,Myoviridae family,T4-like Phagus,which had a genome consisting of 108 114 bp with a GC content of 36.23%.Phage vB_EcoM_GXBP08 genome harbored 8 CDS associated with lysis proteins and lacked virulence genes and drug resistance genes associated with antibiotic resistance,toxins and virulence factors.【Conclusion】 Phage vB_EcoM_GXBP08 had a broad host range,high titer,good thermal stability and acid-base stability,and a strong bactericidal effect in liquid medium.The results provided reference for the development of broad-spectrum phage and its application in the prevention and treatment of Escherichia coli O157∶H7 infection in food industry and breeding industry.

【Objective】 To investigate the pharmacokinetics character of the diflubenzuron (DFB) premix in swine after drenching administration,a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to determine diflubenzuron in swine plasma.【Method】 DFB-¹³C₆ was employed as internal standard.Plasma samples were processed by protein precipitation of methanol.After centrifugation,the supernatant was vacuum evaporation to dry,and the residue was resolved by the solvent of methanol-water (1∶1).The column of ZORBAX Eclipse Plus C18 was used to separate the analyte.Mobile phase was methanol-buffer of 0.1% formic acid and 5 mmol/L ammonium formate with flow rate of 0.4 mL/min under gradient elution.The column temperature was 35 ℃ and injection volume was 10 μL.Determination of DFB was performed using LC-MS/MS with an electrospray ionization source interface operated in the positive-ion scan mode under the multiple reaction monitoring mode.Product ions 311.1→158.1 and 317.1→158.1 were employed as quantifier ion of DFB and DFB-¹³C₆,respectively.The conditions of LC and MS were optimized.The proposed method was validated through specificity,matrix effects,quantitative lower limits,lineral range,precision,accuracy and samples stability.【Result】 The established method had high sensitivity,the limit of detection (LOD) was 0.5 ng/mL and the limit of quantitative (LOQ) was 1 ng/mL.Matrix effects had little influence on the detection of samples.The standard solution had a good linear relationship in the concentration range of 1-1 000 ng/mL (R²≥0.998).The average accuracy was during 97.78% to 115.06% and the intra-assay and inter-assay precisions were both less than 10% when the spiked levels of DFB in plasma were LOQ (1 ng/mL),low (2 ng/mL),medium (100 ng/mL) and high concentration (800 ng/mL).These results could meet the methodological requirements.The standard solution,spiked samples and processed samples had good stability when they stored at the specific conditions except freeze-thaw cycles.【Conclusion】 The established LC-MS/MS detection method was simple,specific,sensitive,accurate and reliable.It could be used to detect the concentration of diflubenzuron in pig plasma,and then applied to the pharmacokinetics of diflubenzuron premix in swine.

【Objective】 This study was aimed to explore the toxicity and functional effects of aristolochic acid Ⅰ (AA-Ⅰ) on the five viscera (heart,liver,spleen,lung and kidney) of rats,so as to provide reference for the clinical use of traditional Chinese medicines of the Aristolochia genus in the future.【Method】 SD rats were randomly divided into aristolochic acid administration and blank control groups.The rats in administration group were gavaged with AA-Ⅰ at 20 mg/kg for 7 days.At 0.25 h,0.5 h,1 h,2 h,24 h,and 15 d after drug withdrawal,the five viscera of rats were taken.HPLC method was used to detect the contents of AA-Ⅰ and aristolactams-Ⅰ (AL-Ⅰ) in five viscera of rats at each time point,and the main physiological functions and histopathological changes of five viscera of rats were detected.【Result】 At 0.25 h after administration,AA-Ⅰ was mainly distributed in heart,liver,spleen and lung,and AL-Ⅰ was detected in liver and spleen.At 0.5 h after administration,AA-Ⅰ was mainly distributed in the heart,liver,spleen,lung and kidney.Except for the heart,the content of AA-Ⅰ in the other four viscera reached a high peak,and AL-Ⅰ was detected in liver,lung and kidney.At 1 h after administration,AA-Ⅰ was mainly distributed in liver and lung,and AL-Ⅰ was detected in liver.At 2 h after administration,AA-Ⅰ was mainly distributed in heart,liver,lung and kidney,and AL-Ⅰ was detected in kidney.At 24 h after administration,AA-Ⅰ was mainly distributed in heart and liver,and AL-Ⅰ was detected in liver.After 15 d of drug withdrawal,AA-Ⅰ and AL-Ⅰ were not detected in five viscera.The results of function tests showed that AA-Ⅰ could cause oxidative stress injury in five viscera.【Conclusion】 AA-Ⅰ was administered to rats at a dose of 20 mg/kg for 7 d, different levels of AA-Ⅰ were detected in five viscera,and AL-Ⅰ was detected in liver,kidney,lung and spleen.AA-Ⅰ could cause different degrees of damage to five viscera,and its damage was related to oxidative stress.

