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20 August 2018, Volume 45 Issue 8
Expression and Identification of H Gene of CDV BJ16B2 Strain in Insect Baculovirus Expression System
CHEN Meirong, LIN Weidong, SONG Tianqi, XIN Ting, GUO Xiaoyu, HUANG Kehe, JIA Hong
2018, 45(8):  2041-2048.  doi:10.16431/j.cnki.1671-7236.2018.08.001
Abstract ( 285 )   PDF (2159KB) ( 257 )  
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To obtain the native conformation and antigenicity of hemagglutinin (H) protein of canine distemper virus (CDV) virulent strain,the H gene which referred to BJ16B2 strain was accurately amplified by RT-PCR and cloned into pFastBacTMHTA vector.The recombinant plasmid was confirmed by restriction enzyme digestion and DNA sequencing,H gene was analyzed and the recombinant plasmid was transformed into E.coli DH10BacTM competent cell,and constructed shuttle vector,which was transfected into Sf9 cell to save baculovirus expressing H protein.To determine the antigenicity of recombinant protein,Western blotting and IFA analysis were used to detect the interesting protein.The results demonstrated that H gene belongs to Asia-1 virulent strain,and the homology of nucleotide and amino acid among the reference strains was 89.8% to 99.3% and 89.6%to 99.5%,respectively.The fusion protein generated by recombinant baculovirus was reacted with both His-tag monoclonal antibodies and CDV positive serum by Western blotting test,and presented specific green fluorescence in IFA test (68 ku),which showed that this protein had sufficient antigenicity.The resluts served as a strong foundation for the the subsequent analysis of protein structure and antigenicity differences,the preparation of monoclonal antibodies and the development of CDV subunit vaccine.

Eukaryotic Expression Vector Construction and Tissue Expression Profile Analysis of GPR1 Gene in Luchuan Pig
XIE Wan, HE Jianxiong, XIA Panjie, WU Yanjun, JIAO Di, CHEN Baojian, GUAN Zhihui, LAN Ganqiu, GUO Yafen, XIE Bingkun
2018, 45(8):  2049-2056.  doi:10.16431/j.cnki.1671-7236.2018.08.002
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This study was aimed to construct the eukaryotic expression vector of G protein-coupled receptor 1 (GPR1) gene in Luchuan pigs,and analyze its tissue expression profile.The CDS region of GPR1 gene was amplified by RT-PCR from the subcutaneous adipose tissue of 10-week-old Luchuan pig,and the eukaryotic expression vector (pEGFP-N1-GPR1) containing the GPR1 gene fragment was constructed.After identification of the recombinant plasmid pEGFP-N1-GPR1 by double digestion and sequencing,it was transfected into 3T3-L1 cells by lipofectamine.Moreover,the fluorescence expression of 3T3-L1 cells was observed by fluorescence microscope after 24 hours and transfected 3T3-L1 cells were collected to extract total RNA.Meanwhile,the expression of GPR1 eukaryotic expression vector was further detected by Real-time PCR.Total RNA was extracted from heart,liver,spleen,lung,kidney,longissimus dorsi and subcutaneous fat of six 10-week-old Luchuan pigs to detect the expression of GPR1 gene mRNA in these tissues.The results indicated that the CDS sequence of GPR1 gene in Luchuan pig was 1 068 bp.The recombinant plasmid pEGFP-N1-GPR1 and the empty vector pEGFP-N1 transfected 3T3-L1 cells all showed green fluorescence,while the blank control group did not display green fluorescence.Real-time PCR result confirmed that the expression level of GPR1 gene in experimental group was extremely significantly higher than that of control group (P<0.01).The expression level of GPR1 gene in liver of 10-week-old Luchuan pig was the highest,and followed by lung,kidney,subcutaneous fat,heart and spleen,but was almost absent in longissimus dorsi.In conclusion,recombined vector pEGFP-N1-GPR1 was successfully constructed,and the tissue expression profile of GPR1 gene was appeared,which laid a foundation for further research about the molecular effect of GPR1 gene on fat deposition in Luchuan pig.

Molecular Characteristics and Cluster of PPRV Guizhou Strains Based on N Gene
WANG Jun, YANG Yuan, ZHANG Yundan, WEN Ming, ZHOU Bijun, CHENG Zhentao, YUN Jun, LI Tao
2018, 45(8):  2057-2066.  doi:10.16431/j.cnki.1671-7236.2018.08.003
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In order to investigate the N gene molecular characteristics and clustering of the peste des petits ruminants virus (PPRV) of Guizhou province,the primers of PPRV N gene were designed and the target gene was amplified from clinical samples and then was cloned to pMD19-T vector.The positive recombinant plasmids were sequenced and nucleotide homologoy, amino acids homology,mutation sites and phylogenetic tree analysis of N gene of Guizhou PPRV strains and reference strains were performed by DANStar software.The results showed that the length of amplified N gene was 1 578 bp and the homology of nucleotide and amino acid sequences among Guizhou PPRV strains were 99.6% to 100.0% and 99.2% to 100.0%,respectively.The homology of nucleotide (97.7% to 99.9%) and amino acids (98.3% to 100.0%) compared with domestic reference strains were higher than those of other countries (88.5% to 97.7% and 92.2% to 98.5%).Compared with the vaccine strain Nigeria 75-1,there were 26 multiple sites in N gene of Guizhou PPRV strains while there was no amino acid loss or increase.The results of phylogenetic analysis showed that Guizhou PPRV strains were in the same evolutionary branch with the domestic reference strains,while the foreign reference strains in a different evolutionary branch.Guizhou PPRV strains belonged to Ⅳ gene group and vaccine strain Nigeria 75-1 belonged to Ⅰ gene group.

Cloning, Expression and Bioinformatics Analysis of sodA Gene of Pasteurella multocida
AN Qi, HUANG Haifeng, ZHANG Zhenxing, LI Baobao, ZHANG Mengmeng, ZHENG Yiying, WANG Chengqiang, ZHANG Luyin, YANG Xiaojian, CAO Ruiyong, NIE Xin, DU Li, WANG Fengyang
2018, 45(8):  2067-2075.  doi:10.16431/j.cnki.1671-7236.2018.08.004
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To investigate the biological functions of superoxide dismutase (SOD) of Pasteurella multocida,the sodA gene was cloned,expressed and analyzed by phylogenetic and bioinformatics,respectively.A pair of primers was designed based on the sodA gene sequence of Pasteurella multocida HN06 strain in GenBank.The PCR products were ligated with pET-28a(+) vector to construct pET-28a(+)-sodA recombinant plasmids.The recombinant plasmids were transformed into E.coli DH5α competent cells for cloning and then transformed them into E.coli BL21(DE3) competent cells for expression.The expressed proteins were induced by IPTG and identified by SDS-PAGE and Western blotting.The results showed that the target fragment with the length of 645 bp was successfully amplified and the target protein which was 28 ku was successfully expressed.Phylogenetic analysis showed that the gene was closely related to the HN07 (GenBank accession No.:CP007040.1) and Pm70 (GenBank accession No.:AE004439.1) strains.Bioinformatics analysis results of the recombinant protein showed that it was a stable acidic hydrophilic soluble fusion protein.The molecular formula and molecular mass were C1085H1651N293O309S9 and 24 032.36 u;The theoretical isoelectric point,extinction coefficient and instability coefficient were 6.19,45 170 and 26.87 (<40),repectively;The half-life of reticulocytes in mammals was estimated to be 30 h;The aliphatic index and total average hydrophobicity (GRAVY) were 82.15 and -0.282,respectively;And the secondary structure was mainly composed of α-helix and random coil.The above results provided references for the further study of the survival mechanism of Pasteurella multocida and the development of a vaccine against pasteurellosis.

Cloning of N-terminal and C-terminal of Pig BLM Gene and Its Expression Analysis in Different Tissues and Diseases
WANG Sainan, XU Houqiang, ZHAO Jiafu
2018, 45(8):  2076-2085.  doi:10.16431/j.cnki.1671-7236.2018.08.005
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In order to investigate the structure and function of N-terminal and C-terminal of pig BLM gene and its relative expression in different tissues and diseases,the N-terminal and C-terminal of BLM gene from Congjiang Xiang pig were amplified by RT-PCR method in this study,the sequence of clones was analyzed using bioinformatics softwares.At the same time,the expression of BLM gene in different tissues and diseases of pig was analyzed by Real-time quantitative PCR.The results showed that the N-terminal (1 025 bp) and C-terminal (1 300 bp) sequences of BLM gene from Congjiang Xiang pig were successfully cloned,and the sequence homology with GenBank (accession No.:NM_001123084.1) was 100%;The bioinformatics prediction results showed that the sequence length of pig BLM gene was 4 316 bp,including an open reading frame of 4 280 bp,and the corresponding polypeptide was consisted of 1 426 amino acids.Amino acid sequence comparison result showed that the amino acid sequence of pig BLM gene was the highest homology with cow (85%).The phylogenetic tree analysis showed that the pig BLM helicase had four genetic domains of DEXDc,HELICc,RQC and HRDC;Phylogenetic tree analysis showed that the BLM gene relationship between pig and bovine was the closest;Real-time quantitative PCR result showed that compared with the heart tissue,the expression of BLM gene in Congjiang Xiang pig was detected in all tissues,there was the highest relative expression in testis,followed by the tissues of lung,liver,spleen,heart,kidney,small intestine and large intestine.The expression levels in testis and lung were extremely significantly higher than those in other tissues (P<0.01).The expression of BLM gene in lung of porcine reproductive and respiratory syndrome,liver of chicken leukemia and quail duck was extremely significantly or significantly higher than that in normal tissues (P<0.01;P<0.05).The difference indicated that the change in BLM helicase might also lead to animal diseases.The results provided a theoretical basis for construction of prokaryotic expression vector of livestock and poultry BLM helicase and their occurrence and prevention in major diseases.

