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20 July 2017, Volume 44 Issue 7
Regulatory Mechanisms of Long Noncoding RNAs and Forecasting Methods in Livestock
JIA Chun-yan, JI Xiao-yang, BAI Xue, DAI Hao-yang, WANG Jian-meng, ZHANG Wen-guang
2017, 44(7):  1895-1905.  doi:10.16431/j.cnki.1671-7236.2017.07.001
Abstract ( 205 )   PDF (1953KB) ( 281 )  
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Long noncoding RNA (lncRNA)is a functional RNA segment longer than 200 nucleotides,without protein-coding capacity.lncRNAs can directly regulate gene expression in the form of RNA,at epigenetic, transcription,post-transcription,translation and post-translation level,also plays important roles in various biological processes in the body of livestock.The author reviewed origin,classifications and structural features, four kinds of molecular regulation mechanisms for implement biological functions (signal moleculars,decoy moleculars,guide moleculars,scaffold molecules),five levels of regulation gene expression of lncRNA,and the application of high-throughput RNA-Seq technique to analyze the sequencing data and introduce the methods for forecasting livestock lncRNA,provided a basis for the reasonable study of livestock lncRNA.Predition of the livestock lncRNA includes four sections:Deep sequencing, transcriptome reconstruction,new transcription forecast and lncRNA identification.At the stage of lncRNA identification,the author's laboratory adopt coding potential assessment tool and online sequence coding potential calculator intersection method to remove the coding potential transcript from a large of the predicting candidates rapidly,also got 68 604 lncRNA of the skin tissues of Inner Mongolia cashmere goats,enriched the basic data of goat lncRNA transcript in the NONCODE database.

Analysis of the Differences in Temperature Adaptability Between Buffaloes and Yellow Cattle Based on Transcriptome Data of Skin Tissues
QI Yu, XING Yan-ping, PAN Jing, LING Yu, CAO Yu, MENG Fan-hua, XING Wen-kui
2017, 44(7):  1906-1914.  doi:10.16431/j.cnki.1671-7236.2017.07.002
Abstract ( 184 )   PDF (2027KB) ( 589 )  
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In order to find out signaling pathways that related to the differences in temperature adaptability between buffaloes and Yellow cattle, RNA-sequencing and bioinformatics analysis were performed on the skin tissues of 3 adult buffaloes and 3 adult Yellow cattle. The results showed that 59 369 167 and 69 837 009 reads were obtained from buffaloes and Yellow cattle skin, respectively. The percentages of the uniquely mapped reads in buffaloes and Yellow cattle were 70.76% and 75.55%, respectively. There were 1 611 differentially expressed genes in buffaloes and Yellow cattle skin, 801 of which were up-regulated in buffaloes skin and 810 were down-regulated in buffaloes skin. GO analysis showed that there were 316, 263 and 278 differentially expressed genes classified to the cellular components, molecular functions and biological processes, respectively. KEGG analysis showed that the differentially expressed genes were enriched in 33 signaling pathways, some of which such as neuroactive ligand-receptor interaction, oxidative phosphorylation, cholinergic synapse, serotonergic synapse, GABAergic synapse and calcium signaling pathway might related to the differences in temperature adaptability between buffaloes and Yellow cattle. In summary, the synapse and oxidative phosphorylation might be related to the differences in temperature adaptability between buffalo and Yellow cattle. This study would lay a foundation for further understanding the molecular mechanism of temperature adaptation differences between buffaloes and Yellow cattle and molecular breeding.

Effects of Eucommia ulmoides Leaves on Sheep Metabolism Based on Blood Metabonomics with Liquid Chromatography-mass Spectrometry
YANG Gai-qing, WANG Lin-feng, LIAN Hong-xia, DU Ying-hui, CAO Yu-liang, ZHAO Zhi-wei, GUO Wen-juan, CHEN Shou-bao, LI Ming, DAI Tong-tong
2017, 44(7):  1915-1924.  doi:10.16431/j.cnki.1671-7236.2017.07.003
Abstract ( 248 )   PDF (1577KB) ( 403 )  
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In order to study the effects of Eucommia ulmoides leaves (EUL) on nutrient metabolism and physiological function in sheep,thirty Hu sheep of 70-80 days old and 25-30 kg body weight were chosen and randomly divided into three groups:Control group (diets without EUL,CTL),low level of EUL group (diets with 10% EUL,EUL1) and high level of EUL group (diets with 20% EUL,EUL2).The experiment was last for 90 d with 15 d of adaption.When the experiment approached to the end,the sheep blood sample was collected via jugular vein with 5 mL vacuum tubes of anticoagulant and coagulant,respectively,to extract plasma and serum. The plasma was used to test biochemical indexes, and the serum was used to metabonomics determination base on liquid chromatography-mass spectrometry (LC-MS). The results showed that the level of glucose (GLU),non-esterified fatty acid (NEFA),high density lipoprotein (HDL) and very low density lipoprotein (VLDL) in EUL1 and EUL2 groups were increased at different degree,and these indexes showed significant or extremely significant difference between EUL2 and CTL groups (P<0.05;P<0.01),but there was no significant difference between EUL1 and CTL groups (P>0.05). The cholesterol (TG) in EUL1 and EUT2 groups was significantly increased compared with CTL group (P<0.05),while the urea level was extremely significantly decreased (P<0.01),and there was no significant difference between EUL1 and EUL2 groups (P>0.05). Metabonomics determination of LC-MS found 593 features under positive mode and 1 570 features under negative mode using the serum samples.After analyzing with SIMCA-P software (V.13.0),principal component analysis (PCA),partial least squares discriminant analysis(PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) showed that the score plot of the three groups separated markedly meant that the metabolites among the three groups contribute to differentiate them. Further analysis with t test for the features of variable importance in the projection (VIP) value larger than 1.0 (VIP>1.0) found that there were 24 characteristic metabolites were significantly different (P<0.05).These metabolites were involved in metabolism of glucose,fat and protein,most of them were related with the pathway of TCA (tricarboxylic acid) cycle, and some of them were concerned with improving of animal health and immune.In conclusion, this study demonstrated that some special ingredients in Eucommia ulmoides leaves affected metabolism of sheep and altered nutrient metabolic pathway greatly.Eucommia ulmoides leaves was not only a kind of high quality of forage, but also a functional feed regulated animal metabolic and healthy status.

Isolation, Identification of Four Newcastle Disease Virus Strains and Study on Their Biological Characteristics and Genetic Characteristics
LI Xiao-wen, LIU You-ming, LIANG Guo-zhi, MEI Min-min, HUANG Wen-jing, LI Wen-feng, HUANG Shu-jian, LI Zhi-li
2017, 44(7):  1925-1933.  doi:10.16431/j.cnki.1671-7236.2017.07.004
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In order to study the relationship and evolution between the latest Newcastle disease virus (NDV) epidemic strain and the vaccine strain (Mukteswar,La Sota and Clone 30 strains),four NDV epidemic strains were initially isolated and identified from immunized chicken groups in Guangdong,Guangxi and Hainan provinces. Fusion (F) gene and hemaglutinin neuraminidase (HN) gene of four isolated stains were amplified,cloned and sequenced by RT-PCR method,and the homology analysis of amino acid sequences deduced by F and HN genes of NDV and other strains published in GenBank were studied, the phylogenetic tree were build,and the pathogenicity index of each strain (EID50,MDT, ICPI and IVPI) were determined. The results showed that the EID50,MDT,ICPI and IVPI of the HN-08 strain were 10-8.37/0.2 mL,58.5 h,1.78 and 2.45,that of the XX-08 strain were 10-6.50/0.2 mL,75.0 h,1.61 and 2.41,that of the YS-09 strain were 10-7.75/0.2 mL,64.5 h,1.71 and 2.38,and of the LF-09 strain were 10-7.60/0.2 mL,63.8 h,1.84 and 2.38.In this experiment,the similarity of amino acid sequences of F and HN genes was over 95.8% among four strains of NDV isolated from chicken,the similarity of four NDV strains with chicken epidemic strains (GX11/03 and GM strains),vaccine strains (Mukteswar,La Sota and Clone 30 strains) and standard virulent strain (F48E9 strain) were from 96.8% to 98.6%,86.7% to 90.6% and 88.4% to 91.3%,respectively. The results showed that four NDV epidemic strains were virulent strains,which were closely related to the GX11/03 and GM strains that were popular in China in recent years,but relatively far from the traditional vaccine strains.

Usage of Two-step Red Homologous Recombination Method to Knockout the Gene of Escherichia coli
LI Xin, LI Ya-xin, DAI Jian-jun
2017, 44(7):  1934-1940.  doi:10.16431/j.cnki.1671-7236.2017.07.005
Abstract ( 881 )   PDF (1281KB) ( 976 )  
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Knock-out is the most direct way to explore gene's function. The traditional method of Escherichia coli (E.coli) gene knock-out is to use its own RecA reorganization system, which relies on the specific given cleavage sites of restriction enzymes, requires long homologous arm, furthermore, the operation is complex. Modification of the genome has become simple and rapid since the advent of Red homologous recombination method. And its application in E. coli gene knock-out has been more mature. The structure and functional principle of Red homologous recombination is introduced, besides, this review expounds the operating steps and notices of the common two-step Red-mediated recombination. The Red homologous recombination technology in E.coli has made great contribution in the area of modifications of engineering strain, pathogenicity and bacterial resistance of the pathogenic bacteria. Additionally,the advantages and shortcomings of the method are set forth. According to the disadvantage that the traditional method do leave recombinase recognition site scars and has low efficiency, a sum of classical methods for scar-less gene replacement are described in the final part of the article. These approach can provide better applications for the construction of E.coli genome.