【Objective】 This study was aimed to investigate the protective effects of Polygonum chinense L. (P.chinense,PCL) on the liver of mice infected with Salmonella Typhimurium (S. Typhimurium) and its underlying mechanism.【Method】 48 male BALB/c mice were randomly divided into 6 groups:Normal,model,positive drug,PCL-H,PCL-M and PCL-L groups.Mice in the positive drug group were given gentamicin (20 mg/kg) orally.Mice in the PCL-H,PCL-M and PCL-L groups were orally administered with PCL at 16,8 and 4 g/kg,respectively.The normal and model groups mice were given with the same volume of PBS.The drugs were administered for 8 consecutive days.After 2 days of prophylactic administration,the mice in the normal group were given 0.2 mL PBS,and the mice in the other groups were given 0.2 mL S. Typhimurium solution (5×10⁴ CFU/mL) at one time.After the administration,the mice were killed,and the liver was dissected to make tissue sections and stained with HE to observe the pathological changes of the liver.The liver tissue suspension was cultured in the plate medium to detect the bacterial load in the liver of the mice.Western blotting was used to detect the expression levels interferon alpha (IFN-α),interferon beta (IFN-β),interferon gamma (IFN-γ),tumor necrosis factor α (TNF-α),interleukin 1β (IL-1β),TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) protein.【Result】 The pathological observation of liver showed that the liver of mice in the model group had congestion and a large number of inflammatory cells infiltrated.The hepatocytes were obviously swollen and disordered,the cytoplasmic staining was deepened,and the hepatocytes were necrotic and apoptotic.In PCL-H mice,there were a few inflammatory cells in liver,congestion and necrosis and apoptosis of liver cells were improved.In PCL-M and PCL-L,except for hepatocytes with dark cytoplasmic staining,slight swelling,and disordered arrangement,other lesions were not obvious.Compared with the normal group,the load of S. Typhimurium in liver of the model group mice was significantly increased (P＜0.05),and the expressions of IRF3,P-IRF3,IFN-α,IFN-β and IFN-γ were significantly decreased (P＜0.05),the expressions of TNF-α and IL-1β were significantly increased (P＜0.05).Compared with the model group,the load of S. Typhimurium in liver of mice in PCL-H,PCL-M and PCL-L groups was significantly decreased (P＜0.05).Among them,PCL-M could significantly enhance the phosphorylation of TBK1 and IRF3 proteins and the expression of IRF3 protein (P＜0.05).The protein levels of IFN-α,IFN-β and IFN-γ were significantly increased (P＜0.05),and the secretions of TNF-α and IL-1β were significantly decreased (P＜0.05).【Conclusion】 PCL could induce the production of interferon through enhancing TBK1-IRF3 pathway to alleviate the liver damage of mice infected by S. Typhimurium,and the dosage of 8 g/kg was better.

