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20 September 2016, Volume 43 Issue 9
JSRV-Env Recombinant Plasmid Induced-transfection of TC-1 and TC-1-Hyal2 and Influence of Cytokine VEGF and TGF-α
LUO Shuang, SUN Li-hong, Siriguleng, YAO Hong-qiang
2016, 43(9):  2215-2222.  doi:10.16431/j.cnki.1671-7236.2016.09.001
Abstract ( 238 )   PDF (3466KB) ( 400 )  
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JSRV-Env recombinant plasmid was transiently transfected with mouse lung epithelial cell (TC-1) and ovine Hyal-2 which could stablely express in TC-1 cell (TC-1-Hyal2) by the method of liposome,then we could explore the mRNA and protein expression of TGF-α and VEGF in order to analyze the correlation TGF-α and VEGF with ovine pulmonary adenomatosis (OPA) and what roles they played in the carcinogenesis process.Both cells were cultured in vitro,according to different plasmids,TC-1 and TC-1-Hyal2 cells were set pEGFP-C1-env transfected groups,pEGFP-C1 transfected and non-transfected groups.The expression of TGF-α and VEGF were detected through Real-time quantitative PCR and ELISA.The PCR results showed that compared with control groups (pEGFP-C1 and non-transfected cells groups),the mRNA expression of VEGF was significantly increased in two cell groups of both transfected with pEGFP-C1-env (P<0.05),while the mRNA expression of TGF-α was extremely significantly increased (P<0.01).The ELISA test results showed that compared with two control groups,the protein expression of VEGF and TGF-α were extremely significantly increased in TC-1-Hyal2 cell which transfected with pEGFP-C1-env (P<0.01),and in TC-1 cell which transfected with pEGFP-C1-env,the expression of VEGF protein was extremely significantly increased (P<0.01),the protein expression of TGF-α was significantly increased (P<0.05).There was no significant difference in mRNA and protein expression of two cytokines between pEGFP-C1-transfected and non-transfected cells groups (P>0.05).The results showed that the expression of TGF-α and VEGF both increased in two cell lines which transfected with JSRV-Env.The results suggested JSRV-Env might raise the expression of TGF-α and VEGF,futher we could assume that the TGF-α,VEGF expression changes had a correlation with OPA,and it could provide an important theoretical basis on OPA for gene targeting therapy.

Cloning and Sequence Analysis of PTTG1 Gene in Luchuan Pig
QIN Zhao-xian, NONG Su-qun, GUAN Zhi-hui, LIANG Dan, QIN Qian, LAN Xi, PAN Tian-biao, XIE Bing-kun
2016, 43(9):  2223-2229.  doi:10.16431/j.cnki.1671-7236.2016.09.002
Abstract ( 243 )   PDF (4421KB) ( 516 )  
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This experiment was aimed to clone PTTG1 gene CDS sequence of Luchuan pig,and was analyzed by bioinformatics methods.A pair of special primers was designed according to predicted sequence of porcine PTTG1 in GenBank.The coding sequence of PTTG1 in Luchuan pig was amplified by RT-PCR,its gene sequence characteristics and protein structure was systemically analyzed by bioinformatics techniques.The results showed that the cloned PTTG1 fragment included a 609 bp CDS (coding 202 amino acids).The sequence multi-aligned results showed that Luchuan pig shared 90.15%,87.85%,87.52%,87.03%,76.03%,74.38%,55.74% and 44.48% of similar nucleotide sequence with that of Bos,Pan troglodytes,Homos,Macaca,Rattus,Mus,Gallus and Danio retio,respectively.The protein structure analysis results showed that the protein attributed hydrophilic protein without signal peptide,localized in cell cytoplasm and had 16 phosphorylation sites.The phylogentic tree of amino acid indicated that PTTG1 was highly conserved in the process of evolution of different species.The cloning and analysis of PTTG1 gene provided an important foundation for further study biological function of porcine PTTG1 during early embryonic development.

Establishment and Application of Single and Duplex RT-PCR Method for Differentiation of Wild-type and Vaccine Viruses of Classical Swine Fever Virus
MA Ping, LI Da, TANG De-yuan, ZHANG Yuan-xin, ZHANG Hua, ZENG Zhi-yong, LIU Xia, WEI Guan-dong
2016, 43(9):  2230-2239.  doi:10.16431/j.cnki.1671-7236.2016.09.003
Abstract ( 305 )   PDF (3983KB) ( 1137 )  
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For the distinguishment of wild-type and vaccine viruses of classical swine fever (CSF),a rapid and simple method was established,which laid the foundation for the purification of classical swine fever virus (CSFV) in large-scale farms.It was found that T-rich insertion sequence independently that 3'-NTR in Lapinized vaccine strain according to the genome sequence comparative analysis of CSF wild virus,vaccine virus and near origin virus published in GenBank,on the basis of this,two pairs of specific primers were designed in the upper and lower ends of the insertion sequence and duplex RT-PCR primers were designed.The anneal temperature of polymerese chain reaction (PCR) was optimized,then the two single and duplex RT-PCR method for the differentiation of wild-type and vaccine viruses of CSF were established.The sensitivity and specificity results showed that the minimum amounts of nucleic acid by the single RT-PCR were 2.2 pg (one pair of primers in single RT-PCR)and 1.7 pg (another pair of primers in single RT-PCR),respectively,and that of the duplex RT-PCR were 8.2 pg (wild-type virus of CSF) and 6.7 pg (vaccine virus of CSF), respectively, and no amplification of PRV,PRRSV,JEV,BVDV,PCV2 DNA/RNA were detected by these methods.Then the methods were used to detect 146 suspicious clinical samples,and the results showed that the positive rates of the mixed infection by wild-type and vaccine viruses of CSF in breeding sows,fattening pigs,nursery pigs and suckling piglets were 6.3%,7.4%,8.3%,8.6%, respectively.The results indicated that the single and duplex RT-PCR methods had high specificity,sensitivity,and good repeatability,and it had an important reference value for the purification of CSF in large-scale farms.

Effect of DNA Methylation Inhibitor 5-Aza-CdR on Patterns of Pluripotent-related Genes and Methylation Genes in Handmade Cloning Embryo
LU Xing-rong, LI Zhi-Peng, SUN Jun-ming, LV Ling-yan, XU Zhong-feng, LIU Qing-you, SHI De-shun, CUI Kui-qing
2016, 43(9):  2240-2248.  doi:10.16431/j.cnki.1671-7236.2016.09.004
Abstract ( 200 )   PDF (2524KB) ( 452 )  
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The expression pattern of pluripotent gene Oct4 and Nanog,and methylation related genes Dnmt1 and Tets of handmade cloning (HMC) embryos were studied by Real-time quantitative PCR assay.The effect of genes expression pattern by 5-Aza-CdR on HMC reconstructed embryos was also explored. The results showed that the expression of Oct4,Nanog and Tet3 genes reached peak on 2-cell stage,the expression of Dnmt1 and Tet2 genes declined with the embryo development,while the expression of Tet1 gene increased.The use of 5-Aza-CdR didn't change the expression pattern of Oct4,Tet1 and Tet3 genes,but increased the expression of Nanog gene at the beginning of embryo development,while decreased the expression of Dnmt1 and Tet2 genes.The expression pattern of Oct4,Nanog,Dnmt1 and Tets genes in the development of HMC embryo was established,the use of 5-Aza-CdR could influence the methylation process of HMC embryo.

