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20 March 2020, Volume 47 Issue 3
Biotechnology
Prokaryotic Expression and Bioinformatics Analysis of NYD-SP27 Gene in Sheep
LI Na, WU Yuhong, YUAN Liming, ZHANG Xiaoxiao, Saiwujafu
2020, 47(3):  645-654.  doi:10.16431/j.cnki.1671-7236.2020.03.001
Abstract ( 295 )   PDF (2392KB) ( 146 )  
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This study was aimed to clone and express the NYD-SP27 gene in sheep,and analyze the bioinformatics of its expressed protein.A pair of specific primers were designed based on the sequence of NYD-SP27 gene (accession No.:KX905090) in GenBank,and the NYD-SP27 gene fragment was amplified by PCR method.The recombinant plasmid pMD19-T-NYDSP27 was constructed and then transformed into E.coli DH5α competent cells.The plasmid was identified by restriction enzyme digestion.The recombinant plasmid pET-22b(+)-NYDSP27 was constructed and then transformed into E.coli BL21(DE3) competent cells.The expressed protein was induced by IPTG and identified by SDS-PAGE and Western blotting.The amino acid sequence encoded by NYD-SP27 gene was analyzed by bioinformatics methods.The results showed that the 1 617 bp gene fragment was successfully amplified,and two fragments of 5 400 and 1 617 bp were digested by Nde Ⅰ and Xho Ⅰ,indicating that the recombinant plasmid pET-22b(+)-NYDSP27 was successfully constructed.The expressed recombinant protein was about 60 ku.The NYD-SP27 recombinant protein was an inclusion body with a molecular formula of C2798H4319N737O819S19.The total number of atoms was 6 892,the theoretical isoelectric point (pI) was 6.16,which was an acidic protein,the instability coefficient was 46.96,which was an unstable protein,and the average hydrophilicity was -0.403.The protein had no signal peptide and transmembrane structure,and had 39 potential phosphorylation sites and 24 epitopes.In the secondary structure of NYD-SP27 protein,alpha helix,beta turn,extended chain and random coil accounted for 26.21%,3.90%,18.03% and 51.86%,respectively.This results might provide reference data and material basis for further study of regulatory mechanisms of NYD-SP27 protein in sheep.
Cloning,Bioinformatics and Expression Analysis of CAPN1 Gene in Guangling Donkey
ZHAO Jingwei, SUN Yutong, LI Wufeng
2020, 47(3):  655-665.  doi:10.16431/j.cnki.1671-7236.2020.03.002
Abstract ( 248 )   PDF (2267KB) ( 96 )  
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This study was aimed to clone the calcium protease 1 (CAPN1) gene CDS and analyze its bioinformatics,and identify its expression in different tissues of Guangling donkey.The CAPN1 gene was amplified,cloned and sequenced,the physicochemical,subcellular localization,modified structure and protein structure of CAPN1 protein were predicted using bioinformatic methods.The expression level of CAPN1 gene in heart,liver,spleen,lung,kidney and longissimus dorsi muscle of Guangling donkey were analyzed by Real-time PCR.The results showed that the length of CAPN1 gene CDS was 2 148 bp,and encoded 715 amino acids,which had been submitted to GenBank,the accession number was MN158194.The homology of nucleic acid sequence of CAPN1 gene in Guangling donkey was 99.7%,92.3%,92.5%,92.0%,92.5%,85.9% and 92.9% with Equus caballus,Ovis arise,Bos taurus,Homo sapiens,Capra hircus,Mus musculus and Sus scrofa,respectively.The molecular weight,isoelectric point,instability index of CAPN1 protein were 82.01 ku,5.59 and 36.42,respectively,and the average hydrophobic index was -0.374.CAPN1 protein had no signal peptide and no transmembrane region.The secondary structure of CAPN1 protein was consisted of random coil,alpha helix,beta turn and extended chain.The CAPN1 gene was expressed in all six tissues,the expression in longissimus dorsi muscle was the highest,followed by liver,and the expression in heart was the lowest.This experiment successfully cloned CAPN1 gene of Guangling donkey and analyzed its expression in different tissues,it provided a theoretical basis for further study of the expression and regulation function of CAPN1 gene in the muscle tenderness and developing the meat products industry of local species Guangling donkey.
Construction of Enterotoxigenic E.coli LT Knockout Strain Using CRISPR/Cas9 and λ-Red Cascaded Technology
TAN Keqin, MA Xianyong, CUI Yiyan, TIAN Zhimei, DENG Dun
2020, 47(3):  666-675.  doi:10.16431/j.cnki.1671-7236.2020.03.003
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The aim of this study was to use the CRISPR/Cas9 and λ-Red cascade technology to perform a seamless knockout the heat-labile toxin (LT) gene of enterotoxigenic Escherichia coli (ETEC) K88 and obtain a K88 LT deficient strain.The homologous sequences of boundary sequences at both ends were obtained by sequence alignment,and a donor fragment was constructed containing the LT boundary sequences,the chloromycetin selection marker,sgRNA sequences and the LT homology arm.The donor fragment was transformed into ETEC K88,and LT gene was knocked out using λ-Red homologous recombination system and CRISPR/Cas9 gene editing system,respectively.The K88 LT-deficient strain was obtained by PCR,and the hemolysis ability and growth curve of the knockout strain were determined.The results showed that LT gene was successfully replaced by the corresponding donor fragment using λ-Red homologous recombination system,and the chloromycetin selection marker was efficiently deleted using CRISPR/Cas9 gene editing system.The gene editing system successfully performed a seamless knockout of LT gene of ETEC K88.In vitro experiment results showed that the K88 LT deficient strain had no capacity to hemolysis and grew slowly than the wild-type strain.It indicated that LT affected the pathogenicity and growth performance of the strain.Therefore,the results suggested that λ-Red and CRISPR/Cas9 cascade knockout methods could be used for knockout of LT toxin genes,and could also be used for other E.coli knockouts.The construction of K88 LT-strain laid the foundation for further study on the pathogenesis of LT toxin.
Expression of miRNA from Canine UC-MSC-Exo and the Effect of cfa-miR-34a/-143 on Proliferation of Vascular Endothelial Cells
LUO Huina, LUO Dongzhang, FAN Quanbao, WANG Bingyun, ZHAN Xiaoshu, CHEN Shengfeng, CHEN Zhisheng, BAI Yinshan, LIU Canying, JI Huiqin
2020, 47(3):  676-685.  doi:10.16431/j.cnki.1671-7236.2020.03.004
Abstract ( 303 )   PDF (2268KB) ( 90 )  
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This study was aimed to isolate and identify exosomes secreted by canine umbilical cord mesenchymal stem cells(UC-MSC-Exos) and study the expression of miRNAs derived from UC-MSC-Exo and its effect on proliferation of vascular endothelial cells (VECs).Exosomes were isolated from the culture supernatant of canine umbilical cord mesenchymal stem cells by ultra-high speed centrifugation and identified by Western blotting,transmission electron microscopy and nanoparticle tracking analysis (NTA).Two miRNAs (cfa-miR-34a/-143) were screened by sequencing miRNAs in canine UC-MSC-Exo,and the corresponding mimic and inhibitor were synthesized and then transfected into canine VECs.The mimic and inhibitor transfections and their transfection efficiency were detected by fluorescence microscopy and Real-time PCR,and its effect on the proliferation of canine VECs was detected by CCK-8 method.Finally,the target genes were predicted.Canine UC-MSC-Exo expressed specific exosomal surface proteins CD9,CD63 and CD81 with a particle size of 100-200 nm and a typical cup shape.The results of immunofluorescence assay showed that both miRNA mimic and inhibitor were transfected into cells and accumulated in the cytoplasm;Real-time PCR results showed that the expression of cfa-miR-34a in cells increased about 400-fold after transfection of cfa-miR-34a mimic,while cfa-miR-143 increased about 78-fold;Whereas transfection of cfa-miR-34a inhibitor,the expression of cfa-miR-34a in cells decreased by 77%,while that of cfa-miR-143 decreased by 83%,and the cfa-miR-34a and cfa-miR-143 could extremely significantly promote the proliferation of VECs in vitro by CCK-8.The target genes of cfa-miR-34a predicted that there were 195 conserved target sites,and 69 cross-target genes were shared with miRDB prediction results,while cfa-miR-143 had 447 conserved target genes,128 cross-target genes were shared with miRDB prediction results.Canine UC-MSC-Exo was successfully isolated,and it was confirmed that the target miRNAs cfa-miR-34a and cfa-miR-143 could extremely significantly promote VECs proliferation in vitro.