【Objective】 The aim of this study was to isolate Bacillus and Lactobacillus from goose feces,so as to provide references for the study of goose-derived functional bacteria and rational drug use.【Method】 The rectal feces of 30 50-week-old healthy Northern White geese were collected,and the strains were isolated and screened by specific culture medium and biochemical reaction test.The selected strains were identified by DNA extraction and PCR amplification 16S rDNA sequencing.The phylogenetic tree was established by comparing with the reference strains in GenBank and the strains were identified by homology comparison.After 48 h of culture,the growth characteristics,optimum pH and temperature of bacteria were detected.The growth status of bacteria under pH 7.0 and no bile salt environment was used as control.The ability of acid and bile salt tolerance was detected,and the tolerance of antibiotics was detected by drug sensitivity test.【Result】 After isolation and screening,9 strains of bacteria were obtained,and 2 strains were finally selected by biochemical test.According to the evolutionary distance and homology comparison of the phylogenetic tree,they were Bacillus tequilensis CC2FG2 and Weissella paramesenteroides JY0R2.The results of growth characteristics showed that Bacillus tequilensis CC2FG2 had the best proliferation effect under the condition of medium pH 6.5 and temperature 30 ℃ for 16 h,which could tolerate 1.2% bile salt,the number of viable bacteria was 69.2% of that without bile salt,and the acid resistance was strong.The number of viable bacteria under the strong acid environment of medium pH 3.5 was 81.5% of that of pH 7.0.The results of antibiotic tolerance showed that it was sensitive to ofloxacin,norfloxacin,kanamycin,streptomycin,clarithromycin,erythromycin,compound sulfamethoxazole and vancomycin,but not sensitive to cefuroxime,cefazolin,cefotaxime,ampicillin,nitrofurantoin,ciprofloxacin,gentamicin and tetracycline.In the medium with pH of 6.0 and temperature of 36.5 ℃,Weissella paramesenteroides JY0R2 had the best prolifervation effect when cultured for 18 h.Under 1.2% bile salt,the number of viable bacteria was 50.6% of that without bile salt.Under strong acid environment with pH 3.5,the number of viable bacteria was 87.6% of that with pH 7.0.It was sensitive to ciprofloxacin,ofloxacin,norfloxacin,clarithromycin and erythromycin,moderately sensitive to kanamycin,nitrofurantoin and tetracycline,and insensitive to cefuroxime,cefazolin,cefotaxime,ampicillin,compound sulfamethoxazole,vancomycin,gentamicin and streptomycin.【Conclusion】 The results of the above studies showed that Bacillus tequilensis CC2FG2 and Weissella paramesenteroides JY0R2 isolated from geese had good acid resistance and bile salt resistance,and Bacillus tequilensis could be used in combination with some β-lactams,quinolones,aminoglycosides,nitrofurans and tetracyclines,and Weisseria paramesenteroides could be used in combination with some β-lactams and aminoglycosides,providing a reference for the development of goose-derived functional bacteria.

【Objective】 This study was aimed to establish the epidemiological cut-off values of Enterococcus isolated from animals for antimicrobial growth promoters and further to understand the resistance rate of animal-derived Enterococcus to antimicrobial growth promoters from 2019 to 2021.【Method】 The MICs of Enterococcus isolated from breeding farms in Beijing,Hebei,Sichuan,Shanxi,Shaanxi and Inner Mongolia from 2019 to 2021 to seven antimicrobial growth promoters (kitasamycin,flavomycin,enramycin,nasipeptide,averamycin,viginiamycin and bacitracin) were tested.According to the method specified in CLSI-VET,the non-linear regression simulation of MIC distribution data was carried out using the statistical tool ECOFFinder.The drug resistance rate of Enterococcus from animals to 7 kinds of antibacterial growth promoters was calculated by the epidemiological cut-off values under 95% confidence interval.【Result】 The epidemiological cut-off values of kitasamycin,flavomycin,enramycin,narceptide,averamycin,virinomycin and bacitracin were 8,8,8,0.25,8,8 and 32 μg/mL,respectively.Based on this,the drug resistance rates calculated were 64.38%,34.48%,80.06%,14.69%,19.06%,50.69% and 24.96%,respectively.Among them,Enterococcus was most resistant to enramycin,kitasamycin and virginiamycin,but most sensitive to nazeptide.In the statistics from 2019 to 2021,the drug resistance rates of flavomycin and bacitracin decreased relatively obviously.【Conclusion】 This study established the epidemiological cut-off values of animal-derived Enterococcus against seven antimicrobial growth promoters commonly used in aquaculture. It could be used as a preliminary criterion for determining the sensitivity and resistance of animal-derived Enterococcus to antimicrobial growth promoters and provide a scientific basis for further establishing drug resistance points and monitoring the changes of drug resistance of animal-derived Enterococcus to antimicrobial growth promoters.

