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05 June 2024, Volume 51 Issue 6
Biotechnology
Identification and Analysis of Lipid Profile in DHAV-3 Resistant and Susceptible Lines of Pekin Duck
CHANG Zhuo, LIANG Suyun, WANG Shuaiqin, YANG Yuze, XUE Zhenhua, ZHAO Chunying, LIN Xiaoran, LU Yongqiang, HOU Shuisheng
2024, 51(6):  2253-2260.  doi:10.16431/j.cnki.1671-7236.2024.06.001
Abstract ( 75 )   PDF (6245KB) ( 72 )  
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【Objective】 The purpose of this experiment was to study the lipid metabolism profiles of the resistant Pekin duck line (Z7-R) and susceptible line (Z7-S) of Pekin ducks to Duck hepatitis A virus genotype 3 (DHAV-3),and investigate lipid markers with significant differences between this two lines.【Method】 6 2-day-old Z7-R and 6 2-day-old Z7-S Pekin ducks were selected and their blood and liver tissues were collected for blood biochemical index determination and non-targeted liver lipidomics detection based on liquid chromatography-mass spectrometry (LC-MS).t-test,partial least squares-discriminant analysis (PLS-DA) and fold change (FC) were used to comprehensively screen the significantly different lipids between the two lines.【Result】 The contents of total cholesterol,low density lipoprotein and phospholipid in the plasma of Z7-R Pekin duck were significantly higher than those in Z7-S (P<0.05).A total of 1 532 lipid metabolites were identified by lipidome analysis,covering five major categories:Fatty acyl (FA),glycerolipids (GL),glycerophospholipids (GP),sphingolipids (SP),and sterol lipids (ST).A total of 84 significantly different lipids were identified.Triglyceride (TG) was the main component in glycerolipids,and the content of TG in Z7-R Pekin duck was significantly higher than that in Z7-S (P<0.05).Lysophosphatidylcholine (LPC),lysophosphatidylethanolamine (LPE) and free fatty acid (FFAs) were the significantly different glycerophospholipids,and the contents of these lipids in Z7-R Pekin duck were significantly lower than those in Z7-S (P<0.05).A total of 10 significantly different lipids were screened and they were significantly enriched in sphingolipid metabolism,glycerophospholipid metabolism,glyceride metabolism,linoleic acid metabolism and α-linolenic acid metabolism.【Conclusion】 Lipidomics technology could be used to differentiate Z7-R lines from Z7-S lines of Beijing ducks.The 10 significantly different lipids could be used as potential biomarkers related to DHAV-3 resistant and susceptible traits.
Bioinformatics and Expression Characteristics Analysis of MGP Gene in Qinchuan Cattle
JIANG Lei, WANG Yuxin, WAN Yuan, ZAN Linsen, WANG Hongbao
2024, 51(6):  2261-2272.  doi:10.16431/j.cnki.1671-7236.2024.06.002
Abstract ( 48 )   PDF (15957KB) ( 47 )  
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【Objective】 This study was aimed to predict the fundamental functions of matrix Gla protein (MGP),reveal the tissue expression patterns of MGP gene in Qinchuan cattle and its expression characteristics during different stages of preadipocyte differentiation,so as to provide a theoretical foundation for studying the regulatory mechanisms of MGP gene in the adipose development of Qinchuan cattle.【Method】 The similarity alignment and phylogenetic tree construction of MGP protein in various species were conducted,and the physicochemical properties and subcellular localization of bovine MGP protein were predicted using bioinformatics online software.The differentiation effect of preadipocytes in Qinchuan cattle was examined using oil Red O staining.The expression of MGP gene in different tissues of Qinchuan cattle and its characteristics during different stages of preadipocyte differentiation were detected by Real-time quantitative PCR.【Result】 The similarity alignment results showed that the similarity of MGP protein was the highest (99%) between Bos taurus and Capra hircus,while the similarity was the lowest (31%) with Danio rerio.The phylogenetic tree revealed that Bos taurus and Capra hircus shared the closest relationship,whereas the relationship was the most distant with Danio rerio.MGP gene encoded 103 amino acids with a theoretical isoelectric point of 9.27,indicating alkalinity.MGP was considered a hydrophilic protein with a total average hydrophobicity index of ―0.630.MGP was classified as a secretory protein due to the presence of a Sec/SPⅠ-type signal peptide and the absence of transmembrane structures.Additionally,MGP protein included 5 serine and 4 threonine phosphorylation sites,and it possessed a GLA_domain containing γ-carboxyglutamic acid (Gla).The alpha helix constituted the highest percentage of the secondary structure at 55.34%.The oil Red O staining confirmed the well-differentiated state of isolated and cultured preadipocytes.Real-time quantitative PCR results showed that the expression of MGP gene in heart of Qinchuan cattle was significantly higher than that in other tissues (P<0.05).Elevated expression were also observed in large intestine and perirenal fat,while the expression in small intestine was significantly lower than that in other tissues (P<0.05).MGP gene exhibited a trend of initial upregulation followed by downregulation during different stages of preadipocyte differentiation,with significantly higher expression on the 2nd day of preadipocyte induction compared with other days (P<0.05),and significantly lower expression on the 10th day compared with other days (P<0.05).【Conclusion】 This study predicted the basic structure and function of MGP protein.The expression of MGP gene was the highest in heart of Qinchuan cattle and was the lowest in small intestine.MGP gene exhibited a trend of initial upregulation followed by downregulation during different stages of preadipocyte differentiation.
Construction of ACTA1 Gene Knockout PEFs Cell Lines by CRISPR/Cas9 Editing System
ZHANG Xueping, LIU Jiayi, WANG Yanfang, WU Tianwen
2024, 51(6):  2273-2284.  doi:10.16431/j.cnki.1671-7236.2024.06.003
Abstract ( 45 )   PDF (7539KB) ( 28 )  
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【Objective】 The purpose of this study was to establish actin alpha 1 (ACTA1) gene knockout porcine embryo fibroblasts (PEFs) by CRISPR/Cas9 system,laying the foundation for subsequent research on the function of ACTA1 gene and exploration of harbor locus in pigs.【Method】 Expression of ACTA1 gene in seven tissues (heart,liver,spleen,lung,kidney,dorsal subcutaneous fat and longissimus dorsi muscle) of Bama Miniature pigs was detected by Real-time quantitative PCR.3 single guide RNAs (sgRNAs) were designed in the exon 7 region of the porcine ACTA1 gene by the CRISPOR online website and were ligated into the pX330 vector.The activity of different sgRNA vectors was detected by T7E1 digestion assay,and vector plasmids with higher efficiency and meeting the target were selected for co-transfection into PEFs.Monoclonal cells were screened by limited dilution method and conducting genotype identification and off-target analysis.【Result】 The results of Real-time quantitative PCR showed that the expression of ACTA1 gene was extremely significantly higher in the longissimus dorsi muscle than other tissues (P<0.01).The gene editing efficiencies of 3 sgRNAs were 24.87% (sgRNA1),39.59% (sgRNA2) and 36.93% (sgRNA3),respectively.Based on the cutting position and gene-editing efficiency chose sgRNA1 and sgRNA2 for co-transfected into cells.Genotyping results showed that gene editing occurred in 20 of the 69 monoclonal cells were obtained,of which 1 was biallelic fragment knockout cells and 3 were monoallelic fragment knockout cells,with a fragment knockdown efficiency of 5.8% (4/69).Off-target analysis showed that no off-target effects were detected at the predicted off-target sites.【Conclusion】 This study successfully constructed a PEFs with a biallelic knockout of ACTA1 gene using CRISPR/Cas9 editing system,which provided technical and theoretical bases for further research on the regulation of ACTA1 gene on porcine skeletal muscle development,and also provided new idea for exploration of tissue-specific harbor locus in pigs.
Cloning of the SMIT1 and SMIT2 Genes in Gymnocypris przewalskii and Its Response to Alkaline Environment
WEI Wei, GAO Zihan, YUE Miao, ZUO Yang, WANG Tonggang, WEI Fulei, YU Luxian, LIANG Jian
2024, 51(6):  2285-2299.  doi:10.16431/j.cnki.1671-7236.2024.06.004
Abstract ( 35 )   PDF (11746KB) ( 13 )  
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【Objective】 The sodium/myo-inositol cotransporter (SMIT) in mammalian is a solute carrier (SLC) superfamily protein that has two homologs:SMIT1 and SMIT2.The experiment was aimed to investigate the response of SMIT1 and SMIT2 genes in Gymnocypris przewalskii to alkaline environment,so as to lay the foundation for understanding the molecular mechanisms of SMIT1 and SMIT2 genes in myo-inositol transport under alkaline condition.【Method】 The CDS region of SMIT1 and SMIT2 genes were amplified using PCR and cloned,followed by bioinformatics analysis.The expression of SMIT1 and SMIT2 gene in eight tissues,such as gill,kidney and brain,of Gymnocypris przewalskii in freshwater environment was detected using Real-time quantitative PCR.Additionally,the expression of the two genes in gill and kidney of Gymnocypris przewalskii under different alkaline stress (0,J25,J50,J75 and J100) conditions were investigated,and the changes in myo-inositol content were detected using gas chromatography-mass spectrometry (GC-MS) technology.【Result】 The CDS sequence of SMIT1 gene was 2 130 bp,encoding a total of 709 amino acids,the amino acid sequence of SMIT1 gene showed the highest similarity of 86.5% with Sinocyclocheilus rhinocerous,indicating a closer phylogenetic relationship with Onychostoma macrolepis.The CDS sequence of SMIT2 gene was 2 016 bp,encoding a total of 671 amino acids,the amino acid sequence of SMIT2 gene showed the highest similarity of 90.3% with Onychostoma macrolepis,indicating a closer phylogenetic relationship with Sinocyclocheilus rhinocerous and Onychostoma macrolepis.SMIT1 and SMIT2 both belonged to the SLC5 family members of the solute superfamily,SMIT1 protein contained two conserved domains of the SLC family.Real-time quantitative PCR results indicated that both SMIT1 and SMIT2 genes were expressed in various tissues of Gymnocypris przewalskii in freshwater environment,the expression of SMIT1 gene was higher in brain and kidney,while the expression of SMIT2 gene was highest in kidney,both of which were significantly higher than that in other tissues (P<0.05).With increasing alkalinity,compared with control group,the expression of SMIT1 gene in gill of Gymnocypris przewalskii at J25,J50 and J100 was significantly increased (P<0.05),and the overall expression in kidney was significantly increased (P<0.05).On the other hand,the expression of SMIT2 gene in gill of Gymnocypris przewalskii at J25 was significantly increased,and the expression in kidney at J50,J75 and J100 was significantly decreased (P<0.05).The GC-MS results showed that the myo-inositol content in gill of Gymnocypris przewalskii was significant higher than that in control group (P<0.05),but there was no significant change of the myo-inositol content in kidney with control group(P>0.05).【Conclusion】 The CDS length of SMIT1 and SMIT2 genes were 2 130 and 2 016 bp,encoding 709 and 671 amino acids.SMIT1 and SMIT2 genes were highly expressed in brain and kidney of Gymnocypris przewalskii,respectively. The expression patterns of SMIT1 and SMIT2 genes in gill and kidney of Gymnocypris przewalskii under different alkalinity stresses. The myo-inositol content in gill was significantly increased and reached its peak at J50,with no significant change in kidney.The results provided a reference basis for further exploring the molecular mechanism of Gymnocypris przewalskii adaptation to alkaline environment.
Study on the Targeted Regulation of Wnt7a Gene Expression by miR-192 in Pigs
FU Binbin, WANG Dongsheng, HE Fan, LI Qingchun, QI Mengfan, ZHANG Huapeng, HUANG Tao
2024, 51(6):  2300-2307.  doi:10.16431/j.cnki.1671-7236.2024.06.005
Abstract ( 30 )   PDF (2950KB) ( 12 )  
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【Objective】 The targeting relationship between procine miR-192 and Wnt family member 7A (Wnt7a) genes was identified to establish a basis for further construction of miR-192-mediated embryonic communication.【Method】 The miRNA pull-down was used to identify the interactions between miR-192 and Wnt7a gene.Bioinformatics software RNAhybrid 2.2 was utilized to predict the binding sites of miR-192 and Wnt7a gene 3'-UTR.Fragments of Wnt7a gene 3'-UTR containing miR-192 binding sites were cloned into the dual luciferase reporter gene plasmid and subjected to identification through NotⅠ and XhoⅠ digestion and sequencing.The identified plasmids were co-transfected with miR-192 mimics and mimics NC into porcine endometrial epithelial cells to detect dual-luciferase activity.miR-192 mimics,mimics NC,miR-192 inhibitor,inhibitor NC were separately co-transfected with constructed Wnt7a gene 3'-UTR wild-type and mutant dual-luciferase reporter gene plasmids into porcine endometrial epithelial cells.The mRNA and protein expression of Wnt7a gene were detected by Real-time quantitative PCR and Western blotting.【Result】 Pull-down results showed that the relative expression of Wnt7a gene was extremely significantly increased in miR-192 mimics group compared with its negative control group (P<0.01),and extremely significantly decreased in the miR-192 input group compared with its negative control group (P<0.01).The binding site (UGACCUA) between miR-192 seed region and Wnt7a gene 3'-UTR was predicted by RNAhybrid 2.2 software.The dual luciferase activity was significantly lower in miR-192 mimics group transfected with Wnt7a wild-type plasmid compared with miR-192 mimics NC group (P<0.05),and there was no significant difference in dual luciferase activity between miR-192 mimics group transfected with Wnt7a mutant plasmid and miR-192 mimics NC group (P>0.05).Real-time quantitative PCR results showed that the mRNA relative expression of Wnt7a gene in miR-192 mimics group was significantly decreased than that in its negative control group (P<0.05),and miR-192 inhibitor group was significantly higher than that in its negative control group (P<0.05).Western blotting results showed that the expression of Wnt7a protein was extremely significant inhibited after overexpression of miR-192 (P<0.01),and the expression of Wnt7a protein was extremely significant increased after inhibiting miR-192 (P<0.01).【Conclusion】miR-192 was able to target Wnt7a gene 3'-UTR and inhibit its expression.The results of this study could provide a theoretical basis for future in-depth research on the regulatory mechanism of miR-192 in the process of pregnancy implantation.