Cloning and Sequence Analysis of Nsp9 Gene of Porcine Reproductive and Respiratory Syndrome Virus FS Strain
ZHAO Meng-meng, SONG Zhong-bao, FENG Song-lin, WANG Wen-jia, XING Xing, FENG Jia-ping, ZHANG Gui-hong
2016, 43(9):  2249-2256.  doi:10.16431/j.cnki.1671-7236.2016.09.005
Abstract ( 293 )   PDF (2424KB) ( 323 )  
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In order to evaluate the function of Nsp9 gene,the target gene of porcine reproductive and respiratory syndrome virus (PRRSV) FS strain was amplificated and sequenced after being cloned into the pMD18-T vector.The physical and chemical properties,homology,hydrophilicity,surface probability plot,antigenic index,secondary structure and subcellular localization were predicted by various softwares.The results showed that the length of Nsp9 was 1 929 bp,its predicted molecular weight was 70.5 ku and pI was 7.78,and it was unstable protein.There were many antigen sites,and the flexibility and hydrophilicity of Nsp9 were ideal.The study showed that Nsp9 possessed potential antigenicity,and it fits for preparation of monoclonal antibodies.The results of subcellular location showed that it might exist in the cytoplasm.Nsp9 of FS strain shared 96.9% to 98.9% amino acid homology with other strains of the some hypotype.Some amino acid mutations were found between the parent strains and vaccine strains,and insertions and deletions could be found among the European strains and the American strains.Whether these insertions and deletions correlate with the virulence and polymerase needed further research.

Study on Expression Difference of IGFBP-5 Gene in Different Tissues of Kazakh and Yanqi Horses
TUERXUNJIANG·Wumueraili, MENG Jun, WANG Jian-wen, ZENG Ya-qi, YAO Xin-kui, LI Lin-ling, YAO Fang-fang, WANG Huan, REN Hai-fan, HUANG Jing-jing, A Mi-na
2016, 43(9):  2257-2264.  doi:10.16431/j.cnki.1671-7236.2016.09.006
Abstract ( 201 )   PDF (1829KB) ( 371 )  
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The study aimed to explore the mRNA expression pattern of insulin-like growth factor binding protein-5 (IGFBP-5) gene in different tissues of Kazakh and Yanqi horses.The expression of IGFBP-5 gene in different tissues of heart,liver,spleen,lung,kidney,small intestine,large intestine,cecum,intercostal muscles,longissimus dorsi muscles,brachialis muscle and gluteus in two horses were detected by Real-time quantitative PCR and compared the mRNA expression in the same tissues of two breeds.The results showed that the expression of IGFBP-5 in longissimus dorsi muscle and brachialis muscle of two breeds were significantly higher than other tissues including heart,liver,spleen,lung,kidney,small intestine,large intestine and cecum (P<0.05),and was the lowest in large intestine.The expression of IGFBP-5 in kidney,small intestine,large intestine,cecum,longissimus dorsi muscle and brachialis muscles of Yanqi horse were higher than in the same part of Kazakh horse,and among those in longissimus dorsi muscle and large intestine of Yanqi horse were extremely significantly higher than in the same part of Kazakh horse (P<0.01),and in small intestine and cecum of Yanqi horse were significantly higher than in the same part of Kazakh horse (P<0.05).The test was for further researching the biological function of IGFBP-5 gene,and it could provide a theoretical basis for genetic improvement of production performance of horse in our country.

Expression Pattern of HOXA1 Gene and Its Putative Regulating Mechanism of Yak
ZHANG Ya-nan, SHI Xian, XIONG Xian-rong, LAN Dao-liang, LI Jian
2016, 43(9):  2265-2271.  doi:10.16431/j.cnki.1671-7236.2016.09.007
Abstract ( 214 )   PDF (2048KB) ( 351 )  
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The aim of study was to obtain the yak HOXA1 gene sequence and related bioinformatics information,and analyze its expression spectrum and temporal expression profiles for providing related information of the formation of multiple vertebrae and its mechanism.RT-PCR technology was applied to clone the yak HOXA1 gene,semi-quantitative RT-PCR technique was used to detect the gene expression level in multiple organisms,and Real-time quantitative PCR technique was used to detect the gene expression level in the organisms in different development periods.The results showed that the obtained HOXA1 gene was 885 bp including the ORF of 870 bp,encoding 290 amino acids.The yak HOXA1 gene shared a homology of 75.4% to 98.1% with ordinary cow,wild yak,human,wild boar,horse,mice,chimpanzees,jungle fowl and wild boar.There were different expression level of HOXA1 gene in heart,liver,spleen,lung,kidney,large intestine,small intestine,muscle,stomach,ovaries,uterus,fallopian tubes,breast and testicular tissue.With the growth of the yak,HOXA1 gene expression level in tissue also increased.

Cloning and Sequence Analysis of Zfx/Zfy Gene in Cervus elaphus
WEI Li-min, SUN Li-rong, ZHANG Yong-sheng, XI Ji-feng, LI Chao-cheng, WANG Xu-hai, WANG Xiang-zu, JIA Bin
2016, 43(9):  2272-2278.  doi:10.16431/j.cnki.1671-7236.2016.09.008
Abstract ( 281 )   PDF (1597KB) ( 383 )  
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Specific primers were designed according to the mRNA sequence of Bos taurus Zfx/Zfy genes provided in GenBank.Zfx/Zfy sequences of Cervus elaphus were cloned and analyzed.The results showed that the cloned Zfx gene sequence of Cervus elaphus was 2 115 bp (GenBank accession No:KP257294.1) that encoded 696 amino acids,the cloned Zfy gene sequence was 2 406 bp (GenBank accession No:KU041539) that encoded 801 amino acids.The phylogenetic trees of Zfx and Zfy amino acid were obtained using the Neighbor-Joining method of DNAStar software.Analysis showed that Zfx of Cervus elaphus was closely related to Homo sapiens,compared with Bison,Pantholops hodgosoniiand,Panthera tigris,Felis catus,Bos taurus,Canis lupus familiaris,Ovis aries.Zfy of Cervus elaphus was closely related to Bos taurus,Bison,Ovis aries,Pantholops hodgosonii compared with others.Zfx/Zfy genes of Cervus elaphus were cloned and sequenced for the first time,and this research provided a foundation for further studying the structure and biological functions of the proteins.

Establishment and Application of a Double RT-PCR Method for Detection of PEDV and TGEV
HU Hong-hui, NAN Wen-jin, HUANG Jian-qiang, WU Jing-bo, PENG Guo-liang
2016, 43(9):  2279-2284.  doi:10.16431/j.cnki.1671-7236.2016.09.009
Abstract ( 261 )   PDF (1336KB) ( 503 )  
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The study was aimed to establish a rapid one-step duplex RT-PCR detection method,which could be used to identify and diagnose PEDV and TGEV in clinical diarrhea cases.According to the gene sequences of PEDV and TGEV from GenBank,two pairs of specific primes were designed.Through optimizing and selecting of the best reaction conditions,we finally pinpointed the duplex one-step RT-PCR detection method with strong specificity,which could detect 1×10-5 diluent degree of vaccine. Suspected samples,which were collected from different pig farms in 2015,were detected of PEDV with 100% positive rate.The method was rapid,high sensitivity and specificity,which could be used for clinical detection of PEDV and TGEV,and also for epidemiological investigation.

Establishment and Application of Nested RT-PCR Assay for Detection of Classical Swine Fever Virus
LIU Mei-fen, WANG Shu-juan, YAN Ruo-qian, WANG Dong-fang, CAO Wei-wei, ZHAO Xue-li, LI Qin-nan, LI Ning
2016, 43(9):  2285-2290.  doi:10.16431/j.cnki.1671-7236.2016.09.010
Abstract ( 229 )   PDF (2294KB) ( 443 )  
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A nested RT-PCR for detection of classical swine fever virus (CSFV) was developed using the total RNA of CSFV as a reverse transcription template,and two pairs of specific primers were designed based on the conserved region of CSFV E2 gene.The specificity,sensitivity and repetition of CSFV nested RT-PCR were tested,and 35 samples taken from clinical suspicious CSFV infected pigs were tested by the established nested RT-PCR assay.The results indicated that the nested RT-PCR assay was successfully established.The sensitivity and specificity of the nested RT-PCR assay revealed that the minimum detectable virus content limit was 1 TCID50,and no products were amplified from the nucleic acid of ST normal cell or other 8 pathogenic viral or bacterial microorganisms.The repetition test indicated the nested RT-PCR assay showed a good repeatability.Meanwhile,19 positive samples were detected in 35 suspected samples,which was consistent with the results tested by sequencing.The study suggested that the established nested RT-PCR assay was specific,sensitive and suitable for early detection of CSFV.