【Objective】 This study was aimed to learn the resistance of clinical isolates of Streptococcus suis in Guangdong to tetracyclines and the carriage of resistance gene.【Method】 The drug-sensitive disk diffusion method was used to analyze the tetracycline antibiotics resistance of 34 strains of Streptococcus suis isolated from Guangdong during 2016 to 2020.The tetracycline resistance genes tetA,tetC,tetD,tetM,tetO and tetX were detected by PCR.【Result】 The resistance rate of 34 strains of Streptococcus suis to tetracycline was as high as 100% (34/34),followed by minocycline 82.4% (28/34) and doxycycline 47.1% (16/34).Among 34 strains of Streptococcus suis,15 strains were triple-resistant,accounting for 44.1% (15/34),14 strains were double-resistant,accounting for 41.1% (14/34),and 5 strains were resistant to one drug,accounting for 14.7% (5/34).The results of drug resistance gene detection showed that the carrying rates of tetA,tetC,tetD,tetM,tetO and tetX genes were 0,0,0,14.7% (5/34),100% (34/34) and 0,respectively.【Conclusion】 The resistance rate of tetracycline antibiotics in clinical isolates of Streptococcus suis in Guangdong was relatively high,and the main tetracycline resistance genes carried were tetO and tetM genes.

【Objective】 The aim of this study was to screen the cyprin-derived lactic acid bacteria with excellent probiotics as candidate bacterial strains for aquaculture probiotics.【Method】 In this experiment,the intestinal contents and mucous membrane of carp were used as the sources of bacterial isolates.MRS medium was used for bacterial isolates and purification.After identification by 16S rRNA sequencing,the biological characteristics of isolated strains,such as growth characteristics,acid production capacity,acid tolerance,bile salt tolerance,antibacterial characteristics,drug sensitivity and safety were analyzed.【Result】 The results showed that a strain of Lactobacillus plantarum was successfully isolated and named YY001.On MRS medium,the colony morphology of the isolated strain was round,smooth surface,neat edge,milky white,Gram-positive,spun-free,blunt short-bacillus.The strain began to grow in logarithmic phase after 4 h,and reached a stable phase after 12 h.The strain had a strong acid production capacity.The acid production curve showed that pH decreased rapidly from 0 to 12 h,and pH reached 3.8 after 16 h culture.The strain had a certain tolerance to acid and bile salt at pH 3.0 and 0.3% bile salt.The strain had bacteriostatic effect on Flavobacterium columnar,Aeromonas vibrii and Aeromonas hydrophila,and the bacteriostatic effect on Flavobacterium columnar was very significant,and the diameter of bacteriostatic zone was 34.19 mm.The drug sensitivity test showed that the strain was resistant to kanamycin,streptomycin and vancomycin.Once a day for one week,10⁸ CFU isolated strain had no toxic effect on mice after feeding.【Conclusion】 The strain YY001 isolated in this study had excellent biological characteristics,which could be used as a candidate strain for aquaculture probiotics,and laid a foundation for the subsequent preparation of probiotics for carp.