Cloning, Bioinformatic and Expression Analysis of SERPINE1 Gene in Longissimus Dorsi Muscle of Yaks with Different Tenderness
JING Kemin, ZHANG Peng, LI Yuqian, TIAN Yuan, DONG Wenjing, CHAI Zhixin, WANG Jikun, ZHONG Jincheng, CAI Xin
2024, 51(6):  2308-2318.  doi:10.16431/j.cnki.1671-7236.2024.06.006
Abstract ( 32 )   PDF (7511KB) ( 27 )  
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【Objective】 This study was aimed to clone the serpin family E member 1 (SERPINE1) gene sequence in yak,and explore the nucleotide sequence difference of SERPINE1 gene in longissimus dorsi muscle of yak with different tenderness,so as to provide experimental data for further study on its effect on the tenderness of yak meat.【Method】 Based on the yak SERPINE1 gene sequence in GenBank,specific primers were designed to amplify the full length of SERPINE1 gene sequence in longissimus dorsi muscle of yak with high and low tenderness by PCR,and their structure and encoded protein were analyzed by bioinformatics.Real-time quantitative PCR was used to detect SERPINE1 gene expression in longissimus dorsi muscle of yak with different tenderness.【Result】 The total length of SERPINE1 gene in longissimus dorsi muscle of yak with high and low tenderness was 8 315 and 8 318 bp,respectively,encoding 402 amino acids with identical amino acid sequence.There were 3 base deletions and 22 base mutations (4 base translocations and 18 base transitions) of SERPINE1 gene in high and low tenderness longissimus dorsi muscle.The nucleotide sequence of SERPINE1 gene in yak showed 99.26%,99.26%,99.59%,96.94%,97.02%,90.49%,86.52% and 80.10% similarity to Bos taurus,Bos indicus,Bison bison bison,Ovis aries,Capra hircus,Sus scrofa,Homo sapicns and Mus musculus,respectively.SERPINE1 protein was a hydrophilic stable extracellular protein containing 23 amino acid signaling peptides, and located in the extracellular with 34 potential phosphorylation sites,including 1 reactive center loop (RCL).The secondary structure of SERPINE1 protein was mainly composed of alpha helix (44.78%) and random coil (35.32%).Real-time quantitative PCR results showed that the expression of SERPINE1 gene in longissimus dorsi muscle of yak with high tenderness was extremely significantly higher than that in longissimus dorsi muscle of yak with low tenderness (P<0.01).【Conclusion】 The full length of SERPINE1 gene was successfully cloned from longissimus dorsi muscle of yak with high and low tenderness and the bioinformatics characteristics were analyzed,which provided theoretical reference for the subsequent research on the mechanism of SERPINE1 gene involved in the regulation of yak meat tenderness.
Application of Mass Spectrometry Techniques in the Characterization and Quantitative Analysis of Therapeutic Protein Drugs in Veterinarian
QIU Jicheng, YANG Yuxin, ZHANG Lu, WEN Zeyu, CHEN Sumeng, CAO Xingyuan
2024, 51(6):  2319-2329.  doi:10.16431/j.cnki.1671-7236.2024.06.007
Abstract ( 33 )   PDF (8971KB) ( 12 )  
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Therapeutic protein drugs have evolved into a crucial branch of drug research and development.Due to their remarkable clinical efficacy and safety profile,they have become the preferred choice for treating conditions like cancer and autoimmune disorders.Therapeutic protein drugs produced through in vitro cell expression are often highly complex.Therefore,it is essential to establish a series of quality analysis and standards to ensure consistency and safety during the development and production processes.Mass spectrometry technology,known for its high sensitivity,excellent selectivity,specificity and high throughput capabilities,has been widely employed in the development of small molecule drugs.It has also emerged as a vital tool for the quality analysis of therapeutic protein drugs,contributing to structural characterization,post-translational modification assessment,quantitative analysis,impurity analysis and interaction studies.This article offers a comprehensive overview of protein analysis through mass spectrometry,including ionization techniques,dissociation methods and mass analyzers.Moreover,it delves into the sample preparation and methodologies for protein structural characterization and post-translational modification analysis such as glycosylation and disulfide bonds.The article also explores techniques for protein quantification via mass spectrometry,covering sample pre-processing and standard analytical procedures.Moreover,it also provides an outline of general strategies for characterizing and quantifying antibody-drug conjugate (ADC) and application in other way.By comprehensively exploring these topics,this article aims to provide valuable insights and reference to advance veterinary therapeutic protein drug research and development through mass spectrometry technology.
Identification and Efficient Expression in Ovarian Granulosa Cells of circFDXR in Goat
LIU Jie, LI Zhihan, GUO Conghui, FENG Guanghang, XUE Wenzhe, LIU Dewu, LIU Guangbin, SUN Baoli, GUO Yongqing, DENG Ming, ZOU Xian, LI Yaokun
2024, 51(6):  2330-2341.  doi:10.16431/j.cnki.1671-7236.2024.06.008
Abstract ( 35 )   PDF (18660KB) ( 15 )  
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【Objective】 The purpose of this experiment was to identify the expression of circular RNA (circRNA) circFDXR in goat and establish its efficient expression mode in goat primary ovarian granulosa cells.【Method】 The cyclic properties of circFDXR were identified by PCR amplification,DNA sequencing,RNase R digestion and Real-time quantitative PCR.Cytoplasmic separation and fluorescence in situ hybridization (FISH) were used to detect the subcellular localization of circFDXR.The linear sequence of circFDXR was seamlessly cloned into the Lentivirus vector pLC5-ciR by homologous recombination,and transfected into 293T cells to package the virus,then the virus titer was determined,and the optimal multiplicity of infection (MOI) was determined.The optimal MOI was used to infect goat primary ovarian granulosa cells.Real-time quantitative PCR was used to detect the overexpression efficiency,and sequencing verified whether circFDXR was cyclized correctly.【Result】 Goat circFDXR was mainly localized in the cytoplasm of ovarian granular cells and tolerates digestion of RNase R enzymes.The circFDXR lentiviral overexpression vector was successfully constructed,the concentrated virus titer was 5.49×109 TU/mL,and the optimal MOI for infecting goat ovarian granulosa cells was 100.Real-time quantitative PCR results showed that the expression level of circFDXR in overexpressed group was extremely significantly higher than that in control group (P<0.01),and the sequencing results showed that the overexpressed circFDXR could be cyclized correctly.【Conclusion】 circFDXR was mainly localized in the cytoplasm of granular cells and had higher stability than linear RNA.The circFDXR packaged by Lentivirus could be cyclized correctly in the primary ovarian granulosa cells of goats.The experimental results laid a theoretical basis for further study of the function of circFDXR in the primary ovarian granulosa cells of goats.
Cloning,Bioinformatics Analysis and Spatiotemporal Expression of CKMT2 Gene in Jianzhou Big-eared Goats
SUN Shiyu, LI Jinlan, XING Jiani, DONG Yaohui, LI Yanyan, WANG Youli, LIN Yaqiu
2024, 51(6):  2342-2353.  doi:10.16431/j.cnki.1671-7236.2024.06.009
Abstract ( 33 )   PDF (7071KB) ( 18 )  
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【Objective】 This study was aimed to clone the creatine kinase mitochondrial 2 (CKMT2) gene and explore its biological characteristics,and clarify its expression characteristics in different tissues and myoblasts of different differentiation stages of Jianzhou Big-eared goats.【Method】 The samples of heart,liver,spleen,kidney,longissimus dorsi muscle and triceps brachii muscle in Jianzhou Big-eared goats were collected.The CKMT2 gene was amplified by PCR method,cloned and sequenced,and bioinformatic analysis was conducted by online software.Real-time quantitative PCR was used to detect the expression of CKMT2 gene in different tissues and myoblasts of different differentiation stages in Jianzhou Big-eared goats.【Result】 The total length of CKMT2 gene in Jianzhou Big-eared goats was 1 314 bp,including 1 259 bp in CDS region,encoding 419 amino acids,which was closely related to Ovis aries and Oryx dammah.CKMT2 protein was an alkaline hydrophophilic stable protein without signal peptide and transmembrane domain,mainly located in mitochondria,peroxasome and cytoplasm,which contained 32 phosphorylation sites,1 N-glycosylation site and 4 O-glycosylation sites.The secondary structure of the protein mainly consisted of alpha helix (39.14%),random coil (36.28%),extended chain (16.23%) and beta turn (8.35%),and the tertiary structure was basically consistent with the predicted secondary structure.The results of protein interaction analysis showed that CKMT2 protein was associated with cysteine and glycine-rich protein 3 (CSRP3),creatine kinase M-type (CKM) and phosphoglycerate mutase 2 (PGAM2) and other proteins.The results of tissue expression profile showed that the expression of CKMT2 gene was the highest in heart of Jianzhou Big-eared goats,which was significantly higher than that in other tissues (P<0.05).The expression of longissimus dorsi muscle was higher than that of spleen and triceps brachii muscle (P<0.05).The results of time series expression profile showed that the expression of CKMT2 gene in Jianzhou Big-eared goats reached a peak at 4 days after differentiation,which was significantly higher than 0 and 6 days (P<0.05).【Conclusion】 CKMT2 gene sequence was successfully cloned and its molecular characteristics were clarified,and its expression was higher in heart,longissimus dorsi muscle and myoblast at 4 days of differentiation.The results laid the foundation for further elucidating the molecular mechanism of CKMT2 gene regulating the proliferation and differentiation of myoblasts in Jianzhou Big-eared goats.
Physiological and Biochemical
Identification and Bacteriostatic Activity Analysis of an Antifungal Bacillus velezensis lut-Y1
YUAN Huijun, YU Shiman, XU Yanying, YUAN Yijun, FENG Huan, ZHANG Huanhuan
2024, 51(6):  2354-2364.  doi:10.16431/j.cnki.1671-7236.2024.06.010
Abstract ( 33 )   PDF (9728KB) ( 15 )  
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【Objective】 The aim of this study was to investigate the growth characteristics and antibacterial activity of a strain of Bacillus velezensis lut-Y1 from Lycium barbarum L.,and provide a reference for the development and application of microecologics.【Method】 The strain was identified by morphological observation and 16S rDNA gene sequencing.The growth curve,optimum pH,carbon source,nitrogen source and salt tolerance of the strain were measured.The antagonistic effects of the strain on Alternaria alternate,Bipolaris sorokiniana and Ascochyta leptospora were observed by plate confrontation method.The antibacterial activity of the fermentation broth against three kinds of pathogenic fungi was analyzed.【Result】 The lut-Y1 strain grew well on LB,NA and PDA medium.The strain lut-Y1 was identified as Bacillus velezensis based on its morphology and 16S rDNA gene sequence characteristic.In LB liquid medium,lut-Y1 strain showed a retardation stage from 0 to 3 h,a logarithmic stage from 3 to 18 h,a stable stage from 18 to 62 h,and a decline stage after 62 h.The optimal pH was 7.0,the optimal carbon and nitrogen sources were sucrose and peptone,respectively,and the tolerance to NaCl was no more than 2.0 g/L.lut-Y1 strain had obvious antagonistis effect on Alternaria alternate,but had weak antagonistic effect on Bipolaris sorokiniana and Ascochyta leptospora.At the junction of the two bacteria in the standoff plate,the mycelium in the base of Alternaria alternate was thin and curved.The mycelium cell wall was destroyed and bulbous protrusions were found in the base of Bipolaris sorokiniana.When the fermentation broth was added by 25%,the growth circle diameter were decreased by 72.80% and 83.24%,respectively.【Conclusion】 Bacillus velezensis lut-Y1 had good antibacterial activity,which could provide materials for the development of microecological preparations and biological pesticides with Bacillus as the main active ingredient.
Biochemical Characteristics and Functional Studies of the Sperm-specific Calcium Channel CatSper in Tupaia belangeri
LI Xiang, DAI Xu, YANG Minghua, LI Yahui
2024, 51(6):  2365-2374.  doi:10.16431/j.cnki.1671-7236.2024.06.011
Abstract ( 29 )   PDF (11089KB) ( 11 )  
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【Objective】 The purpose of this experiment was to explore the biochemical characteristics of specific calcium channel CatSper and its subcellular location in Tupaia belangeri sperm,and to clarify the regulatory role of CatSper on the motility of Tupaia belangeri sperm.【Method】 Sexually mature male Tupaia belangeri aged 1 to 2 years were selected,semen was collected after being killed with CO2,sperm motility was observed and protein was extracted.Western blotting and immunohistochemistry were used to detect the presence and subcellular localization of CatSper family in sperm.Different calcium channel inhibitors (CatSper inhibitor HC-056456,transient receptor potential vanillate subtype 1 (TRPV1) inhibitor Capsazepine) co-incubated with progesterone (P4) were used to observe the motility of Tupaia belangeri sperm.Calcium probe method was used to detect the effects of different inhibitors and P4 co-incubation on calcium ion concentration of Tupaia belangeri sperm.【Result】 CatSper and TRPV1 channel protein were present in Tupaia belangeri sperm,and CatSper was located in the sperm head and flagella.Compared with control group,sperm motility and calcium ion concentration were significantly decreased when HC-056456 concentrations were 100,200,400 and 1 000 nmol/L (P<0.05),and Capsazepine concentrations were 10,20 and 40 μmol/L,sperm motility and calcium ion concentration were significantly decreased (P<0.05).Compared with Capsazepine group,when HC-056456 was co-incubated with P4,the calcium ion concentration of Tupaia belangeri sperm was significantly increased,and there were significant differences between the two groups when P4 concentrations were 10,20 and 30 μmol/L (P<0.05).When Capsazepine and HC-056456 were co-incubated with P4,there was no significant difference in sperm motility between the two groups (P>0.05).【Conclusion】 CatSper channel played an important role in the regulation of sperm motility and calcium ion transport in Tupaia belangeri,and the calcium ion concentration in sperm was increased through P4 activation.The results were of great significance for further research on the regulatory mechanism of sperm function and sperm fertilization ability.