Expression Analysis of TGFRⅠ Gene in Ovary of AoHan Fine-wool Sheep at Different Tissues and Estrous Cycles
YU Shun-yu, LIU Kai-dong, LUAN Zhao-jin, WANG Guo-yi, LIU Nan, HE Jian-ning
2017, 44(7):  1941-1946.  doi:10.16431/j.cnki.1671-7236.2017.07.006
Abstract ( 187 )   PDF (1800KB) ( 243 )  
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This study was aimed to investigate the expression of transforming growth factor-beta receptor Ⅰ(TGF-βR Ⅰ) in main tissue of AoHan Fine-wool sheep,the expression variations of ovary in different estrous states,and the mechanism of TGFRⅠ gene regulation in seasonal estrus of AoHan Fine-wool sheep. Twenty-four seasonal estrus AoHan Fine-wool sheep were chosen and divided into 4 groups of anestrus, proestrus, estrus and dioestrum with 6 sheep per group. First of all,the expression of TGFRⅠ gene in twelve tissues (thyroid,hypothalamus,hypophysis,ovary,adrenal gland,heart,liver,spleen,lung,kidney,pancreas and muscle) of AoHan Fine-wool sheep in the estrous stage and its expression variations in ovary of anestrus,proestrus,estrus and dioestrum were detected by Real-time PCR. The results showed that TGFRⅠ gene expressed in all the 12 tissues,and its expression in ovary and thyroid were extremely significantly higher than that in others (P<0.01). The expression of TGFRⅠ gene in hypothalamus,hypophysis,pancreas,adrenal gland, spleen and lung were high,which were significant higher than heart,liver,kidney and muscle (P<0.05).The expression levels of TGFRⅠgene in proestrus were extremely higher than other stages (P<0.01). The expression in dioestrum were the lowest which extremely lower than other stages (P<0.01). The above results indicated that TGF-βRⅠ might play an important role in turning on the maturation of follice.

Cloning,Prokaryotic Expression and Bioinformation Analysis of dhbC Gene of Brucella melitensis
LI Bao-bao, NIE Xin, YANG Xiao-jian, CAO Rui-yong, ZHU Shu, HUANG Hai-feng, ZHANG Zhen-xing, PENG Dong-mei, LI Guo-hua, LI Ya-ying, WANG Feng-yang, DU Li
2017, 44(7):  1947-1953.  doi:10.16431/j.cnki.1671-7236.2017.07.007
Abstract ( 190 )   PDF (2100KB) ( 243 )  
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This study was aimed to clone and express dhbC gene of Brucella melitensis, and analyze the bioinformatics of its expressed protein. A pair of primers were designed by referring to dhbC gene sequence information of Brucella melitensis M5-90 strain in GenBank, and the dhbC gene fragment was amplified by PCR method. The obtained dhbC gene was ligated into pMD20-T vector to construct pMD20-T-dhbC recombinant plasmid and transformed into E.coli DH5α competent cells. The plasmid was identified by restriction enzyme digestion. The recombinant plasmid pET28a-dhbC was constructed and transformed into E.coli BL21 (DE3) competent cells. The expression was induced by IPTG. The expressed product was analyzed by SDS-PAGE and Western blotting. Bioinformatics analysis of the amino acid sequence encoded by dhbC gene was carried out using bioinformatics software DNAMAN and related online sites ProtParam, SOPMA and Protscale. The results showed that dhbC gene was cloned with the length of 1 093 bp, and protein expression was expressed. The expressed fusion protein was about 47 ku, and was mainly in the form of inclusion body. The molecular weight of the dhbC protein was C1866H2968N544O562S15, the molecular mass was 42 496.3 u, the theoretical isoelectric point (pI) was 5.81, the extinction coefficient was 33 835, the instability coefficient was 36.76, the hydrophobic index was 86.19, the total average hydrophobicity (GRAVY) was -0.215. The half-life of reticulocytes in mammals was predicted to be 30 h, and the secondary structure was dominated by α-helix (41.94%) and random coil (31.46%).

Cloning and Subcellular Localization of ApoA1 Gene in Congjiang Xiang Pig
ZHAO Jia-fu, DUAN Zhi-qiang, YANG Yuan-qing, JI Xin-qin, WANG Yuan-yuan, SUN Cheng-juan
2017, 44(7):  1954-1960.  doi:10.16431/j.cnki.1671-7236.2017.07.008
Abstract ( 221 )   PDF (2099KB) ( 280 )  
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This experiment was aimed to clone apolipoprotein A1(ApoA1) gene of Congjiang Xiang pig, and study the subcelluar localiztion of ApoA1 gene in eukaryocyte. The recombination plasmid pEGFP-C1-ApoA1 was constructed with RT-PCR and other methods, and detected by colony PCR,double digestion and sequencing, after successful construction of the recombination plasmid pEGFP-C1-ApoA1,the subcellular localization of ApoA1 protein were analyzed by fluorescence co-localization technique in the 36 h-transfected HEK-293T cells. Compared with ApoA1 gene of Sus scrofa submission in GenBank, the results showed that six base mutations were found in ApoA1 gene of Congjiang Xiang pig, five of above mentioned mutations were sense mutations, causing alanine to glutamic acid, histidine to glutamine, valine to leucine and aspartic acid to glycine in 180,185,186 and 209 amino acid residues, respectively. Using PSOR Ⅱ Prediction and fluorescence co-localization, it was found that the expression of ApoA1 protein was observed mainly in the extracellular matrix (77.8%). In conclusion, ApoA1 gene of Congjiang Xiang pig was cloned successfully, and the expression of ApoA1 protein was mainly concentrated in the extracellular matrix. These results would provide a knowledge for further constructing the ApoA1 gene transgenic animal models, and contribute to understanding the relation between ApoA1 gene and the human obesity-induced diseases.

Effect of LDL on the Mitochondrial Transmembrane Potential and ZP Protein Ubiquitination in Vitrified-warmed MⅡ Oocytes
JIANG Ze, XUAN Biao, LI Zuo-chen, WANG Yi-nan, JIN Yi
2017, 44(7):  1961-1966.  doi:10.16431/j.cnki.1671-7236.2017.07.009
Abstract ( 195 )   PDF (1403KB) ( 320 )  
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The purpose of this study was to determine the effect of LDL addition on oocyte developmental rate,mitochondrial membrane potential and ZP protein ubiquitination level in MⅡ oocytes from vitrified-warmed GV oocytes.Mitochondrial membrane potentials were labeled with specific probe JC-1,and ZP protein samples of different treatment groups in MⅡ oocytes were analyzed by SDS-PAGE and Western blotting methods. The results showed that normal rate and in vitro maturation of MⅡ oocytes in the 10 mg/mL LDL group (71.92% and 69.86%) were significantly higher than those in control group (58.26% and 54.55%)(P<0.05),that were nearest to non-vitrified group.The mitochondrial membrane potential of MⅡ oocytes in the 10 mg/mL LDL group was significantly higher than those in control group and 1,20 mg/mL LDL groups (P<0.05).The ubiquitinated ZP1,ZP2 and ZP3 of MⅡ oocytes in non-vitrified group and LDL treated groups were labeled at 61,80 and 106 ku,respectively.The ubiquitinated ZPs (ZP1,ZP2 and ZP3) level of MⅡ oocytes in the 10 mg/mL LDL group was significantly higher than those in 1 and 20 mg/mL LDL groups (P<0.05),while significantly lower than that of non-vitrified group (P<0.05). In conlusion, LDL could improve the maturation rate, mitochondrial membrane potential and ubiquitinated ZPs level of MⅡ oocytes from vitrified-warmed GV oocytes.

Research Progress on Cytoprotection of Bcl-xL and PTD-FNK Protein
WANG Ying-qun, SHI Bo-mei, LI Xun, XU Chun-rong, LI Mei-zhen, TANG Rong-fu, WANG Xiao-ye, HU Chuan-huo
2017, 44(7):  1967-1974.  doi:10.16431/j.cnki.1671-7236.2017.07.010
Abstract ( 188 )   PDF (1177KB) ( 238 )  
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In this article,firstly,the structure and functions of Bcl-xL protein is briefly introduced. It is believed that Bcl-xL protein as a member of the Bcl-2 family,has the same head folding structures as Bcl-2 family,which plays an important role in suppressing apoptosis of cell and protecting various cells from injury. Then the author introduces the biological properties,functions, mechanisms and clinical application of PTD-FNK protein (a gain-of-function mutant of Bcl-xL). It is thought that PTD-FNK protein is able to rapidly penetrate into cell in vitro and in vivo, and can protect various types of cells from many damage through the inhibition of apoptosis in the path of mitochondrion,so being a potent protein with various therapeutic applications. Finally,the author points out that the difficulty of mass production and practical application of two proteins is that prokaryotic expression of the proteins is inclusion body,which need the complex refolding process in order to obtain soluble proteins,and proteins' loss in the refolding process is great. The priorities and direction of future research are to realize the soluble expression and purify the two proteins in large quantities by optimizing expression conditions of induction,laying the foundation of their application to clinical medicine and cell preservation under room temperature, low temperature and frozen condition.