Establishment and Application of A Double TaqMan MGB Real-time PCR Assay for Detection of Canine Distemper Virus and Canine Parvovirus
ZHAO Xueli, LIU Yi, BAN Fuguo, YAN Ruoqian, WANG Huajun, WANG Shujuan, MA Zhenyuan, WANG Dongfang
2018, 45(8):  2086-2094.  doi:10.16431/j.cnki.1671-7236.2018.08.006
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This study was aimed to establish a double TaqMan MGB Real-time PCR assay to simultaneously and specifically detect canine distemper virus (CDV) and canine parvovirus (CPV) in one reaction.Two pairs of specific primers for CDV and CPV,along with two TaqMan MGB probes for each virus were designed in the assay basing on CDV H gene and CPV VP2 gene sequences.The specificity,sensitivity and repetition of the double TaqMan MGB Real-time PCR assay were tested,and 48 samples taken from clinic suspicious CDV and CPV infected canines had been testified by the established double TaqMan MGB Real-time PCR.The results indicated that the doulde TaqMan MGB Real-time PCR assay was successfully established,and the number of standard curve correlation (R2) of CDV and CPV were 0.997 and 0.993,respectively.The specificity of the double TaqMan MGB Real-time PCR assay revealed that amplifications were showed on CDV and CPV samples,but other pathogens and negative controls had no amplifications;The sensitivity of CDV and CPV were both 10 copies/μL.Meanwhile,14 CDV positive samples,19 CPV positive samples and 4 CDV/CPV double positive samples were detected,which were consistent with the results of the sequencing.Therefore,the established double TaqMan MGB Real-time PCR assay had high sensitivity,specificity and flux accurate quantitative,which could be applied to clinical CDV/CPV infection each periods.

Cloning and Molecular Characterization Analysis of E2 Gene of Classical Swine Fever Virus
LUO Dandan, LIAO Dangjin, YE Yonggang
2018, 45(8):  2095-2103.  doi:10.16431/j.cnki.1671-7236.2018.08.007
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To study the genetic variations of classical swine fever virus (CSFV) E2 gene in Sichuan province,we used RT-PCR method to amplify E2 gene and then cloned into pMD18-T vector,and strongly confirmed by PCR amplification,restriction digestion and sequencing,then the CSFV E2 gene was analyzed by bioinformatics softwares.The results showed that the CSFV E2 gene was 1 125 bp,and encoded a polypeptide of 375 amino acid residues.Moreover,the nucleobtide sequence of CSFV E2 had higher homology with its homologous protein of other CSFV strains GXWZ02,CSFV/2.1,HEBZ,YC11WB,SXYL2006 and 0406/CH/01/TWN in GenBank,the homologies were 98.3%,96.5%,94.7%,92.4%,92.1% and 90.9%,respectively,but it had lower homology with HCLV E2 gene (87.6%).The ENc value of E2 gene was 53.161,and there were distinctly differences of 33,25 and 25 codons frequency comparing CSFV E2 with E.coli,yeast and human,respectively.Besides,there were total 34 rare codons in CSFV E2 gene and serial rare codons being strung together.The results suggested that E2 gene was not obvious in codon preference,and was closest to eukaryotes in codon usage pattern.The nucleotides of E2 gene was partially mutated compared to vaccine strains,but the main antigenic domain didn't change.The results provided basic data for the further study of CSFV E2 function and the development of genetic engineering seedlings at later stages.

Cloning and Bioinformatics Analysis of EDC3 Gene from Marc145 Cells
LI Changhong, WEN Guilan, ZHANG Shengbo, CHEN Shaopin, LIN Hanqing, XU Li, GUAN Guodan, WANG Desheng, JI Xinqin, WEN Ming, ZHOU Bijun, CHENG Zhentao
2018, 45(8):  2104-2112.  doi:10.16431/j.cnki.1671-7236.2018.08.008
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In order to obtain the sequence of EDC3 gene from Marc145 cell,and analyze the sequence characteristics of EDC3 gene and the structure and function of EDC3 encoding protein,EDC3 gene from Marc145 cells was amplified and cloned by RT-PCR method.The nucleotide and amino acid sequences of EDC3 gene were analyzed by DNAStar software,the homology was analyzed by BLAST,and the phylogenetic tree was constructed.The secondary and tertiary structures,B cell epitopes,transmembrane domain and signal peptide of EDC3 protein were predicted by bioinformatics softwares.The results showed that the length of EDC3 gene was 1 527 bp,which encoded 507 amino acids.The CDS sequence and amino acid sequence of EDC3 gene from Marc145 cell shared 91.5% to 99.2% and 95.3% to 99.6% identity with Chlorocebus sabaeus,Sus scrofa,Pan paniscus,Papio anubis,Macaca mulatta,Homo sapiens,Orcinus orca,Ovis aries,Ailuropoda melanoleuca,Equus caballus and Loxodonta africana,respectively,the shared lowest homology was 81.2% and 88.8% with Gallus gallus.The results of phylogenetic tree showed that EDC3 gene from Marc145 cell was closely relative with Chlorocebus sabaeus,followed by primates.The alpha helix and radom coil were dominant in secondary and tertiary structures,which was 23.38% and 47.35%,respectively.The results predicted that the protein had multiple B cell dominant antigen epitopes,and there was no transmembrane domain and signal peptide region.The results might provide references for further research on the function of EDC3 gene from Marc145 cell.

Construction and Eukaryotic Expression of Recombinant Adenovirus Shuttle Plasmid of Mycoplasma suis ENO Gene
WANG Miao, NI Tingting, WU Shengjun, ZHAO Yun, SONG Jianchen, DONG Liang, XUE Shujiang
2018, 45(8):  2113-2118.  doi:10.16431/j.cnki.1671-7236.2018.08.009
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To construct recombinant adenovirus shuttle plasmid of ENO gene of Mycoplasma suis and express it in eukaryotic cells,a pair of primers was designed and synthesized according to the ENO gene sequence of Mycoplasma suis in GenBank (accession No.:CP002525.1) in this study.ENO gene was amplified by PCR method,and the products were cloned into the pMD19-T vector.The pMD19T-ENO plasmid was digested with Kpn Ⅰ and Xho Ⅰ, and then subcloned into the adenovirus shuttle vector PCR259.The PCR259-ENO plasmid was constructed and then identified by PCR and enzyme digestion.The PCR259-ENO recombinant shuttle plasmid was transfected into 293 cells by liposome-mediated transfection,and the expression of ENO gene in 293 cells was detected by IFTA technology.The results showed that the cloned ENO gene was 1 182 bp in length and encoded 393 amino acids with a homology of 99% with the ENO gene sequence in GenBank (accession No.:CP002525.1).The constructed recombinant adenovirus shuttle plasmid PCR259-ENO was identified by PCR and restriction enzyme digestion correctly, and was able to be expressed in 293 cells,indicating that ENO gene was successfully inserted into adenovirus shuttle plasmid PCR259 and the recombinant shuttle plasmid was successfully constructed.

Effects of SNAT2 on Amino Acid-stimulated Milk Protein and Milk Fat Synthesis in Bovine Mammary Epithelial Cells
MENG Chunyu, HUANG Xin, LIU Lijie, CHEN Dongying, GAO Xuejun
2018, 45(8):  2119-2127.  doi:10.16431/j.cnki.1671-7236.2018.08.010
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This experiment was aimed to investigate the effect of amino acid transporter the sodium-dependent neutral amino acid transporter 2 (SNAT2) on amino acid-stimulated milk protein and milk fat synthesis in bovine mammary epithelial cell (BMEC).After stimulating BMEC with methionine (Met),lysine (Lys) and leucine (Leu),the expression level of SNAT2 and β-casein gene was detected by Real-time quantitative PCR and Western blotting,and the secretion of triglyceride in culture supernatant was detected using BMEC triglyceride kit.The N-flag-SNAT2 eukaryotic expression vector and SNAT2 siRNA were transfected into the cells for SNAT2 overexpression and knockdown experiments,respectively.The expression level of SNAT2,β-casein,the mammalian target of rapamycin (mTOR),and the content of triglyceride in the culture supernatant of BMEC were detected by Western blotting and triglyceride kit,respectively.The results showed that all three amino acids (Met,Lys and Leu) could significantly increase the content of milk protein and milk fat secreted by BMEC and activate the mTOR signaling pathway.Met and Lys also up-regulated significantly the expression of SNAT2 gene.SNAT2 regulated positively the synthesis of milk protein and milk fat in BMEC,and activated the mTOR signaling pathway,indicating that amino acid activation of the mTOR signaling pathway was mediated through SNAT2,which in turn regulated BMEC milk protein and milk fat synthesis.