Nutrition and Feed
Effects of Guanidine Acetic Acid and Alpha-lipoic Acid Supplementation on Rumen Microbita and Metabolites in Sheep
JIANG Lu, LIANG Bencong, XU Zejun, YANG Gaiqing, YANG Sihan, CHEN Qixin, WANG Xianwei, HU Yeyong, WANG Linfeng, GAO Tengyun
2024, 51(6):  2375-2387.  doi:10.16431/j.cnki.1671-7236.2024.06.012
Abstract ( 38 )   PDF (11067KB) ( 19 )  
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【Objective】 This experiment was aimed to investigate the effects of adding guanidine acetic acid (GAA) and alpha-lipoic acid (LA) to diets on rumen microbiota and metabolites in sheep.【Method】 Twenty-four healthy Dorper×Hu hybrid male sheep with similar body weight and good physical condition were randomly divided into four groups:Control group (CTL,fed with basic diet),GAA group (fed with basic diet supplemented with 1 500 mg/kg GAA),LA group (fed with basic diet supplemented with 600 mg/kg LA),and MIX group (fed with basic diet supplemented with 1 500 mg/kg GAA and 600 mg/kg LA).The experiment lasted for 60 days,and rumen fluid samples were collected after the experiment for 16S rRNA sequencing and untargeted metabolomics analysis of rumen microbiota and metabolites.【Result】 At the phylum level of the rumen microbial community in sheep,compared with control group,the abundance of Patescibacteria in GAA and MIX groups was significantly increased (P<0.05),while the abundance of Verrucomicrobiota in LA and MIX groups was significantly decreased (P<0.05).At the genus level of the rumen microbial community in sheep,the abundance of Quinella in LA group was significantly decreased (P<0.05);The abundance of Candidatus_Saccharimonas in all groups was significantly increased (P<0.05),while the abundance of Turicibacter was significantly decreased (P<0.05);The abundance of Veillonellaceae_UCG-001 in GAA group was significantly decreased (P<0.05);The abundance of Eubacterium_Ruminantium_group in MIX group was significantly decreased (P<0.05). Metabolic pathway analysis of the rumen microbial community in sheep showed that the differential metabolites in GAA group,such as 11-hydroxyperoxyeicosatetraenoic acid and 8-methoxyeicosatetraenoic acid,primarily participated in the tryptophan metabolism pathway,The differential metabolites in LA group,such as sphingosine-1-phosphate and dihydrosphingosine,mainly participated in the sphingolipid signaling pathway and sphingolipid metabolism pathway.Meanwhile,the differential metabolites in MIX group,such as 8-methoxyeicosatetraenoic acid,guanosine monophosphate,and 3-methylindole,primarily participated in the tryptophan metabolism pathway.【Conclusion】 The addition of GAA or LA alone or in combination in the feed could regulate the composition and metabolic functions of the rumen microbial community in sheep. The results were helpful to reveal the underlying principles of GAA and LA as additives.
Effects of Dietary n-6/n-3 PUFA Ratio on Fatty Acid Composition in Duck Eggs and Quality of Salted Egg Yolk in Laying Duck
LAI Yue, ZHANG Yanan, WANG Shuang, LIANG Mingqi, JIANG Liying, CHEN Wei, JIA Xuchao, GUO Liang, ZHENG Chuntian
2024, 51(6):  2388-2398.  doi:10.16431/j.cnki.1671-7236.2024.06.013
Abstract ( 26 )   PDF (2393KB) ( 16 )  
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【Objective】 This study was conducted to investigate the effects of dietary n-6/n-3 PUFA ratio on fatty acid composition in duck eggs and quality of salted egg yolks in laying ducks.【Method】 A total of 180 healthy 21-week Fujian Longyan laying ducks were randomly divided into 2 treatments,with 6 replicates of 15 ducks each.They were fed corn-soybean meal and corn-soybean meal-linseed diets,and the n-6/n-3 PUFA ratio in diets were 16 and 8,respectively.The trial lasted for 12 weeks.At the end of week 12,fresh duck eggs were collected to determine the fatty acid composition,and the salted egg yolks were prepared to determine the yolk interior properties,texture properties,odor and flavor.【Result】 ①Compared with the diet with the ratio of n-6/n-3 PUFA was 16,the diet with the ratio of n-6/n-3 PUFA was 8 increased the contents of C18∶0,C20∶0,C22∶0,C22∶1n9,C18∶2n6c,C18∶3n3,C20∶3n6,C20∶4n6,n-6 PUFA and n-3 PUFA in egg yolk,and decreased the ratio of n-6/n-3 PUFA in egg yolk (10.20 to 5.65,P<0.05).②Dietary decreased n-6/n-3 PUFA ratio reduced the hardening ratio and its weight of salted egg yolk (P<0.05),but had no significant effect on oil output and sandiness (P>0.05).As to the texture properties,the hardness was decreased,the springiness was increased (P<0.05),and there were no significant differences in adhesiveness,cohesiveness,gumminess and chewiness of salted egg yolk (P>0.05).The odor of alcohol,ether,aldehyde and ketone in salted egg yolk was decreased with dietary decreased n-6/n-3 PUFA ratio (P<0.05).As to the flavor,dietary decreased n-6/n-3 PUFA ratio reduced the bitterness and umami,increased the richness of salted egg yolk (P<0.05),and no significant effect was observed in astringency and saltiness (P>0.05).【Conclusion】 Overall,dietary decreased n-6/n-3 PUFA ratio (8 vs 16) could decrease the ratio of n-6/n-3 PUFA in the fresh egg yolk,improve the quality and flavor of salted egg yolk.
Effects of Restaurant Plate Leftovers with Different Inclusion Ratios on Growth Immunity and Meat Quality of Broiler in Late Growing Period
LI Shiqiang, DONG Yingchao, LI Junguo, YANG Jie, WANG Jiqing, NIU Libin, MA Ying
2024, 51(6):  2399-2408.  doi:10.16431/j.cnki.1671-7236.2024.06.014
Abstract ( 35 )   PDF (1240KB) ( 20 )  
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【Objective】 This study aimed to provide data support for the application of restaurant plate leftovers (RPL) in the replacement of corn and soybean meal with reduced feed production and broiler breeding by studying the effects of RPL inclusion ratio on growth performance,slaughter performances,meat quality,immune-organ indices,and serum biochemical indices of broilers in late growing period.【Method】 504 1-day-old White feather broilers were selected and randomly divided into 4 groups,with 6 replicates in each group and 21 broilers in each replicate.In the early stage of the experiment (1 to 21 days of age),the same diet was fed.In the later stage (22 to 42 days of age),four types of diets were designed.The broilers in control group were fed the corn-soybean meal basal diet,and in experimental groups were fed diets inclusion with 10%,20% and 30% RPL (1/2 RPL instead of corn,1/2 RPL instead of soybean meal),respectively.The growth performance of broilers was recorded,slaughter performance,meat quality,immune-organ indices and serum biochemical indices were measured after slaughter at 42 days of age.【Result】 Compared to control group,① The inclusion of 10% and 20% RPL had no significant effect on the growth performances of broilers (P>0.05),while the inclusion of 30% RPL significantly reduced average body weight,average daily feed intake,average daily gain (P<0.05),and extremely significantly increased the ratio of feed to gain (P<0.01).② The inclusion 10% and 20% RPL had no significant effect on the dressing percentage,eviscerated yield percentage,breast muscle rate,and leg muscle rate of broilers (P>0.05),while inclusion 30% RPL significantly reduced breast muscle rate (P<0.05).③ The inclusion of different proportions of RPL to the feed had no significant effect on the immune-organ indices of broilers (P>0.05).The serum IgG content of broilers significantly increased with the inclusion of 10% and 20% RPL (P<0.05),while the serum SOD activity of broilers was significantly decreased with the inclusion of 30% RPL (P<0.05).④ The inclusion of different proportions of RPL did not significantly affect the color,drip loss at 24,48 and 72 hours,acidity,cooking loss,and shear force of broiler breast and leg muscles (P>0.05).【Conclusion】Dietary inclusion of 10% to 20% RPL had no negative impact on broiler growth performances,slaughter performances,meat quality,immune-organ indices,and serum biochemical indices of broilers in late growing period.RPL could replace corn and soybean meal in conventional diets in poultry farming.
Effects of Dihydromyricetin on Immune Function of Chicks
HAO Shuangshuang, CAO Chuanbao, GONG Yingchao, FAN Xianan, JIANG Xinru, JI Zhenghua, CHANG Yicong, LI Rui, LI Changwen, LIU Fangping
2024, 51(6):  2409-2417.  doi:10.16431/j.cnki.1671-7236.2024.06.015
Abstract ( 32 )   PDF (17698KB) ( 17 )  
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【Objective】 The purpose of this study was to explore the effects of dihydromyricetin (DHM) on immune organ index,histological morphology of immune organ and serum immune factor content in chicks.【Method】 150 Hy-line White chicks of 7-day-old were selected and randomly divided into five groups with 30 per group.The control group was given the basal diet,and the administration groups were given the basal diet supplemented with 0.025%,0.05%,0.1% and 0.2% DHM,respectively.At 7,14,21,28 and 35 d after administration,six chicks were randomly selected from each group for heart blood collection and the immune organs (thymus,spleen and bursa of Fabricius) were collected and weighed separately.The immune organ index of chicks in different treatment groups were calculated.The serum immunoglobulin IgG and complement (C3 and C4) content were measured,and HE staining was performed on the immune organs of chicks at 35 d of administration to observe tissue morphology.【Result】 Compared with the control group,the thymus index of chicks in 0.2% DHM group was significantly increased at 35 d after administration (P<0.05).The spleen index of chicks in 0.2% DHM group was significantly increased at 28 and 35 d after administration (P<0.05).The bursa of Fabricius index of chicks in 0.1% and 0.2% DHM groups were significantly increased at 7 d after administration (P<0.05).After adding DHM to the feed,the immune organ cells of chicks were arranged neatly,the number of lymphocytes increased,and the structure became denser.Compared with the control group,the serum IgG content of chicks in 0.05%,0.1% and 0.2% DHM groups were significantly increased at 28 and 35 d after administration (P<0.05). Compared to each dose group,the serum IgG content of chicks in 0.2% DHM group was significantly higher than that in other groups at 21 and 28 d,and was significantly higher than that in 0.025% and 0.05% DHM groups at 35 d (P<0.05).Compared with the control group,the content of serum complement C3 of chicks in 0.1% and 0.2% DHM groups were significantly increased at different administration times (P<0.05). Compared to each dose group,the serum complement C3 content of chicks in 0.2% DHM group was significantly higher than that in other groups at 14 and 28 d,and significantly higher than that in 0.025% and 0.05% DHM groups at 21 and 35 d (P<0.05).Compared with the control group,the serum complement C4 content of chicks was significantly increased in 0.05%,0.1% and 1.2% DHM groups at 14,28 and 35 d (P<0.05). Compared to each dose group,the serum complement C4 content of chicks in 0.2% DHM group was significantly higher than that in other groups at 14,21 and 28 d,and significantly higher than that in 0.025% DHM group at 35 d (P<0.05).【Conclusion】 Adding 0.2% DHM to the diet could promote the growth and development of immune organs,increase the expression of immune factors of chicks,and enhance immune function of chicks.
Effects of Wheat Particle Size on Growth Performance,Intestinal Morphology, Digesta Enzyme Activities and Serum Biochemical Indices of Broilers
OU Jiancun, CHEN Zhiyong, CEN Mingzhu, ZHENG Chaojun, QIU Ting, ZHANG Huihua
2024, 51(6):  2418-2428.  doi:10.16431/j.cnki.1671-7236.2024.06.016
Abstract ( 33 )   PDF (3366KB) ( 13 )  
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【Objective】 To study the effects of wheat particle size on growth performance,intestinal morphology,digesta enzyme activities and serum biochemical indices of 817 broilers aged 1-20 days.【Method】 A total of 360 1-day-old 817 broilers (male) were randomly divided into 3 groups with 6 replicates per group and 20 chickens per replicate according to the principle of no difference in body weight.The broilers in control group were fed corn-soybean meal diet,and in two experimental groups (1.5 and 2.5 mm groups) were fed diets supplemented with wheat and non-starch polysaccharide enzyme,and the wheat supplemental amount was 25%,and were crushed by 1.5 and 2.5 mm screen size respectively.The trial lasted for 20 d. At 20 days of age,the growth performance of broilers was measured,intestinal tissues and contents,pancreatic tissues and blood of broilers were collected,intestinal morphology,digestive enzyme activity and serum biochemical indexes were determined.【Result】 Compared with the control group,①There were no significant differences in growth performance of broilers in 1.5 and 2.5 mm groups (P>0.05),but the final body weight and average daily gain of broilers in 1.5 mm group were significantly higher than those in 2.5 mm group (P<0.05).②The intestinal morphology of broilers in two experimental groups was significantly improved (P<0.05),and the improvement effect in 1.5 mm group was better than that in 2.5 mm group. ③The activities of duodenal α-amylase,lipase and jejunal trypsin in 1.5 mm group were significantly increased (P<0.05),the activities of duodenal α-amylase and jejunal trypsin in 2.5 mm group were significantly increased (P<0.05),and the activities of duodenal lipase were significantly decreased (P<0.05).The jejunal trypsin activity in 1.5 mm group was significantly higher than that in 2.5 mm group (P<0.05).④The serum total cholesterol content of broilers in 1.5 mm group was significantly decreased (P<0.05),and the activities of alanine transaminase (ALT) and aspartate transaminase (AST) in 2.5 mm group were significantly increased (P<0.05);Serum total cholesterol content,alanine transaminase and aspartate transaminase activities in 1.5 mm group were significantly lower than those in 2.5 mm group (P<0.05).【Conclusion】 Under the conditions of this experiment,replacing corn with different particle size of wheat (25% replacement) had no adverse effect on 817 broilers aged 1-20 days.When the particle size of wheat was 1.5 mm,the growth performance,intestinal morphology, digestive enzyme activities and serum biochemical indexes of broilers were the best.