Culture of Dairy Cow Primary Mammary Epithelial Cells and Expression of β-casein mRNA
PU Jun-hua, ZHU Xiao-rui, XU Xin, LU Xu-bin, MAO Yong-jiang, YANG Zhang-ping
2017, 44(7):  1975-1981.  doi:10.16431/j.cnki.1671-7236.2017.07.011
Abstract ( 181 )   PDF (1600KB) ( 238 )  
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The aim of this study was to construct the culture method of primary mammary epithelial cells in vitro using mammary tissue and to test expression of β-casein mRNA in mammary gland epithelial cells. The fresh lactating mammary gland tissue was obtained, microscope observation was used for cell morphology and cell growth counting,and the Real-time PCR method was used for detection of β-casein mRNA expression. The results showed that purified primary mammary gland epithelial cells formed island-like and grew with typical shape of slabstone and cobblestone; Primary mammary gland epithelial cells growth curve was "S" type and accorded with rule of general cell growth. Mammary epithelial cells expressed β-casein mRNA successfully. In conclusion, the primary mammary epithelial cells with normal physiology function had been established successfully. The study provided favourable cell model for further research of the functions of mammary epithelial cells.

Genetic Variation Analysis of S Gene of Porcine Epidemic Diarrhea Virus Isolated in Shanxi Provine
LIU Wen-jun, YAO Jing-ming, WU Xin, MENG Fan, HAN Yi-chao, WANG Juan-ping, FAN Zhen-hua, XUE Yi-peng, MI Rui-juan, LI Hong-li, ZHAO Yue
2017, 44(7):  1982-1989.  doi:10.16431/j.cnki.1671-7236.2017.07.012
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In order to investigate the variation in S gene of porcine epidemic diarrhea virus (PEDV), the 4 strains of PEDV S gene nucleotide sequences were obtained, through RT-PCR amplification of tissue samples from Shanxi province. The obtained sequences and the deduced amino acid were analyzed and compared with the other published PEDV strains. Sequence analysis showed that compared with CV777 vaccine, there were 12 nucleotides insertions between 170 to 171 bp, 3 nucleotides insertions between 401 to 402 and 454 to 455 bp, 6 nucleotides deletion between 461 to 468 bp. The nucleotide and amino acid homologies were 99.2% to 99.8% and 98.6% to 99.7% respectively among 4 strains of PEDV S gene; Comparing with the strains isolated from China in 2011 to 2015, CV777 vaccine, attenuated DR13 and CV777, the nucleotide homologies were 95.0% to 98.5%,93.2% to 93.6%,92.1% to 92.9%,93.7% to 94.4%,respectively.The amino acid homology were 96.2% to 98.9%,91.9% to 92.9%,91.9% to 92.6%,92.9% to 94.0%, respectively. Phylogenetic analysis revealed that 4 strains of PEDV S gene belonged to the first group and had high correlative genetic relationship with the PEDV strains which isolated after 2010 in China, and had far correlative genetic relationship with the PEDV strains which isolated before 2010 in China, 2 strains of Japanese, 7 strains of South Korea, 2 vaccine strains. The results suggested that the prevalence of PEDV in Shanxi province had a more obvious variation. Therefore, it was necessary to develop a new vaccine to control the outbreak of PEDV.

Effects of Duodenal Infusions of Essential Amino Acid on Nitrogen Balance and Plasma Urea Nitrogen of Growing-finishing Lambs
ZHANG Fan, GAO Xin-mei, XIAO Min-min, TANG Fu, GAO Wei
2017, 44(7):  1990-1996.  doi:10.16431/j.cnki.1671-7236.2017.07.013
Abstract ( 206 )   PDF (1440KB) ( 523 )  
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This study was aimed to explore the effects of duodenal infusions of essential amino acid (EAA) on nitrogen balance and plasma urea nitrogen (PUN) of growing-finishing lambs.Five Kazakh male lambs with (29.40±1.76) kg body weights and 4 months old were chosen,fitted with duodenal cannulae and raised in metabolic cage individually. The 5×5 Latin square design was used in this experiment and 7 d for per period. The duodenal infusions in control group contained no EAA,that in 8EAA group contained 8 kinds of EAA,while that in -Arg,-Lys and -Met groups were contained 8 kinds of EAA without Arg,Lys and Met,respectively. The results showed that the fecal N excretion in 8EAA group was no significant difference compared with control group (P>0.05),while the urinary N,N retention,N apparent digestibility and PUN concentration were significantly increased (P<0.05).Compared with 8EAA group,the N retention in -Arg,-Lys and -Met groups were significantly decreased by 30.62%,11.56% and 12.99% (P<0.05),and the daily gain were decreased by 13.89%,7.78% and 6.58% (P>0.05),respectively. PUN concentration was increased in response to the removement of Met,Arg and Lys. In conclusion,Arg,Met and Lys had restricted effect to Kazakh lamb in actual fattening conditions.

Effects of Replacing the Corn Silage with Puelia sinese Roxb Silage on Production Performance, Composition of Milk and Economic Benefits in Dairy Cows
HUANG Xiao-fei, MENG Qing-xiang, YANG Jia-xuan, XIE Xiang-xue
2017, 44(7):  1997-2002.  doi:10.16431/j.cnki.1671-7236.2017.07.014
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This experiment was conducted to study the effects of replacing the corn silage with Puelia sinese Roxb silage on the production performance, composition of milk and economic benefic in dairy cows.90 healthy Holstein cows in middle or late lactation were chosen and randomly divided into 3 groups (control group,groups Ⅰ and Ⅱ). The control group was fed with diet based on corn silage,groups Ⅰ and Ⅱ were fed diets with Puelia sinese Roxb silage instead of 50% and 100% corn silage, respectively. The experiment lasted for 8 weeks,including 2 weeks pre-trail period and 6 weeks formal period.The results showed that:The ether extract,neutral detergent fiber,acid detergent fiber,Ash and nitrogen-free extract of Puelia sinese Roxb silage were 192.3%,45.9%,34.1%,128.6% and 194.3% higher than corn silage respectively (P<0.05),and the crude protein of Puelia sinese Roxb silage was 15.3% lower than that of corn silage (P>0.05).The milk yield of group Ⅰ was significantly increased (2.38 kg/d higher) compared with control group (P<0.05),while that in group Ⅱ was decreased (0.87 kg/d lower)(P>0.05).With the increase of Puelia sinese Roxb silage,the butter-fat percentage, lactoprotein and lactose contents were increased (P>0.05).More profits (7.64 yuan/d) could get in group Ⅰcompared with control group. It was concluded that Puelia sinese Roxb silage had a high feeding value,which could be used to fulfill the requirement of dairy cows in high feeding value silage,and the effect of 50% substitution group was the best.

Effect of Fermented Corn Stalks on Growth Performance and the Growth Promotion Mechanism in Otter Rabbit
QI Cong-yan, LI Hong-ya, WANG Shu-xiang, WANG Quan, LI Shu-na
2017, 44(7):  2003-2008.  doi:10.16431/j.cnki.1671-7236.2017.07.015
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This experiment was conducted to research the growth promoting effects of fermented corn stalks with compound bacteria on otter rabbit. The daily feed of otter rabbit was based on fermented corn stalks (roughage ration composed mostly of fermented corn stalks). The growth performance, intestinal tract bacteria and the digestive enzyme activities of otter rabbit were studied. A total of 150 health otter rabbits, 9 weeks old, weighted about (1.90±0.14) kg, were allotted to 2 groups (control group and experimental group) for 10 days preliminary trial period and 30 days trial period. The results were as follows:Compared with control group, the average daily gain (ADG) increased by 14.60% (P<0.05) and F/G decreased by 10.50% (P<0.05) of the experimental group; In intestinal tract of otter rabbit, the number of E.coli were reduced by 46.13% (P<0.01), the number of Lactobacillus, Bifidobacteria and Bacillus increased by 30.64% (P<0.05), 29.27% (P>0.05) and 39.23% (P<0.01),respectively; The activities of protease, amylase, cellulose enzyme and glucanase were increased by 23.20%(P<0.05), 32.96% (P<0.05), 12.63% (P<0.01) and 21.09% (P<0.05), respectively. In conclusion, the fermented corn stalks could promote the growth and improve the intestinal tract ecological environment of the otter rabbit. The results established the theoretical foundation for fermented corn stalks feeding on otter rabbit.

Effects of β-lactam Antibiotic-contaminated Milk on Growth Performance and Blood Immune Parameters in Holstein Calves
HAN Yun-sheng, QU Yong-li, WU Jian-hao, LI Wei, YUAN Xue, PAN Qi-qi, GAO Yan
2017, 44(7):  2009-2015.  doi:10.16431/j.cnki.1671-7236.2017.07.016
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This experiment was conducted to investigate the effect of feeding antibiotic-contaminated milk on growth performance and blood immune parameters of Holstein calves. Eighteen calves (3 days old) with similar body weight were selected and randomly assigned into two feeding groups, which were fed bulk milk (control group) or antibiotic-contaminated milk (experimental group) until 60 days old. The experiment period lasted for 180 d. Bulk milk and antibiotic-contaminated milk were respectively collected on 10, 20, 30, 40, 50 d, which were used to detect milk composition content, E.coli and antibiotics in milk. Body weight and body measurement were severally measured on 30, 60, 90 and 180 d, and then average daily gain (ADG) was calculated. On 7, 15, 30, 60, 90 and 180 d, serum samples were collected from Holstein calves, and used for measure of immunoserologic indexes. The results showed that β-lactam antibiotic residues were 268.65 μg/L in antibiotic-contaminated milk, and the milk protein content, somatic cell counts (SCC), and E.coli number in antibiotic-contaminated milk were all significantly higher than that in bulk milk (P<0.05). Feeding antibiotic-contaminated milk significantly decreased body weight on 30 d and ADG in 60 d, as well as body length on 90 d and heart girth on 60 and 90 d (P<0.05). Compared with control group, the calves'IgA levels on 30, 60, and 90 d, IgM levels on 7 d, IgG levels on 30 and 60 d of experimental group were all significantly increased (P<0.05). Likewise, IL-2 and TNF-α levels on 15 and 30 d of experimental group were also significantly increased (P<0.05). It was concluded that feeding antibiotic-contaminated milk could obviously decrease calves'ADG of early lactation, and to some extent enhance the levels of serum immune parameters before or after weaning.