Study on Eukaryotic Expression of ORF059 Gene of Orf Virus
ZHU Shu, PANG Feng, LI Guo-hua, LI Ya-ying, PENG Dong-mei, YANG Xiao-jian, NIE Xin, CAO Rui-yong, WANG Feng-yang, DU Li
2016, 43(9):  2291-2295.  doi:10.16431/j.cnki.1671-7236.2016.09.011
Abstract ( 266 )   PDF (1200KB) ( 622 )  
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The study was aimed to clonie and express ORF059 gene of Orf virus (ORFV).The genome of ORFV HCE attenuated vaccine strain was extracted and used as the template.Based on the referenced gene sequences of ORF059 gene on GenBank,the specific primers were designed,the fragment of ORF059 gene was amplified by PCR.After being purified,ORF059 gene was inserted into pEGFP-N1 vector to construct recombinant plasmid pEGFP-ORF059-N1.After being sequenced,the recombinant plasmid pEGFP-ORF059-N1 was transfected into NIH3T3 cells with liposomes.The expression of fusion protein was identified by the fluorescence microscope observation and Western blotting.The results showed that the ORF059 gene was 1 005 bp.The recombinant plasmid pEGFP-ORF059-N1 was constructed correctly.Green fluorescence could be observed in NIH3T3 cells transfected by pEGFP-ORF059-N1 under the fluorescence microscope.The expressed fusion protein was about 64 ku.The results laid a foundation for the further research on the mechanism of ORF059.

Prokaryotic Expression and Development of an Indirect ELISA Detection Method of Porcine Transmissible Gastroenteritis Virus N Protein
GUO Hong-wei, ZHENG Ming, QIAO Hong-xing, MA Hui, ZHAO Xu-yong, WANG Yong-fen
2016, 43(9):  2296-2301.  doi:10.16431/j.cnki.1671-7236.2016.09.012
Abstract ( 210 )   PDF (1179KB) ( 357 )  
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In the study,the recombinant expression plasmid pET28a-TGEV-N was constructed,and expression and purification of the TGEV N protein was completed.Through SDS-PAGE and Western blotting analysis,the N protein could be identified by transmissible gastroenteritis virus (TGEV) positive serum.Using the TGEV N protein as diagnosis antigen,we established an indirect ELISA method for detecting TGEV serum antibodies.The coefficients of variation of intro-batch and inter-batch duplicability test were less than 5%.The specificity was 100%,and the rate of false positive and the false negative rate were 0 and 5%,respectively.No cross reactions with seven porcine diseases positive serum were detected.Using this method,the clinical serum samples were detected.The results showed 100% coincidence rate compared with neutralization test.The method could detect TGEV antibody quickly and effectively,and had good repeatability and specificity,which laid the foundation for the development of standardized diagnostic kits.

Study on Formula Screening and Mixing Technology of Microbial Solid-state Fermentation Feed for Mutton Sheep
ZHANG Li-ming, GUO Peng, ZHANG Zheng, ZHANG Qi-wei, LIU Da-cheng
2016, 43(9):  2302-2309.  doi:10.16431/j.cnki.1671-7236.2016.09.013
Abstract ( 216 )   PDF (1150KB) ( 434 )  
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The experiment was designed to develop a microorganism fermentation feed for mutton sheep,and determined its solid-state mixing technology.In this experiment,the wheat bran,rice bran and jujube fruit residues were chosen as the solid-state fermentation media,and combined the Saccharomyces cerevisiae strains BC,XR4 and Bacillus subtilis strain A15 as the fermentation strains.The DPS software was applied to design the uniform mixture experiment,and 8 formulas were selected for solid-state fermentation experiments,and evaluated the optimal formula by measuring the number of live bacterium and three kinds of nutritional active substances (β-glucan,mannan and peptide).Meanwhile,we studied the mixing technology on the selected formula,and the control group was without turned mixing,the experimental group 1 was turned mixing once and the experimental group 2 was turned mixing twice.The optimal mixing technology was determined by monitoring the feed temperature,the number of live bacterium and the content of three kinds of nutritional active substances (β-glucan,mannan and peptides) during the solid-state fermentation.The results showed that the number of live bacterium of 8 formulas all increased first and then decreased rapidly.Three kinds of nutritional active substances were significantly increased with the extension of fermentation (P<0.05).The formula 7 had the optimal comprehensive index,of which the living bacterium number reached 37.30×105 CFU/g and the contents of three nutrient active substances were 97.41 mg/100 mg,37.66 mg/100 mg and 20.17 μg/100 mg.In the mixing technology experiment,the number of live bacterium and nutritional active substances of the experimental groups were significantly higher than the control group (P<0.05).Comparing with the control group,the number of live bacterium and the content of nutritional active substances in experimental group 2 increased by 10.1%,7.5%,7.6%,3.0%,respectively.

Effect of Low Crude Protein Diet Supplemented with Amino Acids on Reproductive Performance and Protein Metabolism in Pregnant Sows
CUI Jia-jun, ZHANG He-liang, ZHANG Zhao-qin, LI Ya-kui, LI Guang-dong, ZHAO Xiu-cheng
2016, 43(9):  2310-2316.  doi:10.16431/j.cnki.1671-7236.2016.09.014
Abstract ( 224 )   PDF (966KB) ( 728 )  
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In order to study the effect of low crude protein (CP) diet supplemented with amino acids on the reproductive performance and protein metabolism in pregnant sows, a single-factor experimental design was used and twenty-four pregnant sows were randomly divided into three treatments with 8 replicas each according to parity,due date and body weight of sows. The pregnant sows in control group were fed conventional feed with CP level of 14.5%, and the pigs in experimental groups Ⅰ and Ⅱ were fed 12.5% and 10.5% of CP diets supplemented with amino acids,respectively. All diets contain similar amino acid concentration. The experiment began at 20 days after breeding and finished after farrowing. The results showed that:① Low CP diets supplemented with amino acids had no significant effect on the reproductive performance of sows,including the total litter size,number of live litter size,weight at birth and backfat thickness of sows (P>0.05).② The serum urea nitrogen of sows in the control group had no significant difference with experimental group Ⅰ (P>0.05),while significantly higher than experimental group Ⅱ (P<0.05).And there was no significant difference between experimental group Ⅰ and Ⅱ (P>0.05). Serum glucose content also had a downward trend with the decrease of CP level,and that of the control group had no significant difference with experimental group Ⅰ (P>0.05),while significantly higher than experimental group Ⅱ (P<0.05).There was no significant difference between experimental group Ⅰ and Ⅱ (P>0.05). ③ Apparent digestibility of CP showed a trend of increase with the decrease of dietary CP levels. CP apparent digestibility of experimental group Ⅰ had no significant difference with control group (P>0.05),while that of experimental groupⅡ was significant higher than other two group (P<0.05).In conclusion,the low CP diet supplemented with amino acids had no significant effects on the reproductive performance and backfat thickness in pregnant sows.