Research Progress on Effect of γ-aminobutyric Acid on LPS-mediated Inflammation to the Body
SUN Shiping, ZHANG Chengyu, LI Heping, XU Chunmei, CHEN Peige, ZHONG Kai, WANG Yueying
2018, 45(8):  2128-2134.  doi:10.16431/j.cnki.1671-7236.2018.08.011
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γ-aminobutyric acid (GABA) is an important neurotransmitter in mammals,it is mainly distributed in the central nervous system,with a wide range of regulatory functions.GABA can participate in the regulation of body homeostasis through the hypothalamic-pituitary-adrenocortical (HPA) axis,and it has a certain mitigation effect on the inflammatory response caused by harmful stimuli outside.The body will causes the increase of blood lipopolysaccharide (LPS) content under the stimulation of external stress and activate the Toll-like receptor 4 (TLR4)/NF-κB signaling pathway,resulting in the increase of the expression of inflammatory factors such as tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6) and interleukin-8 (IL-8),eventually leading to the body's inflammatory response.GABA can reduce the damage by regulating the expression of the corresponding inflammatory cytokines to the organism when the inflammatory reaction occurs in the body.This article provides a brief overview of the physiological effect of GABA,the types of GABA receptors,and the function of GABA in the inflammatory response to the body,analyzes the effect of GABA on inflammatory response in the body and its relationship with LPS-induced inflammatory response,which provide a theoretical basis for the clinical use of GABA in the treatment of inflammatory response.

Nutritional Value of Cassava and Its By-products and Their Application in Animal Production
CUI Yiyan, TIAN Zhimei, LI Zhenming, MA Xianyong
2018, 45(8):  2135-2146.  doi:10.16431/j.cnki.1671-7236.2018.08.012
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Cassava is resistant to drought and easy to manage.Its stem,leaf,root and industrial by-products are rich with nutrients,which is a good non-conventional feed.The contents of crude protein,crude fat,ash,Ca,Fe, Zn and Mn in cassava leaves are rich.Compared with cassava leaves,cassava roots have a lower level of crude protein,crude fat,minerals and vitamins that is a good source of energy.Cassava residues are mainly composed of the skin and broken cells wall of cassava,which have very low content of crude protein,but rich with crude fiber,neutral detergent fiber,acid detergent fiber,mineral and amino acids.Ca,P and amino acids in cassava residue are imbalanced.The species,growth conditions,harvest methods,harvest time,processing technology will affect the nutrients contents of cassava and its by-products.Cassava contains cyanide,tannin,phytic acid and other anti-nutritive factors.Cyanide is the most important factor limiting cassava applications.Cyanide can bind cytochrome C oxidase,block the mitochondrial electron transport chain,inhibit aerobic respiration,and cause central nervous system depression and death.Tannin and phytic acid can combine with protein,which hinder the digestion and absorption of nutrients.Cassava can be detoxified by the physical and chemical methods,such as boiling, baking and drying,as well as fermentation,breeding and so on.Cassava and its by-products can be widely used in animal production, and have no adverse effects on animal's growth performance and meat quality.The development and utilization of unconventional feed is beneficial to alleviate the shortage of feed resources and reduce the cost of breeding.The nutritional value,anti-nutritional factors and detoxification methods,as well as its application in animal production of cassava and its by-product were introduced in order to provide theoretical basis and references for the comprehensive utilization of cassava in livestock and poultry breeding.

Study on Alkali Storage Effect of Spent Mushroom (Hypsizygus marmoreus) Substrate
XU Jincong, MIAO Jing, HE Xiangbo, HUANG Dingrui, GAN Qianfu, LIANG Xuewu
2018, 45(8):  2147-2156.  doi:10.16431/j.cnki.1671-7236.2018.08.013
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In order to study the effect of alkali storage on spent mushroom substrate,CaO,Ca (OH)2,CaO+3% urea and Ca(OH)2+3% urea were adopted in the experiment.Each treatment of alkali (CaO/Ca(OH)2) had four levels (3%,4%,5% and 6% additive amount),that was named CO3,CO4,CO5 and CO6;CH3,CH4,CH5 and CH6;COU3,COU4,COU5 and COU6;CHU3,CHU4,CHU5 and CHU6,with 4 replicates per treatment.There were 2 blank groups,one group (CK group) was only spent mushroom substrate,the other one (CKU) was added 3% urea in spent mushroom substrate.Alkaline storage were fermented for 30 d at room temperature.The results showed that compared with the CK group,the content of ADF and free gossypol in CO3,CO4,CO5 and CO6 groups decreased 8.61%,12.69%,13.74%,16.63% and 29.58%,33.92%,39.04%,41.31%;The contents of NDF and ADL in CO5 and CO6 groups were significantly decreased,while the CB2 and RFV were significantly increased (P<0.05).Compared with the CK group,the content of ADF and free gossypol in CH3,CH4,CH5 and CH6 groups were decreased 7.30%,10.90%,13.90%,17.14% and 28.47%,32.69%,40.48%,43.59%,respectively;The contents of NDF and ADL in CH5 and CH6 groups were significant decreased,while the CB2 and RFV were significant increased (P<0.05).Compared with the CKU group,the contents of NDF,ADF and free gossypol in COU3,COU4,COU5 and COU6 groups decreased 6.72%,8.86%, 9.78%,12.10% and 14.63%,16.37%,18.19%,20.21% and 5.49%,14.24%,23.63%,23.66%;RFV increased 18.97%,23.19%,25.86%,30.79%,respectively.Compared with the CKU group,the contents of NDF,ADF,ADL and free gossypol in CHU3,CHU4,CHU5 and CHU6 groups decreased 8.34%,11.87%,14.08%,16.97% and 11.94%,15.19%,18.21%,19.06% and 7.40%,14.93%,19.47%,19.30% and 6.84%,15.60%,22.39%,24.10%,RFV was increased 18.88%,26.20%,34.00%,37.88%, respectively.The effect of CaO and Ca(OH)2 alkali storage was similar.Adding 3% urea alkali to storage much more better than adding CaO or Ca(OH)2,and it could be preserved for a long time.

Study on Metabolizable Energy, Crude Protein, Ca and Available P Contents in Black Muscovy Ducks Aged from 22 to 42 Days
ZHANG Ling, SUN Guobo, DUAN Xiujun, GU Wenjie, WANG Jian, QIN Haorong, GE Yuzhu, ZHANG Kai
2018, 45(8):  2157-2166.  doi:10.16431/j.cnki.1671-7236.2018.08.014
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In order to obtain the adequate contents of metabolic energy,crude protein,calcium and available phosphorus in diet of Black Muscovy duck aged from 22 to 42 days,288 22-day-old healthy Black Muscovy ducks were chosen and separated to 9 groups, with 4 repeats per group and 8 ducks per repeat,and L9 (34) design was selected to engage in orthogonal experiment of four factors at three levels.The ducks were fed 9 experimental diets of B1 to B9 with different levels of ME (11.92,12.32,12.72 MJ/kg),CP(16%,18%,20%),Ca(0.6%,0.8%,1.0%) and available P (0.30%,0.45%,0.60%).The growth performance,slaughter performance and serum biochemical indexes of the ducks were detected by feeding and slaughter test.The result showed that the nutritional advantages of B3 group were more obvious in weight gain and slaughter performance, the average final weight, average daily gain and F/G of B3 group were 1 556.64 g,46.43 g/d and 2.39,respectively,of which the average final weight was significantly higher than that of B1 and B9 groups (P<0.05).The slaughter rate,semi-eviscerated rate,eviscerated rate and leg muscle rate of ducks in B3 group were 73.43%,64.81%, 55.26% and 26.56%,respectively, showing a good growth and development trend.The result of body size measurements and serum biochemical indexes indicated the nutrition level and feed formula of B3 group was the best.Therefore,the recommend nutrient levels of diet for 22 to 42 days old Black Muscovy duck were metabolic energy 11.92 MJ/kg,crude protein 19.25%,calcium 1.03% and effective phosphorus 0.60% under the presented experiment condition.