Effects of Dietary Protein Levels on Urinary Sediments of Early Weaned Tibetan Lambs
CUI Xiao-peng, HOU Sheng-zhen, WANG Zhi-you, MA Liao-wei
2017, 44(7):  2016-2021.  doi:10.16431/j.cnki.1671-7236.2017.07.017
Abstract ( 174 )   PDF (1611KB) ( 199 )  
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This research was aimed to study the effects of dietary protein levels on urinary sediments of early weaned Tibetan lambs. Ninety healthy two months old male Tibetan lambs with weaning weight (20.80±2.75) kg were chosen and randomly divided into three groups with thirty replicates per group and one lamb per replicate. The lambs in three groups were fed based diets with 10.8%(LP group), 12.0% (MP group) and 13.2%(HP group) crude protein, respectively,and the trial period lasted for 127 days. The results showed that:In HP group,the bladder and urethra of three lambs were damaged,and one lamb had bladder calculi,while there was no lamb had the above phenomenon in LP and MP groups,which meant that the relative low protein levels could reduce the incidence of urinary system diseases,so as to improve the health of lambs.The white blood cells and conductivity in LP group were volatile and there was no cast.The indexes of urine sediments in HP group were volatile,and the counts of white cell,red cell,crystals and yeast-like fungi were increasing with the increase of days.While the urine sediments in MP group were slight oscillation within the normal range,and there was no cast.Under the experimental condition,the dietary protein levels did had an impact on the urinary sediments in early weaned Tibetan lambs. When the dietary protein levels was 12.0%,the urinary sediments were best,and the urinary tissues were more complete,and conducive to production.

Research Progress on the Evaluation Methods of Safety and Efficacy of Probiotics
FENG Yuan-yuan, QIAO Lin, YAO Hong-ming, LIU Rui, JIE Lin-xia, GAO Chang-bin
2017, 44(7):  2022-2032.  doi:10.16431/j.cnki.1671-7236.2017.07.018
Abstract ( 287 )   PDF (1155KB) ( 703 )  
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Probiotics are live microorganisms. When they are administrated in adequate amounts, they can have beneficial effects on the host. They are widely applied in food fermentation and feed industry. With the appearance of commercial probiotics, more concerns are raised on the safety and efficacy evaluation of probiotics. At present, the evaluation of probiotics are mainly about the safety and efficacy, and both of them can be done in vivo and in vitro. When developing probiotic products, many companies rarely make a comprehensive safety evaluation of strains, and the evaluation index and methods used by various researchers from different countries are different. Besides, standards in efficacy evaluation of probiotics are not uniform either. In order to solve the problems of probiotics evaluation, it is necessary for evaluating the safety and effectiveness of the probiotics systematically and scientifically to establish a comprehensive evaluation method. From the aspects of potential harm and efficacy of probiotics, the authors summarizes recent developments in some aspects of safety evaluation in vitro including hemolytic, evaluation of virulence factors, antibiotic resistance and toxic metabolites-producing ability, in functional characteristics evaluation including resistance to acid and bile, tolerance to simulated gastrointestinal tract, adhesion capacity to gastrointestinal mucosa and epithelial cells, antimicrobial activity, and in safety and functional characteristics in vivo. The aim of this paper is to provide references to build a comprehensive technical system and standard of evaluation of probiotics.

Effects of Maternal Exposure to Bisphenol A on Survival Rate, Reproductive Hormones and Relative Genes of Offspring Mice
MA Shuang, SONG Peng-yan, ZHAI Fu-zhan, ZHONG Xiu-hui
2017, 44(7):  2033-2041.  doi:10.16431/j.cnki.1671-7236.2017.07.019
Abstract ( 188 )   PDF (3818KB) ( 191 )  
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This study was aimed to investigate the reproductive toxicity of bisphenol A (BPA) exposed to the mother on the offsprings mice. Forty pregnant Kunming mice were randomly divided into 4 groups, i.e. groups A, B, C and D with 10 mice in each group. Group A was the control group and the mice received conventional feeds, mice in groups B, C and D were given 50,500 and 2 500 mg/kg BW BPA in feedstuffs during the whole gestation period (from 1 d to parturition), respectively. The death rates of the offsprings were calculated every week. The offspring mice were sacrificed at 56 days of age (at puberty). The morphology of ovary and testicular tissues were observed with hematoxylin-eosin (HE) staining. The levels of estradiol (E2), follicle stimulating hormone (FSH), testosterone (T) in mice serum were detected with ELISA Kit. The protein levels of Bax and Bcl-2 in ovary or testicular tissues were detected with immunohistochemistry, and the StAR,CYP11a mRNA levels in testicular tissues, the AMH, Kitlg mRNA levels in ovary were measured using Real-time PCR. The results showed that exposure of BPA to the mother extremely significantly increased the mortality (P<0.01),and significantly reduced the testicular weight of offspring mice (P<0.05). Maternal exposure to BPA extremely significantly reduced the levels of T (♂) and FSH(♀) (P<0.01),and extremely significantly elevated E2 (♀) level in offspring mice (P<0.01). BPA exposure damaged the testicular with less leydig cells and ovarian tissues with more vacuoles and less corpus granules in offspring mice. Immunohistochemistry results revealed that maternal exposure to BPA increased the Bax protein level and decreased the Bcl-2 protein level of testicular and ovary tissues in offspring mice. BPA significantly reduced the StAR mRNA expression in male offsprings (P<0.05). However, the mRNA level of CYP11a in groups B and D extremely significantly decreased while group C showed an significant elevation in male offsprings (P<0.01). The expression levels of Kitlg mRNA in groups C and D were decreased extremely significantly in female offsprings (P<0.01), the AMH mRNA expression in groups C and D increased significantly (P<0.05). The conclusion indicated that pregnant mice exposed to different doses of BPA had harmful effects on survival rate in offspring mice, and impact the reproductive hormones, proteins and genes expression.

Effects of Antioxidants on the Quality of Hypothermic Preservation and Frozen-thawed Equine Semen
XU Wen-hui, ZHENG Xin-bao, YU Wei-hao, MENG Jun, LUO Yong-ming, LI Hai, ZENG Ya-qi, WANG Jian-wen, WU Zhuang-yuan, YAO Xin-kui
2017, 44(7):  2042-2049.  doi:10.16431/j.cnki.1671-7236.2017.07.020
Abstract ( 189 )   PDF (1279KB) ( 467 )  
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This study was aimed to evaluate the effects of various antioxidants, namely glutamine (0.015 g/mL), glycine (0.019 g/mL), cysteine (0.024 g/mL), methionine (0.015 g/mL), taurine (0.063 g/mL), vitamin C (0.4 mg/mL), vitamin E (0.5 mg/mL) and melatonin (0.001 mg/mL) on equine sperm quality after chill or freeze-thaw.Semen were collected from 6 adult thoroughbred stallions, INRA82 was used as the base extender (control group), adding INRA82 with different antioxidants was used in experimental group. Assess the effect of antioxidants on semen by detecting motion parameters after storage at 5℃ for 48 h. Motion parameters, plasma membrane integrity (PMI) and mitochondrial membrane potential were used to evaluate semen quality after thawing. The extender supplemented with 25 mmol/L taurine led to higher TM and PM, and supplemented with 0.4 mg/mL vitamin C obtained significant higher PM compared with control group (P<0.05) after storage at 5℃ for 48 h. The freeze extender supplemented with 0.5 mg/mL vitamin E or 0.015 g/mL methionine significantly increased the mitochondrial membrane potential compare with control group (P<0.05). No significant differences were observed for PMI and acrosomes integrity rate after frozen-thawed (P>0.05), but there was a trend that PMI of adding methionine and glycine group was higher than control group. The results suggested that extender supplemented with taurine and vitamin C could improve the semen preservation effect, and the extender supplemented with methionine could improve plasma membrane integrity, mitochondrial membrane potential of thawing sperm, and also could prolong the survival time of frozen thawed sperm.