Antigen Epitope Prediction and Recombinant Protein Design and Verification of ORFV B2L Protein
SUN Zhengnan, ZHANG Huanrong
2020, 47(3):  686-695.  doi:10.16431/j.cnki.1671-7236.2020.03.005
Abstract ( 244 )   PDF (2915KB) ( 118 )  
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To obtain the possible antigenic epitopes of ORFV B2L protein and express the recombinant protein efficiently after truncation.DNAstar,Mega 7.0 and other softwares were used to analyze partial ORFV B2L gene sequences recorded by NCBI.The signal peptide,transmembrane region,cytotoxic T cell epitope,helper T cell epitope,B cell epitope and secondary structure of protein were predicted by different online servers.At the same time,tertiary structure prediction was carried out by referring to multiple templates.Antigenic site analysis and recombinant protein design were carried out by synthesizing major predictive data such as epitope,hydrophilicity,surface accessibility and antigen index,the fusion protein was double His·tag.Several possible high-quality epitopes were obtained,which were 88-93,133-138,172-175,251-254,311-313 and 370-377 amino acids,respectively.A recombinant protein containing major predictive epitopes was designed and expressed by fusing two His·tags to link flexible proteins.The purified and renatured expressed proteins existed in the form of polymers.Western blotting results showed that the purified and renatured recombinant proteins had good reactivity.This study acquired the ORFV B2L antigenic sites and constructed the highly efficient prokaryotic expression vector of recombinant B2L protein,providing the bases for further explore the diagnosis reagent and subunit vaccine.
Cloning,Bioinformatics Prediction and Tissue Expression Analysis of PPARGC1A Gene in Jinhua Pigs
LIU Yufang, GUO Siwu, ZHANG Qingyang, ZHANG Xin, YANG Junqi, BAI Ying
2020, 47(3):  696-705.  doi:10.16431/j.cnki.1671-7236.2020.03.006
Abstract ( 242 )   PDF (2183KB) ( 100 )  
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The aim of this study was to investigate the genetic characteristics and expression patterns of PPARGC1A gene in Jinhua pigs and Yorkshire pigs.The Jinhua pigs and Yorkshire pigs were used as samples to extract total RNA from the dorsal lipid tissue,and the coding region and real-time quantitative PCR primers were designed according to the sequence of porcine PPARGC1A gene in GenBank (accession No.:NM_213963.2).The porcine GAPDH gene and β-actin protein were used as internal parameters,and the PPARGC1A gene-encoded protein was applied by various bioinformatics methods.Functional analysis was performed and its expression level in dorsal lipid tissue of Jinhua pigs was detected by Real-time quantitative PCR and Western blotting assay.The results showed that the full length of PPARGC1A gene CDS region was 2 361 bp and encoded 786 amino acids.The protein had a molecular size of 90 336.01 u,with serine (Ser) accounting for the highest proportion (13.7%) and tryptophan (Trp) accounting for the lowest proportion (0.8%).Homology alignment results showed that PPARGC1A gene in Jinhua pigs had high homology with goats,cattle and sheep,which were 95.0%,94.9% and 94.9%,respectively,and was highly conserved in species evolution.The PPARGC1A protein of the instability index in Jinhua pigs was 74.88,belonging to unstable hydrophilic protein,and no transmembrane structure.Its secondary structure consists of alpha helix,extended chain,beta turn and random coil,and the proportion of them were 26.59%,5.73%,5.73% and 61.96%,respectively.The PPARGC1A protein homology modeling obtained a series of complex processes such as folding and bending to obtain a three-level structural model.The results of the Real-time quantitative PCR and Western-blotting were consistent,indicating that the expression of PPARGC1A gene in Jinhua pigs with dorsal lipid tissue was significantly lower than that of lean pigs (P<0.05).This study provides a theoretical basis for the discovery of the molecular biological function of PPARGC1A gene in pig fat deposition.
Characterization Analysis of HSPA6 Protein in Goat and Construction of Interactive Protein Network
YANG Jiadong, LIU Yueqin, ZHANG Yingjie
2020, 47(3):  706-713.  doi:10.16431/j.cnki.1671-7236.2020.03.007
Abstract ( 228 )   PDF (1304KB) ( 115 )  
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In order to investigate the structure and functional characteristics of HSPA6 belongs to heat shock protein 70 family in goat,the amino acid composition,physical and chemical properties,hydrophobicity,transmembrane structure,signal peptide,possible phosphorylation modification sites,subcellular localization,secondary structure and tertiary structure of HSPA6 were analyzed using bioinformatic methods.HSP70 protein sequences of various organisms were selected to construct phylogenetic tree,at the same time,the protein interaction network was analyzed.The results showed that HSPA6 in goat had higher homology sequences with mammalian (cattle,pig);the molecular weight of HSPA6 protein in goat was 70.9 ku;The isoelectric point was about 5.78,which indicated it belonged to acidic protein and hydrophilic protein;Without transmembrane structure and signal peptide.There were 10 phosphorylation sites with high scores,which located in N-terminal(nucleotide binding domain,NBD) and C-terminal (peptide binding domain,PBD),and C-terminal contains EEVD motif,which was the conserved motif of HSP70 family proteins located in the cytoplasm,indicating that it was mainly distributed in cytoplasm.The secondary structure of protein was mainly composed of α-helix (41.52%) and random coil (33.75%).The construction of protein interaction network indicated that HSPA6 might interact with HSP70 family members,and might play a role in the formation of complex with HSP40 and HSP90 in cells.Based on the above,this study provided a theoretical basis for further exploring the functional mechanism of HSPA6 in goat in response to environmental stress.
Physiology and Biochemistry
Effect of Ciglitazone on Adipogenic Transdifferentiation of Skeletal Muscle Satellite Cells in Yanbian Yellow Cattle
YAN Yan, ZHANG Junfang, SUN Bin, WANG Ying, SUN Jianfu, SUN Xiaojiao, CUI Yan, JIN Xin, YAN Changguo, LI Xiangzi
2020, 47(3):  714-721.  doi:10.16431/j.cnki.1671-7236.2020.03.008
Abstract ( 215 )   PDF (1965KB) ( 99 )  
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This study was aimed to investigate the effect of ciglitazone,a peroxisome proliferator-activated receptor (PPARγ) agonist,on the adipogenic transdifferentiation of skeletal muscle satellite cells in Yanbian Yellow cattle.The skeletal muscle satellite cells were treated with ciglitazone at concentrations of 0 (CON group),5 (CL group),10 (CM group) and 20 (CH group) μmol/L for 96 h.The formation of lipid droplets was evaluated by oil red O staining and triglyceride amounts was determined by triglyceride determination kit.Real-time quantitative PCR and Western blotting were employed to detect whether adipocytogenesis related factors were involved.The results showed that the area of oil red O and the amounts of triglyceride were both significantly increased after treated with ciglitazone compared with control group (P<0.05).Moreover,the area of oil red O and the amounts of triglyceride were positively correlated with the concentration of ciglitazone.Compared with control group,the mRNA and protein expression of adipocytic transcription factors,including PPARγ,CCAAT/enhancer binding protein alpha (C/EBPα) and peripoietin-2 (PLIN2),were significantly upregulated in ciglitazone addition groups (P<0.05).However,the expression of myogenic-related factor MyoD was downregulated after treated with ciglitazone (P<0.05).The results indicated that ciglitazone could promote the transdifferentiation of skeletal muscle satellite cells to adipocytes in cattle.
Effects of Mitochondrial Dynamics Balance on Cardiac Structure and Function
CUI Yunli, SONG Xiaochao, CHEN Lingli, GE Yaming, NING Hongmei, HU Dongfang
2020, 47(3):  722-728.  doi:10.16431/j.cnki.1671-7236.2020.03.009
Abstract ( 232 )   PDF (771KB) ( 119 )  
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The heart is an organ that consumes a lot of energy and oxygen,its normal contraction and normal transmission of electrical signals also need a lot of energy supply,so cardiomyocytes contain a large number of mitochondria.Under normal physiological conditions,the number,morphology and function of mitochondria in cells are relatively stable.When the energy production of cells in the body is insufficient or there are different defects in two independent mitochondria,the mitochondria will fuse under the regulation of Mfn1/2 and OPA1.After fusion,the content of mitochondrial matrix mixes with each other to form a new mitochondrion with perfect function,which is called mitochondrial fusion.In order to maintain the stability of mitochondrial DNA and mitochondrial membrane potential,mitochondria divide under the regulation of Drp1 and its receptors.Division can make damaged mitochondrial DNA and depolarized mitochondrial membrane aggregate into one daughter mitochondrion during division.And it can be eliminated by ubiquitination protease system or autophagy to maintain the normal function of mitochondria.Mitochondrial fusion and division is a continuous fluctuating process,which has been considered to be the key process to maintain the normal function and morphology of mitochondria and cells.In recent years,it has been found that the imbalance of mitochondrial fusion and division in cardiomyocytes will lead to the disorder of their own morphology and function,and then damage the structure and function of the heart.Therefore,maintaining the homeostasis of cardiomyocytes requires the dynamic balance between mitochondrial division and fusion,while maintaining the dynamic balance of mitochondria needs to mediate mitochondrial fusion and division related dynamic proteins.In this paper,the functions of key proteins involved in mitochondrial fusion and division were reviewed,and the effects of mitochondrial dynamic balance on cardiac structure and function were discussed,which would provide a theoretical basis for later research.