Effect of Slaughter Weight on Carcass Traits,Meat Quality,Amino Acids and Fatty Acids in Muscle of Jiangkou Luobo Pigs
YANG Zhong-cheng, GONG Yu, YANG Mao-lin, LIAO Qiao-ping, YANG Chang-ping, WU Yu-ping, PAN Dao-xing, LIU Ruo-yu
2016, 43(9):  2317-2325.  doi:10.16431/j.cnki.1671-7236.2016.09.015
Abstract ( 256 )   PDF (1153KB) ( 483 )  
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In order to study the effect of slaughter weight on carcass traits,meat quality,amino acids and fatty acids in muscle of Jiangkou Luobo pigs,36 Jiangkou Luobo pigs (6 pigs of 60,70,80,90,100 and 110 kg body weight,respectively) were selected to determine carcass quality,meat quality characteristics and nutritious component and analyze their changes with weight.The results showed that before the body weight reaching to 80 kg,the slaughter rate and loineye area were increased fast,the contents of various amino acids were increased obviously.And when the body weight was 80 kg,the fatty acids were rich,including oleic acid and linoleic acid.When the body weight was more than 90 kg,there were excessive fat deposition in porcine somatic and the content of lysine,valine,threonine and other essential amino acids were extremely significant or significant differences (P<0.05;P<0.01).Generally Jiangkou Luobo pigs with the 80 kg body weight had the better carcass traits and meat quality,rich and ideal amino acid and fatty acid composition in muscle,especially had the high content of essential amino acids and flavor amino acid,and suitable proportion of unsaturated fatty acid and saturated fatty acid.Therefore,the reasonable slaughter weight of Jiangkou Luobo pigs was about 80 kg.

Effect of Dietary Grape Pomace on Growth Performance,Organ Index and Blood Biochemical Indexes in Sheep
JIN Ya-qian, HAO Song-hua, ZHAO Jun-xing, MA Xue-hao, SU Rui, REN You-she, ZHANG Chun-xiang, ZHANG Jian-xin
2016, 43(9):  2326-2332.  doi:10.16431/j.cnki.1671-7236.2016.09.016
Abstract ( 276 )   PDF (964KB) ( 477 )  
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This experiment was aimed to investigate the effect of grape pomace on growth performance,organ index and blood biochemical indexes in sheep.A total of 24 hybrid 5-month-old Dorper (♂)×Small Tail Han sheep (♀) F1 male lambs weighted at 25 kg were randomly divided into 4 groups with 6 lambs per group.All these sheep were fed diets with the addition of grape pomace at 0,5%,10%,20% on a dry weight basis, respectively.The results showed that the ADG in group 10% was extremely significantly higher than that in group 0 and 5% (P<0.01),and the weights of liver and kidney in group 10% were notably higher than that in group 0 and 5% (P<0.05).The weights of large intestinein in group 10% and 20% were obviously higher than that in the other two groups (P<0.05).The weights of abomasum were extremely significantly increased in grape pomace supplemental groups (P<0.01).Grape pomace could significantly improve serum HDL-C,and it was the highest in group 10% (P<0.05).ALT and AST had the same variation trend,they were the lowest in group 5%,and the values in group 5% were significantly lower than that in group 0 and 20% (P<0.05).Compared with group 0,the serum CRE in group 5% was significantly decreased (P<0.05),while the serum T-AOC significantly improved (P<0.05).The CAT enzyme activity was highest in group 20%,it was higher than the other groups (P<0.05).In conclusion,adding grape pomace properly could improve ADG,promote development of liver,kidney and digestive organs,increas serum HDL-C level,and improv the level of serum antioxidant.Taking all factors into account,dietary supplementation with 10% of grape pomace was advisable.

Comparison Research on Nutrient Composition of Meet between Yanbian Yellow Bulls and Steers
GUO Pan-pan, MAO Yuan-ting, LI Xiang-zi, YAN Chang-guo
2016, 43(9):  2333-2338.  doi:10.16431/j.cnki.1671-7236.2016.09.017
Abstract ( 320 )   PDF (957KB) ( 881 )  
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In order to compare the nutrient composition of meet between Yanbian Yellow bulls and steers,16 healthy beef cattles from same breed group with similar age and body weight were chosen and randomly divided into two groups with 8 cattles per group.The experimental group was made up by steers and control group was bulls,all cattles were slaughtered after feed for 373 d.The results showed that:① At the early stage of experiment,the total protein,globulin,A/G,blood glucose,triglyceride,aspertate aminotransferase,alanine aminotransferase levels of the two groups had no significant difference (P>0.05),and albumin,high-density lipoprotein cholesterol, urea nitrogen and cholesterol of experimental group were extremely significantly or significantly higher than that of control group (P<0.01;P<0.05);At the end of the experiment,albumin,A/G and cholesterol of experimental group were significantly higher than the control group (P<0.05),and the triglycerides content was extremely significantly higher than control group (P<0.01);There was no significant difference in other indexes between the two groups (P>0.05);② The oleic acid content and C18:1 n9/C18:2 n6 in longissimus dorsi of experimental group were 6.51% and 229.42% higher than that in control group,the difference were significant or extremely significant (P<0.05;P<0.01),respectively.The linolic acid and octadecoic acid in control group were extremely significantly higher than that of experimental group (P<0.01);③The proportion of Pro,Gly,Ala and Arg in longissimus dorsi of control group were significant higher than that in experimental group (P<0.05),while other amino contents were no significant difference between two groups (P>0.05).In conclusion,the fat deposition,unsaturated fatty acid,especially oleic acid content in Yanbian Yellow steers were significantly higher than bull and it had good meat quality and good taste which was suitable for the production of high-grade beef.

Determination and Correlation Analysis on the Egg Quality of Egg-laying Ducks Reared in Cages and on Floor
LI Jian-dong, CHEN Xi-ping, HUANG De-chun, LIAO Yi-shu, JING Dong-lin
2016, 43(9):  2339-2343.  doi:10.16431/j.cnki.1671-7236.2016.09.018
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The purpose of this test was to evaluate the egg quality from two breeds of ducks reared in cages and on floor.30 eggs were chosen from each type of eggs,which were from Jinding (green shell) and Shan Partridge ducks (white shell)at 40 weeks-old reared in cages and on floor for egg quality evaluation.The results showed that the egg weight of caged duck was extremely significantly larger than the floor duck (P<0.01) in the same breed,there were very significant positive correlations between the egg weight and eggshell weight,egg-white weight and egg yolk weight (P<0.01),but no significant difference among the egg yolk weights of different types of ducks (P>0.05).The shell strength of green egg was obviously higher than that of white shell eggs (P<0.05),and the shell strength of green egg from caged ducks was higher than that of the green egg ducks on floor.The correlation coefficients of the egg specific gravity and eggshell thickness,the specific gravity and eggshell strength were 0.503 (P<0.01) and 0.536 (P<0.01) respectively.Haugh units of four types of ducks all reached 72 or more.Although there was significant positive correlation between egg-white weight and egg yolk weight (P<0.01),significant negative correlation was existed between egg-white weight and egg yolk ratio (P<0.01).There were significant negative correlations between egg specific gravity and egg yolk weight (P<0.05) and egg yolk ratio (P<0.05).In conclusion,the mode of cage feeding and the breed with green shell were appropriate choices in laying-egg duck production for high egg quality.

Research Progress on Rapid Determination Technology of Avermectins in Feeds and Animal Products
NI Teng-teng, PENG Da-peng, XIE Shu-yu, CHEN Dong-mei, WANG Yu-lian, PAN Yuan-hu, TAO Yan-fei, YUAN Zong-hui
2016, 43(9):  2344-2351.  doi:10.16431/j.cnki.1671-7236.2016.09.019
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Currently,avermectins are the most widespread anti-parasite drugs,and it is urgent to develop the simple,faste,high sensitive high-throughput and low cost methods for residues rapid detection because of its residual in animal body for a long time.In order to provide certain reference for the choice and application of current multi-residues detection method,the rapid detection technology of avermectins in feeds and animal products were reviewed in this paper,such as biochip,fluorescent immunoassay,immune affinity chromatography and enzyme linked immunosorbent assay.At the same time,this paper described the prospects of the development in residues detection methods.