Study on Expression of Bcl-2 and PCNA Genes in Testicular Tissue of Chickens, Quails and Chicken-quail Hybrids
ZHANG Mei, ZHOU Ling-ling, ZHANG Miao-miao, ZHONG Xin-xian, SHAO Jun, FAN Li-na, GENG Peng-rui, LI Yi-dan, LI Yan, LIAO He-rong
2017, 44(7):  2050-2056.  doi:10.16431/j.cnki.1671-7236.2017.07.021
Abstract ( 179 )   PDF (1842KB) ( 181 )  
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In order to investigate the molecular mechanism of stunted growth testis of chicken (♂) and quail (♀) hybrids. 84 testicular tissue samples of New Roman cocks, male Korean quails and chicken-quail hybrids at different developmental stages were collected,the mRNA expression of Bcl-2 and PCNA genes in testicular tissue of cocks, quails and chicken-quail hybrids at different growth stages were detected using Real-time PCR. The results showed that the Bcl-2 and PCNA genes mRNA expression patterns in testes of chickens and quails at different growth stages were similar.The Bcl-2 gene was fluctuated in a whole, and decreased to the lowest value, then recovered rapidly and remained at a high level. The PCNA gene had a very significant peak value.Compared with the chicken and quail,the expression of Bcl-2 and PCNA genes mRNA in hybrids had no obvious change, which indicated that the pattern of the Bcl-2 gene regulation apoptosis in testis was different from chicken and quail at the hybrid testicular development process, and there was no obvious cell appreciation process.It was suggested that the molecular influencing factors of testicular dysplasia of chicken and quail hybrids were related to the abnormal expression of Bcl-2 and PCNA genes mRNA in testes.The results provided an important reference for the further study of the mechanism of testicular dysplasia between chicken and quail interspecific hybrids.

A Comparative Study on the Carcass and Meat Quality Traits of Jiangquan Black Pig and DLY Pig
LI Wen-tong, LIU Ying, ZHOU Rong, SHEN Zi-quan, DONG Sheng-hua, SONG Xiao-tao, WANG Yue-jiang, TANG Hui
2017, 44(7):  2057-2064.  doi:10.16431/j.cnki.1671-7236.2017.07.022
Abstract ( 283 )   PDF (1021KB) ( 427 )  
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To further understand the germplasm characteristics of Jiangquan Black pig,100 kg Jiangquan Black pigs (10 barrows, 10 sows) and 10 Duroc×Landrace×Yorkshire (DLY) pigs under the same conditions were randomly selected to analyse the slaughter performance and meat quality according to related rules. After slaughtered,the carcass index (carcass weight, loin muscle area, carcass length and backfat thickness etc), meat quality physical indicators (pH, meat color, marbling, drip loss etc), gerneral chemical index (the contents of crude protein, intramuscular fat, moisture and ash etc) and the proportion of amino acids in Jiangquan Black pig were analyzed. The results showed that, Jiangquan Black pig carcass lean meat rate was relatively low, the loin muscle area was reduced by about 30% (P<0.05) compared with DLY pig, while the backfat thickness was higher than that of DLY pig (P<0.05). The meat color, marbling, drip loss, cooking loss, water loss rate index and intramuscular fat content of Jiangquan Black pig were significantly better than that of DLY pigs (P<0.05), the rate of water loss and drip loss and cooking loss were decreased by 4.0%, 3.1% and 2.7%, intramuscular fat content increased by 150.0%. Therefore, The lean yield of Jiangquan Black pig was less than DLY pig, but water retention property, intramuscular fat content, meat color, marbling, meat quality indexes were significantly better than that of DLY pig. In conclusion, Jiangquan Black pig could provide good quality pork for consumers.

Cloning and Bioinformatic Analysis of Goats CLAⅠα Chain Gene
DENG Yang, WU Guo-hua, YAN Xin-min, ZHAO Zhi-xun, LI Yang, LI Jian, DOU Yong-xi, ZHANG Zhi-dong, ZHANG Qiang
2017, 44(7):  2065-2070.  doi:10.16431/j.cnki.1671-7236.2017.07.023
Abstract ( 208 )   PDF (1907KB) ( 190 )  
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In order to explore the polymorphism of goat CLAⅠα chain gene, the primers were designed according to goat CLAⅠα chain gene in GenBank. Goat CLAⅠα chain gene was amplified from white blood cells of ten varieties of goats by RT-PCR,then the sequences were analyzed by bioinformatics. The results showed that the length of CLAⅠα chain gene was 1 074 bp, which encoded 357 amino acids. The nucleotide sequence analysis indicated that the homology of CLAⅠα chain genes of ten varieties of goats was 82.8% to 99.5%. The deduced amino acid sequence analysis revealed that the mutations were concentrated in α1 region (22-110 amino acids) and α2 region (111-203 amino acids). Genetic evolution analysis showed that the goat CLAⅠα chain gene in GenBank was close to the CLAⅠα chain gene of HSH-goat, HX-goat,SB-goat and AEBS-goat on genetic relationship, but with far relation to other 6 breeds, including JYDE-goat, JTH-goat, XNSN-goat, MG-goat, DZH-goat and NBY-goat. This study laid a theoretical foundation for the further study on polymorphism of CLAⅠα chain gene.

Effect of Different Crossbred Combinations on Fatting Performance in Beef Cattle
WANG Jun, LIU Xiao-fei, JIA Ya-xiong, GUO Jiang-peng, LU Yong-qiang, QI Zhi-guo, CHANG Liang, YANG Jian-jun, XU Meng
2017, 44(7):  2071-2078.  doi:10.16431/j.cnki.1671-7236.2017.07.024
Abstract ( 182 )   PDF (1222KB) ( 240 )  
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This study was aimed to determine the effects of different crossbred combinations on fatting performance in beef cattle. Using single factor randomized block design, 30 bulls (health, similar weight and age) were chosen, Simmental cattle×Kerqin beef cattle (group A), Charolais cattle×Kerqin crossbred beef cattle (group B) and Kerqin beef cattle (group C) were divided into three groups with 10 bulls per group. After 95 d, the body weight, slaughter performance, meat quality and the economic benefits of fattening were measured. The results showed that there were no significant influence on dry matter intake among trials (P>0.05). The F/G, ethanolic extract (EE) of muscle in crossbred groups (groups A and B) were significant lower than group C (P<0.05). The weight of carcass, weight of meat, rate of carcass, and the weight of shank, plate, chine and rump in crossbred groups were significant higher than group C (P<0.05). There were no significant difference in content of amino acids among groups (P>0.05),and it had difference in the content of palmitoleic acid (PAA),stearic acid(SA) and linoleic acid (LA). In this trial, it had better fattening performance and the economic benefits in crossbred cattle groups than Kerqin beef group cattle, but the ethanolic extract and unsaturated fatty acids of muscle in Kerqin beef cattle were higher than the crossbred groups, and it had better fattening performance by crossbred.

Expression and Bioactivity Identification of eis Gene of Mycobacterium bovis in Mycobacterium smegmatis
ZHANG Tong-ming, SONG Tian-qi, XIN Ting, ZHU Hong-fei, JIA Hong
2017, 44(7):  2079-2085.  doi:10.16431/j.cnki.1671-7236.2017.07.025
Abstract ( 229 )   PDF (1473KB) ( 269 )  
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This study was aimed to construct a shuttle expression vector of Mycobacterium bovis(M.bovis) eis gene and identify its bioactivity in recombinant Mycobacterium smegmatis (M.smegmatis). M.bovis eis gene was cloned by PCR and the shuttle expression vector pMV261-Mbeis was constructed, then it was identified by double digestion and sequencing. The recombinant plasmid was transformed into M.smegmatis mc2155 by electroporation. The expression of M.bovis eis gene in M.smegmatis was detected by SDS-PAGE and Western blotting, and the amino acids sequence of the target protein was identified by mass spectrometry. The growth curve of recombinant M.smegmatis mc2155 containing pMV261-Mbeis was successfully constructed.The results showed that pMV261-Mbeis did not affect the growth of M. smegmatis in vitro. The results of SDS-PAGE and Western blotting confirmed that the M. bovis eis gene expressed the eis protein which was about 44 ku in M. smegmatis. Mass spectrometry proved that the protein was the eis protein of M. bovis.The expression vector pMV261-Mbeis was successfully constructed and the expressed recombinant protein was proved to be have biological activities in M. smegmatis, which laid a foundation for the further study of the function of eis protein in M. bovis.

Isolation and Identification of Variant HeNZK-2014 of Pseudorabies Virus and Sequence Analysis of gE Gene
MA Zhen-yuan, LI Ning, YAN Ruo-qian, BAN Fu-guo, WANG Shu-juan, ZHAO Xue-li, XIE Cai-hua, WANG Dong-fang, WANG Hua-jun, CAO Wei-wei
2017, 44(7):  2086-2095.  doi:10.16431/j.cnki.1671-7236.2017.07.026
Abstract ( 210 )   PDF (6575KB) ( 215 )  
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In order to learn the situation of pig pseudorabies virus (PRV) variant in this study, tissue samples such as lymph nodes which were collected from clinical pigs with suspected PRV infection were identified by PCR. PRV positive sample were inoculated on PK-15 cells after grinding and degerming, with further experiment including virus isolation, plague purification, PCR and IFA identification, TCID50 confirmed by Reed-Muench method, inoculation test and observation of clinical symptoms in mice. The gE gene of purified PRV and brain tissue samples of dead mice were identified by sequencing analysis. The results showed that the virus grown on PK-15 cells could produce typical cytopathic effect (CPE) after 24 h; After three rounds of plaque purification,the isolate was PRV positive identified by PCR and IFA, and nominated as HeNZK-2014; The TCID50 of the isolate was 10-9.77/0.1 mL; The virus in 1×108 TCID50 inoculation was able to cause itching, tearing, death in infected mice, and PRV could be detected in tissues of dead mice; The molecular genetic variation analysis of gE gene by PCR amplification and clone sequencing indicated that the gE gene from brain tissue of infected mice shared 100.0% homology with HeNZK-2014, and located in a relatively independent branch with newly pandemic isolates in recent years after 2011, but was far from the classical strains before 2011, and both had two insertion of aspartic acid (D) at sites of 48 and 496 amino acids, which were considered to be the typical characteristics of PRV variants. This study successfully isolated a PRV variant, which laid a foundation for further research on vaccine development, prevention and control against PRV variants.