Prediction of Dairy Cow Production Performance by in vitro Fermentation Technology
YANG Jinshan, YUE Kuizhong, LIU Xin, NIKITA, ZHANG Guangning, LIU Yan, ZHANG Yonggen
2018, 45(8):  2167-2174.  doi:10.16431/j.cnki.1671-7236.2018.08.015
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The object of this experiment was to predict the production performance of dairy cows by in vitro fermentation parameters of total mixed ration (TMR).The rumen fluid that was collected from two Holstein dairy cows with 550 kg body weight and permanent fistula,and 40 kinds of TMR were collected and used in the in vitro fermentation experiment,the contents of acetic acid,propionic acid,butyric acid, NH3-N,CH4,MCP,IVDMD,IVCPD and IVOMD of fermented liquid were determined when fermented for 24 h.The production performance of dairy cows fed TMR was recorded (milk yield,milk fat rate and milk protein rate),and the prediction model was established between them and the production performance of dairy cows.The results showed as follows:①Milk production and propionic acid content (r=0.37,P<0.05),MCP (r=0.40,P<0.05) were significantly positively correlated.However,it was extremely significantly negatively correlated with the IVCPD (r=-0.44,P<0.01).There was a significantly or extremely significantly positive correlation between milk fat rate and acetic acid content (r=0.55,P<0.01),CH4 emission(r=0.36,P<0.05),MCP(r=0.40,P<0.05).There was an extremely significantly positive correlation between milk protein rate and MCP (r=0.91,P<0.01),IVDMD (r=0.44,P<0.01),IVCPD (r=0.45,P<0.01).However,it was extremely negatively correlated with the IVOMD (r=-0.56,P<0.01).②The production performance of Holstein dairy cows could be predicted using in vitro fermentation parameters.Milk production (kg/d)=29.72-0.86IVCPD (R2=0.73,RSD2=47.26,P<0.001).Milk fat rate (%)=0.40+0.06AA (R2=0.91,RSD2=0.15,P<0.001).Milk protein rate (%)=1.52+0.06MCP (R2=0.78,RSD2=0.01,P<0.001).The results showed the error between the predicted value and the actual value of production performance was within the allowable range,indicating that the equations were feasible.

Effects of L-theanine on Growth Performance and Blood Biochemical Indicators of Oxidative Stress Weaned Piglets
YAN Jun, LI Zeqing, WANG Wenjie, LIU Tong, MA Yanxuan, LI Ning, MA Yong, ZHENG Zi, MU Shuqin
2018, 45(8):  2175-2181.  doi:10.16431/j.cnki.1671-7236.2018.08.016
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To explore the effects of L-theanine on growth performance,antioxidant and immune response of oxidative stress weaned pigs,a two-factor (2×2) experimental design was employed.160 weaned pigs (7.53 kg±0.51 kg) were divided into 4 treatment groups:Treatment 1 (without L-theanine,intraperitoneal injection of 5 mL normal saline),treatment 2(without L-theanine,intraperitoneal injection of 8 mg/(kg·BW) diquat),treatment 3(diet added 1 000 mg/kg L-theanine,intraperitoneal injection of 5 mL normal saline) and treatment 4 (diet added 1 000 mg/kg L-theanine,intraperitoneal injection of 8 mg/(kg·BW) diquat).7 d for preliminary feeding and 28 d for formal feeding.The results showed that:① Compared to treatment 1,the average daily gain (ADG) and average daily feed intake (ADFI) of weaned pigs in treatment 2 decreased significantly (P<0.05),while F/G increased significantly (P<0.05);Compared to treatment 2,L-theanine significantly increased ADG and ADFI in treatment 4 (P<0.05).②Compared to treatment 1,diquat significantly increased serum malondialdehyde (MDA) content,while the total antioxidant capacity (T-AOC) and the activity of glutathione peroxidase (GSH-Px) significantly decreased in treatment 2 (P<0.05),serum MDA content in treatment 3 significantly increased while the activity of GSH-Px significantly tended down (P<0.05);Compared to treatment 2,L-theanine significantly decreased serum MDA content while increased the activity of T-AOC and GSH-Px in treatment 4 (P<0.05).③Compared to treatment 1,the levels of serum IgA,IgG and IL-2,IL-4 significantly increased,while C3 and C4 significantly decreased (P<0.05) in treatment 2,and the levels of serum IgA,IgG and IL-4 were significantly decreased,while the concentration of C3 and C4 significantly increased in treatment 3 (P<0.05);Compared to treatment 2,the levels of serum IgA,IgG and IL-2,IL-4 significantly decreased,while C3 significantly increased in treatment 4 (P<0.05).In conclusion,injection of diquat could cause oxidative stress in piglets,resulting decrease of growth performance and humoral immunity;Diets added 1 000 mg/kg L-theanine could relieve oxidative damage,improve the growth performance,the capacity of antioxidation and humoral immunity in oxidative stressed piglets.

Effects of Enterococcus faecium on Antioxidant Function in Serum and Liver of Arbor Acres Broilers
MAO Junzhou, DONG Li, WANG Shunan, PENG Zhong, ZHONG Zhaoxin, YU Lihuai
2018, 45(8):  2182-2189.  doi:10.16431/j.cnki.1671-7236.2018.08.017
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The experiment was aimed to investigate the effects of dietary Enterococcus faecium on antioxidant capacity in serum and liver of Arbor Acres (AA) broilers.A total of 600 AA broilers (roosters) with 1-day-old were randomly allocated into 5 groups with 6 replicates per group.Treatments in control group (CON group) were fed a basal diet,antibiotic group (Ant group) was fed a basal diet supplemented with 0.1% chlorotetracycline,the three groups (LEF,MEF and HEF groups) were fed the basal diet supplemented with 50,100 and 200 mg/kg Enterococcus faecium,respectively.The results showed that:① At 21 days of age,the activity of total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px) activity in serum of HEF group were significantly higher than that of CON group (P<0.05),T-AOC in the serum of Ant group was significantly higher than that of CON group (P<0.05).At 42 days of age,the contents of malondialdehyde (MDA) in serum of MEF and HEF groups were significantly lower than that of CON group (P<0.05);The activity of superoxide dismutase (SOD) in serum of LEF group was significantly higher than that of CON and Ant groups (P<0.05).② At 21 days of age,the activity of GSH-Px and T-AOC in MEF and HEF groups were significantly higher than that of CON group (P<0.05),and T-AOC in liver of HEF group was significantly higher than that of Ant group (P<0.05).At 42 days of age,the activity of CAT in liver of HEF group was significantly higher than that of CON group (P<0.05).The results showed that the diet added Enterococcus faecium could significantly increase the activity of GSH-Px and T-AOC and decrease the content of MDA in serum and liver,increase the activity of antioxidant enzyme,improve anti-oxidative stress ability of broiler,and the basal diet supplemented with 200 mg/kg Enterococcus faecium was comparable to that of antibiotics in improving the antioxidant capacity of serum and liver.

Effects of Nutrient Level During Late Pregnancy on Reproductive Performance and Blood Biochemical Indexes of Primiparous Sows
CHEN Ling, HUANG Dapeng, WEI Guosheng
2018, 45(8):  2190-2196.  doi:10.16431/j.cnki.1671-7236.2018.08.018
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To study the effects of nutrient level during late pregnancy on the body condition, reproductive performance and blood biochemical indexes of primariparous sows,36 sows with similar weight and backfat thickness were chosen and randomly divided into 3 groups (groups Ⅰ,Ⅱ and Ⅲ) according to the body condition and breeding time with 6 replicates per group and 2 sows per replicate.The pregnant sows in groups Ⅰ,Ⅱ and Ⅲ were fed the diets with 1.5, 2.0 and 2.5 times as much as maintenance requirement.The test began at 81 d after pregnancy,and end with the parturition,which last for 33 d.The results showed that the backfat thickness of sows in group Ⅰ was significantly lower than that in group Ⅲ (P<0.05).There was no significant difference in backfat thickness between all groups at weaning (P>0.05).The loss of backfat of lactating sows in group Ⅰ during the lactation period was significantly lower than that in group Ⅲ (P<0.05).The body weight after delivery and body weight before delivery in group Ⅲ were significantly higher than that in group Ⅰ (P<0.05).There was no significant difference of body weight at 81 days after pregnancy and body weight loss during lactation among all groups (P>0.05).There were no significant difference of litter size,number of piglets born alive per litter,average body weight at weaning and weaning-oestrus interval among all groups (P>0.05).The average birth weight,average litter weight at weaning and litter weight gain during lactation of group Ⅲ were significantly higher than those in group Ⅰ (P<0.05).The level of estrogen in the groups Ⅱ and Ⅲ was significantly higher than that of group Ⅰ (P<0.05),and there was no significant difference of prolactin and progesterone levels among all groups (P>0.05).The triglyceride level in group Ⅰ was significantly lower than that in groups Ⅱ and Ⅲ (P<0.05).The content of urea nitrogen and glucose in each test group increased in turn with the increase of nutrient level and the difference was significant (P<0.05).The content of insulin in group Ⅲ was significantly higher than that in group Ⅰ (P<0.05).The total protein content of each group was not significant different (P>0.05).The results showed that the feeding level of sows was 2.0 times as much as maintenance requirement in the later stage of pregnancy could make the body condition reach the standard level,while the nutritional level was 2.5 times as much as maintenance requirement could raise the performance of the sows farrowing.