Research on the Effect of Sheep Embryonic Fibroblast Feeder for Culturing Human Induced Pluripotent Stem Cell
ZHANG Shiqiang, LI Mengxin, HAN Gaolian, GUO Lirong, ZHAO Dipeng, LI Junling, GUO Tianyan, DU Rong, QIN Jian
2020, 47(3):  729-735.  doi:10.16431/j.cnki.1671-7236.2020.03.010
Abstract ( 230 )   PDF (7023KB) ( 55 )  
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The aim of this study was to investigate the feasibility of using the isolated and mitomycin C-treated sheep embryonic fibroblast (SEF) in vitro as the feeder layer of the induced pluripotent stem cells (iPSCs).Taking SNL feeder layer cells as the control group and the human iPSC (hiPSC) as the experimental object,the effects of SEF cells and SNL cells as feeder layer of stem cells were compared by the morphological observation,the alkaline phosphatase (AP) staining,and the qRT-PCR and immunocytochemistry detection of the mRNA and protein expression for hiPSC marker genes.The results demonstrated that,being similar to hiPSC on SNL feeder layer,hiPSC on SEF feeder layer showed rapid growth and proliferation as colonies during the experimental period.Meanwhile,the AP staining was blue-purple,the undifferentiated state was maintained,and the pluripotent marker genes were normally expressed.The expression of hiPSC pluripotent marker genes c-myc,Klf4,OCT4,and SOX2 mRNAs,as well as the expression of OCT4,SOX2,SSEA4,and TRA-1-60 proteins were not significantly different for hiPSCs on SEF and SNL (P>0.05).The results indicated that SEF could be used as feeder layer cells for culturing iPSC in vitro,which laid a foundation for the further establishment of the gene-modified SEF cell lines that could express the growth-promoting factors.
Animal Nutrition and Feed Science
Effects of Different Dietary Fiber Source on the Meat Quality and Amino Acid Content of Sichuan White Geese
ZHANG Jie, WU Qiushen, JIE Xiaodie, CHENG Yating, MU Zhiping, HE Hang, LIU Anfang
2020, 47(3):  736-743.  doi:10.16431/j.cnki.1671-7236.2020.03.011
Abstract ( 254 )   PDF (752KB) ( 88 )  
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This experiment was aimed to study the effects of dietary fiber sources on the meat quality,amino acid content and nutritive value of Sichuan White geese.120 Sichuan White geese at similar body weight of 28 days old were randomly divided into 4 groups,3 replicates in each group with 10 geese,used floor rearing,and separately fed with alfalfa,ryegrass,oat grass and peanut dietary.Geese had access to feed and water ad libitum in the experiment period.The feeding experiment lasted for 42 days.The results showed that shear force,meat color,drip loss rate,crude fat and amino acid content were significantly affected under same nitrogen,energy and feed intake level (P<0.05),the shear forces of the alfalfa and peanut groups were significantly lower than that of ryegrass and oat grass groups (P<0.05),the drip loss rate of alfalfa,oat grass and peanut groups were significantly lower than that of ryegrass group (P<0.05),the crude fat of oat grass group was significantly higher than that of other groups (P<0.05),while the peanut group was significantly lower than that of other groups (P<0.05).There was no significant effect of different fiber source dietary on moisture,ash and crude protein content (P>0.05).Adding alfalfa meal powder to the diet could improve the water retention of the goose meat,increase its tenderness,and also increase the umami amino acid content in the goose meat.So,it was recommended to add alfalfa powder to the meat goose diet in order to improve its meat quality and nutritive value.
Research Progress on Animal Amino Acid Transporter
HE Yue, ZHAO Shengguo, LUO Chaochao, ZHENG Nan, WANG Jiaqi
2020, 47(3):  744-753.  doi:10.16431/j.cnki.1671-7236.2020.03.012
Abstract ( 425 )   PDF (1672KB) ( 197 )  
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The amino acid transporter is an important protein that mediates the transport of amino acids from the outsides to the inside of cells and also is an crucial nutrient-sensing molecule that can regulate amino acid-related signaling pathways,which has significant effects in many aspects,including body's growth and metabolism,nutrition and health.There are many types of AAT in animal organisms that can sense changes of amino acid levels in the body,mediating cellular amino acid sensing signaling pathways-mammalian rapamycin target protein complex 1 (mTORC1) and general control nonderepressible 2(GCN2) to active,which causes the downstream of the hair pathway to function.The leading AAT exists diversity in different tissues,indicating that AAT is tissue-specific,simultaneously,AAT is also affected by many factors,such as the animal body itself,nutrient levels,hormone levels,and so on.This review focuses on four aspects to understand AAT macroscopically,including the types and transport mechanisms of AAT,mediating the initiation of nutrient signaling、mTORC1 pathway and GCN pathway,the role of ATT in different tissues,and the regulation of AAT expression,in order to provide some useful knowledge for AAT research.
Current Situation for Effect of Dietary Amino Acids on Growth Performance and Immune Function in Calves
KONG Fanlin, DIAO Qiyu, TU Yan
2020, 47(3):  754-762.  doi:10.16431/j.cnki.1671-7236.2020.03.013
Abstract ( 208 )   PDF (946KB) ( 77 )  
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The immune system of calves is immature and sensitive before weaning.However,the calves can grow rapidly after weaning.In both stages,it is necessary to provide a diet containing high quality protein to ensure the growth of calves.Considering the current situation of protein shortage in China,optimizing amino acids supply to calves is important for improving health status and utilization rate of protein in the calf stage.In addition,amino acid can play an important role in improvement of immune function in the calves.Recently,some studies found that amino acids would have some special effects on susceptible bacteria and virus in digestive tract of calves.According to the physiological characteristics of calves,such as the rapid growth,imperfect immune function,high mortality and rapid changes in physiological structure and nutritional requirements before and after weaning,the authors reviewed the effects of different levels of single amino acid and different proportions of multiple amino acids on the growth performance of calves before and after weaning.The mechanisms about the effects of amino acids on the health of calves from the aspects of improvement of autoimmunity of calves and inhibiting microbial reproduction was summarized also.The paper was to provide theoretical basis for promoting the application of amino acid in calf production.
Effects of Dietary Protein and Ether Extract Levels on Nutrient Digestibility and Nitrogen Metabolism of Growing Raccoon Dogs
GONG Wenyang, LI Mo, NIU Yibing, DONG Xueyu, LI Yong, ZHANG Guixian, WANG Xiguo, GONG Yuanfang, SU Xin, YANG Liu, XING Guanzhong, FENG Minshan, LI Sufen
2020, 47(3):  763-770.  doi:10.16431/j.cnki.1671-7236.2020.03.014
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The purpose of this study was to study the effects of dietary protein and ether extract levels on nutrient digestibility and nitrogen (N) metabolism of growing raccoon dogs.A total of 63 healthy 11 weeks old raccoon dogs were randomly divided into 9 groups according to a complete random design with 3×3 factorial arrangement.The 3 dietary protein levels were 22%,24% or 26%,and 3 ether extract levels were 5.5%,7.5% or 9.5%,respectively.The diets contained same levels of lysine,sulfur-containing amino acids and threonine.The experiment lasted for 56 d.The results showed that interaction between protein and ether extract did not affected the indices of either energy and nutrient digestibilities or nitrogen metabolism (P>0.05).The DM digestibility was affected by dietary protein level (P<0.05),in which the dogs fed diet with 22% protein had significantly higher value than those fed diet with 24% or 26% protein (P<0.05).The ether extraxt digestibility increased with the increasement of dietary ether extract level (P<0.05) and tend to increase with the increasement of dietary protein level (0.05 < P < 0.10).The N intake,fecal and urine N outputs,biological value (BV) and availability were affected by dietary protein level significantly (P<0.05),in which the BV values in groups of dietary protein levels of 22% and 24% were significantly higher than the values in groups of dietary protein levels of 26% (P<0.05).No differences were detected in N retentions among dietary protein levels (P>0.05).The urine N output,N retention,BV and N availability were affected significantly by dietary ether extract level (P<0.05),in which the N retention,BV and N availability in groups of dietary ether extract levels of 9.5% and 11.5% were significantly higher than the values in groups of dietary ether extract levels of 7.5% (P<0.05).These results suggested that diet containing lower protein and higher ether extract could decrease urine N output and improve protein availability and BV.
Advances in Research on Calcium Metabolism and Regulation Mechanism in Pregnancy-Lactating Sows
YANG Li, MA Longteng, HE Zhixiong, CHEN Yuguang
2020, 47(3):  771-779.  doi:10.16431/j.cnki.1671-7236.2020.03.015
Abstract ( 216 )   PDF (1260KB) ( 105 )  
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The performance of sows is affected by many factors,among which dietary mineral level is one of the main factors.Calcium plays an important role in regulating the performance of sows.There are three ways of calcium absorption,i.e.active transport,passive transport and vesicle transport.Hormones or other factors that affect calcium absorption mostly affect the utilization of dietary calcium by regulating calcium absorption.On the one hand,the appropriate dietary calcium level can promote the sow to play the maximum production potential and improve the breeding efficiency;on the other hand,when the dietary calcium level is insufficient or the utilization rate of calcium is low,first of all,the potential production performance of the sow cannot be fully developed,such as the number of litter or live litter is low,and the growth speed of the piglet is slow.Secondly,the lack of calcium utilization in sows leads to the occurrence of bone diseases,especially in the late pregnancy and lactation period,which ultimately leads to a high elimination rate,which ultimately leads to a significant reduction in the production efficiency of the breeding industry.In recent years,scholars at home and abroad have made a lot of research on the factors affecting the utilization of dietary calcium by sows,and made great progress.For example,the feeding time of calcium,the ratio of dietary calcium to phosphorus,vitamins,hormones and pH all affect the absorption and utilization of dietary calcium by sows.Therefore,it is of great practical significance to study the calcium absorption characteristics of sows and the influencing factors to improve the performance of sows.In this paper,the mechanism of calcium absorption in the body and the main factors affecting calcium absorption in sows are briefly introduced in order to provide theoretical basis for the feeding of dietary calcium in sows.