Soluble Expression, Purification and Activity Analysis of Capsid Protein of Very Virulent Infectious Bursal Disease Virus
GAO Xiang, LI Hui, ZHANG Li-zhou, LU Zhen, WANG Yong-qiang, GAO Li, WU Tian-tian, GAO Yu-long, LIU Chang-jun, WANG Xiao-mei, QI Xiao-le
2017, 44(7):  2096-2102.  doi:10.16431/j.cnki.1671-7236.2017.07.027
Abstract ( 202 )   PDF (1764KB) ( 384 )  
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To obtain the capsid VP2 with high quality, VP2 gene of very virulent infectious bursal disease virus (vvIBDV) Gx was cloned and inserted into pCold-Ⅰ and the prokaryotic expression plasmid pCold-Ⅰ-GxVP2 was constructed. In engineering bacteria Transetta(DE3), the induction conditions of protein VP2 expression were optimized.With affinity chromatography and gel filtration, protein VP2 was purified. With the monoclonal antibody directed Western blotting, protein VP2 was identified. Using SPF chicken, immunocompetence of VP2 was evaluated. The results showed that the dissoluble protein VP2 was expressed successfully in Transetta(DE3) in cold-shock conditions; Protein VP2 was purified and the concentration was 542 μg/mL; The purified protein VP2 not only reacted with the monoclonal antibody against protein VP2, but also induced specific immune response in immunized chickens. In general, with 15℃ of cold-shock condition, 120 r/min of shaking culture, 1 mmol/L of IPTG,inducting for 24 h, soluble capsid VP2 of IBDV with immunocompetence was successfully expressed and purified.The preparation of highly purified, soluble capsid protein with functional activity laid the foundation for further researches on the pathogenic mechanism.

Research Progress on Salmonellosis Prevention and Control in Livestock and Poultry
ZHANG Lu, ZHANG Xue-wei, YUAN Zong-hui, HUANG Ling-li, WANG Yu-lian
2017, 44(7):  2103-2111.  doi:10.16431/j.cnki.1671-7236.2017.07.028
Abstract ( 187 )   PDF (1046KB) ( 339 )  
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Salmonella is a member of the Salmonella family of Enterobacteriaceae and has about 2 600 different serotypes. As a widely distributed conditional pathogen,it has a serious impact on animal health, public safety and food safety.Pathogenic Salmonella can cause paratyphoid fever, pullorosis, avian typhoid and avian paratyphoid, non-pathogenic Salmonella may cause the contamination of animal-derived food. With the rapid development of aquaculture in recent years, the adverse effects of Salmonella to livestock and poultry breeding industry is more and more terrible.Antibiotic resistance of Salmonella resulting from abuse of antimicrobial agents is becoming more serious,thereby increasing the difficulty of Salmonella control. In this paper, Salmonella clinical diagnosis, laboratory diagnosis, the problems of prevention and treatment were reviewed in order to provide a reference for the improvement of salmonellosis prevention and control of livestock and poultry.

Establishment and Application of Real-time Quantitative PCR Assay for Detection of Classical Swine Fever Virus
WANG Shu-juan, LIU Mei-fen, YAN Ruo-qian, BAN Fu-guo, ZHAO Xue-li, MA Zhen-yuan, WANG Hua-jun, WANG Cui, ZHAO Ming-jun
2017, 44(7):  2112-2118.  doi:10.16431/j.cnki.1671-7236.2017.07.029
Abstract ( 201 )   PDF (1787KB) ( 229 )  
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A Real-time quantitative PCR assay for detection of classical swine fever virus (CSFV) was developed using the specific probe and primers designed basing on the E2 gene of CSFV. The Real-time quantitative PCR assay was established using the total RNA of CSFV as template. The specificity, sensitivity and repeatability of the assay were tested, and samples taken from clinic suspicious CSFV infected pigs had been testified by the established assay. The results indicated that the Real-time quantitative PCR assay was successfully established, and showed a good linear relationship at a template range of 101 to 106 copies/μL with a coefficient correlation of 0.999; The specificity of the assay revealed that amplifications were showed on CSFV samples, but other pathogens had no amplifications; The sensitivity of the assay was 10 copies/μL nucleic acid and 1 TCID50/mL virus; Meanwhile,19 positive samples were detected, which were consistent with results of CSFV detected by Nested RT-PCR, cloning and sequencing. The eatablished Real-time quantitative PCR assay was specific, sensitive rapid and suitable for early detection and epidemiological study of CSFV.

Prokaryotic Expression and Immunoreactivity Analysis of VP2 Protein of Bluetongue Virus Serotype 1
SONG Jian-ling, LI Hua-chun
2017, 44(7):  2119-2125.  doi:10.16431/j.cnki.1671-7236.2017.07.030
Abstract ( 179 )   PDF (1647KB) ( 238 )  
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The study was aimed to test the immunoreactivity of the VP2 protein of bluetongue virus serotype 1 (BTV-1) in vitro. Based on the published BTV-1 L2 gene of Y863 strain, specific cloning PCR primers were designed and synthesized. The L2 gene was amplified through RT-PCR method and then was purified and cloned into the expressing vector pEASY-Blunt E1. The cloned recombinant plasmids were identified. The positive recombinant L2 plasmid was cloned into BL21(DE3) competent cells to express VP2 protein. The acquired purified recombinant BTV-1 VP2 protein was analyzed through the methods of Western blotting, ELISA and blocking ELISA. The results showed that:BTV-1 VP2 protein was expressed as the inclusion bodies in the pEASY-Blunt E1 vector; 160 and 200 mmol/L glyoxaline were the best condition to wash down the expressed protein. The molecular weight of this purified recombinant protein with N-terminal His-tag was about 105 ku. Through the results of Western blotting, ELISA and blocking ELISA, it had been proved that this recombinant protein could combine with BTV-1 specific antibody and this combination could be blocked by BTV-1 virus. The study showed that the recombinant BTV-1 VP2 protein, expressed through the prokaryotic expression vector pEASY-Blunt E1, possessed good immunoreactivity and this study had established foundation for locating the serotypic epitopes of the BTV-1 VP2 protein.

Development of a Duplex PCR Assay for Detection of Avian Influenza Virus and Chicken Parvovirus
HE Ying, XIE Zhi-xun, LUO Si-si, LI Meng, XIE Li-ji, DENG Xian-wen, XIE Zhi-qin, FENG Bin, HUANG Jiao-ling, ZHANG Yan-fang
2017, 44(7):  2126-2131.  doi:10.16431/j.cnki.1671-7236.2017.07.031
Abstract ( 158 )   PDF (1308KB) ( 268 )  
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In order to establish a method to simultaneously detect avian influenza virus (AIV) and chicken parvovirus (ChPV),two pairs of specific primers were designed according to the sequences of AIV M gene and ChPV NS gene in GenBank. The duplex PCR assay was established by optimizing the reaction conditions.The tests showed that this method had high specificity, could simultaneously detect AIV and ChPV and no specific band was amplified for other subtypes avian pathogenic virus. The sensitivity result showed that the lower detection limit of this method was 100 fg. The results of 159 clinical samples were consistent with the sequencing results of PCR positive product. The double PCR methods for detection of AIV and ChPV established in this study had the characteristics of good specificity and high sensitivity, which was of great significance to the prevention control of AIV and ChPV.

Research Progress on Prevention and Treatment of Necrotic Enteritis in Livestock
WANG Fang, XIE Shu-yu, PANG Yuan-hu, QU Wei, DAI Meng-hong, HUANG Ling-li
2017, 44(7):  2132-2138.  doi:10.16431/j.cnki.1671-7236.2017.07.032
Abstract ( 175 )   PDF (1030KB) ( 257 )  
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Necrotic enteritis is an acute gastrointestinal infectious disease with high morbidity and mortality which caused by Clostridium perfringens. The disease not only harms the livestock health and animal welfare,but also has been an emerging threat for breeding industry and human health. Antibacterial drugs played a positive role on preventing this disease,however,drug-resistant strains were increasing with irrational use of antibiotics,the incidence of necrotic enteritis has drastically increased, prevention and treatment of it faced severe challenges. The author reviewed the characteristics of necrotic enteritis include the etiology,physicochemical properties, epidemiology and clinical signs,at the same time, the control measures and common drugs were summarized,and the new development trend and direction for prevention and treatment necrotic enteritis was objective analyzed, aiming to establish a comprehensive understanding of the disease,provide references for prevention and treatment of the disease.

Research Progress on Molecular Etiology and Pathogenesis for African Swine Fever Virus
OU Yun-wen, YAN Chuan-zhong, ZHANG Jie, MA Bing, DAI Jun-fei, ZHANG Yong-guang, JIA Ning
2017, 44(7):  2139-2146.  doi:10.16431/j.cnki.1671-7236.2017.07.033
Abstract ( 421 )   PDF (1048KB) ( 1357 )  
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African swine fever (ASF) is a devastating disease caused by African swine fever virus (ASFV), characterized by acute feature, high fever, high mortality and other characteristics. ASF mainly outbreaks in African, Eastern Europe countries, Russia and Caucasus region. At present, there are no vaccine available and effective control strategies against ASFV spread, therefore, ASF has a serious impact on the pig industry in the affected countries. The major target cells of the virus are swine reticuloendothelial cells and monocyte-macrophage cells. ASFV can result in apoptosis and affect the host's immune system, and then show the characteristics of the corresponding disease. ASFV has the characteristics of large genome, more genotype and more variability. In this paper, the molecular etiology and pathogenesis of ASFV are reviewed, so as to provide theoretical basis for prevention and control of ASF.