Application of Straw Steam Explosion Technology in Livestock Production
ZHOU Tao, CHEN Wan-bao, MENG Qing-xiang, ZHOU Zhen-ming, REN Li-ping
2016, 43(9):  2352-2357.  doi:10.16431/j.cnki.1671-7236.2016.09.020
Abstract ( 270 )   PDF (966KB) ( 553 )  
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Steam explosion technology is a comprehensive fiber processing craft of physical and chemical,which through vapor phase heating and exploding effect to achieve the purpose of changing fiber chemical composition and structure.It can improve the potential of animal feed intake and digestibility of straw,and contribute to the straw resources utilization in China.The author introduced the principle and processing technology of steam explosion,summarized the difference of steam explosion making on straw nutritional value and the problems of this techndogy in straw feed development,and its application prospects in livestock production,hoping to provide the reference for its application in livestock.

Research Progress on Characteristics of Oat Hay Supplementary Feeding on Tibetan Sheep in Cold Season
LI Fu-hou, WANG Zhao-feng, HOU Fu-jiang, CHENG Yun-xiang
2016, 43(9):  2358-2365.  doi:10.16431/j.cnki.1671-7236.2016.09.021
Abstract ( 259 )   PDF (1132KB) ( 447 )  
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Lack of forage and lower nutrient level are limiting factors for Tibetan sheep production in cold season.It is imperative to improve the productivity of both alpine grassland and Tibetan sheep.In the light of research progress on oat hay supplementary feeding on Tibetan sheep in cold season,the authors reviewed the urgency of supplementary feeding,oat hay production and the research status,the effect of oat hay supplementary feeding on digestibility,reproduction,productive and meat quality in Tibetan sheep.Furthermore,we put forward some suggestions for the shortcomings of oat hay supplementary feeding on feeding value,supplementary feeding regime and the popularity of cold season supplementary feeding technology,in order to provide a reference for further research.

PCV2 Regulates PAMs Secreting IL-1β through the NF-κB/NLRP3 Signalling Pathway in vitro
LI Hai-hua, ZHANG Lei, YANG Chun-lei, ZHAO Xiang-hua, QIAO Jia-yun, WANG Wen-jie
2016, 43(9):  2366-2372.  doi:10.16431/j.cnki.1671-7236.2016.09.022
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This study investigated the molecular mechanism of interleukin-1β (IL-1β) production in porcine alveolar macrophages (PAMs),which were infected by porcine circovirus 2 (PCV2).Two 6-week-old piglets were selected as experiment animals,which were free of PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) antibody and antigen.The alveolar macrophages were isolated aseptically and cultured in vitro as this study object.IL-1β in the supernatants from PCV2-infected PAMs was assayed by ELISA.The expressions of NLRP3 and ASC in the PCV2-infected PAMs were detected by Real-time quantitative PCR.The regulatory function of NLRP3 and NF-κB in IL-1β secretion by the PCV2-infected PAMs was analyzed by small interfering RNA (siRNA) and NF-κB inhibitor BAY11-7082,respectively.The results showed that the expressions of IL-1β,NLRP3 (except for 1 h) and ASC (except for 1 and 3 h) in cell culture supernatants or PCV2-infected PAMs were significantly increased(P<0.05;P<0.01).siRNA could lead to 58.3% of NLRP3 gene silencing.IL-1β secretion in the PCV2-infected PAMs,which were companied by NLRP3 gene silencing,was significantly decreased (P<0.05).The IL-1β level in the PCV2-infected PAMs,which were treated with inhibitor BAY11-7082,was also significantly decreased.These results suggested that PCV2 regulated PAMs secreting IL-1β through the NF-κB/NLRP3 signal pathway in vitro.

Research Progress on Regulation of microRNAs in Animal Skin and Hair Follicle
WU Yue-hong, LI Yong, YANG Yi, HE Yu-long
2016, 43(9):  2373-2378.  doi:10.16431/j.cnki.1671-7236.2016.09.023
Abstract ( 223 )   PDF (1017KB) ( 630 )  
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The regulation of gene expression is a decisive factor in the growth and development of animal hair.microRNA,as a newly discovered gene regulatory element,is expressed in many kinds of mammalian skin and hair follicles,regulating the growth and development of skin and hair in post-transcriptional level.Analysising the characteristics and mechanisms of hair growth in sheep,goats,alpacas and other mammals,in microRNA level,may provide the new ideas in improving economic animal hair quality and yielding,and the more in-depth research the function of microRNA in skin tissue.In the present review,we focus on the regulatory role of microRNA in sheep,goats,alpaca and other mammalian skin and hair follicle development.

Research Progress on the Mechanisms of Inflammation-induced Muscle Protein Degradation
WANG Sheng-jun, LIU Yan, LIU Yu-lan, WANG Wen-jun, ZHANG Jing
2016, 43(9):  2379-2387.  doi:10.16431/j.cnki.1671-7236.2016.09.024
Abstract ( 205 )   PDF (1192KB) ( 1148 )  
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Skeletal muscle wasting is a consequence of the decrease of muscle protein synthesis and/or degradation acceleration,and often accompanies with various serious diseases,such as pathogen infection,injury and tumor.At present,many studies have shown that the systemic inflammation is playing an important role in the process of muscle wasting.Based on the current literature,we present an overview of the molecular and cellular mechanisms that contribute to the inflammation-induced muscle wasting,mainly including the ubiquitin-proteasome pathway (UPP),autophagy-lysosomal pathway (ALP),PI3K/Akt pathway,p38/MAPK pathway and NF-κB pathway,which hope to offer the guidance for the treatment of skeletal muscle wasting.

Protective Effects of Oleuropein on Cerebral Ischemia-reperfusion Injury in Mice
WANG Yu, QIN Xu
2016, 43(9):  2388-2394.  doi:10.16431/j.cnki.1671-7236.2016.09.025
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To explore the protection role and mechanism of oleuropein and edaravone on cerebral ischemia-reperfusion injury in mice.Fifty mice were divided into five groups:Sham operation group,model group,oleuropein group,edaravone group,oleuropein+edaravone group.The model was established by ligating common carotid artery.After modeling,the mice were administrated with oleuropein,edaravone and oleuropein+edaravone for 21 d,respectively.The contents of TNF-α,IL-1β and IL-10 were detected by radioimmunoassay.The activities of ATPase,MPO,SOD and CAT and MDA content were measured by spectrophotometry.The changes of BDNF expression in cerebral cortex were analyzed by immunohistochemistry and Western blotting.The results showed that compared to sham operation group,the contents of TNF-α,IL-1β,MDA and MPO activity in model group were extremely significantly increased (P<0.01),and the levels of IL-10,ATPase,SOD and CAT in model group were extremely significantly reduced (P<0.01),the BDNF expression in model group was extremely significantly decreased (P<0.01).Compared to model group,the contents of TNF-α,IL-1β,MDA and MPO activity in oleuropein,edaravone,and oleuropein+edaravone groups were extremely significantly decreased (P<0.01),the levels of IL-10,ATPase,SOD and CAT were extremely significantly increased (P<0.01),the BDNF expression was extremely significantly up-regulated in cerebral cortex of treatment group (P<0.01).Furthermore,the oleuropein+edaravone combined administration showed a better effect.The treatments of oleuropein and edaravone had a protective effect on cerebral ischemia reperfusion injury,the mechanisms of which might depend on improving neurological function,reducing free radical lesion and inhibiting inflammatory response.Moreover,the oleuropein+edaravone combination therapy might have an additive effect.

Research Advance on Antioxidant Function Roles and Its Mechanism of Total Phenols from Magnolia officinalis
CAO Rong, XIE Kun, HOU De-xing, HE Xi
2016, 43(9):  2395-2400.  doi:10.16431/j.cnki.1671-7236.2016.09.026
Abstract ( 273 )   PDF (964KB) ( 603 )  
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Total phenols from Magnolia officinalis,one kind of phenolic mixtures,is extracted from Magnolia officinalis and mainly contains Magnlo and Honokial,which has a wide range of biological functions,such as anti-viral,anti-microbial,anti-inflammatory and anti-diarrhea activities,ect.The antioxidant activity is thought as a key foundation of these above-mentioned function roles.In this paper,antioxidant function roles of total phenols from Magnolia officinalis are reviewed through three mechanisms which included eliminating superoxide anion free radical,increasing the activity of antioxidation enzyme and inhibiting lipid peroxidation.This review will provide a reference for further researching about its antioxidant function roles and being used in livestock production as a green antioxidant.