The Effect of Compound Plant Essential Oil on LPS-induced Immune-stressed Weaned Pigs
XU Haiwang, YU Kui, WU Mengjun, LIU Wenkai, LI Peng, YI Dan, WANG Lei, HOU Yongqing
2018, 45(8):  2197-2203.  doi:10.16431/j.cnki.1671-7236.2018.08.019
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The purpose of this research was to study the effect of OCT on the immune stress of weaned piglets induced by LPS.A total of 24 weaned piglets (Duroc×Large white×Landrace) were randomly assigned to three treatments at 28 days:Control group (based diet,injection of saline),LPS group (basic diet,injection of LPS) and OCT group (basal diet+50 mg/kg OCT,injection of LPS).Each treatment included 8 piglets that were subdivided equally to 8 pens,each pen had 1 piglets.On 21th day of the experiment,piglets in the LPS and OCT groups were injected intraperitoneally with LPS (100 μg/(kg·BW)),whereas piglets in the control group were injected intraperitoneally with the same volume of physiological saline.Blood samples were obtained 3 h after injection for analysis of white blood cell differential counts.6 h after LPS challenged,the pigs were slaughtered and thymus and spleen were taken for measuring the related genes IL-4,IL-6,IL-10,TNF-α,NF-κB,and STAT3 mRNA.The result showed that:①Compared with control group,LPS stimulation could lead to a significant reduction in the number of the eosinophils,neutrophils,lymphocytes,monocytes,white blood cells in the blood (P<0.05).Compared with LPS group,adding OCT could significantly increase blood levels of white blood cells (P<0.05).②Compared with control group,after injection of LPS,mRNA levels of IL-6 and STAT3 genes significantly increased in spleen and thymus (P<0.05);mRNA levels of IL-4,TNF-α in spleen and IL-4,TNF-α,NF-κB genes mRNA levels in thymus had been significantly reducted (P<0.05).Compared with LPS group,adding OCT could significantly reduce IL-6 and STAT3 mRNA levels in both spleen and thymus (P<0.05),and significantly increase IL-4,IL-10,TNF-α and NF-κB genes mRNA levels in thymus (P<0.05).In conclusion,LPS could induce immune stress in weaned pigs,whereas OCT could improve its immunity and alleviate inflammation to some extent.

Effects of Different Addition Amounts of Portulaca oleracea L.Silage on Lactation Performance of Dairy Goats
LIANG Qi, GAO Zhanjun, YU Zhu, XU Qingfang, CHENG Long, SONG Wenqi, GAO Pan, PAN Xiaoyan
2018, 45(8):  2204-2211.  doi:10.16431/j.cnki.1671-7236.2018.08.020
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The study was aimed to explore the effects of different addition amounts of Portulaca oleracea L.silage on lactation performance of dairy goats.12 healthy dairy goats with (62±3) kg body weight,(1.90±0.15)kg/d milk yield and similar lactation period (0 to 60 d postpartum) were chosen and randomly divided into 4 groups with 3 replicates per group and 1 dairy goat per replicate.The dairy goats were fed with total mixed rotation (TMR) adding 0 (CK group),10 (group A),20 (group B) and 30 g/d DM (group C) Portulaca oleracea L.silage,respectively.The trial period was 75 d,of which the pre-test period was 15 d and the trial period was 60 d.The dry matter intake,milk yield,milk quality and blood biochemical indexes were determined.The results showed that the amount of dry matter intake of dairy goats increased with the increase of Portulaca oleracea L.silage adding amount.The milk yield in Portulaca oleracea L.silage adding groups were improved compared with control group.The pH range of goat milk was 7.10 to 7.24,showing a downward trend with the increase of additive,and CK group was highest while the group C was lowest.The content of lactose,TPC,TFC and β-carotene in the milk increased with the increase of Portulaca oleracea L.silage dosage,while the total cholesterol content in the serum showed the opposite trend.In conclusion,there was no adverse effect on dairy goats by adding Portulaca oleracea L. silage in TMR, which could increase the feed intake of dairy goats,milk yield and milk quality,meanwhile decrease the total cholesterol.

Effect of Glucose Oxidase on Growth Performance and Serum Biochemical Indexes of Piglets
MU Shuqin, LI Ning, YAN Jun, ZHENG Zi, MA Yong, LI Qianjun, ZHANG Chunhua
2018, 45(8):  2212-2218.  doi:10.16431/j.cnki.1671-7236.2018.08.021
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This study was conducted to elucidate the effects of glucose oxidase (GOD) on growth performance and serum biochemical indicator of piglets.Sixty piglets were randomized in three groups,control group (basal diet),experimental group 1(basal diet added 100 g/t olaquindox,75 g/t aureomycin,60 g/t oxytetracycline calcium) and experimental group 2 (basal diet added 400 g/t GOD).Each group had four replicates and each replicate had five piglets.The experiment lasted 30 d.The growth performance during the experiment and the serum biochemical indexes at the ending of the experiment were measured.The results showed that:For the pigs in experimental group 2,the final body weight,ADG,the level of IL-2,IL-4,IgA,lgG,IgM and TNF-α were all significantly higher (P<0.05),while the ADFI,F/G and surem AST activity were significantly lower (P<0.05) than that in control group and experimeatal group 1;The serum level of MDA was significantly lower than that in control group (P<0.05),while the IFN-γ level was significantly higher than control group (P<0.05);The other serum biochemical indexes,such as the activities of ALT,GSH-Px and SOD,the level of TP,ALB and GLB,and T-AOC had no significantly change(P>0.05).In conclusion,adding 400 g/t GOD in diets could improve the growth performance and antioxidant and immune ability,alleviate liver damage of piglets.

Effects of Compound Probiotics and Antibiotics on Growth Performance, Intestinal Flora and Immune Function of Broilers
YU Jiamin, CHE Zhen, QI Xiuye, XIE Quanxi, ZHENG Junhong, LIU Xuejiang, GU Wei, SHAN Baolong
2018, 45(8):  2219-2226.  doi:10.16431/j.cnki.1671-7236.2018.08.022
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This trial was conducted to evaluate the effects of compound probiotics and antibiotics on growth performance,intestinal microflora and immune function of broilers.Six hundred one-day-old female broiler chickens were randomly divided into 5 groups with 6 replicates per group and 20 broilers per replicate.The broilers in groupⅠ were fed basal diet,and the basal diet+1.0‰ T3(compound probiotics),the basal diet+20 g/t colistin sulphate,the basal diet+50 g/t bacitracin zinc,the basal diet+50 g/t bacitracin zinc+10 g/t colistin sulphate were provided to the broilers in group Ⅱ,Ⅲ,Ⅳ and Ⅴ,respectively.The pre-trial period lasted for 3 days,and the experiment lasted for 35 days.The results showed as follows:Compared with group Ⅰ,Ⅲ,Ⅳ and Ⅴ,the average daily gain (ADG) at each trial stage,the number of Lactobacillus in cecal and rectal were significantly increased (P<0.05),while the feed gain ratio (F/G),the number of Escherichia coli in cecal and rectal were significantly decreased in T3 group.Compared with group Ⅰ,Ⅲ,Ⅳ and Ⅴ,the spleen index of the group treated with T3 increased 27.44%,40.27%,29.01% and 29.81% (P<0.05);The bursa of Fabricius index increased significantly by 12.82%,37.50%,33.33% and 38.95%,respectively (P<0.05);The content of serum endotoxin in T3 group was lower than that of group Ⅰ(P>0.05) and group Ⅲ (24.77%, P<0.05).Compared with the groupⅠ and Ⅲ,the content of secreted immunoglobulin A (sIgA) in T3 group was significantly increased by 32.37% and 25.79%(P<0.05).In conclusion,adding 1.0‰ T3 could improve the growth performance and intestinal flora,promote the development of spleen and bursa in broilers.And the role of increasing the content of sIgA in cecal contents,decreasing the content of serum endotoxin of 1.0‰ T3 was better than the use of 20 g/t of clostridium sulfate.

Effects of Dietary Energy and Crude Protein Levels on Growth Performance, Serum Biochemical Indexes and Slaughter Performance of Graylag
YUAN Yingliang, TANG Dan, LU Ying, RAN Guixia
2018, 45(8):  2227-2235.  doi:10.16431/j.cnki.1671-7236.2018.08.023
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This experiment was conducted to study the effects of dietary energy and crude protein levels on growth performance,serum biochemical indexes and slaughter performance of graylag to provide theoretical foundation for scientific feeding.A double factors experimental design was used with metabolic energy (ME) levels of 11.0,12.0,13.0 MJ/kg and crude protein (CP) levels of 14.0%,15.0%,16.0%,respectively.Four hundred and fifty 28-day-old graylags were randomly assigned into 9 groups with 5 replicates per group and 10 graylags per replicate.Graylags in each group were fed one kind of diet.The experiment lasted for 92 days including 12-day pretest period and 80-day formal test period.The results showed as follows:①The average daily gain of graylag with metabolic energy level of 12.0 MJ/kg was significantly higher than those with metabolic energy level of 11.0 and 13.0 MJ/kg (P<0.05);The average daily gain with crude protein level of 14.0% was significantly lower than those with crude protein level of 15.0% (P<0.05).②As the metabolic energy level reached 13.0 MJ/kg,the serum triglyceride of graylag was significantly lower than those with metabolic energy level of 11.0 MJ/kg (P<0.05);The uric nitrogen of graylag with crude protein level of 16.0% was significantly higher than those with crude protein level of 14.0% (P<0.05).③As the metabolic energy level reached 11.0 MJ/kg,the semi-eviscerated percentage, eviscerated percentage,breast muscle percentage and leg muscle percentage of graylag were lower than those with metabolic energy level of 12.0 and 13.0 MJ/kg;As the metabolic energy level reached 13.0 MJ/kg,the abdomen fat percentage was significantly higher than those with metabolic energy level of 11.0 and 12.0 MJ/kg (P<0.05);As the crude protein level was 14.0%,the breast muscle percentage and leg muscle percentage was lower than those with crude protein level of 15.0% and 16.0%.In conclusion, the dietary metabolic energy level had significant effects on final weight,abdominal fat percentage,triglyceride of graylags,and excessive metabolic energy level could increase abdominal fat percentage,which resulted in higher feed cost.The crude protein level had significant effects on final weight and uric nitrogen of graylag.It was suggested that the optimal metabolic energy should be 12.0 MJ/kg and crude protein should be 15.0% for 5 to 16-week-old graylags.