Comparative Study on Fattening Performance,Slaughter Performance and Meat Quality of Black Goats and Their Hybrid Offspring
XIAO Wen, HAN Yong, YANG Hongwen, LONG Yong, SU Chaozhi, YUAN Chao, YANG Yang, WANG Defeng, ZHAO Yanpin, QIN Yang, YIN Fuyue
2024, 51(6):  2429-2439.  doi:10.16431/j.cnki.1671-7236.2024.06.017
Abstract ( 18 )   PDF (2631KB) ( 12 )  
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【Objective】 Under the same fermented total mixed ration (TMR) fattening conditions,the differences in fattening performance,slaughter performance,meat quality traits and nutrient apparent digestibility of Black goats and their hybrid offspring were comparatively analysed,so as to provide some data support for the evaluation of crossbreeding effect between Guizhou Black goats and Black Nubian goats,as well as the research on the fattening efficiency of hybrid new lines.【Method】 A total of 15 healthy Guizhou Black goats,Black Nubian goats,and their hybrid F1 generation goats (Guizhou Black goats♀×Black Nubian goats♂) were selected from 7 to 8 months of age in the fattening experiment.The experiment lasted for 90 days,including 10 days of pre-test and 80 days of trial.During the experiment,body weight at 10 d intervals,body size index at the beginning and end of the test and apparent digestibility of nutrients at the end of the test were measured. At the end of the fattening period,three goats from each breed/line were selected for slaughtering to determine the slaughter performance,and the biceps femoris muscle of the hind limb and triceps brachii muscle of the fore limb were also taken for the measurement of meat quality traits.【Result】 For hybrid F1 generation goats,the average daily weight gain was significantly higher than that of Guizhou Black goats (P<0.05),the feed to gain ratio was significantly lower than that of Guizhou Black goats (P<0.05),and the changes in body weight gain and body size were similar to those of Black Nubian goats (P>0.05).The apparent digestibility of crude protein,crude fat and neutral detergent fibre were significantly higher than that of Guizhou Black goats (P<0.05).Carcass weight,net meat weight,net meat ratio,meat-bone ratio and rib thickness (GR value) were significantly higher than that of Guizhou Black goats (P<0.05),shear force of hind limb biceps femoris and forelimb triceps brachii muscles were significantly lower than that of Guizhou Black goats and Black Nubian goats (P<0.05),meat L* values were significantly higher than that of Black Nubian goats (P<0.05),the steaming loss of hind limb biceps femoris was significantly lower than that of Guizhou Black goats and Black Nubian goats (P<0.05),the steaming loss of forelimb triceps brachii was significantly lower than that of Guizhou Black goats (P<0.05),and the pH45 min of forelimb triceps brachii was significantly higher than that of Guizhou Black goats and Black Nubian goats(P<0.05).【Conclusion】 After crossbreeding with Black Nubian goats,the changes in body weight gain and body size during the fattening period of the F1 generation of Guizhou Black goats were similar to those of the Black Nubian goats,and slaughter performance,meat quality traits,and apparent digestibility were increased and improved to different degrees compared with Guizhou Black goats.
The Application of Macroalgae in Ruminant Production
SUN Yi, LI Shuai, TAN Sheng, YANG Yufeng, LI Dagang, MIN Li
2024, 51(6):  2440-2450.  doi:10.16431/j.cnki.1671-7236.2024.06.018
Abstract ( 28 )   PDF (1799KB) ( 16 )  
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China has a long coastline and a rich variety of macroalgae,including red seaweeds,brown seaweeds and green seaweeds.The output of macroalgae cultivation in China is about 19.4 million tons,and the main varieties are kelp,wakame,laver,gracilaria and so on.Macroalgae is a kind of sustainable marine resource,rich in nutrients such as protein,fatty acids,minerals,and functional substances including polysaccharides,polyphenols,halogenated compounds,and terpenoids.It is beneficial for ruminant nutrition and physiological health,and is helpful for reducing methane emissions of ruminants and alleviating global warming.Macroalgae has unique advantages of no residue and no biological resistance.Hence,it has great potential in developing green and healthy animal feedstuff.The article reviews the bioactive substances and physiological functions of macroalgae,while combined with their applications in rumen environmental regulation,improving antioxidant and immune properties,enhancing production performance and reducing greenhouse gas emissions of ruminants,in order to provide scientific basis for the development and utilization of macroalgae resources.
Effects of Walnut Green Husks Powder and Its Extracts on Growth Performance,Carcass Traits and Meat Quality of Finishing Pigs
WANG Jing, JIA Mingyang, CHEN Junfeng, REN Qiaoling, LIU Fujiu, MO Delin, ZHANG Jiaqing, WANG Lei, XING Baosong
2024, 51(6):  2451-2459.  doi:10.16431/j.cnki.1671-7236.2024.06.019
Abstract ( 25 )   PDF (1283KB) ( 11 )  
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【Objective】 The aim of this experiment was to investigate the effects of walnut green husks powder (WGH) and its extracts (WGHE) on growth performance,carcass and meat quality traits of fattening pigs.【Method】 A total of 90 healthy cross-bred (Duroc×Landrace×Yorkshire) castrated male finishing pigs (around 80 kg live weight) were randomly divided into 3 groups with 3 replicates in each group,with 10 pigs in each replicate.The pigs in control (Ctr) group were fed with basal diet,and the pigs in the walnut green husk (WGH) and walnut green husk extract (WGHE) groups were fed the basal diet supplemented with 0.1% WGH and 0.1% WGHE,respectively.The experiment had a 10-day pre-experimental period and 40-day of experimental period.The initial weight,final weight of each pig,and feed intake of each group were determined on the 41 st day.After test,two test pigs with medium weight and similar weight in each replicate were selected for slaughter test,the carcass trait,meat quality trait and the content of amino acid and fatty acid in longissimus dorsi muscle were detected.【Result】 Compared with Ctr group,adding WGH and WGHE to the diet had no significant effects on the final body weight,average daily gain,average daily feed intake,F/G,carcass length,carcass slanting length,carcass weight,dressing percentage,loin muscle area,leaf fat weight,as well as pH45 min,drip loss,pressurization loss,marbling score,and a* of the longest back muscle in fattening pigs (P>0.05),but could significantly improve the backfat thickness,b* and intramuscular fat content of the longest back muscle in fattening pigs (P<0.05).Compared with Ctr group,the content of amino acids in the longissimus dorsi muscle of fattening pigs in WGH group were increased except for histidine,while the content of amino acids in WGHE group was similar with Ctr group.The total content of polyunsaturated fatty acids in WGH and WGHE was higher than that in Ctr group,respectively.【Conclusion】 There was no negative effects of 0.1% WGH and 0.1% WGHE to growth and carcass traits of finishing pigs,but meat quality traits were increased,and walnut green husk powder was more cost-effective.
Research Progress on the Regulation of Animal Intestinal Microbiota by Mulberry Leaves and Its Active Substances
FU Bing, ZHOU Donglai, LI Qingrong, XING Dongxu
2024, 51(6):  2460-2470.  doi:10.16431/j.cnki.1671-7236.2024.06.020
Abstract ( 39 )   PDF (1283KB) ( 22 )  
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Mulberry leaves not only have high nutritional value,but also are rich in a variety of biologically active substances such as mulberry leaf flavonoids,mulberry leaf polysaccharides,alkaloids and γ-aminobutyric acid.These active substances have multiple functions for microorganisms.On the one hand,active substances in mulberry leaves can disrupt the structure and genetic material of bacteria by binding or competing with them for substrates required for growth,thereby inhibiting the proliferation of harmful bacteria.On the other hand,microorganisms in intestine can decompose and convert these active substances into small molecules with higher biological activity or lower toxicity with the assistance of enzymes,improving the relative abundance of beneficial bacteria in intestine,increasing the generation of short chain fatty acids such as acetic acid and butyric acid in intestine,promoting the synthesis of tight junction proteins,thereby promoting digestion and absorption in intestine,enhancing the body’s antioxidant and immune abilities,and maintaining the intestinal barrier function of animals.Good intestinal status is an important guarantee for the healthy growth of animals.In the context of today’s highly intensive breeding model and the ban on antibiotics in feed,mulberry leaves and its active substances have important functions such as maintaining animal intestinal health.They are ideal substitutes for antibiotics and can be used to develop green and efficient feed additives.The authors provide a comprehensive explanation of the main active substances in mulberry leaves,their interaction with microorganisms,and the impact of mulberry leaves and their active substances on the intestinal microorganisms of livestock,poultry,and aquatic animals.Furthermore,the authors also put forward his own opinions on the focus of future research on mulberry leaf resources.This paper aims to provide valuable reference material for the application of mulberry leaves in livestock,poultry and aquatic animal feed.
Interactions and Their Mechanisms Between Stress and Poultry Intestinal Microbiota
HU Xiaodi, ZHEN Wenrui, BAI Dongying, ZHONG Jiale, ZHANG Ruilin, ZHANG Haojie, ZHANG Yi, MA Yanbo
2024, 51(6):  2471-2480.  doi:10.16431/j.cnki.1671-7236.2024.06.021
Abstract ( 29 )   PDF (3124KB) ( 12 )  
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Poultry grown under intensive breeding conditions are exposed to a variety of challenges,including immune stress,heat stress,cold stress,oxidative stress and density stress.Stress factors have negative impacts on growth performance,immunity,disease resistance,meat and egg quality.The gut microbiota is essential for metabolism of nutrients,regulation of the immune system,and pathogen clearance.There is an interactive relationship between stress and intestinal microbiota.Stress can alter the structure and function of the intestinal microbiota,resulting in gut microbiota dysbiosis,which finally affect the health and performance of poultry.Intestinal microbiota can influence brain function via microbiota-gut-brain axis connections at the same time,so as to change behavior and emotions.The microbiota-gut-brain axis is a complex neural loop in which the gut,nerve,immune system and endocrine systems interact.Gut microbiota mediates the function of the microbiota-gut-brain axis by producing metabolites,regulating immune function and modulating neurotransmission.Furthermore,gut microbiota is able to affect brain function through interfering with the metabolism of neurotransmitters.In order to provide a theoretical foundation for alleviating stress through regulation of the gut microbiota,the author reviewed the effects of several major stresses on the intestinal microbiota of poultry and the bidirectional communication between stress and intestinal microbiome through the microbiota-gut-brain axis.
Effects of Tetrastigma hemsleyanum Leaves Powder on Growth Performance,Serum Biochemical Index and Jejunum Morphology of Chongren Patridge Chickens
SONG Qiongli, CHEN Jiang, WU Dong, ZOU Zhiheng, GONG Jianping, CHEN Xiaolian, SONG Wenjing, XIONG Pingwen, XU Chuanhui, LI Mengchu, SU Weide, AI Gaoxiang
2024, 51(6):  2481-2488.  doi:10.16431/j.cnki.1671-7236.2024.06.022
Abstract ( 28 )   PDF (1201KB) ( 9 )  
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【Objective】 The aim of this study was to evaluate the effects of Tetrastigma hemsleyanum leaves powder (THL) on growth performance,serum antioxidant,immune function and jejunum morphology of Chongren patridge chickens.【Method】 A total of 360 50-day-old Chongren patridge female chickens were randomly divided into 5 groups with 6 replicates per group and 12 hens per replicate.Hens in control group were fed a basal diet,and hens in experimental groups were fed the basal diet supplemented with 0.5%,1%,2% and 4% THL,respectively.The experiment lasted for 56 days.After the completion of the experiment,weight was utilized as the repetition unit,and feed intake was recorded for calculating the growth performance index.Blood and intestinal tissue samples were collected,serum antioxidant and immune indexes were detected,jejunum tissue sections were prepared and jejunum morphology was observed.The villus height and crypt depth of jejunum were measured,and the V/C ratio was calculated.【Result】 Compared with control group,the ratio of feed to gain (F/G) of Chongren patridge chickens supplemented with 1% THL was significantly decreased (P < 0.05),the contents of immunoglobulin A (IgA),IgM,transforming growth factor β (TGF-β) and the activity of glutathione peroxidase (GSH-Px) in serum were significantly increased (P<0.05).The contents of tumor necrosis factor-α (TNF-α) and malondialdehyde (MDA) were significantly decreased (P<0.05).The contents of IgA and TGF-β in serum were significantly increased (P<0.05),and the contents of interleukin-1β (IL-1β) and TNF-α in serum were significantly decreased (P<0.05) after adding 2% THL.The serum TGF-β content was significantly increased (P<0.05),and the serum IL-1β content was significantly decreased (P<0.05) after the supplementation of 4% THL.The villus height and V/C of jejunum were significantly increased after diets supplemented with 0.5% and 1% THL (P<0.05).【Conclusion】 Dietary supplementation of 0.5% to 4% THL could promote the growth,enhance body immunity and improve intestinal tisste morphology of Chongren patridge chickens to a certain extent.Under the conditions of this experiment, the effect of 1% to 2% THL supplementation was better.