Polymorphism of LAMB1 Gene and Its Association Analysis with the Fiber Diameter in Fine-wool Sheep
ZHAO Bing-ru, HUANG Xi-xia, TIAN Ke-chuan, FU Xue-feng, XU Xin-ming, BAI Yan, TIAN Yue-zhen
2016, 43(9):  2401-2411.  doi:10.16431/j.cnki.1671-7236.2016.09.027
Abstract ( 197 )   PDF (3059KB) ( 419 )  
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This study aimed to research the single nucleotide polymorphism (SNPs) of LAMB1 gene exon and its correlation with the fiber diameter in Fine-wool sheep.Based on DNA pools with the Re-sequencing technology to gain SNPs data,totally screened 20 SNPs of LAMB1 gene exon regions,at the same time,combining with direct sequencing method,PCR-SSCP and bioinformatics software to verify the accuracy of 10 missense mutations.The genetic effects of LAMB1 on fiber diameter at Xinjiang Kunes farm were analyzed by the GLM of SAS,totally 300 sheep.The results showed that 20 SNPs of LAMB1 gene were screened,which included 10 synonymous mutation SNPs and 10 non-synonymous mutation SNPs,after the validation,there were 7 missense mutation SNPs which led to the nature of protein change,3 SNPs were not mutated.LAMB1 protein became increasingly hydrophobic after mutating,predicting that was insoluble protein.TT genotype in the fiber diameter of the individual value was significantly higher than TC and CC genotypes on SNP5(P<0.05),GG and TT genotypes in the fiber diameter of the individual value was significantly higher than GT genotype on SNP9 (P<0.05).Although,all of the SNPs were completely detected by DNA pool with the Re-sequencing technology and minimized the false positives,it still need to be validated by direct sequencing.The results revealed that LAMB1 gene existed highly genetic diversity,mutated SNPs were unevenly distributed in exons,SNP5(rs159769941) and SNP9(rs159769901) could be considered as an effective genetic markers of Fine-wool sheep on fiber diameter.

Cloning of GABRP Gene and Its Expression in Buffalo Mammary Tissue
HAO Wen-yan, CHEN Pei-fang, DAI Xiao-li, WANG Ping, WU Da-ping, SHI De-shun, LI Xiang-ping
2016, 43(9):  2412-2417.  doi:10.16431/j.cnki.1671-7236.2016.09.028
Abstract ( 174 )   PDF (2268KB) ( 343 )  
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GABA is an important inhibitory neurotransmitter in the brain,beside the function of transmissing information,which plays an important role in endocrine tissues.In this study,the CDS of buffalo GABRP gene was cloned and its biological information was analyzed,the expression of GABRP in buffalo mammary tissue was also examined by Real-time quantitative PCR and immunohistochemistry.The RT-PCR results showed that GABRP CDS fragment was obtained successfully from Guangxi local buffalo,which shared 99% with that of river buffalo,its length was 1 401 bp.The phylogenetic tree showed that GABRP had high conservation in different species.The immunohistochemistry results showed that buffalo GABRP protein located at the alveolar epithelial cells of buffalo mammary.The expression of GABRP gene in non-lactation buffalo mammary was significantly higher than that of lactation tissue (P<0.05).The results laid a foundation for further study the function of GABRP gene in the development of buffalo mammary gland.

Single Nuclease Polymorphism Analysis of Leptin Gene in Buffalo
PANG Chun-ying, DENG Ting-xian, LU Xing-rong, ZHU Peng, DUAN An-qin, LIANG Xian-wei
2016, 43(9):  2418-2424.  doi:10.16431/j.cnki.1671-7236.2016.09.029
Abstract ( 221 )   PDF (1625KB) ( 403 )  
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This study was aimed to detect the polymorphisms of Leptin gene in buffalo that provided a fundamental for further study on marker assisted breeding in buffaloes.The single nuclease polymorphisms (SNP) of Leptin gene were identified and genotyped by using DNA pooled sequencing and high-resolution melting (HRM) method in three buffalo breeds with 182 buffalo individuals,respectively.The results showed that seven SNPs of Leptin gene were identified in studied population that were located in the intron 1,intron 2 and exon 3 regions,respectively.All the SNPs loci were moderate polymorphism except for the SNP5 and SNP6.The χ2-test indicated that all the SNPs loci were in agreement with Hardy-Weinberg equilibrium in Nili-Ravi population (P>0.05).Our findings revealed that seven SNPs of Leptin gene in buffalo were identified and genotyped in population,which provide the data support for further analyzing the associations between these polymorphism and production traits in buffaloes.

Function and Research Methods of Long Noncoding RNA in Livestock and Poultry
LU Qi-rong, LIU Meng-ke, LI Li, CHENG Gu-yue, HAO Hai-hong, WANG Xu, DAI Meng-hong, YUAN Zong-hui
2016, 43(9):  2425-2434.  doi:10.16431/j.cnki.1671-7236.2016.09.030
Abstract ( 272 )   PDF (1718KB) ( 960 )  
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Long noncoding RNA (LncRNA) is a class of RNA molecules,which is more than 200 nt in length and not capable of encoding proteins.It mainly participates in various biological processes including lipid metabolism,muscle development,embryonic development,sex determination,differentiation and diseases control.The establishment and application of research methods plays a critical role in the research on LncRNA.The review introduces the biological characteristics and biological function of LncRNA in livestock and poultry,focusing on the research methods of LncRNA.Currently used methods in LncRNA research include gene chip,RNA-seq,Northern blotting,fluorescence in situ hybridization,Real-time quantitative PCR,parallel analysis of RNA structure (PARS) and fast predictions of RNA and protein interactions and domains (CatRAPID).In addition,the methods for analyzing the interaction of LncRNA with DNA,RNA,and protein including RNA-pulldown,RNA binding protein immunoprecipitation (RIP),photoactivatable ribonucleoside enhanced cross-linking and immunoprecipitation (PAR-CLIP),RNA antisense purifyication (RAP),chromatin isolation by RNA purification (ChIRP),capture hybridization analysis of RNA targets (CHART) and crosslinking,ligation and sequencing of hybrids (CLASH) are highlighted in order to provide a reference for research of LncRNA in livestock and poultry.

Effect of Different Yolk Composition,Soybean Lecithin and Cholesterol-loaded Cyclodextrin on Equine Sperm Cryopreservation
WU Zhuang-yuan, LUO Yong-ming, DONG Hong, HUO Fei, ZHENG Xin-bao, YU Wei-hao, XU Wen-hui, CHEN Jing-bo
2016, 43(9):  2435-2440.  doi:10.16431/j.cnki.1671-7236.2016.09.031
Abstract ( 238 )   PDF (966KB) ( 538 )  
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In the present study,we investigated the effect of different yolk components (whole egg yolk (EY),egg yolk plasma proteins (EYP),low-density lipoprotein (LDL) and livetin),soy lecithin (SL) and cholesterol-loaded cyclodextrin (CLC) on equine sperm cryopreservation.Semen was collected from 4 adult thoroughbred stallions,experiments 1:Adding 0,2%,4% and 8% concentration of EY,LDL,EYP or livetin in INRA82+3.5% combination cryoprotectants;Experiments 2:Adding 1%,2% and 4% SL with 4% EY as control group;Experiment 3:Adding 0,0.75,1.5 and 3 mg/mL concentration of CLC.The results showed that EYP,LDL and livetin had a significant cryoprotective function(P<0.05),but no significant difference was observed between different components of the yolk and whole egg yolk (P>0.05);Addition of 1% SL obtained similar cryopreservation result with the control group (4% egg yolk) (P>0.05),but post-thaw TM,PM and RAP supplemented with 2% and 4% SL group were significantly lower than the control group (P<0.05);There was no significant difference between different concentrations of CLC group and the control group in thawed sperm motility (P>0.05).In conclusion,EYP,LDL and livetin had a significant cryoprotective function,but no significant difference with EY;Addition of 1% SL could obtain similar cryopreservation result with the 4% EY group;Supplying CLC in extender contain egg yolk could not improve post-thaw equine semen quality.