Genetic Polymorphism Analysis of BMP15 and GDF9 Genes in Six Sheep Breeds
WU Cuiling, ZONG Xinglong, ZHAO Zhuo, ZHAO Yunhui, ZHAI Bo, ZHANG Mingxin, WANG Chunxin
2018, 45(8):  2236-2246.  doi:10.16431/j.cnki.1671-7236.2018.08.024
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This study was aimed to detect the gene polymorphism of bone morphogenetic protein 15(BMP15) and growth differentiation factor 9 (GDF9) genes in Xinji Fine-wool sheep,Northeast Fine-wool sheep,Chinese Merino(Mulley type) sheep,Dorper×Han crossbred sheep (F1),Dorper×Han crossbred sheep (F3) and White Dorper sheep,which would provide a theoretical basis for marker-assisted selection and breeding for prolificacy in sheep.A total of 378 individuals from six sheep breeds were used to study the polymorphism of BMP15 and GDF9 genes by PCR-SSCP.Secondary structure and tertiary structure of proteins were predicted using DNAStar and I-TASSER.Gene frequency,genotype frequency,Hardy-Weinberg equilibrium,heterozygosity (He),homozygous (Ho),number of effective alleles (Ne) and polymorphic information content (PIC) were calculated.The correlation was analyzed between BMP15 gene polymorphism and litter size in Xinji Fine-wool sheep by GraphPad Prism 6 software.The results showed that the polymorphism was found in P1 primer amplification fragment of BMP15 gene,there were three genotypes of AA,AC and CC in six breeds.This mutation was a 58-60 bp (CTT) deletion in exon 1 of BMP15 gene.According to χ2 test,Xinji Fine-wool sheep,Dorper×Han crossbred sheep (F3) and White Dorper sheep were not in Hardy-Weinberg equilibrium.There was no significant difference in the average litter size of different genotypes in Xinji Fine-wool sheep (P>0.05).The polymorphism was found in P2 primer amplification fragment of GDF9 gene.There were three genotypes of DD,DE and EE in six breeds,and there were only one genotype in Xinji Fine-wool sheep.This mutation was a T→C conversion at 477 bp on GDF9 gene exon 2 which didn't lead to amino acid change.According to χ2 test,five breeds (except for Xinji Fine-wool sheep) were in Hardy-Weinberg equilibrium.The results indicated that the GDF9 gene T477C mutation couldn't be used as a genetic marker for multiple fetal traits in Xinji Fine-wool sheep.

Effects of Gallic Acid on in vitro Maturation of Bovine Oocytes
HUANG Yongjun, LI Xiaofeng, WEN Dongmei, SHE Chun, DENG Kai, PANG Erke, SHAO Qiming, SHI Deshun, WEI Yingming, LU Fenghua
2018, 45(8):  2247-2254.  doi:10.16431/j.cnki.1671-7236.2018.08.025
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The aim of this study was to investigate the effects of adding various concentrations of gallic acid (GA) to the maturation medium on the maturation of bovine oocytes and early embryo development in vitro in order to further optimize culture system of bovine oocytes in vitro maturation.Oocytes were matured in vitro on culture medium with different concentrations (0,10,30,50,100 μmol/L) of GA in incubator for 22 to 24 h.Subsequently,the maturation rate of oocyte and the expansion of cumulus cell were calculated,and the cleavage rates,the blastocyst rates,the number of blastomere and the apoptosis rate of blastomere were evaluated after in vitro fertilization (IVF).According to the above test results,oocytes were matured in vitro on culture medium with the optimal concentration of GA for 24 h,and then the intracellular reactive oxygen species (ROS) level and total glutathione level were detected.The results showed that the mature medium with 30 μmol/L GA could significantly increase the oocytes maturation rates and the sore of expansion of cumulus cell than those of the control group (P<0.05),but there was no significant difference between other treatment groups and control group (P>0.05).The cleavage rates of 10 and 30 μmol/L GA groups were significantly increased than 0 and 100 μmol/L GA groups(P<0.05),while there were no significant differences among 0,50,100 μmol/L GA groups (P>0.05).The blastocyst rate of 30 and 100 μmol/L GA groups were significantly improved than that of control group (P<0.05),but 10 and 50 μmol/L GA groups had no significant differences with control group (P>0.05).The No.of blastomere in 30 and 100 μmol/L GA groups were significantly higher than that of the control group (P<0.05),however,the apoptosis rate of blastomere in each group had no significant difference (P>0.05).The detection of the ROS level and total glutathione content in oocyte showed that the levels of ROS were significantly decreased and the content of total glutathione increased significantly in 30 μmol/L GA group (P<0.05).These results suggested that adding GA during IVF might reduce oxidative stress by decreasing ROS levels directly and increasing total glutathione levels in oocytes,thus improve the quality of oocyte maturation and its subsequent IVF embryos developmental ability.

Effect of Lactogenic Hormones on the Transcriptional Activity of FASN Gene in Goats
LI Jun, QUAN Kai, ZHANG Guizhi, ZHAO Jinyan, DENG Hongyu, HE Qiuya, SHI Huaiping
2018, 45(8):  2255-2262.  doi:10.16431/j.cnki.1671-7236.2018.08.026
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This study was aimed to investigate the effects of lactogenic hormones on transcriptional activity of fatty acid synthase (FASN) gene in goat mammary epithelial cells cultured in vitro.The goat mammary epithelial cells were treated with different concentrations of insulin (INS),estradiol (E2),prolactin (PRL) and hormone combinations (INS+PRL,E2+INS+PRL) for 24 h,respectively.The relative mRNA expression of FASN gene was measured by Real-time quantitative PCR.Moreover,other cells were transfected with goat FASN gene promoter reporter vectors.Then,the cells were treated with INS,E2,PRL and hormone combinations (INS+PRL,E2+INS+PRL) for 24 h,respectively.The promoter activity of FASN gene was detected by dual-luciferase reporter assay system.The results showed that the promoter activity and mRNA level of FASN gene were extremely significantly increased in 10 and 100 μmol/L E2 group (P<0.01),and also extremely significantly or significantly increased in 0.1 and 1 μg/mL PRL group (P<0.01;P<0.05).However,different concentrations of INS had no effect on the transcriptional activity of FASN gene (P>0.05).In addition,hormone combinations (INS+PRL,E2+INS+PRL) significantly increased the promoter activity and mRNA expression level of FASN gene (P<0.05).In conclusion,E2 and PRL could regulate the transcriptional activity of FASN gene,which provided a theoretical basis for further study of the molecular regulation mechanism of FASN gene during lactation.

Analysis of Differences in Milk Yield of Chinese Holstein Dairy Cows with Different Heat Resistance
LUO Hanpeng, LI Yuantao, LIU Jiali, XU Wei, ZHANG Hailiang, DONG Ganghui, LI Xizhi, HUANG Xixia, WANG Yachun
2018, 45(8):  2263-2269.  doi:10.16431/j.cnki.1671-7236.2018.08.027
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In this study,Chinese Holstein lactating cows were assigned into two groups,namely heat stress tolerant cows (2 271 heads) and heat stress sensitive cows (2 024 heads) by respiratory and drooling score during heat stress period,and fixed model and mixed model including factors of farm,parity and milk stage were used to compare rectal temperature (RT) and milk production of these 2 groups of cows when they were in heat stress period and comfortable period.The results showed that the RT of heat stress tolerant cows were significant lower than that of heat stress sensitive cows (P<0.05).During comfortable period (May),the average daily milk yield of heat stress tolerant cows were slightly lower than that of heat stress sensitive cows,the difference was 0.68 kg and was not significant (P>0.05).During the period of heat stress,the heat stress tolerant cows performed better than heat stress sensitive cows in terms of the magnitude of decrease in milk production,especially during August when cows suffered severe heat stress (comparing to May,the decrease were 18.06% and 23.01%,respectively).In this study,two physiological performance indicators (respiratory and drooling score) were used for judging the heat stress resistance of lactating cows,and proved to be effective.The results could be helpful for assessing heat stress response and quantification of cow's heat stress,and adopting appropriate measurements to cool the cows at the right time to reduce economic losses in dairy farming.