Genetics and Breeding
Effects of Feeding Risperidone During Late Pregnancy on Mammary Gland Development of Maternal Mice and Growth of Offspring Mice
WANG Shunbo, WANG Hao, LIANG Yunyi, WANG Wenjing, LI Zicong, XU Zheng
2024, 51(6):  2489-2500.  doi:10.16431/j.cnki.1671-7236.2024.06.023
Abstract ( 30 )   PDF (20445KB) ( 12 )  
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【Objective】 In this study,mice were used as experimental objects to explore whether risperidone could promote mammary gland development and offspring growth in mammals.【Method】 Twenty Kunming female mice were divided into two groups with 10 mice in each group.Mice in experimental group were fed with risperidone containing 0.25 mg/L in the later stage of pregnancy for 4 days,and mice in control group were fed with drinking water without risperidone.Mammary gland tissue and blood were collected from 5 mice in each group on the 3rd and 18th day of lactation.PRL levels in serum were detected by ELISA,breast tissues were analyzed by HE staining and transcriptome sequencing techniques,the differences of breast weight of maternal mice,and average weight and survival rate of offspring mice between two groups were calculated by weighing and counting methods,and GO,KEGG and GSEA enrichment analysis were performed on the sequencing data.【Result】 On the 3rd day of lactation,PRL levels in serum of risperidone-treated maternal mice were extremely significantly higher than that of control group (P<0.01).The mammary gland richness on the 3rd and 18th day of lactation risperidone-treated maternal mice was higher than that of control group.On the 18th day of lactation,breast weight of risperidone-treated maternal mice, average weight and survival rate of offspring mice were significantly higher than that of control group (P<0.05).GO enrichment analysis showed that the differentially expressed genes (DEGs) on the 3rd day of lactation were mainly enriched in biological processes such as fatty acid metabolism and adipocyte differentiation,and the DEGs on the 18th day of lactation were mainly enriched in biological processes such as negative regulation of fatty acid metabolism and tissue remodeling.KEGG pathway enrichment analysis showed that the DEGs on the 3rd day of lactation were mainly enriched in the PPAR signaling pathway,cAMP signaling pathway,fat digestion and absorption and other pathways,while the DEGs on the 18th day of lactation were mainly enriched in the PPAR signaling pathway,regulation of adipocyte lipolysis and other pathways.GSEA showed that the estrogen and PRL signaling pathways were enriched in the risperidone-treated maternal mice in both periods.【Conclusion】 Feeding risperidone to female mice in late pregnancy could promote the development of maternal mammary glands and growth of offspring mice.This study revealed the promoting effect of risperidone on mammary gland development in mice,which laid the foundation for the establishment of a new method of adding risperidone to the drinking water of female animals in the later stage of pregnancy to improve female milk production and offspring growth.It was of great significance to promote the reproduction and breeding efficiency of mammals.
Estimation of Genetic Parameters for Health Traits in Chinese Holstein
ZHAO Xiaofeng, GUO Gang, CAO Jie, WANG Yachun, ZHANG Yi
2024, 51(6):  2501-2507.  doi:10.16431/j.cnki.1671-7236.2024.06.024
Abstract ( 27 )   PDF (1232KB) ( 10 )  
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【Objective】 The objective of this study was to perform a genetic analysis using farm production data to determine the genetic parameters of five common diseases in Chinese Holstein.The findings would guide the formulation of dairy cattle breeding strategies that incorporate health traits,thereby facilitating an enhancement in the health status of the dairy cattle population in China.【Method】 Quality control was performed on 230 826 calving records from 95 710 cows and 333 038 disease records from 121 811 cows,collected from 30 national dairy farms during the period of 2019 to 2022.These records were then matched and compiled into a phenotypic dataset.The phenotype for each individual cow in each calving event was defined based on the presence or absence of disease,with 0 representing healthy and 1 representing diseased.The diseases considered were mastitis (MAST),clinical ketosis (KET),retained placenta (RETP),metritis (MET),and displaced abomasum (DA).A single-trait repeatability model was used to estimate the variance components for each trait,followed by the calculation of heritability and repeatability.A two-trait repeatability model was employed to estimate the phenotypic correlation and genetic correlation between traits.Both models incorporated fixed effects for farm-year,parity,and calving season.The effect of selection on health traits was investigated by comparing the disease incidence in the offspring of bulls ranked in the top 20 and bottom 20 for estimated breeding values (EBV).This comparison was performed using a chi-square test.【Result】 The results showed that the heritability for the traits MAST,KET,RETP,MET,and DA was estimated to be 0.063±0.005,0.051±0.005,0.015±0.002,0.033±0.003,and 0.020±0.002,respectively,all of which were characterized as traits with low heritability.Genetic correlations among these traits varied from ―0.110 to 0.684,with notably strong positive correlations observed between KET and DA as well as MET and RETP.For all traits,the average incidence rate of the offspring of the top 20 bulls of EBV was significantly lower than that of the offspring of the bottom 20 bulls (P<0.01),and the difference in incidence rate reached 2.2 to 7.4 times.【Conclusion】 In this study,all five health traits exhibited low heritability,but they could be improved by selective breeding.Thus,it’s validated to incorporate the heallth traits in Chinese Holstein genetic improvement program to achieve balanced breeding objectives.
Polymorphisms of KRT10 Gene and Its Association Analysis with Reproductive Traits in Kele Pigs
ZHAO Yong, YING Qixin, LI Wei, XIONG Li, YANG Hongwen, WANG Chunyuan, WU Yan, XIANG Jin, ZHANG Yiyu
2024, 51(6):  2508-2516.  doi:10.16431/j.cnki.1671-7236.2024.06.025
Abstract ( 30 )   PDF (4160KB) ( 17 )  
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【Objective】 The aim of this study was to investigate the effect of the polymorphism of keratin 10 (KRT10) gene on reproductive traits in Kele pigs,in order to improve the fertility of Kele pigs.【Method】 There were 255 Kele pigs used in this experiment.The single nucleotide polymorphism (SNP) of KRT10 gene was identified using a combination of PCR product sequencing,DANMAN sequence alignment software and manual verification.The population genetic characteristics of SNP were analyzed by SHEsis online software,and the bioinformatics analysis of SNP was conducted by RNAfold online tool.The general linear model of SPSS 22.0 software was used to analyze the correlation between SNP of KRT10 gene and reproductive traits in Kele pigs.【Result】 Three SNPs were identified in KRT10 gene of Kele pigs:g.21643703 C>T and g.21643714 G>A in intron 4,and g.21643741 G>A in exon 5.g.21643741 G>A mutation caused a codon mutation from AAG to AAA,encoding amino acids of lysine (K),which was a synonymous mutation and caused a change of the secondary structure in mRNA.Three genotypes were detected at all three mutation sites.g.21643703 C>T and g.21643741 G>A belonged to moderate polymorphism sites (0.25<PIC<0.5),and g.21643714 G>A belonged to low polymorphism site (PIC<0.25).g.21643703 C>T and g.21643741 G>A were in Hardy-Weinberg equilibrium (P>0.05),g.21643714 G>A was significantly deviated from Hardy-Weinberg equilibrium (P<0.05).There were no strong linkage imbalance among three SNPs.Three SNPs produced a total of 4 haplotypes and 10 diploids.The results of correlation analysis showed that the effect of g.21643703 C>T on newborn litter weight,weaned piglets and weaned litter weight in Kele pigs reached a significant level (P<0.05),the effect of g.21643714 G>A on total litter size and weaned litter weight in Kele pigs reached a significant level (P<0.05),and the effect of g.21643741 G>A on total litter size,live litter size,weaned piglets and weaned litter weight in Kele pigs reached a significant level (P<0.05).The diploid H3H3 had the most significant effects on total litter size,live litter size,weaned piglets and weaned litter weight in Kele pigs.【Conclusion】 Three SNPs of KRT10 gene had significant effects on reproductive traits in Kele pigs,and H3H3 was advantageous diplotype,which might be used as a genetic marker for reproductive trait selection in Kele pigs.
Genetic Parameter Estimation of Growth and Developmental Traits in Angus Cattle During Weaning Stage
WANG Suwan, FENG Xiaofang, TONG Lijia, LI Desheng, WANG Yu, FENG Yuan, JIANG Qiufei, XU Jun, CHEN Yafei, GU Yaling, ZHANG Juan
2024, 51(6):  2517-2523.  doi:10.16431/j.cnki.1671-7236.2024.06.026
Abstract ( 32 )   PDF (1230KB) ( 14 )  
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【Objective】 The aim of this study was to measure the growth and development potential of beef cattle by accurately estimating the genetic parameters of body weight and body size traits during weaning stage,so as to provide a theoretical basis for the genetic improvement of Angus cattle.【Method】 The genetic parameters of body weight and body size traits of 3 173 weaned Angus cattle from seven Angus beef farms in Ningxia were estimated by constrained maximum likelihood method (AI-REML) in combination with the EM algorithm,using the DMUAI module of DMU software.【Result】 According to the criteria for high and low heritability estimates,the heritability of body weight (BW),body height (BH),hip cross height (HCH),body length (BL),chest girth (CG),abdomen circumference (AC) and cannon circumference (CC) in Angus cattle during weaning stage were classified as high heritability traits (h2≥0.40).The correlation results showed that the genetic correlations (0.31-0.79) and phenotypic correlations (0.30-0.81) among the traits were positive,except for the genetic correlations (―0.06-0.44) and phenotypic correlations (―0.07-0.35) between CC and the other traits,which were negative or weakly positive.【Conclusion】 There was potential for improvement in growth traits of Angus cattle.It was recommended to consider the growth and development traits during weaning stage when formulating the breeding plan for Angus cattle.
Research Progress on Vitrification Injury of Oocytes in Livestock
MO Xianhong, LI Junjie, GUO Cheng, WEN Zhaoyu, ZOU Yuzhu, ZHAO Wenbo, XU Zhenjun
2024, 51(6):  2524-2532.  doi:10.16431/j.cnki.1671-7236.2024.06.027
Abstract ( 25 )   PDF (1241KB) ( 12 )  
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Vitrification cryopreservation of oocytes is crucial for accelerating the livestock breeding,increasing the production efficiency,maintaining the species diversity,and rescuing endangered species.At present,the vitrification technique has been widely used in the oocyte cryopreservation of various domestic animals including pig,cow,and sheep.Nevertheless,compared with fresh oocytes,the competence of maturation,fertilization,cleavage,and blastocyst formation from vitrified oocytes was still greatly compromised.It dramatically discourages the practical application of oocyte cryopreservation.The freezing injury is the key factor determining the efficiency and quality of oocyte cryopreservation.It includes broken zona pellucida,membrane lipid phase transition,DNA damage,chromosomal abnormalities,aberrant distribution of cortical particles,impaired structure and function of endoplasmic reticulum and mitochondria,and abnormal spindle morphology.Furthermore,vitrification could cause the disorders of cell physiological state,such as oxidative stress and calcium dyshomeostasis,which are related with the compromised oocyte development.To further increase the effectiveness of oocyte vitrification and facilitate the improvement of cryopreservation technology,it is necessary to clarify the underlying mechanisms of freezing injury.The author reviews the types,origins,and repair strategies of various freezing injuries to provide reference for improving the efficiency and quality of oocyte vitrification.
Genetic Diversity and Phylogenetic Analysis of Goat Population in Karakoram-Pamir Region
HUANG Xianghui, ZHANG Tianneng, ARZUGUL·Musha, WANG Yutao
2024, 51(6):  2533-2544.  doi:10.16431/j.cnki.1671-7236.2024.06.028
Abstract ( 31 )   PDF (6662KB) ( 8 )  
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【Objective】 This experiment was conducted to further clarify the genetic diversity and phylogeny of goat population in Karakoram-Pamir region,and to provide theoretical basis for the conservation,excavation and application of biological resources.【Method】 The mitochondrial (mtDNA) D-loop region and cytochrome b (Cytb) gene sequences were amplified by PCR and sequenced of goats in Karakoram-Pamir region.Mega 7.0.26 software was used to analyze the base composition and draw the phylogenetic tree by combining the D-loop region and Cytb gene of goats downloaded from GenBank,and DNASP 5.10 software was used to analyze haplotype distribution and neutral test,and haplotype media-joining network was constructed by Network 10.2 software.【Result】 The AT content in mtDNA D-loop region and Cytb gene sequences of goats in Karakoram-Pamir region was 60.1% and 58.7%,respectively,and the AT content was higher than the GC content,showing a preference for base composition.There were 163 SNPs in D-loop region and 84 SNPs in Cytb gene,and 40 and 30 haplotypes were defined,respectively,with haplotype diversity (Hd) of 0.985 and 0.883 and nucleotide diversity (Pi) of 0.01570 and 0.00425,respectively.The Tajima’s D value and Fu’s Fs value of mtDNA D-loop region and Cytb gene were all less than 0 by the neutrality test,and significantly deviate from neutrality.Phylogenetic tree analysis showed that goats in Karakoram-Pamir region were distributed in 4 lineages:A,B,C and G.【Conclusion】 Goats in Karakoram-Pamir region had experienced at least three population expansion events and had rich genetic diversity.There was gene infiltration from goats in Western Asia,while Capra ibexs in the same region had no genetic contribution.