Correlation Research of Beijing-You Chicken between Polydactyly Characteristic and Product Quality
LV Xue-ze, JIA Ya-xiong, HU Yan-peng, ZHAO Chun-ying, LI Xing-min, CHEN Ji-lan, CHEN Yu
2016, 43(9):  2441-2446.  doi:10.16431/j.cnki.1671-7236.2016.09.032
Abstract ( 217 )   PDF (950KB) ( 406 )  
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The purpose of this study was to explore differences of the Beijing-You chicken product quality between different dactyly.Through the production performance,egg quality and meat characteristics comparative analysis research,we could get the correlation between the Beijing-You chicken polydactyly characteristic and its product quality.So this research could provide the reference to characteristic breeding and the development of the product quality of Beijing-You chicken.In this test,we selected Beijing-You chicken (for meat and eggs),and divided into 3 groups (four-toed (4F),single five toes (SF) and double five toes (DF)) with 4 replicates per guoup and 60 hens per replicate, 720 hens totally.Then,the production performance (weight,laying rate and egg weight),egg quality (haugh unit) and muscle quality (pH,waterholding capacity,shear force,TPA,andnucleotide metabolites) were determined,respectively.The results showed that the weight of DF Beijing-You chicken in 120 days and 240 days of age were slightly higher than that of 4F and SF Beijing-You chicken,but no significant difference (P>0.05);The egg weight of DF Beijing-You chicken in 160 days of age was significantly higher than other Beijing-You chicken (P<0.05).As for DF Beijing-You chicken,the waterholding capacity in leg muscles was better than the other types (P<0.05),hardness,conglutination and chewing of leg muscle were significantly lower than other types (P<0.05),while the cohesiveness and elasticity were significantly higher than other toe types (P<0.05).The shear force in leg muscles of DF and SF Beijing-You chickens were significantly higher than other types (P<0.05),the flavor substance shared no difference between different dactyly Beijing-You chicken (P>0.05).The performance of body weight and egg weigh of DF Beijing-You chicken were superior than 4F and SF Beijing-You chicken in the early stage,but had no significant difference.In addition,the muscle quality of different dactyly Beijing-You chicken had their own advantages,overall,the correlation between leg muscle quality and different dactyly was greater than the brisket.

Analysis of the Immune Efficacy of Attenuated Live Vaccine in the Pig Herds Previously Infected with Wild-type Porcine Reproductive and Respiratory Syndrome Virus
XU Xin-ting, SHI Kai-chuang, LI Chang-ting, LONG Fei-xiang, MO Sheng-lan, CHEN Han-zhong
2016, 43(9):  2447-2454.  doi:10.16431/j.cnki.1671-7236.2016.09.033
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To evaluate the immune efficacy of porcine reproductive and respiratory syndrome (PRRS) attenuated live vaccine,this study selected 4 litters from two small pig farms (two litters each farm) and vaccinated with porcine reproductive and respiratory syndrome virus (PRRSV) attenuated live vaccine.Sera were collected on different days post vaccination (dpv) to detect PRRSV nucleic acid by RT-PCR,and PRRSV antibody using two different ELISA Kits,i.e.N-ELISA and G-ELISA.The results showed that PRRSV nucleic acid were positive on 0 dpv until 30 dpv in piglets and also positive on 0 dpv in the corresponding sows.All piglets were negative for PRRSV antibody on 0 dpv,but were positive on 30 dpv until 150 dpv.The positive rates detected by N-ELISA Kit were higher than those of G-ELISA Kit on 30 and 60 dpv,but lower than those of G-ELISA Kit on 120 and 150 dpv.A total of 216 sera were detected respectively by two ELISA Kits and the coincidence rate of the results was 95.83%.The P value of χ2 test was more than 0.05,showing there was no significant difference between the results of two Kits.The Kappa value was 0.87,showing there was strong consistence between them.The correlation coefficient was 0.605,showing there was significantly linear correlation between them.The results indicated that the wild-type PRRSV in the previously infected pig herds could be eliminated by vaccination with attenuated live vaccine and both N-ELISA and G-ELISA Kits could be used to estimate the immune efficacy of the attenuated live vaccine effectively.

Establishment and Application of Duplex RT-PCR Assay for Detection of Porcine Epidemic Diarrhea Virus and Pseudorabies Virus
SUN Yan-qin, MA Zhen-yuan, YAN Ruo-qian, WANG Dong-fang, CAO Wei-wei, GUO Yu-pei
2016, 43(9):  2455-2460.  doi:10.16431/j.cnki.1671-7236.2016.09.034
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In order to establish an assay for detecting porcine epidemic diarrhea virus (PEDV) and pseudorabies virus (PRV),two pairs of primers were designed basing on the M gene of PEDV and gE gene of PRV,respectively.The total RNA of standard PEDV and PRV strains were used as templates to establish the duplex RT-PCR assay.The specificity,sensitivity,repetition and clinic detection of the established assay were tested.The result revealed that the threshold of duplex RT-PCR was 10 TCID50/mL of PEDV and PRV,and no products were amplified from the cell or the nucleic acid of other 7 kinds of pathogenic viral or bacterial microorganism.The detection results for 26 clinical suspicious PEDV or PRV infected pigs were consistent with the results tested by sequencing.This study suggested that the duplex RT-PCR method was highly specific,repeatable and sensitive,and was suitable for clinic rapid differential diagnosis of PEDV and PRV.

Identification of Pseudorabies Virus Variant and Control of Pseudorabies
HUANG Yuan, CHEN Jing, ZHAO Cui-ling, CHEN Jin-liang, XIANG Rong, WANG Xiao-hu, XIANG Hua, HUANG Zhong
2016, 43(9):  2461-2467.  doi:10.16431/j.cnki.1671-7236.2016.09.035
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Due to variant strain,pseudorabies virus (PRV) has broken out again and spread in China since 2011.A swine farm in Guangdong province was found pseudorabies (PR) symptoms-like miscarriage after introduction.The study was carried out to identify and control the PR.Serum of sows with and without miscarriage were randomly collected and the PRV gB and gE were detected by ELISA method,and brain tissues of sick piglets were sampled and the PRV gH gene was tested by PCR.All the sows in the farm were emergently inoculated PRV variant strainin activated vaccine.Serum before and after immunization were collected and detected by ELISA and micro-serum neutralization test.ELISA results showed that gE antibody of all the breeding sows with miscarriage were positive,and that of sows without miscarriage showed weekly positive;The average gB ELISA S/P value of sows with miscarriage was as high as 4.0,while that of sows without miscarriage was over 3.0.PCR of 3 sick piglets were all positive and the sequence of gB gene was 100% identical to BJ-YT-2012,a wide variant stain in 2012. The result of detection of the sows serum at before and after immunization showed that the S/P value of gB rose up from 1.603 before immunization to 2.88 at four weeks after immunization,and the neutralizing antibody rose up from 1:24 to 1:213.This agreed with the results that the sows showed less probability of miscarriage since the first week after immunization and almost no miscarriage after two weeks after immunization.This study suggested that classical PRV vaccine was not effective in this case,while the vaccine made from the variant PRV strain was.