Preliminary Study on a Novel Atypical Congenital Tremor of Piglets and Its Etiology
ZHANG Wen-bo, WU Song-song, DENG Shun-zhou, ZHOU He-tian, ZHANG Zhi-qing, YANG Dan-feng, CAO Liang-liang
2017, 44(7):  2147-2154.  doi:10.16431/j.cnki.1671-7236.2017.07.034
Abstract ( 232 )   PDF (2738KB) ( 1036 )  
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To investigate the infection of atypical porcine pestivirus (APPV) in China, autopsywas carried out for clinical suspected piglet of congenital tremor, viscera pathologic changes were observed. Two pairs of primersfor detection of the NS2-3 and NS5B genes by RT-PCR were designed according to the genome of APPV. The positive product was sequenced using DNAStar and Mega 7.0 softwares,and drawing the molecular evolution trees. The results showed that the lesions were similar to classical swine fever, but without infarction of the spleen and peripheral hemorrhage of the mandibular lymph node.Epidemic materials amplified products were consistent with the expected size, the sequences were APPV fragments by BLAST. APPV and other pestivirus belonged to two branch with the homologies about 70%. The homologies of NS2-3 and NS5B genes among the isolates and the APPV reference strains were about 76.9% to 98.8%, and the homologies of NS2-3 gene of 7 isolates were 89.7% to 94.8%,the homologies of NS5B gene of 12 isolates were 82.2% to 100.0%. This was the first report for the infection of APPV in piglets in China.

Preparation and Identification of Monoclonal Antibody Against Swainsonine
JIA Qi-zhen, CHEN Gen-yuan, WANG Lian-qun, YASEN·Maimaiti, WANG Shuai
2017, 44(7):  2155-2159.  doi:10.16431/j.cnki.1671-7236.2017.07.035
Abstract ( 184 )   PDF (1517KB) ( 181 )  
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In this test,BALB/c mice were immunized by SW-OVA for the preparation of monoclonal antibody against swainsonine (SW). Hybridoma cells that could secrete specific antibody against SW were prepared by hybridoma technique,and then the strain that secreted monoclonal antibody against SW designated 1F10 was prepared,and the chromosome average number of 1F10 cell was 45 to 50 couples. The ELISA titers of cell supernatant were 1:25 600, and that of ascites were 1:80 000.The subclasses of monoclonal antibody was IgG1,and the affinity constant was 1.14×1010,the purity of ascites antibodies was up to 98%,and the recovery rate was 80%.The result of sodium dodecyl sulfate polyacrylamide gel electropheresis proved that purified antibody had been obtained that the molecular weight of H-chain and L-chain of antibody was about 50 and 25 ku. The monoclonal antibody against SW specifically bound to SW determined by Western blotting. The linear range was 4 to 128 μg/mL (R2=0.9969) and no cross-reactivity was detected with BSA,gelatin,polylysine,me-Gal,etc. The result laid the foundation for immunodetection on SW and immunological prevention of animal toxic disease.

Establishment of the Rex Rabbit Fluorosis Model and Detection of the Fluoride Content in Organs
WANG Liang, FAN Hong-jie
2017, 44(7):  2160-2164.  doi:10.16431/j.cnki.1671-7236.2017.07.036
Abstract ( 175 )   PDF (1071KB) ( 172 )  
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In order to study the influence of different doses of sodium fluoride on rex rabbit,rex rabbit fluorosis model was established and the fluorine contents of blood,bone,liver,kidney and other organs in rex rabbit were examined to provide a theoretical reference for the further research. Rex rabbits (age (30±5) days,weight (1.1±0.2) kg) were divided into three groups:Control group,test group Ⅰ (10 mg/(kg·d) NaF group) and test group Ⅱ (20 mg/(kg·d) NaF group). The tests lasted 100 d. The results showed that,on the 100th day,fluoride contents of bone and tooth were significantly different (P<0.05),and they were positively correlated with the time and intake of fluoride;The fluoride contents of kidney and liver were slightly different (P>0.05),and they were not significantly different between both test groups (P>0.05).The fluoride contents of blood were not significantly different between group Ⅰ and group Ⅱ (P>0.05),but both had no significant changes compared with control group (P<0.05). Sodium fluoride as a fluoride source could be used to simulate the natural state of rex rabbit fluorine poisoning. The fluoride accumulating ability in bone and tooth were stronger while the ability in blood,liver and kidney were weaker.

Investigation and Analysis of Porcine Epidemic Diarrhea Virus, Transmissible Gastroenteritis Virus and Pseudorabies Virus of Swine Diarrhea Cases in Shandong Province
WANG Song, ZENG Hao, CHEN Zhi, JIAO An-qi, ZHANG Shu-jin, YU Jiang, SUN Wen-bo, ZHANG Yu-yu, CHEN Lei, DU Yi-jun, LI Jun, WU Jia-qiang, WANG Jin-bao
2017, 44(7):  2165-2170.  doi:10.16431/j.cnki.1671-7236.2017.07.037
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To understand porcine viral diarrhea prevalence in the large-scale pig farms of Shandong province, a total of 3 035 clinical samples were detected by PCR from January, 2014 to December, 2016.Those samples were detected for porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and pseudorabies virus (PRV). The results showed that the detection rate of PEDV, PRV and TGEV were 67.49%, 9.33% and 3.29%, respectively.During the past three years, the lowest detection rate of PEDV was 48.15% in the fourth quarter of 2014,and the highest was 88.57% in the fourth quarter of 2015.In 2016,the detection rate represented fluctuate declining compared with 2015.The highest positive rate of TGEV was 18.52% in the fourth quarter of 2014,and in the third quarter of 2015 was 15.38%.The lowest positive rate of TGEV was 6.67% in the first quarter of 2016 and TGEV was not detected in the other quarters. The highest detection rate of RPV was 15.68% in the second quarter of 2016,and the lowest was 2.56% in the second quarter of 2014,except the first quarter of 2014 that the PRV was 0. By detecting three kinds of viruses in 69 clinical samples collected passively, the results showed that the detection rate of PEDV,TGEV and PRV were 86.96%,5.80% and 37.68%,respectively. The total single infection rate was 69.57%,the single infection rates of PEDV,TGEV and PRV were 57.97%,1.45% and 10.14%, respectively;The total mixed infection rate was 30.43%,the mixed infection rates of PEDV/PRV,PEDV/TGEV and TGEV/PRV were 26.09%,2.90% and 1.45%, respectively;Obviously, the total single infection rate was higher than the total mixed infection rate. The results showed that the PEDV, PRV and TGEV were prevailing in Shandong province. There were PEDV/TGEV, TGEV/PRV, PEDV/PRV mixed infection, and the number of PEDV/PRV mixed infection was in the majority. However, there was no PEDV/PRV/TGEV mixed type infection. At present, PEDV was the major pathogen of porcine viral diarrhea and the test results could provide the reference to the diagnosis of porcine viral diarrhea.

Development of an Allergic Murine Model Induced by Recombinant Blo t 5 Allergen of Blomia tropicalis
LI Xiao-ye, XIAO Zheng-pan, LIU Chu, WEI Shuang-shuang, WANG Da-yong, PEI Ye-chun
2017, 44(7):  2171-2177.  doi:10.16431/j.cnki.1671-7236.2017.07.038
Abstract ( 221 )   PDF (1861KB) ( 267 )  
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To development an allergic murine model induced by the recombinant Blo t 5 (rBlo t 5) allergen, the Blo t 5 gene sequences were optimized, synthesized and inserted into the prokaryotic expression vector PQE80L. The Blo t 5 was expressed in E.coli, purified by NI-NTA agarose,and tested by Western blotting. The results showed that the rBlo t 5 could specially react with monoclonal antibodies against Blo t 5. Twelve BALB/c mice were randomly divided into control group (PBS group, n=6) and rBlo t 5-induced group (rBlo t 5 group, n=6). After sensitization by intraperitoneal injection (i.p) and challenged by intratracheal instillation of rBlo t 5, the total serum IgE levels were measured by ELISA; The mice in rBlo t 5 group were challenged with rBlo t 5 by intraperitoneal injection, and the body temperature was measured at regular intervals; The lung was fixed and stained with haematoxylin and eosin (HE).The result showed that compare with PBS group, the total serum IgE levels rised significantly in rBlo t 5 group (P<0.05). The body temperature dropped significantly and a severe allergic reaction (anaphylaxis) was occurred in mice of rBlo t 5 group (P<0.05).The mice in rBlo t 5 group showed inflammatory cell infiltration in airway by HE.These results showed that the allergic murine model was induced successfully by rBlo t 5 allergen.