Genetics and Breeding
Effect of the Polymorphism of GnRHR and INHA Genes and Its Interaction on Litter Size in Wuhu Hybrid Sheep
LIU Haixia, HAN Dayong, ZHU Aiwen, YAN Wei, WANG Buzhong, CAI Zijian
2020, 47(3):  780-788.  doi:10.16431/j.cnki.1671-7236.2020.03.016
Abstract ( 220 )   PDF (1443KB) ( 83 )  
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This study was aimed to analyze the influence of genetic variation of GnRHR and INHA genes and its interaction effect on the number of lambing in sheep.Primers were designed according to DNA sequence of GnRHR and INHA genes (GenBank accession No.:AH004943 and U16237).PCR-SSCP was used to detect the genetic polymorphisms of GnRHR and INHA genes in Wuhu sheep,Hu sheep and Black bone sheep,the correlation between polymorphic loci and litter size and the interaction effect between GnRHR and INHA genes were analyzed.The results showed that AA,AB and BB genotypes,GG,GC and CC genotypes were detected in GnRHR and INHA genes of Wuhu sheep and Hu sheep,no polymorphism site was found in Black bone sheep with few samples.G→C mutation occurred at 198 bp of GnRHR gene CDS,resulting in glycine to arginine,which was missense mutation;T→C mutation occurred at 877 bp of INHA gene,which was synonymous mutation (still aspartic acid).Genetic analysis results showed that GG and AA were dominant genotypes,and G and A were dominant alleles.The Chi-square test results showed that GnRHR and INHA genotypes distribution was in Hardy-Weinberg equilibrium (P>0.05).By analyzing the interaction effect between GnRHR and INHA genes,it was found that there was a significant difference in the interaction effect between Wuhu and Hu sheep (P<0.05).The interaction effect of ABCC genotype was the largest,and AAGG genotype was the smallest.The average lambing number of ABCC genotype were 1.68 and 1.32 in Wuhu sheep and Hu sheep,respectively,which was higher than that of AAGG genotype,and the lambing number of ABCC genotype was higher than that of AB and CC genotypes,which indicated that the interaction effect was positively correlated with lambing number.This results suggested that interaction genotype of INHA (T877C) and GnRHR (G198C) was closely related to the lambing number of sheep,which had a significant effect on the litter size in sheep.
Association Analysis Between PRKAA2 Gene Polymorphisms and Growth Traits in Murrah Buffalo
ZHENG Haiying, YANG Chunyan, ZHENG Wei, YU Nongqi, WEN Chongli, WEI Kelong, LI Lingyu, HUANG Chunli, HUANG Jiaxiang, SHANG Jianghua
2020, 47(3):  789-798.  doi:10.16431/j.cnki.1671-7236.2020.03.017
Abstract ( 236 )   PDF (1451KB) ( 81 )  
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This study was aimed to investigate the genetic polymorphisms of PRKAA2 gene in Murrah buffalo and its associations with growth traits,which could help to mine significant genetic markers for molecular marker assisted selection of Murrah buffalo.SNPs located in exon 4 and intron 3 were detected in PRKAA2 gene of Murrah buffalo by random DNA sequencing method.Genetic variation of SNPs and the association of different genotypes with growth traits of Murrah buffalo were analyzed by bioinformatics method and statistical software.The results showed that 4 SNPs were detected in PRKAA2 gene,namely c.462 G>A,IVS3.557 T>C,IVS3.560 C>T and IVS3.565 G>A,respectively,there were 3 genotypes.The fitness test showed that PRKAA2 gene on polymorphic loci in Hardy-Weinberg equilibrium.All of 4 SNPs were moderate polymorphism,and could be constructed 3 haplotypes,T-C-G-G type was the dominant haplotype in Murrah buffalo.There was a complete linkage disequilibrium between IVS3.560 C>T and c.462 G>A.Association analysis results indicated that H2 haplotype was significantly correlated with body length (BL)and hip width (HW) in Murrah buffaloes (P<0.05).c.462 G>A was significantly correlated with the withers height (WH),height at hip cross (HHC),rump length (RL),pin bone width (PBW) and HW of Murrah buffalo,WH and HHC of GG and AA genotypes were significantly higher than that of GA genotype,RL of GG genotype were significantly higher than that of GA and AA genotypes,and PBW and HW of AA genotype were significantly higher than that of GA genotypes (P<0.05).There was no significant correlation between IVS3.557 T>C and body size traits of Murrah buffalo (P>0.05).IVS3.560 C>T was significantly correlated with WH,HHC and RL of Murrah buffalo,WH and HHC of CC and TT genotypes were significantly higher than that of CT genotype (P<0.05),and RL of CC genotype was significantly higher than that of CT genotype (P<0.05).IVS3.565 G>A was significantly correlated with BL of Murrah buffalo,BL of GG and GA genotypes were significantly higher than that of AA genotype (P<0.05).In conclusion,3 SNPs (c.462 G>A,IVS3.560 C>T and IVS3.565 G>A) of PRKAA2 gene had potential effects on growth traits of Murrah buffalo, and could be used for candidate gene and marker-assisted selection in selection of new varieties of Murrah buffalo.
Detection of InDel of FRZB Gene and Its Association Analysis with Growth Traits in Sheep
CHEN Pingbo, ZHAO Haidong, WU Mingli, WEI Yanpei, SUN Xiuzhu, WANG Shuhui
2020, 47(3):  799-806.  doi:10.16431/j.cnki.1671-7236.2020.03.018
Abstract ( 235 )   PDF (1472KB) ( 84 )  
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In this study,FRZB gene was selected as a candidate gene,582 sheep were selected as experimental materials from four breeds of Tong sheep,Small-tailed Han sheep,Lanzhou Big-tailed sheep and Hu sheep.DNA pool sequencing combined with agarose gel electrophoresis was used to screen the InDel of FRZB gene in order to find the sites of InDel related to the growth traits of four sheep breeds.The results showed that a 14 bp InDel mutation was detected in the first intron of FRZB gene in sheep;The InDel mutation site in the intron region of FRZB gene had three genotypes (II,ID and DD) in Tong Sheep,Small-tailed Han sheep,Lanzhou Big-tailed sheep and Hu sheep populations;Association analysis showed that the InDel mutation detected in FRZB gene had significant effects on the growth traits in Tong sheep.The body length,back height,hip height,talus width,tail length and tail width of II genotype were significantly superior to DD genotype in Tong sheep (P<0.05),it could be used as a candidate molecular marker for the molecular marker-assisted selection of Tong sheep.These results suggested that a novel 14 bp insertion mutation in the first intron of FRZB gene in sheep could be significantly affecting the growth trait of Tong sheep,and could be used as a potential molecular marker for growth traits in sheep breeding.
Expression of RBP4 Gene in Epididymis Before and After Sexual Maturation in Sheep
HAN Yue, LIU Hang, CHEN Yang, JIANG Huaizhi
2020, 47(3):  807-813.  doi:10.16431/j.cnki.1671-7236.2020.03.019
Abstract ( 206 )   PDF (6636KB) ( 116 )  
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The purpose of this study was to investigate the expression of retinol binding protein 4 (RBP4) gene in epididymis of sheep before and after sexual maturity and its role in the regulation of reproductive function.The expression of RBP4 gene mRNA in epididymis before and after sexual maturity was detected by Real-time quantitative PCR,and the expression of RBP4 gene in epididymis head and epididymis tail before and after sexual maturity was detected by immunohistochemistry.The results showed that the expression level of RBP4 gene was 1.3368 and 0.6450 before and after sexual maturity,respectively.Although there was no significant difference between them (P>0.05),the expression level before sexual maturity was higher than that after sexual maturity.RBP4 protein was expressed in the epididymal cortex,smooth muscle layer and interstitial tissue before and after sexual maturity,mainly in the epithelial cytoplasm and smooth muscle cytoplasm,and in the epididymal cortex and interstitial tissue,mainly in the epithelial cytoplasm.It was suggested that RBP4 gene might be related to the secretory function of epididymal main cells and the contractile function of epididymal head,indicating that RBP4 might participate in the regulation of epididymal microenvironment,which was conducive to sperm maturation,movement and storage.