Mechanism of circRNA and Its Effect on Development of Animal Muscles
XIE Yueqin, CHEN Ting, LUO Junyi, XI Qianyun, ZHANG Yongliang, SUN Jiajie
2018, 45(8):  2270-2275.  doi:10.16431/j.cnki.1671-7236.2018.08.028
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circRNA is a non-coding RNA (ncRNA) that does not have a 5' end cap structure and 3' end poly (A) tail structure but can stably exist in eukaryotic cells.circRNA that formed by reverse splicing is the hottest ncRNA in the past few years.Much more researches have proved that circRNA plays an extremely important regulatory roles in the life activities of organisms.According to the different composition,circRNA can be divided into exon candidates,intron candidates and candidates of the intron-exons.In addition,the function of circRNA depended on its cellular site-specification.circRNA located in the nucleus always regulates the transcriptional activity of hosting genes mainly at gene transcriptional level with RNA polymerase Ⅱ;circRNA in the cytoplasm plays a major function in the role of competitive endogenous RNA,and mainly competitive inhibition of miRNA and their target 3'UTR to regulate the expression levels of miRNA and their targets;Recent studies have shown that circRNA can also be translated into proteins or peptides.At present,the related circRNA research on animal muscle growth and development is still at the initial stage,and the related research is mainly about circRNA as a competitive endogenous RNA to play an important regulatory role.This paper summarized the discovery,classification,production mechanism,mode of action of circRNA and its effect on myocyte proliferation and differentiation in recent years.Finally,we proposed a prospective study of circRNA on muscle growth and development,and we hope our research could offer some useful experience for other researches.

Effect of Caffeine and Hypotaurine on Sperm Function of Bovine
LI Xiaoxia, CAO Pinghua, YU Xueli, LI Yinghua
2018, 45(8):  2276-2281.  doi:10.16431/j.cnki.1671-7236.2018.08.029
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The effect of caffeine and hypotaurine were evaluated on sperm function of bovine in this study.Frozen-thawed semen from Holstein bulls was used in sperm capacitation media with different concentrations caffeine (0,2.5,5.0 and 7.5 mmol/L) or hypotaurine (0,5,10,20 and 40 μmol/L),200 μL semen for each treatment was submitted to swim-up for 45 min in order to evaluate the effect of caffeine and hypotaurine on bovine sperm motility,acrosome integrity and plasma membrane integrity.Furthermore,the effect of hypotaurine as substitutes for caffeine in sperm capacitation media was investigated.The results showed that compared with control group,2.5 and 5.0 mmol/L caffeine resulted in greater sperm motility and the rate of acrosome integrity(P<0.05),the greatest sperm motility was observed with 2.5 mmol/L caffeine.The sperm motility,the rate of acrosome integrity and plasma membrane integrity of 10 and 20 μmol/L hypotaurine were significantly higher than those of control group (P<0.05),20 μmol/L hypotaurine was the best on sperm function parameters.The effect of 2.5 mmol/L caffeine and 20 μmol/L hypotaurine on sperm function were evaluated.Compared with 2.5 mmol/L caffeine and control group,20 μmol/L hypotaurine resulted in greater the rate of acrosome integrity and plasma membrane integrity (P<0.05).In summary,2.5 mmol/L caffeine might be replaced by 20 μmol/L hypotaurine in sperm capacitation media,it was beneficial to improve sperm function parameters.

Preliminary Evaluation of Eleven Mycobacterium bovis Antigens in the Diagnostic Methods of Bovine Tuberculosis
GAO Xintao, JIA Hong, HOU Shaohua, YANG Hongjun, GUO Xiaoyu, YUAN Weifeng, JIANG Yitong, ZHU Hongfei, XIN Ting, DING Jiabo
2018, 45(8):  2282-2292.  doi:10.16431/j.cnki.1671-7236.2018.08.030
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To screen and evaluate antigens used for diagnosis of bovine tuberculosis (bTB),eleven Mycobacterium bovis (M.bovis) antigens of CFP-10,ESAT-6,TB10.4,TB27.4,MPT51,MPT63,MPT64,MPB70,MPB83,Rv3872 and Ag85B were used as coated antigen to establish indirect ELISA,the detection rates of ELISA based on each antigen were calculated.The potential of these recombinant proteins used as stimuli in skin test were assessed by skin test in guinea pigs and cattle.Besides,to evaluate the potential of these proteins used as stimuli in IFN-γ release assay,the heparinized blood from bTB positive and negative cattle were stimulated by recombinant proteins for 24 h,and IFN-γ in plasma were detected.The results indicated recombinant proteins showed virous reactivities to bTB positive serum,MPB70-based ELISA showed highest detection rate (59.7%),and followed by Ag85B,ESAT-6 and MPB83 (>45%).MPT51-based ELISA showed the lowest detection rate,which was only 2.2%.The results of skin tests on guinea pigs and cattle showed single recombinant protein as stimulus in skin test couldn't induce satisfactory delayed type hypersensitivity (DTH),while TB10.4,TB27.4,MPT64,MPT63 or Rv3872 as supplementary antigen for CFP-10 or ESAT-6,could induce high DTH in bTB positive cattle,and showed no difference with PPD-B (P>0.05).Whole blood from bTB positive cattle could release a certain level of IFN-γ induced by the recombinant protein CFP-10,ESAT-6,TB10.4 and MPT51.The IFN-γ induced by CFP-10,CFP-10-ESAT-6 and MPT51 were significantly higher in bTB positive cattle than negative cattle (P<0.05).Therefore,these eleven M.bovis antigens were not good for singly used in serological diagnosis,skin test,or IFN-γ release assay for bTB.However,TB10.4,TB27.4,MPT64,MPT63,Rv3872 or MPT51 as supplementary antigen for CFP-10 or ESAT-6 could improve the sensitivity,and be used as specific stimuli in skin test and IFN-γ release assay for the diagnosis of bTB.

Preparation, Purification and Activity Factors Analysis of IgY Against Variant of Porcine Epidemic Diarrhea Virus
LEI Dan, LI Anqi, LUO Suxian, YE Yu, SONG Deping, TANG Yuxin
2018, 45(8):  2293-2302.  doi:10.16431/j.cnki.1671-7236.2018.08.031
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The purpose of the experiment was to prepare the yolk antibody (IgY) against variant of porcine epidemic diarrhea virus (PEDV) and explore methods that could efficiently,easily prepare and purify the prepared IgY.In this study,hens were immunized by variant PEDV (CH/JX01) and then the eggs were collected.Three different methods,water dilution method,polyethylene glycol 6000 (PEG-6000) method and water dilution-ammonium sulfate secondary precipitation method were introduced to purify the IgY targeted to PEDV,and the extraction quantity were evaluated by indirect ELISA and Western blotting.The results showed that the concentration of IgY extracted by water dilution method was the highest,reaching 6.204 mg/mL,followed by PEG-6000 method,4.673 mg/mL,and the concentration of IgY by water dilution-ammonium sulfate secondary precipitation method was 3.359 mg/mL.Based on the results of ELISA,the titer of IgY targeted PEDV from water dilution-ammonium sulfate secondary precipitation method was the highest with a titer of 1:12 800,while the titers of IgY from PEG-6000 and water dilution methods were 1:6 400 and 1:1 600,respectively.Compared the three different methods of purified IgY concentration and titer,it concluded that the purification effect of the water dilution-ammonium sulfate secondary precipitation method was better than the other two methods.The activity factors analysis results indicated that IgY purified by the water dilution-ammonium sulfate secondary precipitation method was stable at 37 to 60℃ and had good activity at pH 4.0 to pH 11.0.Under acidic conditions,20% sucralfate had a good protective effect on IgY activity.The results would provide an effective reference for the large-scale production and storage of the yolk antibody in the future.

Research Progress on Pathogenesis of Bovine Viral Diarrhoea Virus
LI Xinpei, ZHOU Weiguang, GUAN Pingyuan, CHENG Shipeng, WEN Yongjun
2018, 45(8):  2303-2311.  doi:10.16431/j.cnki.1671-7236.2018.08.032
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Bovine viral diarrhea (BVD) and mucosal disease (MD) are all infectious diseases caused by the infection of bovine viral diarrhea virus (BVDV).The diseases threaten the development of the cattle industry seriously.This article briefly summarized the genotyping and molecular biological features of BVDV.BVDV can be divided into two genotypes and two biotypes according to sequence conservation and cytopathic effect.The newly discovered ‘HoBi’ strain is classified as Pestivirus genus,and the BVDV gene has evolved rapidly.The genome encodes four structural proteins and eight non-structural proteins.Coding proteins play an important role in the virus replication,translation and the host pathogenic process.The pathogenesis of BVDV is complicated.In this review,we summed up BVDV current researches on pathogenesis,including acute infection,uterine or placenta infection,persistent infection and mucosal disease,which was useful for discovery of new methods of control propagation.Acute infections can cause viremia,reproductive disorders,immunosuppression,and so on.The causes of diarrhea in acutely infected cattle are associated with the infection of the gastrointestinal muscularis,submucosa and normal function of the intestinal nerves by BVDV,and non-cytopathic (NCP) BVDV is the cause of acute infection.The mechanism of BVDV infection in embryos is more complicated,which depends on the fetal growth stage in the uterus during the first invasion of BVDV.NCP BVDV has the ability to inhibit the production of type Ⅰ interferon in the fetus causing the virus to survive in the host and forming a persistent infected cow.Mucosal disease is directly induced when the persistently infected cow re-infects a cytopathic (CP) strain that is highly homologous to NCP BVDV.Both biotypes are important factors in the development of persistent infection and mucosal disease.NCP BVDV can be transformed into CP BVDV.These fundamental researches are important for eliminating of BVD-MD and providing a basis for the development of new vaccines.