Preventive Veterinary Medicine
Effect and Mechanism of Interferon Stimulated Gene ISG15 on Porcine Epidemic Diarrhea Virus Replication
LIU Lili, BIAN Yuan, WU Shenglong, BAO Wenbin, WU Zhengchang
2024, 51(6):  2545-2555.  doi:10.16431/j.cnki.1671-7236.2024.06.029
Abstract ( 35 )   PDF (14086KB) ( 16 )  
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【Objective】 The aim of this study was to investigate the role and mechanism of interferon-stimulated gene ISG15 in the replication of Porcine epidemic diarrhea virus (PEDV).【Method】 Real-time quantitative PCR was used to detect the tissue expression profile of ISG15 gene and the differential expression in intestinal tissues.Meanwhile,porcine small intestine epithelial cells (IPEC-J2) were used as the study model to detect the expression of PEDV M gene mRNA and N protein of PEDV CV777 infected cells at different time points.The expression of ISG15 was detected at RNA and protein levels.Porcine ISG15 gene interference and overexpression cells were constructed,and the influence of ISG15 gene expression on PEDV replication level was detected by Real-time quantitative PCR,Western blotting and indirect immunofluorescence assay.Transcriptome sequencing was performed before and after the overexpression of ISG15 gene to screen its downstream regulatory genes and signaling pathways.【Result】 ISG15 gene was specifically highly expressed in intestinal tissue of piglets,with significantly higher expression in jejunum and ileum compared with other tissues (P<0.01).The expression of ISG15 gene in duodenum,jejunum and ileum of PEDV infected group was significantly or extremely significantly higher than that of healthy group (P<0.05 or P<0.01).The expression of PEDV M gene mRNA and N protein showed an upward trend,and compared with 0 h,the expression of ISG15 gene was extremely significantly upregulated at 24 h (P<0.01).After overexpression of ISG15 gene,there was a significant or extremely significant decrease in PEDV replication (P<0.05 or P<0.01),while after interference with ISG15 gene,PEDV replication was extremely significantly upregulated (P<0.01).Transcriptome sequencing revealed 1 532 differentially expressed genes before and after overexpression of the ISG15 gene,which were mainly enriched in signaling pathways such as autophagy,MAPK signaling,and endocytosis.【Conclusion】 This study revealed the regulatory function and mechanism of ISG15 during PEDV infection and the increase of ISG15 gene expression could significantly inhibit PEDV replication,which improved the understanding of the molecular mechanism of the interaction between PEDV and host cells.
Prokaryotic Expression of Porcine Secretory Leukocyte Protease Inhibitor and Preparation of Its Monoclonal Antibody
FU Mingzhi, LI Xinxin, LIU Xuewei, LI Huanrong
2024, 51(6):  2556-2565.  doi:10.16431/j.cnki.1671-7236.2024.06.030
Abstract ( 30 )   PDF (6660KB) ( 7 )  
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【Objective】 The purpose of this experiment was to prepare porcine derived secretory leukocyte protease inhibitor (SLPI) and its monoclonal antibody,laying the foundation for the establishment of porcine SLPI detection method and function research.【Method】 Series connection of porcine SLPI gene was cloned into pET-28a(+) and pGEX-6P-1 vectors to construct the expression vector.The recombinant protein was expressed in Escherichia coli expression system,and the induced expression of recombinant proteins were analyzed by SDS-PAGE.After recombinant bacteria were ultrasonicated,the purified recombinant proteins were identified by SDS-PAGE and Western blotting.BALB/c mice were immunized with the purified recombinant protein,and hybridoma cells were prepared by cell fusion.The monoclonal antibody and subclass were screened and detected by indirect ELISA,and the specificity of monoclonal antibody were identified using Western blotting and indirect immunofluorescence assay (IFA).【Result】 The pGEX-6P-1-SLPI and pET-28a-SLPI recombinant plasmids were successfully constructed,and high purity His-SLPI and GST-SLPI recombinant proteins were obtained with molecular weights of 27 and 50 ku,respectively.His-SLPI was expressed in inclusion body,while GST-SLPI was expressed in soluble form.Three hybridoma cell lines that could stably secrete monoclonal antibodies against porcine SLPI were acquired,namely 9D10 as the IgG3 subclass,9G11 and 10E2 as the IgG2b subclass.All three monoclonal antibodies could bind to SLPI in porcine intestinal epithelial cells.9D10 showed strong fluorescence signal in nucleus,while 9G11 and 10E2 showed fluorescence signals in both cytoplasm and nucleus.【Conclusion】 Recombinant His-SLPI and GST-SLPI proteins were successfully prepared,and three stable secretion monoclonal antibodies against porcine SLPI with good specificity were acquired.The results would provide a technical platform for further studying the characteristics of porcine SLPI and its role in mucosal immunity.
Study on the Pathogenicity of Different Serotypes of Streptococcus suis to New Zealand Rabbits
ZHANG Shuzhi, LIU Fei, WANG Zhenyong, CHEN Zhi, ZHANG Lin, SUN Wenbo, ZHANG Yuyu, DING Luogang, LI Jianda, REN Sufang, YU Jiang, WU Jiaqiang
2024, 51(6):  2566-2575.  doi:10.16431/j.cnki.1671-7236.2024.06.031
Abstract ( 21 )   PDF (10375KB) ( 5 )  
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【Objective】 This study was aimed to investigate the distribution pattern and differences in pathogenicity of different serotypes of Streptococcus suis (SS) in New Zealand rabbits,so as to provide reference for the prevention and control of porcine streptococcal disease.【Method】 In this study,New Zealand rabbits were infected with SS1 ZY194,SS2 QH and SS7 ZY188 strains,respectively,and the clinical symptoms were observed,the pathological tissue changes were analyzed by HE staining.The organ bacterial load was detected using Real-time quantitative PCR,the changes in biochemical indexes of serum and liver were detected by kit,and the expression of related inflammatory factors were detected by ELISA.【Result】 The clinical symptoms and the degree of pathological changes in organs of New Zealand rabbits in SS2 group were the most obvious among 3 groups.All the organs of New Zealand rabbits in experimental groups were bacterial-carrying,and the bacterial load of organs in SS2 group was higher than that in SS1 and SS7 groups,and spleen was the organ with the highest bacterial load.The contents of glucose (GLU),triglyceride (TG),total cholesterol (TCHO),low density lipoprotein (LDL) and uric acid (UA) of New Zealand rabbits in experimental groups were extremely significantly or significantly higher than those in control group (P<0.01 or P<0.05),and the content of high density lipoprotein (HDL) of New Zealand rabbits in SS2 and SS7 groups were extremely significantly lower than that in control group (P<0.01).The contents of glutathione peroxidase (GSH-Px),superoxide dismutase (SOD) and lipase (LPS),and the expression of inflammatory factors IL-6,IL-1β and TNF-α of New Zealand rabbits in experimental groups were extremely significantly or significantly higher than those in control group (P<0.01 or P<0.05),and the levels of each index in SS2 group were the highest.【Conclusion】 The different serotypes of Streptococcus suis could cause disease in New Zealand rabbits,among which the SS2 QH strain was the most pathogenic.The results provided a reference for the pathogenesis research on porcine streptococcal disease and a theoretical basis for clinical control.
Prokaryotic Expression of VP27 Protein of Goose Astrovirus Genotype 1 and Preparation and Identification of Its Polyclonal Antibody
XIANG Yong, LI Linlin, ZHANG Junqin, DONG Jiawen, HUANG Yunzhen, ZHAI Qi, XU Zhihong, LIAO Ming, SUN Minhua
2024, 51(6):  2576-2584.  doi:10.16431/j.cnki.1671-7236.2024.06.032
Abstract ( 27 )   PDF (9909KB) ( 12 )  
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【Objective】 The aim of this study was to obtain a polyclonal antibody that could specifically recognize Goose astrovirus genotype 1 (GAstV-1) and did not cross-react with GAstV-2,so as to provide a material for the subsequent establishment of GAstV-1 immunoassay.【Method】 By comparing the amino acid sequences of GAstV-1 and GAstV-2 capsid proteins,the regions with large differences were found as target genes.According to VP27 gene sequence of GAstV-1 GDYJ-21-01,the gene codons were optimized and synthesized,the prokaryotic expression plasmid pCZN1-GAstV-1 VP27 was constructed and transformed into Escherichia coli Top10 competent cells to induce VP27 protein expression.The purified VP27 protein was used to immunize New Zealand White rabbits,and the antiserum was harvested to prepare GAstV-1 VP27 polyclonal antibody.The titer and purity of the purified antibody were analyzed by indirect ELISA and SDS-PAGE,and the specificity of the polyclonal antibody was evaluated by indirect immunofluorescence assay (IFA).【Result】 The recombinant plasmid pCZN1-GAstV-1 VP27 was successfully constructed.SDS-PAGE results showed that the molecular weight of the recombinant protein was about 35 ku,which mainly existed in the form of inclusion bodies.The results of indirect ELISA showed that the titer of purified GAstV-1 VP27 polyclonal antibody was 1∶9 841 500,and the purity was higher than 85%.IFA results showed that GAstV-1 VP27 polyclonal antibody could specifically recognize GAstV-1 and had no cross-reaction with GAstV-2.The detection results of clinical samples showed that the tissues with GAstV-1 nucleic acid positive exhibited specific fluorescence,while the tissues with GAstV-2 nucleic acid positive but GAstV-1 nucleic acid negative did not show fluorescence.【Conclusion】 In this study,a high titer of VP27 polyclonal antibody was obtained,which could specifically recognize GAstV-1 and had no cross-reaction with GAstV-2,laying a foundation for the establishment of antigen immunoassay for the differential diagnosis of GAstV-1.
Detection of Drug Resistance of Escherichia coli from Ducks in Some Areas of Tongliao and Whole Genome Sequencing Analysis of Multi-drug Resistant Strain YA-1
WANG Zi, SUN Miao, WANG Yongqiang, MENG Linghao, GENG Chao, SHI Jinchuan, CHEN Wei, QIU Jun, LIU Kai
2024, 51(6):  2585-2596.  doi:10.16431/j.cnki.1671-7236.2024.06.033
Abstract ( 28 )   PDF (7825KB) ( 9 )  
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【Objective】 As one of the most common bacterial pathogens in poultry,the increasing resistance of Escherichia coli (E.coli) has attracted wide attention.The purpose of this study was to understand the distribution of drug resistance and drug resistance genes of E.coli in some duck farms in Tongliao area,so as to provide reference for the study of drug resistance and drug resistance genes of E.coli from ducks in this area.【Method】 The pathogenic bacteria were isolated and purified from 15 intestinal samples of dead ducks in each county of Tongliao city by 16S rRNA sequencing,biochemical test and mouse pathogenicity test.The isolated strains were subjected to drug sensitivity test and PCR detection of drug resistance genes,and multi-drug resistant strains were selected for whole genome sequencing.【Result】 All 10 strains of E.coli had different degrees of drug resistance.The resistance rates to streptomycin,ciprofloxacin,ofloxacin and enrofloxacin were all above 80%,and the detection rates of drug resistance genes aphA1, strB and qnrS were 100%.The whole genome sequencing of the multi-drug resistant strain YA-1 confirmed that the chromosome size of the strain was 4 844 827 bp,carrying 3 complete plasmids,which were 144 776 bp (pTLYA-1),113 584 bp (pTLYA-2) and 77 544 bp (pTLYA-3),respectively.The isolated strains carried 70 drug resistance genes such as arr-3,aadA16 and sul1.Among them,pTLYA-1 carried 8 mobile drug resistance genes such as arr-3,dfrA27 and aadA16,pTLYA-3 carried 5 mobile drug resistance genes such as floR,tetA and sul2,and no drug resistance genes were found in pTLYA-2.【Conclusion】 E.coli isolated from some duck farms in Tongliao area had multiple drug resistance,and the resistance level was high.The resistance genes of aphA1,strB and qnrS were prevalent.
Preparation and Application of Polyclonal Antibodies Against the Virulence Protein SptP in Salmonella Typhimurium
HUANG Xinmiao, YAO Min, HE Hao, XIAO Huiping, CHEN Jingzhuo, FENG Hao, ZHANG Li, ZHENG Sunli, ZHANG Mengdie, HUANG Tinghua
2024, 51(6):  2597-2604.  doi:10.16431/j.cnki.1671-7236.2024.06.034
Abstract ( 32 )   PDF (6349KB) ( 9 )  
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【Objective】 The aim of this study was to prepare the polyclonal antibodies against SptP of Salmonella Typhimurium Sal-14028,and apply them in the detection of Salmonella SptP protein.【Method】 Using the Sal-14028 genome DNA as a template,the SptP gene fragment was amplified by PCR and a prokaryotic expression plasmid pET28a-SptP was constructed.The recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells,the protein expression was induced by IPTG,and the recombinant SptP protein was identified by SDS-PAGE and Western blotting.The expression product was purified by nickel column and then was used to immunize the rabbits to prepare polyclonal antibodies.The titer and specificity of the antibodies were detected by indirect ELISA and Western blotting.Cell immunofluorescence was used to detect the distribution of SptP in Raw264.7 cells infected with Salmonella,and the total protein of the infected cells was immunoprecipitated.A 25 ku band was enzymatically hydrolyzed and identified by mass spectrometry.【Result】 The results showed that a SptP gene fragment of 1 173 bp was cloned,and enzyme digestion and sequencing showed that the recombinant plasmid pET28a-SptP was successfully constructed.The recombinant SptP protein with the size of about 45 ku was obtained by inducing expression and purification.Polyclonal antibody with titer of 1∶25 600 was obtained by immunizing rabbits with recombinant SptP protein.The antibody could both recognize recombinant SptP protein and Salmonella SptP whole protein,and also could be used in tracking the distribution of SptP within Salmonella-infected cells.The immunoprecipitation test showed that the antibody could bind to multiple proteins,and the 25 ku protein that the antibody binded was identified as mouse Rac1 protein using peptide mass fingerprint.【Conclusion】 This study successfully prepared polyclonal antibodies with high titer,which could recognize recombinant SptP and SptP whole protein,and could be used in Western blotting,cellular immunofluorescence and immunoprecipitation experiments.The results of this study laid a foundation for the detection of Salmonella SptP protein and the exploration of target molecules of Salmonella SptP.