Effect of Training on Body Angle in 1 km Trotter of Yili Horse
MENG Jun, WANG Jianwen, KONG Qisen, ZENG Yaqi, LI Linling, ZHANG Yaang, REN Wanlu, WANG Chuankun, YAO Runchen, YAO Xinkui
2020, 47(3):  814-821.  doi:10.16431/j.cnki.1671-7236.2020.03.020
Abstract ( 212 )   PDF (657KB) ( 55 )  
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This experiment aimed to analyze the effects of different training stages on the limb angle of the Yili horse in 1 km trotter.8 Two-year-old Yili horses were selected as the test subjects.Through the 1 km trotter training for three months,the trotter test race was organized before and after the training.The video analysis was used to analyze the maximum limb opening angle,the minimum contraction angle and the extreme difference of limb angle of straight and curved horses in the process of exercise,and the difference of limb angle changes in different training stages were analyzed.The results showed that in the straight movement,the maximum opening angle and minimum contraction angle of shoulder angle before training were significantly less than those after three months training (P<0.05).The maximum opening angle and angle range of the elbow and the back fetlock angle before training were extremely significantly smaller than those after the three months training (P<0.01).The carpus and tarsus angle of the minimum contraction angle and angle range were extremely significantly different from those after the three months training (P<0.01).The maximum opening angle,minimum contraction angle and angular range of the front fetlock angle before the training were extremely significantly different from those after the three months training (P<0.01).In the curve movement,the maximum opening angle of the shoulder angle was significantly greater than those before the three months training (P<0.05).The minimum contraction angle and angle range of the elbow angle and the front fetlock angle were significantly different from those before the three months training (P<0.05).The minimum contraction angle of the carpus angle was extremely significantly greater than that of the three months after training (P<0.01),the angle range of the carpus angle before the training was significantly greater than three months after training (P<0.05).The maximum opening angle and angle range of the carpus angle were significantly greater than those before the three months training (P<0.01).The minimum contraction angle of the stifle angle before training was extremely significantly less than that after training for three months (P<0.01),the maximum opening angle and the angle range of the tarsus angle was significant greater than those before the three months training (P<0.05).In summary,the training affects the change of limb angles during the movement of the horse,and improves the stability of the horse movement and reduces the risk of injury.
Effect of Non-Genetic Factors on Population Life of Holstein Dairy Cows in Xinjiang
ZHAO Fanfan, WANG Dan, MA Yanjun, MA Xinbing, GE Jianjun, ZHANG Xiaoxue, ZHANG Menghua, XU Lei, JIANG Hui, SANG Zhagen, HUANG Xixia
2020, 47(3):  822-827.  doi:10.16431/j.cnki.1671-7236.2020.03.021
Abstract ( 183 )   PDF (663KB) ( 90 )  
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In order to explore the influence of various factors on the life span of Holstein dairy cows and understand the utilization of dairy cows,the outlier and performance data of 4 057 Holstein dairy cows in large-scale cattle farms in Changji area of Xinjiang from 2011 to 2018 were collected in this study.The effects of field,year,season and cull type on the stray parity and herd life in Holstein cattle were analyzed by SAS 9.2 software,and the least square and multiple comparative analysis were carried out.The results showed that both the year and the type of cull had a extremely significant effect on the number of stray parity and the herd life in the group (P<0.01),and the effect of the field and the season of strays on the number of stray parity and the herd life in the group was not significant (P>0.05).With the increase of stray years,the number of extraneous fetuses decreased first,then increased and then decreased.The number of stray parity in 2016 was the highest (3.222 births),which was higher than that in other years (P<0.01);The number of the stray parity was the lowest in 2018,and it was extremely significantly lower than that of the other years (P<0.01);In 2015,the herd life in the group was higher than that of other years.In 2018,the herd life in the group was the lowest,and it was 1 890.692 days,and was extremely significantly lower than that of other years (P<0.01);According to the seasons,the stray parity in spring was lower than that in other seasons,and the herd life in the group was lower than that in autumn and winter (P<0.01;P<0.05).The stray parity and herd life in-group days in the cull type of limb-hoof disease was extremely significantly higher than other kinds of diseases (P<0.01),and the breast disease was extremely significantly lower than other kinds of diseases (P<0.01).Therefore,by analyzing the influence of various factors on the stray parity and herd life in-group days,it could provide a theoretical basis for the daily management of dairy cows in large-scale pastures,so as to improve the comprehensive use efficiency of dairy cows and prolong their life.
Preventive Veterinary Medicine
Construction of Swinepox Virus TK,ORF121 and ORF143 Genes Deletion Strains and Determination of Their Biological Characteristics
LIU Changjin, DENG Shunzhou, LUO Feng, ZHONG Luohua, WANG Zhe, GAO Yingxue, LIU Xiaolan
2020, 47(3):  828-836.  doi:10.16431/j.cnki.1671-7236.2020.03.022
Abstract ( 243 )   PDF (3630KB) ( 95 )  
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In order to screen the replication non-essential region of swinepox virus (SWPV) and determine the biological characteristics of the deletion strains,primers were designed and synthesized for TK(ORF063),ORF121 and ORF143 genes of SWPV according to the principle of homologous recombination,and the overlap extension PCR technology was used to splice the gene fragments containing enhanced green fluorescent protein (EGFP) and homologous recombination of the left and right arms.The spliced fragments were transfected into PK15 cells infected with SWPV,respectively.Fluorescence microscope was used to select single plaque with EGFP as the reporter gene,and the recombinant strains were identified by PCR and Western blotting.The plaque size of PK15 cells and the pathogenicity of pigs were measured.The results showed that the gene fragment successfully integrated into recombinant strain genome.These recombinant strains were named as rSWPV-TK,rSWPV-121 and rSWPV-143,respectively.The results of Western blotting showed that the recombinant strains stably expressed the foreign protein on PK15 cells.The formation of pox spots on PK15 cells was smaller than that of the parent strain,and the difference of the ORF121 gene deletion strain was extremely significant (P<0.01).All the strains could lead to the formation of pox spots on nursery pigs,in which the ORF121 gene deletion strain caused the lesion for a short time,produced less pox spots,and the virulence was lower than that of wild strains.In summary,in this study,two non-essential replication regions ORF121 and ORF143 in the SWPV genome for foreign protein insertion were selected,laying a foundation for the construction of SWPV gene engineering vectors.
Research Advances on Drug Prevention and Control Technology for Porcine Respiratory Disease Complex
ZHU Minjuan, MI Kun, PU Shanju, XIE Shuyu, YUAN Zonghui, HUANG Lingli
2020, 47(3):  837-847.  doi:10.16431/j.cnki.1671-7236.2020.03.023
Abstract ( 295 )   PDF (1312KB) ( 188 )  
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Porcine respiratory disease complex (PRDC) is widespread in pig industry,which is the result of a combination of bacteria,viruses,parasites,bad breeding administration conditions and so on,it has caused slow growth and death of pigs so that resulted in huge economic losses.Actinobacillus pleuropneumoniae (APP),Haemophilus parasuis (Hps) and Streptococcus suis (SS) are the main bacterial pathogens,while porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus virus type 2 (PCV2) and pseudorabies virus (PRV) are common viral pathogens.Cefquinome,florfenicol and gammamycin can effectively prevent and control bacterial infections because of broad antibacterial spectrum,strong antibacterial activity and excellent pharmacokinetic characteristics in pigs,and for viral infections the commonly used drugs are cytokines and traditional Chinese medicines,especially traditional Chinese medicines,which not only can resist viruses but also enhance the body's immunity has a very broad application prospect.This article systematically expounds the antibacterial mechanism,pharmacodynamics (PD) and pharmacokinetics (PK) of the above antibacterial drugs,at the same time,the antiviral mechanism and application of the above antiviral drugs are introduced in detail.Through this review in order to provide some suggestions for the rational use of drugs to prevent and control PRDC.
Isolation Identification and Virulence Gene Analysis of a Grass Carp Aeromonas veronii
ZHANG Piao, HU Andong, YANG Xia, PAN Jimai, JIANG Haibo, WEN Ming
2020, 47(3):  848-855.  doi:10.16431/j.cnki.1671-7236.2020.03.024
Abstract ( 252 )   PDF (1762KB) ( 58 )  
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In order to identify the pathogenic factors for the occurrence and death of grass carp of a farm in Qingong county,Guizhou province,this study identified the pathogens through clinical anatomical observation,bacterial isolation and culture,gram staining microscopic examination,biochemical identification,drug sensitivity test,animal regression test,16S rDNA and gyr B gene sequencing and systematic analysis.The results showed that a pathogenic bacteria strain GZQG2019 was isolated from the pathogenic grass carp.The isolated bacteria presented a round,moist and smooth surface with neat and translucent edges in the culture medium,and showed a beta hemolysis ring around the colony.Gram's microscopic examination showed that the isolated bacteria were obtuse short negative bacilli at both ends.The 16S rDNA and gyr B gene sequence analysis showed that the isolates and Aeromonas veronii converged into one branch,and the homology was more than 99%.The results of drug sensitivity test showed that among the 20 antibiotics,the bacteria was sensitive to 10 drugs,such as tetracycline,butylamicarine,polycolistin B and ceftriaxone,resistant to 2 drugs,such as cefalexin and compound xinomin,and moderately sensitive to 8 drugs,such as benzacillin,penicillin,amoxicillin and gentamicin.The results of animal regression showed that the bacteria was highly pathogenic to healthy grass carp.The amplification results of 10 virulence genes showed that the isolated bacteria carried 7 virulence genes including aerolysin,heat-unstable enterotoxin,hemolysin,ribozyme,serine protease,elastase and esterase.In this study,a strain of Aeromonas veronii from grass carp was successfully isolated with strong virulence and multiple virulence factors.The drug sensitivity test results provided a reference for clinical drug use of Aeromonas veronii with sensitivity to tetracycline and butanecanil and resistance to tetracycline and butanecanil.