Acute Toxicity of Aspirin Eugenol Ester on Rats
ZHAO Xiao-le, KONG Xiao-jun, MA Ning, YANG Ya-jun, LIU Xi-wang, SHEN Dong-shuai, LI Jian-yong
2017, 44(7):  2178-2184.  doi:10.16431/j.cnki.1671-7236.2017.07.039
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The acute toxicity study was carried out by intragastric administration in rats to evaluate the toxic characteristics of aspirin eugenol ester (AEE) and target organs and fully analyze the reason of death rats. The experiment was designed in accordance with the method provided by the Kärber arithmetic method. At first, 50 rats (half male and half female)were divided into 5 groups to calculate LD100 and LD0. According to the pre-test results, different doses of AEE (2.62,3.25,4.03,5.00,6.20,7.69 and 9.53 g/kg) and control group of 0.5% CMC-Na were orally administered to 80 rats, half male and half female, divided into 8 groups. After treatment, clinical signs of rats and the change of body weights were observed and recorded. The microscopic pathology of mild damages on organs was conducted. 14 days after adminstration, the death time and total deaths of rats were recorded, and the LD50 and 95% confidence interual of AEE were calculated according to Kärber. Organs were fixed with 10% formaldehyde solution at the end of the experiment. The LD50 of AEE in rats was 5.95 g/kg upon oral administration and its 95% confidence interval calculation was 5.30 to 6.68 g/kg. The death time of rats were centralized at 48 to 96 h after exposing to AEE more than 4.03 g/kg by the analysis of the cumulative mortality-time of AEE in each group. Significant decreases in body weights were noted after treatment of AEE for 3 days, after that body weights gradually grew and recovered at the 5th day. Remarkable damages on livers, kidney and gastrointestinal tract were found at AEE dosage of more than 5.00 g/kg. From the obtained value of LD50>5.00 g/kg, AEE was classified as practically non-toxic compound according to WHO. The target organs of AEE were liver, kidney and gastrointestinal tract.

Protective Effect of Saccharum alhagi Against N-acetyl-para-aminophenol induced Liver Injury in Mice
AILI·Aierken, KUERBANJIANG·Maimaitimin, JIANG Zhi-hui, ZHANG Xiao-ying, MIKEREMU·Shayibuzhati
2017, 44(7):  2185-2190.  doi:10.16431/j.cnki.1671-7236.2017.07.040
Abstract ( 184 )   PDF (2135KB) ( 642 )  
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N-acetyl-para-aminophenol (APAP) overdose use induced liver damage.This study was aimed to evaluate the hepatoprotective effect of Saccharum alhagi against APAP-induced liver injury in mice. The aqueous extract of Saccharum alhagi was prepared, ant its ingredients were measured by phenol-sulfuric acid method and aluminium salt chromogenic method. The mice were randomly divided into seven groups,including normal control group,APAP model group,Saccharum alhagi (150, 300 and 600 mg/kg·BW)+APAP groups,positive control group and Saccharum alhagi control group. Administration once per day for 3 consecutive days,with 300 mg/(kg·BW) of APAP after 1 h from the last administration of Saccharum alhagi. 300 mg/(kg·BW) APAP were used to induce liver injury, and after 24 h from APAP challenge,the experimental animals were sacrificed to collect blood and liver tissue samples. The level of serum transaminase (ALT and AST) and the liver pathological changes were observed. The results indicated that the levels of serum AST and ALT were extremely significant lower in the Saccharum alhagi treated groups than the APAP treated group (P<0.01), and the pathological changes were significantly improved. This study showed that Saccharum alhagi could be an effective therapeutic source in APAP-induced liver injuries in mice.

Effects of Different Milk Samples on Sensitization in BALB/c Mice
BAI Na, MING Liang, GAO Wan-ting, GUO Fu-cheng, QIAO Xiang-yu, CHAOGE Sulide, GUO Yan, YI Ri-gui, LIU Dong-hui, JI Rimutu
2017, 44(7):  2191-2196.  doi:10.16431/j.cnki.1671-7236.2017.07.041
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In order to study the allergenicity of camel milk,cow milk and human milk,sixty BALB/c mice were randomly divided into β-lactoglobulin (β-lg) group (positive control group),cow milk group,camel milk group,human milk group and blank group (negative control group). Each group of mice received 1 mg/g BW samples and 0.3 μg/g cholera toxin (CT) per week,while mice in control group were received PBS and CT. After six weeks of intragastric administration,some allergic symptoms,the serum specific immunoglobulin E (IgE) and immunoglobulin G1 (IgG1) levels,plasma histamine levels and vascular permeability were detected. The results showed that the body weight of mice in camel milk,human milk and blank groups were normally increased,while that in β-lg and cow milk groups tended to slow down. The serum-specific IgE,IgG1 levels and histamine level in β-lg and cow milk groups were extremely significantly higher than blank group (P<0.01),the vascular permeability was increased,and the allergy symptoms were obvious. While the specific IgE and IgG1 levels of camel milk group were extremely significantly lower than cow milk group (P<0.01),but there were no significant difference with human milk group (P>0.05),and the allergy symptoms were mild. This study showed that the sensitization of camel milk was lower than cow milk,and similar to human milk.

Determination of Pinoresinol Diglucoside in Eucommie ulmoides Immunoenhancement Liquids by RP-HPLC
ZHENG Yi, CHEN Xiao-lan, GUAN Yuan-hong, JIA Ji-ping, YANG Hai-feng, LI Shi-yang, LI Ran
2017, 44(7):  2197-2202.  doi:10.16431/j.cnki.1671-7236.2017.07.042
Abstract ( 224 )   PDF (1237KB) ( 381 )  
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The assay was aimed to determine the content of pinoresinol diglucoside (PDG) in Eucommie ulmoides immunoenhancement liquids. The chromatographic conditions were as follows:Inersustain® C18 column (250 mm×4.6 mm,5 μm) was under column temperature of 35℃, the mobile phase consisted of acetonitrile-0.2% phosphoric acid(15:85)with a flow rate of 1.0 mL/min,and the UV detection wavelength was 277 nm. Ethyl acetate was used as extraction solvent. In order to determine PDG of the test solution under the chromatographic conditions, the number of theoretical plates and resolution were used as system suitability indicators. Linear regression on reference substance (PDG),linearity range and the precision, stability, and reproducibility of the analysis method, the recovery test of adding samples were all determined. The results showed that under the content determination method, the number of theoretical plates of PDG in the test solution was 8 588, and the resolution was 3.046. PDG performed good linear relation at the linear range between 6 and 192 μg/mL, and the related coefficient was 0.9996. The precision experiments showed that the relative standard deviation (RSD) of reference substance solution was 0.74%. The RSD of reproducibility and stability of PDG in the test solution was 4.50% and 2.69%, respectively. The average recovery was 98.74% with RSD 0.65% (n=9). Under the chromatographic conditions established above, the contents of PDG in the test sample were between 0.124 and 0.127 mg/mL. The conclusion was that the RP-HPLC method performed well system suitability, precision, reoroducibility, stability, and high recovery rate. Meanwhile this method was quick, simple and reliable. It could be used to determine the content of PDG in Eucommie ulmoides immunoenhancement liquids.

Expression of Chicken Interferon-β in Pichia pastoris and Its Antiviral Effect
LIU Xin-wen, WANG Xiu-li, GUO Dong-chun, GUO Wei-wei, ZOU Min, FAN Gen-cheng
2017, 44(7):  2203-2208.  doi:10.16431/j.cnki.1671-7236.2017.07.043
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In order to study the expression of chicken interferon-β (ChIFNβ) in Pichia pastoris and its antiviral effect,ChIFNβ gene was subcloned into pPICZα-A vector and constructed pPIC-ChIFNβ recombinant plasmid. After linearized by Sac Ⅰ, the plasmid was transferred into Pichia pastoris X-33 strain. The recombinant strain was induced by 1.0% methanol for 4 d,the product of recombinant ChIFNβ was detected by SDS-PAGE,the antiviral activity was determined by classical cell pathological effect inhibition assay. The protein weight was approximate 17 ku and the recombinant protein was secreted effectively in Pichia pastoris. Potency of interferon was 107.5 U/mL. The expressed protein could also inhibit H9N2 avian influenza virus proliferation in chick embryo. Animal experiment suggested that recombinant protein could provide 70% immune protection for aviadenovirus. The results showed that the recombinant ChIFNβ had good antivirus activity,it would be reference for clinical application of ChIFNβ.

Effect of Sanguinarine on Active Capacity of Isolated Rat Uterine Smooth Muscle in vitro and Its Mechanism
JIA Hai-yan, XU Wan-qing, ZHANG Ting-hua, GAO Hui-qian, WANG Li-fang, WANG Hui
2017, 44(7):  2209-2213.  doi:10.16431/j.cnki.1671-7236.2017.07.044
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The study was aimed to explore the effects of the sanguinarine on activity of isolated rat uterine smooth muscle in vitro. The effect of the sanguinarine on activity of the isolated myometrium of non-pregnant Sprague-Dawley rats eight weeks old was recorded by BL-420F four channels physiological recorder. Four antagonists, atropine sulfate, ranitidine hydrochloride, propranolol hydrochloride and diphenhydramine hydrochloride were used to study their mechanism, respectively. The results showed that sanguinarine and Yuan hu painkillers markedly inhibited the frequency, amplitude and activity of uterine contractions induced by oxytocin injection (P<0.05). The inhibitory effect of sanguinarine on uterine muscle contractions was blocked after using diphenhydramine hydrochloride (H1-receptor antagonist) and ranitidine hydrochloride (H2-receptor antagonist). However, after using atropine sulfate (M-receptor antagonist) and propranolol hydrochloride (β-receptor antagonist), sanguinarine also significantly inhibited the contractions of rat uterine smooth muscle (P<0.05). It was concluded that the effect of sanguinarine on activity of uterine smooth muscle in rats was mainly associated with H1 receptor or H2 receptor but not M receptor or β receptor.