【Objective】 The objective of this study was to investigate the effects of different culture and fermentation conditions on the antibacterial ability of Bacillus licheniformis 19148 and the resistance of Bacillus licheniformis 19148.【Method】 Bacillus licheniformis 19148 was cultured under different conditions of temperature,pH,metal ions,shaking speed,fermentation time and inoculation amount in a single factor experimental design,respectively,then centrifuge and filter the cultured liquid into sterile filtrate to study inhibition ability against Escherichia coli K88,Salmonella Typhimurium and Staphylococcus aureus 1882 using oxford cup bacteriostatic test.The drug resistance of Bacillus licheniformis 19148 to 27 antibiotics was studied by drug sensitive paper method.【Result】 Bacillus licheniformis 19148 could grow normally at 25,37 and 45 ℃, and was inhibited at 4 and 75 ℃,and had the best antibacterial activity at 37 ℃.Bacillus licheniformis 19148 could grow normally in the pH ranged from 5.0 to 9.0 and had the best antibacterial activity at pH 6.0.Bacillus licheniformis 19148 was intolerant to copper ions,but could tolerate magnesium ions,and there was no significant difference among treatment groups with different magnesium ion concentrations. In in vitro fermentation,Bacillus licheniformis 19148 showed the best antibacterial activity under the conditions of 3% seed liquid inoculation, and 150 r/min fermetation for 28 h.In the drug resistance evaluation test,Bacillus licheniformis 19148 showed moderate sensitivity to penicillin,tetracyclines,ceftazidine,furazolidone and polymyxin B,and showed high sensitivity to the other 22 antibiotics such as cefoperazone and erythromycin.【Conclusion】 Bacillus licheniformis 19148 had great stability and could be used in the production of industrial fermented feed without antibiotic resistance.

Aflatoxins (AF) are secondary metabolites of Aspergillus species with hightoxicity,and mainly found in milk and dairy products and mouldy cereals.At present,there are a lot of studies on aflatoxin B₁ (AFB₁),AFB₂,AFG₁,AFG₂,AFM₁ and AFM₂ among all aflatoxins.The physical and chemical properties of aflatoxins are relatively stable,it is difficult to destroy aflatoxins by heating,and aflatoxins are currently recognised as one of the most toxic carcinogens.Aflatoxins not only cause nutritional problems such as malnutrition and reduced immunity to animals,but also cause great damage to the human liver when entering the body,and the presence of aflatoxins in raw milk causes serious harm to the whole dairy industry chain.Therefore,there is an urgent need to implement selective,sensitive and highly convenient methods for the determination of aflatoxins in milk and dairy products.This paper introduces the research progress of aflatoxins detection methods in milk and dairy products at home and abroad,summarizes the application of mass spectrometry,electrochemistry,spectroscopy,chromatography and test strips in the detection of aflatoxins in milk and dairy products in recent years,summarises the advantages and challenges of the above methods in the detection of aflatoxins in milk and dairy products,and provides an outlook on the future development of aflatoxins detection methods in milk and dairy products.The future development of aflatoxins detection methods in milk and dairy products is also discussed,which may provide some new ideas for the detection of aflatoxins in milk and dairy products.

【Objective】 The effects of adding high temperature period compost on the degradation of organic components and enzyme activity of sheep manure composting in Qinghai Tibet plateau were studied.【Method】 In Qinghai Tibet plateau,sheep manure composting in high temperature period was added to the mixture of sheep manure and rape straw.Taking no high temperature period compost as the control,a 28 day strip compost experiment was carried out.By measuring the composting temperature,pH,total organic carbon,total nitrogen,ammonium nitrogen,nitrate nitrogen,seed germination index,organic matter,lignocellulose and other indexes as well as various enzyme activities in the composting process,the effect of high temperature composting on the fermentation quality of sheep manure composting in Qinghai Tibet plateau was judged.【Result】 The addition of high temperature period compost could promote the temperature rise of the reactor,increased the maximum temperature of the reactor,and prolonged the high temperature period of the reactor for 6 days.At the end of composting,compared with the control group,adding high temperature period compost could significantly reduce NH₄⁺-N/NO₃^--N,E₄/E₆ and C/N value (P＜0.05).It could significantly improve the germination index of compost seeds (P＜0.05).The degradation rates of organic matter,hemicellulose,cellulose and lignin were significantly increased (P＜0.05).It could significantly improve the activities of the protein enzyme,urease,sucrase,cellulase β-glucosidase,peroxidase and polyphenol oxidase (P＜0.05).【Conclusion】 The studies indicated that adding high temperature period compost in sheep manure composting could improve the fermentation quality and enzyme activity of compost,and could significantly shorten the composting time on the Qinghai Tibet Plateau.