Effects of Lactic Acid Bacteria Preparation on Salmonella Typhimurium Infection of Broiler Chickens
ZHENG Jiangping, LIU Ning, SHANG Yueli, BAI Xuerui
2020, 47(3):  856-863.  doi:10.16431/j.cnki.1671-7236.2020.03.025
Abstract ( 234 )   PDF (821KB) ( 238 )  
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This study was aimed to investigate the effects of lactic acid bacteria (LAB) preparationon on Salmonella Typhimurium (ST) infection of broiler chickens.A total of 480 one-day old AA male broiler chickens without ST infection were randomly allotted into 4 groups with 6 replicates per group and 20 birds per replicate.The treatments included uninfection (a basal diet),ST infection,and based on the infection,supplemented with antibiotic enrofloxacin HCl or LAB at 40 mg/kg or 3×109 CFU/kg,respectively.The LAB contained Lactobacillus plantarum,Lactobacillus acidophilus and Enterococcusfaecium combined at an equal amount.At 5 days old in infected groups the chickens were orally inoculated with 1 mL (105 CFU) ST bacterial fluid.At 7,14 and 21 days old,2 birds per replicate were randomly selected and killed for sampling.The results showed that ST infection decreased average daily feed intake(ADFI) and average daily gain(ADG) of broilers during 1-21 days old,while LAB supplementation compensated the growth performance (P<0.05),and reached the levels of antibiotic and uninfected groups.In terms of organ index and bacterial loads in tissues,LAB exhibited a good control effect on broilers with ST infection (P<0.05).At 14 and 21 day olds,the content of IL-10 in LAB and antibiotic groups were significantly higher than that of uninfected group (P<0.05).At 21 days old,the content of IL-10 in LAB group was significantly lower than that of infected group (P<0.05),the contents of IgG and SIgA were significantly increased (P<0.05).There was no significant difference of the contents of IL-10 and SIgA between LAB and antibiotic groups (P>0.05),but the content of IgG in LAB group was significantly lower than that of antibiotic group (P<0.05).In conclusion,dietary LAB supplementation could depress gut ST proliferation and tissue infection,alleviated inflammation,and improved immunity and growth performance in broilers.
Construction of Brucella abortus A19 VirB Promoter Deletion Strain and Study on Its Biological Characteristics
DENG Xiaoyu, HE Jinke, YANG Qin, YI Jihai, WANG Yueli, XI Jing, WANG Zhen, CHEN Chuangfu
2020, 47(3):  864-872.  doi:10.16431/j.cnki.1671-7236.2020.03.026
Abstract ( 236 )   PDF (2328KB) ( 85 )  
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In order to explore the effects of VirB gene on the virulence of Brucella abortus A19,and learn more about the mechanism of Brucella intracellular survival,in this study,Brucella abortus A19 was used as a template to construct a suicide plasmid using the upstream and downstream homologous arms of VirB gene to fuse the kana resistance gene.The suicide plasmid was electroporated into the cells by means of an instantaneous electric shock,and the VirB promoter was replaced with kana gene by homologous recombination to construct an A19ΔVirB deletion strain.The transcription level of the VirB-associated protein of the deletion strain was detected by Real-time PCR,and the growth curve,in vitro stress,adhesion invasion,and intracellular survival of the deletion strain were analyzed.The results showed that the A19ΔVirB deletion strain was successfully constructed.Real-time PCR results showed that the expression of the VirB-associated protein of the deleted strain was extremely significantly lower than that of A19 strain (P<0.01).Although the growth curve was different from the parent strain,the growth trend was the same.The results of in vitro stress showed that there was no significant change in A19 and A19ΔVirB heat shock stress,but the survival rate of A19 was significantly higher than A19ΔVirB under the stimulation of strong acid,alkali and salt (P<0.05).The results of adhesion and invasion showed that the adhesion and invasion ability of the deletion strain to macrophages was similar to that of the parent strain.The results of intracellular survival showed that the intracellular survival trend of A19 gradually increased with time,while the overall trend of the deletion strain A19ΔVirB gradually decreased and the gap with A19 was larger.In summary,this study successfully constructed the VirB promoter deletion strain,which significantly reduced the expression of related proteins,and laid the foundation for the construction of subsequent related deletion strains and the study of Brucella virulence.
Sequence Analysis of HA and NA Genes of Two H9N2 Subtype Avian Influenza Virus in Yunnan
LI Jiajia, LI Suhua, SONG Jianling, HUANG Jingjun, MIAO Yuan, CHEN Xi, JI Jia
2020, 47(3):  873-883.  doi:10.16431/j.cnki.1671-7236.2020.03.027
Abstract ( 208 )   PDF (2646KB) ( 74 )  
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Based on the detection of H9 subtype avian influenza in 57 chicken farms in Yunnan province from January to June 2019,two H9 subtype avian influenza positive samples from Shilin and Chuxiong were selected for virus isolation.Total RNA was extracted from the isolated embryonic allantoic fluid of H9N2 subtype avian influenza virus,and HA and NA genes were amplified by RT-PCR with specific primers.The PCR products were purified and sequenced.Sequence alignment and phylogenetic analysis showed that the nucleotide sequence homology of HA and NA genes for two H9N2 of Yunnan strain in 2019 was 94.2% and 93.6%,respectively.Phylogenetic analysis showed that HA and NA genes of Yunnan H9N2 subtype avian influenza virus in 2019 belonged to ADKHKY28097 branch (Y280-like) of Eurasian lineage.The homology of HA gene of ACKYN12019 and ACKYN72019 was 94.3%,and the highest homology with the reference strain ACKJX2448 was 95.6% to 98.5%,the low homology with the popular H9N2 representative strains and vaccine strains in China.The cleavage site of HA protein 333-340 was PSRSSR↓GLF,which had the molecular characteristics of low pathogenicity avian influenza virus.The receptor binding sites all had mutations of E198T and Q234L,and had the characteristics of human receptor binding.There were 6 glycosylation sites at 29,141,298,305,313 and 492 amino acids.The homology of NA gene of ACKYN12019 and ACKYN72019 was 93.6%,and the homology with the representative strain of Y280-like were 97.1% to 97.5% and 93.7% to 94.6%,respectively.There were 6 potential glycosylation sites at 44,69,86,146,200 and 234 amino acids deleted in NA protein,and 368-369,399-403 were found by analysis of RBC binding sites of NA protein.There were variations in amino acids at position 432.The results showed that H9N2 subtype avian influenza virus had been mutating continuously,so its monitoring and control should be strengthened.
The Effect of Lactobacillus reuteri LR1 on the Immune Response of Swine Common Virus Vaccine
FENG Junsen, XIONG Yunxia, WU Qiwen, WANG Li, JIANG Zongyong, YI Hongbo
2020, 47(3):  884-891.  doi:10.16431/j.cnki.1671-7236.2020.03.028
Abstract ( 251 )   PDF (830KB) ( 84 )  
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In order to study the effect of Lactobacillus reuteri LR1 isolated earlier on immunological effect of swine common viral vaccines,the whole process of Lactobacillus reuteri LR1 was used instead of feed antibiotics.A total of 144 weaned pigs (21 days old,Duroc×Landrace×Large White) were selected and randomly divided into 3 groups with 8 replicates in each group and 6 pigs in each replicate.The control group (CON) was fed basic diet,and the antibiotic group (OA) was fed basic diet +100 mg/kg olaquindox +75 mg/kg aureomycin,and the probiotics group (LR1) was fed basic diet +5×1010 CFU/kg Lactobacillus reuteri LR1.The use of antibiotics was stopped at 120 d,and the experimental period was 166 d.All pigs were immunized according to the unified immunization procedure.The antibody levels of porcine reproductive and respiratory syndrome virus (PRRSV),Hog cholera virus (HCV),foot-and-mouth disease virus (FMDV),pseudorabies virus (PRV),porcine parvovirus (PPV) and the level of IgG were detected in the serum of pigs at the 14th,42nd,120th,127th,134th and 166th days after the test.The results showed that,compared with the control group and the antibiotic group,the antibody level of FMDV in the serum of probiotics group on the 14th day was significantly increased (P<0.05).Compared with the control group,the antibody level of PPV in the serum of probiotics group and antibiotic group on the 120th day were significantly increased (P<0.05).The antibody level of PRRSV in the serum of the probiotics group on the 166th day was significantly higher than that of the control group (P<0.05).The antibody level of HCV in the serum of the probiotics group on the 166th day was significantly higher than those of the control group and the antibiotic group (P<0.05).In conclusion,dietary Lactobacillus reuteri LR1 supplementation could improve the antibody levels of common viruses in commercial pigs to a certain extent.These data suggested that Lactobacillus reuteri LR1 can be used to develop new substitutes for antibiotic,and has a great potential in the prevention and treatment of diseases during replacing dietary antibiotics.