Research Progress on Cross-species Transmission and Prevention and Control Techniques of Hepatitis E Virus
HE Zhenwen, LIU Dingyu, LIU Baoling, ZHANG Pian, WANG Xiaohu, WANG Gang, HUANG Yuan, CHEN Jing, DU Zongliang, CAI Rujian
2024, 51(6):  2605-2620.  doi:10.16431/j.cnki.1671-7236.2024.06.035
Abstract ( 37 )   PDF (6314KB) ( 18 )  
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Hepatitis E virus (HEV) is a zoonotic pathogen mainly transmitted by fecal-oral route and is considered to be one of the major causes of acute hepatitis in humans.HEV can be divided into 8 genotypes,and the distribution of each genotype is related to the region and level of industrialization,among which HEV-3 and HEV-4 have zoonotic transmission capacity and can cause infection in human and various animal hosts.Besides human,pig is the most common host of HEV.With the deepening of research,it is found that HEV hosts are diverse,including cattle,sheep,deer,rabbit,camel and other animals,and there is a possibility of cross-species transmission among animal hosts.The cross-species transmission of HEV is formed through adaptive evolution in the process of frequent exchanges between human and animal hosts,constrained by the specificity differences of host cell receptors and cytokines.HEV genetic recombination facilitates the emergence of new strains adapted to hosts and may also promote new transmission pathways,thereby facilitating the cross-species transmission of HEV.The study of HEV transmission routes shows that transmission routes vary according to genotype,and consumption of animal products carrying HEV,especially undercooked animal products,is the main cause of human infection with HEV-3 and HEV-4.In addition,water contamination,occupational exposure,blood transfusion and organ transplantation are also important transmission routes of HEV.According to various potential transmission routes,HEV can be prevented by relevant prevention and control measures,including adequate heating of animal food to inactivate HEV,disinfection and purification of water sources,occupational protection and vaccination.In order to better understand and grasp the epidemic law of HEV,local monitoring of HEV should be strengthened and relevant prevention and control strategies should be formulated.This paper reviewed HEV etiology,cross-species infection and prevention and control technology,in order to promote the in-depth understanding of HEV cross-species transmission and provide reference for HEV prevention and control.
Prokaryotic Expression of GA Module-containing Protein in Mycoplasma synoviae and Establishment of Indirect ELISA Method
GAO Le, SI Duoduo, GUO Lei, CHEN Can, WANG Wei, WANG Jianlin, WANG Lingling, LI Jidong
2024, 51(6):  2621-2632.  doi:10.16431/j.cnki.1671-7236.2024.06.036
Abstract ( 32 )   PDF (3165KB) ( 5 )  
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【Objective】 GA module-containing protein of Mycoplasma synoviae (MS) was expressed in vitro.A method for detecting MS antibody was established for serological detection and monitoring of MS antibody level.【Method】 The long-chain conserved domain of GA module-containing protein in MS was analyzed and screened,and the recombinant plasmid pET30a-ΔGA-L was synthesized and transformed into Escherichia coli BL21 (DE3) competent cells for induced expression,and the expression conditions were optimized.The expression of recombinant protein ΔGA-L was detected by SDS-PAGE.The recombinant protein ΔGA-L was purified,and identified by Western blotting.The purified recombinant protein ΔGA-L was used as the coating antigen to establish an indirect ELISA method for detection of MS antibody.The response conditions were optimized,the critical value was determined,the specificity,sensitivity and repeatability were tested,and clinical samples were tested.【Result】 The molecular weight of the recombinant protein ΔGA-L was 45.7 ku,and the optimal expression conditions were 25 ℃ and 0.2 mmol/L IPTG induced expression for 5 h,and the expression was in the form of inclusion bodies.Western blotting result showed that the recombinant protein ΔGA-L could react specifically with MS antibody.An indirect ELISA method for MS antibody detection was established with ΔGA-L antigen coating concentration of 0.5 μg/mL,primary antibody dilution ratio of 1∶400 and secondary antibody dilution ratio of 1∶12 000 as the optimal conditions,and the critical positive value was 0.283.The established method had strong specificity,high sensitivity and high stability.The total coincidence rate was 96% compared with commercial MS antibody detection kit.【Conclusion】 In this study,the MS recombinant protein ΔGA-L was successfully expressed,and the established indirect ELISA method for MS antibody detection had great specificity,sensitivity and repeatability,providing an effective and fast method for antibody detection of MS.
Isolation,Identification,Whole Genome Sequencing and Genetic Evolution Analysis of Goose Astrovirus in Some Areas of Guangdong Province
YU Fuxin, WU Siyu, YU Jieshi, ZHANG Xiaoai, YU Ting, DONG Bo, LI Jianbo, WANG Lei, WEI Wenkang
2024, 51(6):  2633-2643.  doi:10.16431/j.cnki.1671-7236.2024.06.037
Abstract ( 25 )   PDF (5478KB) ( 7 )  
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【Objective】 The aim of this study was to understand the prevalence and genetic variation of Goose astrovirus (GAstV) in some areas of Guangdong,elucidate its genetic evolution and genome-wide characteristics,and provide a theoretical basis for the prevention and control of GAstV.【Method】 Using GAstV specific primers,PCR detection was performed on 36 goose samples suspected of gout in goose farms in Guangdong province from 2021 to 2022.Four representative samples with strong positive GAstV were selected,and specific primers for GPV,TMUV,AIV and NDV were used for detection to exclude the presence of other common exogenous viruses in the samples.On this basis,the four GAstV samples were isolated and purified using goose embryos and chicken liver cancer cell lines (LMH),and the morphology of the virus particles was observed using transmission electron microscopy. 7 pairs of specific primers for whole genome amplification and sequencing of 4 strains of GAstV were designed.The sequences were organized and spliced to obtain the whole genome sequence of GAstV by DNAStar software.The nucleotide similarity was compared and the variation of key amino acid sites were analyzed by MegAlign software.The phylogenetic tree for the entire gene and individual ORF genes (ORF1a,ORF1b and ORF2) were constructed by Mega-X software.【Result】 Through PCR identification,it was found that a total of 30 tested samples were GAstV positive.The four representative GAstV positive samples selected did not contain any other exogenous virus infections.The four strains of GAstV virus were successfully isolated using goose embryos and LMH cells,and four GAstV genome sequences with a total length of 7 131 bp were obtained,including 18 bp of 5'-UTR and 184 bp of 3'-UTR,as well as three reading open frames (ORF1a,ORF1b and ORF2 genes),Among them,the ORF1a,ORF1b and ORF2 genes were 3 255,1 551 and 2 115 bp long,respectively,which was basically consistent with the reported whole genome characteristics of the GAstV.Similarity analysis showed that the nucleotide similarity between the four strains of GAstV was between 99.4% and 99.8%,and the nucleotide similarity with the other 19 strains of GAstV included in GenBank was between 57.6% and 99.6%.The genetic evolution analysis of the entire genome and three ORF genes showed that all four strains of GAstV obtained belonged to GAstV-Ⅱ type.Amino acid sequence analysis showed that there were 22 mutations in the amino acids of the 4 strains of GAstV,including 8 mutations in the ORF1a gene and 14 mutations in the ORF2 gene.【Conclusion】 The four isolated strains of GAstV were GAstV-Ⅱ type strains,which were basically consistent with the genotypes prevalent in other regions of China.The amino acid mutations of these four strains mainly occur in the ORF1a and ORF2 regions,providing reference for subsequent vaccine development,epidemiological investigations,and pathogenic mechanisms.
Infection Status of Giardia duodenalis in Cattle in Yunnan Province Based on gdh Gene
WANG Wenya, XIE Xinyan, YAN Dalong, WEI Penghao, YANG Jianling, BI Run, LI Qing, MA Jun, ZOU Fengcai
2024, 51(6):  2644-2652.  doi:10.16431/j.cnki.1671-7236.2024.06.038
Abstract ( 22 )   PDF (1646KB) ( 3 )  
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【Objective】 This study was aimed to investigate the infection and genotype distribution of Giardia duodenalis (G.duodenalis) in cattle in Yunnan province,and evaluate the transmission risk of giardiasis.【Method】 Nested PCR was used to detect fresh fecal samples of 258 cattle collected from three regions in Yunnan province based on the glutamate dehydrogenase (gdh) gene of G.duodenalis.The positive rates of G.duodenalis in different regions,ages,and breeds of cattle were calculated.Positive samples were sequenced,and the sequencing results were submitted to NCBI for genotype identification.MrBayes 3.1 was used in Bayesian inference analysis and construct a phylogenetic tree,choosing GTR (general time reversible) model.【Result】 23 of 258 faecal samples were positive for G.duodenalis,and the total positive rate was 8.91% (23/258).The positive rates of G.duodenalis in cattle from different regions were extremely significantly different (P<0.01),with the highest positive rate in Heqing county,Dali,at 20.75% (11/53,95% CI=9.47-32.04),the positive rates of Menghai county in Xishuangbanna and Yongshan county in Zhaotong were 8.82% (6/68,95% CI=1.91-15.74) and 4.38% (6/137,95% CI=0.91-7.85),respectively.The positive rates of G.duodenalis in cattle of different ages were extremely significantly different (P<0.01).The positive rate in calves (<5 months old) (26.19%,11/42,95% CI=12.32-40.06) was extremely significantly higher than that in adult cattle (>24 months old) (5.56%,12/216,95% CI=2.48-8.64).There was an extremely significant difference in the positive rate of G.duodenalis among different breeds of cattle (P<0.01).Holstein cows had the highest positive rate of G.duodenalis,which was 20.75% (11/53,95% CI=12.00-39.17).Yellow cattle,Western crossbred cattle (a hybrid of Simmental cattle and local Yellow cattle),and Simmental cattle had lower positive rates,which were 17.86% (5/28,95% CI=2.73-32.98),4.38% (6/137,95% CI=0.91-7.85) and 2.50% (1/40,95% CI=0-7.56),respectively.Based on the analysis of gdh gene sequence,23 positive samples of G.duodenalis were all assemblage E,but with 7 different subtypes.Among them,6 subtypes had a similarity of 99.8% to 100% with known reference sequences (E1,E12,E20,E30,E36 and E38),and another subtype had a similarity of 99.8% with unnamed reference sequences.【Conclusion】 Cattle in Yunnan province were infected with G.duodenalis,with the positive infection rate varying with different regions,ages,and breeds of cattle.Assemblage E dominated the assemblages of G.duodenalis infection in cattle of Yunnan province,which possessed rich genetic diversity in Yunnan province.
Research Progress on Porcine Sapelovirus
HAN Yuanyuan, WANG Jingwen, RUAN Jingxian, WANG Donghan, XIA Lu, ZHU Heshui, HU Hui
2024, 51(6):  2653-2660.  doi:10.16431/j.cnki.1671-7236.2024.06.039
Abstract ( 33 )   PDF (2922KB) ( 13 )  
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Porcine sapelovirus (PSV) belongings to the Sapelovirus genus of the Picornaviridae family,is a non-enveloped single-stranded and positive-sense RNA virus.Similar to other porcine enteric viruses,PSV is mainly transmitted through fecal-oral and can also be transmitted through contact or airborne. Piglets is susceptible to the virus and it can cause functional disorders such as the nervous system,respiratory system,and digestive system of piglets,seriously,it can even cause the piglets death,and there are different pathogenicity and tissue tropism between different strains.In clinical practice,PSV is often mixed with a variety of viruses such as Porcine teschovirus,Porcine epidemic diarrhea virus,Porcine kobuvirus,which increases the complexity of clinical symptoms,the difficulty of diagnosis and accelerates the spread of the virus.In addition,the genetic recombination phenomenon and the risk of cross-race spread of PSV have causing a potential threat to pig industry.At present,there are many detection methods such as nucleic acid levels,protein levels,and serology that can be used for PSV laboratory testing.A variety of cell lines can establish in vitro infection models for related research.At present,the research of PSV mainly focuses on the study of epidemiology and its isolation and identification.There are fewer researches on PSV infection and pathogenesis and no commercially available vaccines and effective antiviral drugs.This article comprehensive describes the research progress of PSV in terms of etiological characteristics,epidemiology,pathogenic mechanism,clinical prevention and control,to provide a theoretical basis for further research on the prevention and control of PSV.
Basic Veterinary Medicine
Study on the Therapeutic Effect of Traditional Chinese Medicine Formula on Diarrhoea Caused by Escherichia coli in Mice
GAO Qingyu, FU Le, SHI Yuxiang, LI Jiefeng, LIU Yanran, QIN Lijia, ZHANG Huaying, HE Tuanyong, WANG Xiaofang
2024, 51(6):  2661-2670.  doi:10.16431/j.cnki.1671-7236.2024.06.040
Abstract ( 29 )   PDF (7387KB) ( 8 )  
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【Objective】 The aim of this experiment was to study the therapeutic effect of traditional Chinese medicine formulas on calf-derived Escherichia coli-caused diarrhoea in mice,and to provide a reference basis for the treatment of Escherichia coli-caused diarrhoea in calves with traditional Chinese medicine.【Method】 162 mice were randomly divided into 9 groups,blank group,model group,traditional Chinese medicine treatment groups 2 formulas (with 3 concentrations (1.5,1.0 and 0.5 g/mL) for each) and Western medicine treatment group,with 3 replicates in each group and 6 mice in each replicate.Except for blank group,the mice in each group were injected intraperitoneally with 0.5 mL Escherichia coli suspension with a concentration of 1×108 CFU/mL,and after diarrhoea appeared in mice,the test mice in traditional Chinese medicine group were gave 0.2 mL extracts of traditional Chinese medicine formulas (Bai Tou Weng Tang plus or minus and Wu Mei San plus or minus),respectively,and the test mice in Western medicine group were gave 0.2 mL enrofloxacin.In model group,mice were fed 0.2 mL distilled water. The test lasted for 7 d.The body weight and food intake were recorded daily,and the diarrhoea index was counted on the 3rd day of treatment,mice were dissected on the 7th day of treatment,and pathological changes in the small intestine were detected,and the content of ileal mucosal immunoglobulins,sIgA and pIgR were detected using ELISA,and duodenal Claudin-1,Occludin and ZO-1 genes mRNA relative expression was detected using Real-time quantitative PCR.【Result】 Compared with model group,treatments with two herbal formulas could significantly increase the body weight and food intake of diarrhoeic mice,significantly reduce the diarrhoea index (P<0.05),improve the pathological and histological lesions of the small intestine,and promote the repair of intestinal villi damage.Compared with model group,both formulas could significantly enhance the contents of mouse intestinal mucosal immunoglobulin sIgA and pIgR (P<0.05),and significantly enhance the relative mRNA expression of mouse duodenal Claudin-1,Occludin and ZO-1 genes mRNA relative expression (P<0.05).1.0 g/mL Wu Mei San plus or minus had the best therapeutic effect among the two traditional Chinese medicine formulas,and had no significant difference in intestinal histomorphology,intestinal mucosal immunoglobulin content and relative mRNA expression of tight junction proteins compared with Western medicine group.【Conclusion】 The addition and subtraction of Wu Mei San plus or minus and Bai Tou Weng Tang plus or minus could reduce the diarrhoea index and improve the pathological damage of the intestinal tract in mice,among which the therapeutic effect of the addition and subtraction of Wu Mei San plus or minus was more significant,and this study could provide reference for the treatment of diarrhoea of calves caused by Escherichia coli by traditional Chinese medicine.