Effects of Compound Chinese Herbal Medicinal Polysaccharides on Immune Signal Molecules Expression in Different MHC B-LβⅡ Genotypes Chickens Lymphocyte
MA Zhao, SHANG Yunxia, CHEN Jie, LIU Xiaoting, LIANG Xiaorui, ZHU Xiaoqing, GU Xinli
2018, 45(8):  2312-2319.  doi:10.16431/j.cnki.1671-7236.2018.08.033
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This study was aimed to explore the effect of compound Chinese herbal medicinal polysaccharides (cCHMPS) on immunomodulatory in different MHC B-LβⅡ genotype chickens.MHC B-LβⅡ genotype was analyzed by PCR-SSCP method in 500 chickens.Collected the blood from different MHC B-LβⅡ genotype chickens,isolated peripheral blood lymphocytes,and added cCHMPS with the final concentration of 200,100,75,50,25 and 0 μg/mL cocultured for 24 h.Then the lymphocyte supernatant cAMP,cGMP,Ca2+,NO and iNOS content were detected by ELISA.The results showed that cCHMPS could increase cAMP,cGMP,Ca2+,NO and iNOS levels in chicken lymphocyte supernatant compared with control group in each MHC B-LβⅡ genotype chickens.And when the concentration of cCHMPS was 50 μg/mL,cAMP,Ca2+,NO and iNOS levels of AA and BC genotypes chicken lymphocyte supernatant were higher than that of the same genotype chicken,cGMP level of BC genotype chicken lymphocyte supernatant was higher than that of the same genotype chicken;When cCHMPS concentration was 75 μg/mL,cGMP level of AA genotype chicken lymphocyte supernatant was higher than that of the same genotype chicken;When cCHMPS concentration was 100 μg/mL,cAMP,cGMP,Ca2+,NO and iNOS levels of BB genotype chicken lymphocyte of the supernatant were higher than that of the same genotype chicken.cCHMPS could increase cAMP,cGMP,Ca2+,NO and iNOS levels in different MHC B-LβⅡ genotypes chickens,and the cCHMPS optimum immunomodulatory doses were different in each MHC B-LβⅡ genotypes chickens.

Cloning, Expression and Purification of NS1 Gene of Epidemic Encephalitis Type B
ZHANG Xiaozhong, LIU Yifan, GU Siying, YU Na, DUAN Yanxiang, LI Youwen
2018, 45(8):  2320-2326.  doi:10.16431/j.cnki.1671-7236.2018.08.034
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In order to express non-structural protein NS1 of epidemic encephalitis type B,NS1 gene of Japanese encephalitis virus (JEV) P3 strain was amplified by PCR method,and the prokaryotic expressing vectors were constructed by homologous recombination that fusion expressed His or GST tag in this study.The correct plasmids tested by PCR,enzyme digestion and sequencing were transformed into E.coli BL21,and NS1 fusion proteins were induced by IPTG.The proteins were identified using SDS-PAGE electrophoresis and Western blotting,and purified by urea modified and renaturation and affinity chromatography of nickel.The results indicated that two prokaryotic expression vectors of NS1 pET-42b-NS1 and pGEX-KG-NS1 were constructed successfully,and the length of NS1 gene of P3 strain was 1 056 bp,which expressed about 40 ku protein in the form of inclusion body,the degeneration and renaturation effect of urea was better,and NS1 protein had high purity and concentration by nickel affinity chromatography.JEV NS1 gene could express the protein with high purity through prokaryotic expression system,and which laid a foundation for the later biological function research.

Intervention Effect of Olive Leaf Extract on PP1-DNA-PK-USF1 Signaling Pathway of Alcoholic Fatty Liver Disease Rats
WANG Yu, HE Jiujun
2018, 45(8):  2327-2333.  doi:10.16431/j.cnki.1671-7236.2018.08.035
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To explore the mechanism of olive leaf extract (OLE) on alcohol hepatic injury,fifty rats were randomly divided into five groups,including blank control group,model group and three different doses groups of OLE.Hepatic injury models of rats were established by using edible alcohol with increasing dosage for 24 weeks.In the process of experimental hepatic injury,the rats in OLE groups were given OLE at doses of 1 000,500 or 250 mg/kg by intragastric administration,respectively.The changes of liver histological structure were observed by bio-microscopy.The levels of serum lipids were determined by automatic biochemistry analyzer.Real-time PCR,Western blotting and ELISA were used to detect mRNA and protein content changes of hepatic protein phosphatase 1 (PP1),DNA-dependent protein kinase (DNA-PK) and upstream stimulating factor 1 (USF1).The results showed that compared with blank control group,hepatic injury in the model group exhibited significant changes,the levels of serum triglyceride (TG) and free fatty acid (FFA) were extremely significantly increased (P<0.01),mRNA levels and protein contents of PP1,DNA-PK and USF1 in liver were extremely significantly increased (P<0.01).Compared with the model group,hepatic pathological changes were meliorated.OLE significantly or extremely significantly reduced the levels of serum TG and FFA (P<0.05;P<0.01),and mRNA levels and protein contents of PP1,DNA-PK and USF1 in liver (P<0.05;P<0.01).The results indicated that OLE could improve relieve fatty degeneration,and prevent the occurrence and development of alcohol hepatic injury by regulation of PP1-DNA-PK-USF1 signaling pathway.

Isolation, Identification and Drug Resistance Analysis of a Bovine Buttiauxella agrestis
DU Lin, WANG Lifang, FENG Xiaohui, ZHANG Sanfen, SONG Jie, YAO Yiping, SHI Pei
2018, 45(8):  2334-2340.  doi:10.16431/j.cnki.1671-7236.2018.08.036
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To understand the biological characteristics and resistance of pathogenic microorganisms of dairy cows with mastitis,a gram-negative bacterium,named HLJ-36,was isolated from dairy cow's mastitis milk.Morphological characteristics,biochemical identification,molecular biological identification,mouse pathogenicity test and drug susceptibility test were performed.Biochemical test results showed that xylose,ONPG,citrate and so on were positive;Oxidase,sputum,urease and so on were negative.The physicochemical characteristics and molecular biological test results showed that the isolate was consistent with the characteristics of Buttiauxella agrestis,and had the highest homology with human Buttiauxella agrestis (GenBank accession No.:NR_041968.1) isolated from intestine,reaching 99%.The pathogenicity test in mice showed that mice in the experimental group did not die within 72 hours,indicating that the isolate was less pathogenic.Drug susceptibility test results showed that the bacteria were resistant to ceftiofur,azithromycin,ciprofloxacin,etc.,and sensitive to cefoxitin,meropenem,chloramphenicol,amoxicillin-clavulanic acid,and gentamicin.The results showed that Buttiauxella agrestis might be a pathogen causing dairy cow mastitis and had a certain degree of drug resistance,due to the highest homology of this strain and human Buttiauxella agrestis (GenBank accession No.:NR_041968.1),there was a possibility of transmission to humans through milk and dairy products,which had certain public health significance.

Isolation, Identification and Drug Resistance Analysis of Enterobacter cloacae from Asian Black Bear (Ursus thibetanus)
SU Haijun, WANG Yeying, HE Xinwei, WAN Run, WEN Ming
2018, 45(8):  2341-2346.  doi:10.16431/j.cnki.1671-7236.2018.08.037
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To determine the cause of the death of an Asian Black bear (Ursus thibetanus) at a park zoo in Guiyang,the death Black bear internal organs were collected for bacterial isolation and culture,cell morphology observation,physiological and biochemical identification,16S rRNA gene sequence analysis and mouse infection tests.The results showed that a round,convex,smooth surface,well-defined,medium-sized milky white colony was isolated from the dead Black bear lung specimen as gram-negative bacillus by microscopy.It could utilize the glucose,sucrose,mannitol,sorbitol,and citrate,non-lactose fermentation,non-generated sulfuretted hydrogen,positive arginine decarboxylation test and negative lysine decarboxylase,consistent with the biochemical characteristics of Enterobacter cloacae.1 419 bp fragments were generated by 16S rRNA primer amplification,and the similarity reached to 99.9% with the sequences of Enterobacter cloacae by BLAST comparison with the sequences on NCBI.Mice were inoculated intraperitoneally with the isolated bacteria,and the mice were lethal.The median lethal dose (LD50) was 7.94×108 CFU/mL.Drug susceptibility testing found that the isolates maintained high sensitivity to commonly used antimicrobial agents such as amikacin,ofloxacin,norfloxacin and ciprofloxacin.The results of this test showed that Enterobacter cloacae caused the death of the Asian Black bear.