Basic Veterinary Medicine
Preparation and Application Analysis of Canine Procalcitonin Protein Polyclonal Antibody
WANG Dong, LIU Ying, ZHANG Kang, ZHANG Hongyan, SU Feng, MA Weiming
2020, 47(3):  892-901.  doi:10.16431/j.cnki.1671-7236.2020.03.029
Abstract ( 258 )   PDF (3111KB) ( 70 )  
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The aim of this study was to obtain high-purity canine procalcitonin recombinant protein and evaluate its clinical application.PCT gene was cloned into prokaryotic expression pET-30a(+) vector using the method of E.coli expressing foreign protein.After identified by enzyme digestion and sequencing,the recombinant plasmid was transferred into E.coli BL21(DE3) and induced by 1 mmol/L IPTG for protein.The recombinant protein was purified by nickel column,and polyclonal antibody was prepared by immunizing mice with purified protein.The antibody titer and specificity of procalcitonin were detected by indirect ELISA and Western blotting,respectively.The serum PCT in healthy and inflammatory dogs were detected by ELISA method.Enzyme digestion and sequencing tests confirmed canine PCT gene was successfully inserted into pET-30a(+).SDS-PAGE result showed that canine PCT protein was soluble expressed with a molecular weight of 14.9 ku.PCT antiserum titer was evaluated by indirect ELISA method and the best titer was 1:51 200.The specificity of polyantibody was checked by Western blotting,and the serum exhibited high affinity to PCT protein.The ELISA result showed the PCT in serum had positive correlation with the degree of inflammation.The results indicated that canine PCT protein polyclonal antibody was successfully prepared,and canine serum PCT protein was a potential new indicator for serious bacterial inflammation infection.
Study on the Pathogenicity of Escherichia coli from Cattle to Caenorhabditis elegans
BAI Huili, PENG Hao, LI Jun, LI Changting, TAO Li, CHEN Zhongwei, WU Cuilan, PAN Yan, ZENG Yun, GONG Yu
2020, 47(3):  902-910.  doi:10.16431/j.cnki.1671-7236.2020.03.030
Abstract ( 277 )   PDF (3163KB) ( 100 )  
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In order to study the pathogenicity of Escherichia coli (E.coli)to Caenorhabditis elegans (C.elegans),13 strains of E.coli were isolated from cattle disease materials collected in Nanning,Guilin,Liuzhou and Guangxi in this study.Then,the E. coli were subjected to serological identification,mouse pathogenicity test and C.elegans pathogenicity test.The O serotype of E.coli were identified by slide agglutination and the 13 E.coli bacterial liquids were intraperitoneally injected into the mice at a concentration of 3.0×109 CFU/mL and 0.2 mL/(10 g·BW).At the same time,these 13 strains of E. coli were infected with N2 wild type C.elegans.The results showed that the O serotypes of these E. coli were O127,O126 and O44,of which O126 was the dominant serotype (4/13).The pathogenicity results showed that 11 strains of E. coli could kill mice (40% to 100%),and 2 strains of E. coli were not lethal to mice (the mortality rates were 0). E.coli,which had strong pathogenicity to mice,also had a high lethal rate to nematodes,and the mortality rate were most significant on the 3rd to 6th day.The median lethal time was 3 to 4.5 d,and the longest survival time was 9 d.Two strains of E.coli which were not lethal to mice also had a low lethal rate to nematodes,the mortality of nematodes decreased slowly,the half lethal time was 5 to 6 d,and the longest survival time was 10 to 11 d.The results of intestinal bacterial count showed that the number of E.coli in the worm was linear with time,and E.coli constantly destroyed the nematode's immune system,which led to the death of nematodes.The results of this study indicated that the pathogenicity test of E.coli was consistent with the results of mouse pathogenicity test,indicating that the pathogenic model of E.coli-C.elegans was successfully established and laid the theoretical foundation for prevention and clinical use of bovine disease.
The Prevention and Treatment of a Chinese Herbal Medicine Compound for Salmonellosis in Chickens
LI Zhengtian, DOU Tengfei, LI Qihua, GE Changrong, JIA Junjing, ZHANG Xi
2020, 47(3):  911-921.  doi:10.16431/j.cnki.1671-7236.2020.03.031
Abstract ( 244 )   PDF (1155KB) ( 129 )  
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In order to evaluate the effect of Chinese herbal medicine compound on the prevention and treatment of S.Enteritidis in chicken,a compound of Chinese herbal medicine complex consisting of 23 Chinese herbal medicines such as Coptidis rhizome,wintergreen,and white-headed medicinal herbs was selected for in vitro and in vivo antibacterial experiments in broiler chicken.The antibacterial diameter of the Chinese herbal medicine complex for the in vivo bacteriostatic experiment was 17.4 mm,indicating that the S.Enteritidis was highly sensitive to it.In in vitro antimicrobial experiment,low (0.1%),medium (0.3%),and high (0.5%) dose of the Chinese herbal medicine complex were used.After orally-infected with S.Enteritidis to the broiler,compared with the control group,the survival rates increased extremely significantly (P<0.01) and the feed to gain ratio (F/G) decreased significantly in three experimental groups (P<0.05).The average daily weight gain (ADG) of the middle and high dose groups were significantly higher (P<0.05),and the average daily feed intake (ADFI) of the middle dose group was significantly lower (P<0.05).The spleen index,white blood cell count (WBC),platelet count (PLT),IgA,IgG,IgM,and complement C4 levels in the three experimental groups were significantly lower (P<0.05),while the thymus,bursa of Fabricius indexes,red blood cell count (RBC),hemoglobin concentration (HGB),hematocrit (HCT),and platelet count (PCT) were significantly higher (P<0.05).The results of Real-time quantitative PCR showed that the gene expression levels of antibacterial peptide gene cathelicidins (CATH1,CATH2,CATH3) in spleen,thymus and bursa of Fabricius in three treatment groups were higher than that in control group.Among Chinese herbal medicine doses,the meddle dose (0.3%) had the best control effect.From above mentioned results,it was concluded that the combination of Chinese herbal medicine in the diet can reduce the mortality of chickens infected with S.Enteritidis,enhance growth performance,immunity and antibacterial activity of the chickens against S.Enteritidis.
The Effect of Formononetin on the Immune Function in Immunosuppressed Mice
ZHANG Yanping, DENG Kang, JIA Ning, FANG Mei, HUANG Weikuan, ZHANG Jianan, LIU Sheng
2020, 47(3):  922-930.  doi:10.16431/j.cnki.1671-7236.2020.03.032
Abstract ( 223 )   PDF (12316KB) ( 24 )  
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To investigate the effect of formononetin on the immune function in immunosuppressed mice,100 Kunming mice were randomly divided into five groups (20 in per group,half male and half female):normal control group,immunosuppression group,test groupsⅠ,Ⅱ,and Ⅲ,and the medicines were consecutively given to mice by means of intragastric perfusion.The experimental period was 28 d.The mice in immunosuppression group,test groupsⅠ,Ⅱ,and Ⅲ were all given cyclophosphamide (CTX) with 40 μg/(g·BW) for 1-7 d.From 8 to 21 d,the mice in test groupsⅠ,Ⅱ,and Ⅲ were given the solution of formononetin with 50,150 and 250 μg/(g·BW),respectively.While the other mice were given 0.6 mL physiological saline.After the test,thymus index,spleen index,serum hemolysin and the content of IL-2,IL-4 in serum were detected.The surface receptors CD3,CD20 of lymphocyte and the surface receptors CD68 of KCs were measured by immunohistochemistry.The results showed that the immunosuppressed mice were copied successfully with cyclophosphamid,and the formononetin could increase the serum hemolysin,thymus index,spleen index,the content of IL-2 and IL-4 in serum in immunosuppressed mice,and the differences between the immunosuppression group and test group Ⅱ were extremely significant (P<0.01).Immunohistochemical analysis showed that the formononetin could increase CD3 positive T lymphocytes and CD20 positive B lymphocytes significantly in thymus and spleen of immunosuppressed mice,and encourage the proliferation of KCs cells in the liver of immunosuppressed mice,and the differences between the immunosuppression group and test group Ⅱ were also extremely significant (P<0.01).The results suggested that formononetin could improve the immune function of immunosuppressed mice by promoting the proliferation of lymphocytes and releasing the cytokines,and promote obviously the proliferation of KCs in liver.