Pathological and Molecular Biological Diagnosis of Avian Leukosis
XIAO Jing, WAN Jinlong, YANG Jiangyu, YANG Mengjiao, JIAO Haihong
2024, 51(6):  2671-2679.  doi:10.16431/j.cnki.1671-7236.2024.06.041
Abstract ( 30 )   PDF (10098KB) ( 3 )  
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【Objective】 The study was aimed to understand the infection and gene molecular characteristics of E-avian leukosis in a chicken flock in a city of Xinjiang.【Method】 Laboratory diagnosis of suspected avian leukosis cases was conducted through pathological dissection,histopathological observation,PCR detection,clone vector linkage,etc,and DNAStar software was used to perform env gene nucleotide sequence similarity alignment,phylogenetic tree construction,and mutation site analysis between the isolated strain and the reference strains.【Result】 The liver,heart,spleen,kidney,lung and other organs of the affected chickens were enlarged with white nodular foci of different sizes.Histopathological observation showed that the foci were uniformly sized adult lymphocytes with focal and diffuse distribution.The PCR amplification results of the Avian leukaemia virus (ALV) genome showed that a target band with a size of 302 bp was obtained,which was positive for ALV.Typing identification was performed on positive samples,which showed ALV-E specific bands with a size of 1 074 bp,named AKS2101 and AKS2102,respectively.The sequence analysis results showed that the sequence similarity between the isolated strains and the subgroup E strains was 91.6% to 98.6%.The phylogenetic tree showed that the obtained sequences were in the same branch as the reference strain of ALV-E subgroup,and was in a different evolutionary branch from the reference strains of subgroups A,B,C,D,J and K,further indicating the presence of ALV-E infection in the chicken population.Genetic variation analysis showed 24 nucleotide variations and 17 amino acid site variations in the isolated strain,with variable sites located in the highly variable region of the sequence.【Conclusion】 ALV-E infection existed in chickens in a city of Xinjiang,and this experiment provided basic data to clarify the prevalence of avian leukosis in this area.
Mechanism of the Active Ingredients of Ephedra sinica Stapf Dried Grass Stems in the Treatment of Vasospasm Based on Network Pharmacology
WU Bowen, YAN Peiyu, WU Mishan
2024, 51(6):  2680-2696.  doi:10.16431/j.cnki.1671-7236.2024.06.042
Abstract ( 30 )   PDF (20578KB) ( 11 )  
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【Objective】 The experiment was to explore the potential mechanism of the active ingredients of Ephedra sinica Stapf dried grass stems in the treatment of vasospasm by integrating network pharmacology and in vitro cell experiments.【Method】 The main active ingredients and targets of Ephedra sinica Stapf dried grass stems were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP),SwissTargetPrediction and PharmMapper databases.The disease targets were obtained from DrugBank,GeneCards and OMIM databases.Then the ingredient-disease intersection targets were obtained.STRING database was used for protein-protein interaction (PPI) analysis,and Cytoscape 3.9.1 software was used to screen the core targets and core ingredients.Metascape platform was used for GO function and KEGG pathway enrichment analysis,and AutoDock Vina platform was used for molecular docking.Subsequently,the effect of active ingredients of Ephedra sinica Stapf dried grass stems on the proliferation of vascular smooth muscle cells(vSMCs)induced by angiotensin Ⅱ was detected by MTT method.【Result】 The active ingredients of Ephedra sinica Stapf dried grass stems compound mainly included luteolin,quercetin,beta-quercetin,eriodictyol,naringenin,stigmasterol,kaempferol,etc.The main targets of these drug ingredients were 488,and 394 were related with vasospasm.Ephedra sinica Stapf dried grass stems had 67 prediction targets for vasospasm,mainly including matrix metallopeptidase Ⅸ(MMP9),MMP2,angiotensin Ⅱ receptor type 1(AGTR1),mitogen-activated protein kinase 1(MAPK1),vascular endothelial growth factor A(VEGFA),epidermal growth factor receptor (EGFR) and nitric oxide synthase 2(NOS2).Enrichment analysis of the above 67 targets found that they were related to biological processes such as positive regulation of cell migration,response to xenobiotic stimulus,response to hypoxia and positive regulation of angiogenesis,cell components such as nucleus,cytoplasm,extracellular space,plasma membrane,postsynaptic membrane,presynaptic membrane,dendrite,membrane raft and axon terminus,molecular functions such as RNA polymerase Ⅱ transcription factor activity,ligand-activated sequence-specific DNA binding,serine-type endopeptidase activity,endopeptidase activity,enzyme binding and zinc ion binding,and calcium signaling pathway,vascular smooth muscle contraction,relaxin signaling pathway,phosphatidylinositol 3-kinase/protein kinase B pathway(PI3K-Akt),sphingolipid signaling pathway and other pathways.The results of molecular docking showed that luteolin and quercetin,the main active ingredients of the Ephedra sinica Stapf dried grass stems compound,had excellent binding activity with the key targets MMP2 and MMP9.The in vitro test results showed that luteolin might inhibit angiotensin Ⅱ-induced proliferation and phenotype transformation of vSMCs through inactivating MAPK1 signaling pathways.【Conclusion】 Various active ingredients of Ephedra sinica Stapf dried grass stems might play a role in the treatment of vasospasm by acting on targets,such as MMP2,MMP9,angiotensin Ⅰ converting enzyme(ACE), NOS2,etc.The study provided a scientific basis for revealing the mechanism of active ingredients of Ephedra sinica Stapf dried grass stems in the treatment of vasospasm.
Isolation, Identification and Pathogenicity Analysis of a Strain of Swine-derived Clostridium perfringens Type A
WU Yuxing, ZOU Weili, CHENG Zixin, MO Yupeng, ZHANG Lingyuan, JI Wantong, CHEN Siyu, ZHENG Haodong, MA Runwen, LI Xun, LI Maoning, WANG Xiaoye
2024, 51(6):  2697-2706.  doi:10.16431/j.cnki.1671-7236.2024.06.043
Abstract ( 28 )   PDF (6039KB) ( 4 )  
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【Objective】 The experiment was aimed to investigate the pathogenicity of Clostridium perfringens type A in piglet diarrhoea and provide experimental data and reference for the diagnosis and treatment of diarrhea caused by Clostridium perfringens type A infection.【Method】 Preliminary identification was carried out by culture characteristics and staining microscopy,suspicious strains were further identified by biochemical tests,PCR amplification of toxin genes,16S rRNA sequence alignment and phylogenetic tree construction.Drug sensitivity and animal regression tests were conducted to assess drug resistance and pathogenicity of the isolated strain.【Result】 The isolated strain displayed black colonies on TSN agar plates,α and β hemolytic zones on blood agar plates,and appeared as large purple rods in Gram staining.The results of biochemical tests showed that glucose,maltose,sucrose,lactose,reductive nitrate test,H2S test and gelatin liquefaction were positive,while mannitol,indole test and salicylic acid test were negative.The 16S rRNA sequence alignment showed more than 99% identity with the 16S rRNA gene sequences of Clostridium perfringens in the NCBI database,the strain was identified as Clostridium perfringens.The plc and cpb2 genes were amplified by PCR,indicating that the strain was Clostridium perfringens type A carrying the β2 toxin.Drug sensitivity test revealed the isolate was resistant to penicillin G,gentamicin,lincomycin,sulfafurazole,ciprofloxacin,and norfloxacin.Animal regression test results showed that the strain could cause diarrhea in piglets,with a mortality rate of 50%.A significant amount of Clostridium perfringens was detectable in the feces post-infection.Necropsy of deceased piglets revealed congestion,hemorrhage,and distension in the small intestine,and Clostridium perfringens type A with high load was isolated from the severely injured jejunal segment.【Conclusion】 This study successfully isolated a strain of Clostridium perfringens type A that could induce disease in neonatal piglets.It exhibited strong pathogenicity,and the results of this study provided a reference for the diagnosis,epidemiological investigation,and treatment of Clostridium perfringens type A infections in piglets.
Whole Genome Sequencing Analysis of a ST515 Strain of Multidrug-resistant Listeria monocytogenes
DU Dongdong, QIAN Jing, CHANG Hengrui, LI Hongmin, WEI Xiangli, LIU Changyong, LUO Ruifeng, WANG Guohong, KANG Lichao
2024, 51(6):  2707-2716.  doi:10.16431/j.cnki.1671-7236.2024.06.044
Abstract ( 35 )   PDF (8153KB) ( 7 )  
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【Objective】 The purpose of this study was to understand the drug resistance gene,virulence gene,mobile element and genetic evolution relationship of a strain of Listeria monocytogenes (LM) isolated from prepared meat products.【Method】 The resistance of LM strain LM5567 isolated from prepared meat products in Xinjiang was detected by VITEK-2 Compact and AST-GP67 susceptibility cards.After the whole genome sequencing,the functional annotation,molecular typing,genetic evolution relationship,drug resistance gene,virulence gene and mobile genetic elements of LM5567 strain were analyzed.【Result】 LM5567 strain was resistant to benzicillin,ampicillin,oxacillin,furantoin,cotrimoxazole,tetracycline and other antimicrobials.The total length of the genome was 2.974 Mb and contained 2 941 coding genes.A total of 2 144 genes were obtained by GO annotation,including 50 functional properties.Evolutionary analysis of MLST showed that the LM5567 strain was ST515 and was closely related to CC1 strain isolated from different countries.SNP analysis showed that LM5567 had the fewest mutation sites with Canadian strains 02_17924b and 02_1103, and one strain 220 from Poland.The LM5567 strain carried 88 virulence genes,6 resistance genes and 1 transposon Tn6009,which carring the tetracycline resistance gene tetM.【Conclusion】 The food-borne strain LM5567 was a multidrug resistant strain of ST515 type,carrying multiple drug resistance genes,virulence genes and 1 transposon in the genome,which had the potential for drug resistance transfer and a high risk of human listeriosis via the food chain.
Effect of Compound Chinese Medicine Preparations on Immune Function of H22 Tumor-bearing Mice
DONG Yuan, WANG Jianing, LIU Jiaqi, MA Guanghui, ZHU Jie, YU Huan, WANG Huiyan
2024, 51(6):  2717-2725.  doi:10.16431/j.cnki.1671-7236.2024.06.045
Abstract ( 34 )   PDF (7848KB) ( 16 )  
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【Objective】 The experiment was to investigate the immunoregulatory effect of the compound Chinese mdicine preparations extracted from Cordyceps militaris,Gantinoderma lucidum,Astragalus membranaceus,Schisandra chinensis,Codonopsis pilosula,Angelica sinensis and Poria Cocos on H22 tumor-bearing mice.【Method】 The model of H22 tumor-bearing was established,and the mice were randomly divided into model group,high (250 mg/(kg·d)),medium (50 mg/(kg·d)) and low (10 mg/(kg·d)) dose groups of compound Chinese medicine preparation,each dose group was administrated intragastric once a day,and the model group was administrated intragastric with the same volume of normal saline.The experiment period was 3 weeks.The normal group was also set up for routine feeding.Whole anticoagulant blood was collected after the last administration,and the number of blood cells,the activity of superoxide dismutase (SOD) and the levels of malondialdehyde (MDA) and cytokines in serum were detected.Peripheral blood CD4+/CD8+ T cell subsets were detected by flow cytometry.The immune activity of tumor-bearing mice was investigated by spleen lymphocyte transformation test and carbon clearance test.【Result】 Compared with normal group,the number of white blood cells (WBC),platelets (PLT) and lymphocytes (W-SCR) in the blood of the model group was extremely significantly or significantly decreased (P<0.01 or P<0.05),the contents of interleukin-2 (IL-2),IL-6 and TNF-α,activities of SOD,Caspase-3 and concentration of cytochrome C (Cyt C) were extremely significantly or significantly decreased (P<0.01 or P<0.05).Compared with model group,the contents of IL-2,IL-6 and TNF-α in serum and the activities of SOD and Caspase-3 in high,medium and low dose groups were extremely significantly or significantly increased (P < 0.01 or P < 0.05),MDA content was extremely significantly or significantly decreased (P<0.01 or P<0.05).The amount of WBC and PLT in the medium dose group was extremely significantly or significantly increased (P<0.01 or P<0.05).The number of PLT in high dose group was extremely significantly increased (P<0.01), and the concentration of Cyt C was significantly increased (P<0.05).Flow cytometry showed that the number of T lymphocyte CD4+ and CD8+ subsets in model group was significantly decreased compared with that in normal group (P<0.05).Compared with model group,the number of T lymphocyte CD4+ and CD8+ subsets in high and medium dose groups was significantly increased (P<0.05),and the stimulation index,median hemolysis value and phagocytosis index of mice in high and medium dose groups were extremely significantly or significantly increased (P<0.01 or P<0.05).【Conclusion】 The compound Chinese medicine preparation could improve the immune activity of H22 tumor-bearing mice,and then promote the apoptosis of liver cancer cells.