Investigation on the Spread of Resistance-related Genes in Broiler Farms
CHEN Xia, CHE Jie, LUO Pengjie, ZHANG Yunfei, ZHAO Xiaofei, YUAN Min, BAI Xuemei, LI Wenge, LI Juan
2020, 47(3):  931-939.  doi:10.16431/j.cnki.1671-7236.2020.03.033
Abstract ( 185 )   PDF (1159KB) ( 79 )  
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To investigate resistance genes and their spread related genes in broilers,totally 273 broiler fecal samples were collected by random sampling from two broiler farms in Hebei province.A total of eight kinds of resistance genes and two kinds of spread related genes including blaNDM,blaOXA,blaCTX-M,armA,fexA,cfr,mcr-1,qnrS,and int1,ISCR1 which closely related to the spread of resistance gene,were detected by Real-time PCR,respectively.According to the results,all of the genes were existed in the colaca swab samples with positive rate 4.03%-97.07%.The aminoglycoside-resistance gene,armA,showed the lowest positive rate,and resistance gene spread related gene,int1,showed the highest positive rate.More than two (including two) kinds of resistance genes were detected in 273 of samples.Three kinds of resistance genes co-existing was the most common.Four samples harbored only one resistance gene,and six harbored none.No sample harbored eight kinds of resistance genes.There were 41 resistance gene type profiles through data analysis.The common resistance gene type profiles included blaCTX-M-fexA-mcr-1 and blaOXA-blaCTX-M-fexA-cfr-mcr-1-qnrS.Based on statistics analysis,the more resistance genes detected,the higher int1/ISCR1 gene positive rate exhibited in samples which carried more than two (including two) kinds of resistance genes.The coexistence of resistance genes blaOXA,blaCTX-M,fexA,cfr,mcr-1,qnrS with int1 gene showed statistical significance (P<0.05),respectively.And,the coexistence of resistance genes blaNDM,blaOXA,blaCTX-M,fexA,cfr,mcr-1,qnrS with ISCR1 gene showed statistical significance (P<0.05),respectively.This study provided new Real-time PCR methods for drug resistance surveillance of animal husbandry and veterinary realms,and gave data support to drug resistance prevention and control in future.
Synergistic Effect of Combination of Chinese Veterinary Drugs and Antibiotics on the Prevention and Treatment of Respiratory Diseases in Broilers
FENG Haipeng, XIN Ruihua, ZHANG Kai, WANG Lei, ZHANG Kang, LI Jianxi, WANG Xuezhi
2020, 47(3):  940-947.  doi:10.16431/j.cnki.1671-7236.2020.03.034
Abstract ( 274 )   PDF (5332KB) ( 61 )  
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In order to explore the synergistic effect of the combination of Chinese veterinary drugs and antibiotics on the prevention and treatment of respiratory diseases in broilers,100 three-day-old White-feather broilers were randomly selected from two chicken houses in large-scale chicken farms,which were divided into two groups (group Ⅰ and group Ⅱ).Group Ⅰ was given antibiotics in drinking water.Group Ⅱ was fed with Chinese veterinary drug Yupingfeng oral liquid (3-7 d),Shegan Dilong granule (10-16 d),Shuanghuanglian oral liquid (22-25 d),Maxingshigan oral liquid (27-31 d) in different stages on the basis of adding antibiotics.Chicken infectious bronchitis live vaccine,Newcastle disease-infectious bronchitis combined live vaccine and Newcastle disease-avian influenza two inactivated vaccine were vaccinated at 8-day-old.Serum and tracheal tissues were collected at 1,7,14,21 and 28 days after immunization to detect antibody levels of Newcastle disease,avian influenza (H9),and infectious bronchitis vaccines,observe pathological changes of tracheal tissues,and calculate the immune organ index and average daily gain,and record the incidence rate.The results showed that the survival rate of group Ⅱ increased by 1.23% compared with group Ⅰ.The final weight and average daily weight of group Ⅱ were extremely significantly higher than that of group Ⅰ (P<0.01),which increased by 561.25 g and 16.63 g/d,respectively.The respiratory symptoms of group Ⅱ were lighter than that of group Ⅰ,the course of disease was short,and there were no obvious lesions in liver and heart.The spleen index was significantly higher than that of group Ⅰ at 36 d (P<0.05).At 21 days after immunization,the antibody levels of Newcastle disease,infectious bronchitis and avian influenza (H9) vaccine in group Ⅱ reached the highest level,which was significantly higher than group Ⅰ (P<0.05).The level of Newcastle disease antibody in group Ⅱ was extremely significantly higher than that of group Ⅰ(P<0.01) at 28 days after immunization.In summary,the combination of four kinds of Chinese veterinary drugs,which clearing heat-toxin,relieving cough and asthma,clearing liver and gallbladder,combined with antibiotic had synergistic effect on promoting growth and prevention of respiratory diseases in broilers.
Advances on Risk Assessment of Tetracycline Antibiotics in the Environment
XU Xiangyue, MA Wenjin, AN Boyu, CHENG Guyue, HUANG Lingli
2020, 47(3):  948-957.  doi:10.16431/j.cnki.1671-7236.2020.03.035
Abstract ( 348 )   PDF (1011KB) ( 471 )  
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Tetracycline antibiotics (TCs) are the most widely used in veterinary medicine,which is due to factors such as their low cost,broad spectrum and high antimicrobial activity.Due to their poor absorption when administered to animals,most of the doses of TCs are excreted as original (not metabolized) compounds in feces and urine.In this study,exposure status,adsorption and degradation behavior and effect assessment of TCs were reviewed.The results showed that TCs were exposed to a large amount in the environment,with strong adsorption capacity and weak migration capacity.Microorganisms,light and temperature affected their stability.The exposure in the environment had a negative impact on plant,aquatic organisms,microbial community structure and quantity and resistance genes.By summarizing the current situation of environmental risk assessment of TCs,people should pay more attention to the pollution of TCs in the environment,which will provide reference for the removal of TCs and resistance genes in the environment.Consequently,it is urgent to carry out a systematic research on environmental risk assessment of TCs.
Clinical Veterinary Medicine
Improvement of Culture Method of Primary Bovine Endometrial Epithelial Cells in Vitro
WANG Shiyu, LI Botong, LIU Jiawei, NI Yaodi, LIU Mingchao
2020, 47(3):  958-964.  doi:10.16431/j.cnki.1671-7236.2020.03.036
Abstract ( 243 )   PDF (3890KB) ( 165 )  
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At present,it has become a common method to study bovine endometritis by using the in vitro culture technology of bovine endometrial epithelial cells to establish a model of bovine endometritis so as to reduce uncontrollable factors.Thus obtaining high purity and identical endometrial epithelial cells are the key link in in vitro research.The methods used at home and abroad are mainly enzymatic digestion,tissue block adherence and tissue block digestion and adherence.In this paper,the previous method was modified to establish a simple and easy-to-use technique for in vitro culture of primary bovine endometrial epithelial cells with short cell culture period and good cell activity and morphology.The healthy non-pregnant cow uterus was used as the test material.The endometrial tissue was removed,and 5 mL of DMEM/F12 medium containing 2% double antibody and 40% trypsin was added,and digested at 4 ℃ for 12 h.The tissue was transferred to a 25 cm2 cell culture flask for adherent culture.After obtaining the primary cells,the cells were subjected to immunohistochemical fluorescence identification using keratin-18 antibody,and the CCK-8 method was used for the third generation cells.The detection wavelength was 450 nm,and the cell growth was drawn according to the D450 nm values at different time points curve.The results showed that the primary cells were basically covered with the bottom of the cell culture flask on the 8th day,which effectively shortened the cycle of the conventional tissue block adherence method.By keratin-18 immunohistochemical fluorescence identification,the positive rate of primary epithelial cells was more than 98%.Compared with the conventional tissue block adherence method,the purity of primary epithelial cells was significantly improved,and the steps of cell purification were omitted.The proliferation of the cells was in line with the normal characteristics of division and growth.The results showed that the conventional tissue block attachment method was optimized and improved,which not only shortened the culture period,but also improved the purity and maintained the cell activity,which provided a certain reference for the improvement of the in vitro culture technique of the primary bovine endometrial epithelial cells.
Research Progress on Oncolytic Viruses in Canine Tumor Treatment
REN Xiaoli, FAN Yuying, JIN Shuangxing, ZHANG Lei, LIU Yun, SHI Dongmei
2020, 47(3):  965-971.  doi:10.16431/j.cnki.1671-7236.2020.03.037
Abstract ( 238 )   PDF (749KB) ( 126 )  
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Oncolytic virotherapy is a promising new biological therapy for the treatment of malignant tumors using viruses.The oncolytic virus not only directly infects and dissolves tumor cells,but also stimulates the immune response at the tumor site,to indirectly dissolve the tumor tissue cells,and has no killing or wake killing effect on normal tissue cells,thus achieving satisfying anti-tumor effect.In this paper,the development history of oncolytic virus and the mechanism of tumor ablation mediated by oncolytic virus were reviewed.Then we analyzed the killing effect on various canine tumor cells in vitro,in xenotransplantation tumor animal model and in dogs with tumor of eight oncolytic viruses including measles virus,canine distemper virus,Newcastle disease virus and Sendai virus and so on.Some encouraging results suggested that oncolytic virotherapy might become a safe and reliable new method for the treatment of malignant tumors.However,in order to apply oncolytic virotherapy in the clinical treatment of canine tumors,many problems still need to be solved,such as how to ensure the specificity of the virus,and how to determine the dose of the virus.In summary,this article provided new ideas and technical approaches for the safe use of oncolytic viruses in the clinical treatment of canine tumors.