Colonization Detection of Recombinant Attenuated Salmonella ΔcrpC79-13 (pcDNA3-HN) in Major Organs of Chicks
JIN San-jun, DONG Jia-qi, WU Hong-zhi, SONG Jian-lou, WANG Chuan-qi, GUO Zhao-zhou, DIAO Xin-ping, CHENG Xiang-chao
2016, 43(9):  2468-2473.  doi:10.16431/j.cnki.1671-7236.2016.09.036
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The present study was designed to detect ΔcrpC79-13(pcDNA3-HN) which was a recombinational attenuated Salmonella embedded status in chicks major organs,120 one-day chicks were randomly assigned into 3 treatments according to a single factorial arrangement,which were immunized by ΔcrpC79-13,recombinational ΔcrpC79-13(pcDNA3-HN) and PBS,respectively.After immunization for 4,24,48,72,96,120,144 and 168 h,the embedded status of ΔcrpC79-13and recombinational ΔcrpC79-13(pcDNA3-HN) was detected in the heart,liver,spleen,cecum and blood of chicks.The results showed that the amount of attenuated immune Salmonella ΔcrpC79-13 reached a peak in the blood within 24 h,blood bacterial population decline in 24 to 48 h, number of bacteria in the blood rose at 48 to 96 h and reached another peak in 96 h,and the bacteria of the blood fell at 96 to 168 h.In the heart,liver,spleen and cecum,the number of bacteria in these organs at 4 to 72 h had an upward trend and reached a peak at 72 h,viscera bacterial population declined at 72 to 96 h,and the count rose at 96 to 120 h and reached a peak at 120 h,then the number of bacteria decreased.It was similar to the change in each viscera strain of group ΔcrpC79-13(pcDNA3-HN).These data indicated that both of the recombinational bacterial strains ΔcrpC79-13(pcDNA3-HN) and parental bacterial strain ΔcrpC79-13 could be embedded in a series of organ of chicks such as heart,liver,spleen,and cecum.Meanwhile,there was a consistent declining trend of bacterial strain amount in each viscus of chicks.

Study on the Effect of Infectious Bronchitis Virus and Newcastle Disease Virus on Proliferation and Immune Interference
ZHANG Dong-tao, SONG Zhen-wei, ZHOU Xue-zhang
2016, 43(9):  2474-2482.  doi:10.16431/j.cnki.1671-7236.2016.09.037
Abstract ( 205 )   PDF (1943KB) ( 375 )  
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This experiment aimed to investigate the proliferation interference effects of the infectious bronchitis virus (IBV) and Newcastle disease virus (NDV),analyze the immune interference of two vaccines in the immune process,and provide a basis for determining vaccine immune program.This experiment was in a complete randomized design,10-day-old allantoic cavity of the chick embryo was inoculated with different concentrations of IBV and NDV and with a mixture of different concentrations of IBV and NDV,and then the allantoic fluid was collected for determination of the titer of hemagglutination (HA).Furthermore,BALB/c mice and SPF chicken were immunized with different concentrations of IBV and NDV and also with the mixed IBV and NDV with different concentrations,the blood of mice and chicken were collected,and antibody titer and inhibitory titer of IBV and NDV were determined by indirect enzyme linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test.Experimental results showed that in the homeomorphic cultivation research,whether IBV and NDV were inoculated in a different order or inoculated at the same time,the inoculation of IBV behaved interference to the NDV,while the inoculation of NDV had no interference effects to IBV;Mice were inoculated with IBV and NDV,IBV and NDV antibody titers were lower than single immunized group,immune procedures and immune intervals often had effect on the titer of IBV and NDV antibody,IBV had interference on the immune effect of NDV,different needle mixed immunization can improve the antibody titer of IBV and NDV.In the group of SPF chicken inoculated with IBV and NDV,the order of the inoculation and the immune interval had effect on the titer of IBV and NDV antibody,IBV had a interference on the immune effect of NDV;In the immune serum inhibitory test,the data showed that the IBV and NDV antibody production were affected by the immune order of the inoculation and the interval,the effect on the NDV antibody was more obvious,but with the increase of the immune interval,the antibody level of IBV and NDV showed an increasing trend,the chicken was more sensitive than the mice to reflect the immune interference of IBV on NDV.IBVand NDV antibodies titer was also reduced in the groups that was inoculated with the mixed IBV and NDV,IBV had a interference on the immune effect of NDV.In the homeomorphic cultivation research and animal research,IBV behaved interference to the NDV,while NDV had no interference effects to IBV.IBV and NDV antibody titers decreased when mixed immunization,IBV had a interference on the immune effect of NDV.The order of the inoculation and the immune interval had effect on the antibody titer of IBV and NDV,but with the increase of the immune interval,the antibody level of IBV and NDV showed an increasing trend.

Research Progress on Pseudorabies and Its Influence on Fur-bearing Animals
LI Xin-tong, LIU Hao, LIU Shuai, YAN Xi-jun
2016, 43(9):  2483-2489.  doi:10.16431/j.cnki.1671-7236.2016.09.038
Abstract ( 241 )   PDF (983KB) ( 455 )  
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Pseudorabies outbreak and spread both at home and abroad since 2010.It is reported that some vaccinated swine are infected with pseudorabies virus,even a certain amount of death happened in not only China but European and American area.From about 7% in 2010 to 26% in 2014,the prevalence of field strain pseudorabies virus(PRV) rose year by year.At the same time,in the Northeast and North China including Shangdong province,fur-bearing animals as mink and fox also were attacked by this virus with a high virus carried rate reaching 30.6% to 37.2%.In addition,the PRV adapt to fur-bearing animals would cause high incidence and mortality rates.In order to study the causes of the disease and develop the prevention and control measures in time,pseudorabies epidemical situation,test method,the use of vaccine were reviewed in this paper.What's more,the endemism of the mink was expounded,and gave analysis and prospects to prevention as well as treatment of this disease on fur-bearing animals.

Research Progress on Fish Streptococcus agalactiae Disease
DENG Yong-qiang, WANG Kai-yu
2016, 43(9):  2490-2495.  doi:10.16431/j.cnki.1671-7236.2016.09.039
Abstract ( 232 )   PDF (1036KB) ( 767 )  
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Streptococcus agalactiae is common zoonotic bacteria,which cause neonatal pneumonia,meningitis,septicemia and mastitis in dairy cows.In recent years,it was also be isolated from aquatic animals frequently.Especially since 2007,diseases caused by the bacteria outbreak in Guangdong,Hainan,Fujian,Guangxi and other tilapia aquaculture areas and had a serious threat to the healthy development of tilapia aquaculture in china.In this paper, research progresses on fish Streptococcus agalactiae disease were reviewed.The etiology,epidemiology,symptoms,pathological changes,virulence factors (capsular polysaccharide,CAMP factor,Sip,protein alpha,C5a peptidase) and prevention and control were included in the review,which would provide references for the fish Streptococcus agalactiae disease prevention and control.

Research Progress on Effects of Dietary Crude Protein Levels on Intestinal Barrier Function in Weaned Piglets
MENG Hong-jiao, ZHU Shi-xin, XIANG Dong, KONG Xian-jie, JIANG Hai-long
2016, 43(9):  2496-2501.  doi:10.16431/j.cnki.1671-7236.2016.09.040
Abstract ( 233 )   PDF (967KB) ( 482 )  
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In recent years,many studies have indicated that appropriate reduction of dietary crude protein (CP) level is an effective way to maintain intestinal health and integrity of weaned piglets,and it is very important to protect the intestinal tract of piglets after weaning.In this paper,the effect of dietary CP level on the intestinal barrier,the permeability of intestinal mucosa,micro ecological and enteropathogenic Escherichia coli infection of weaned piglets were summarized to provide theoretical references for further study of the reasonable application of the dietary CP level on weaned piglets.

Detection and Analysis of the Pig Sperm Quality by Flow Cytometry
WU Meng, DING Yu-chun, LIU Zuo-hua, LIU Wen, LUO Lin, GE Liang-peng
2016, 43(9):  2502-2507.  doi:10.16431/j.cnki.1671-7236.2016.09.041
Abstract ( 245 )   PDF (1422KB) ( 503 )  
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The flow cytometry was used in this trail to determine the pig sperm quality,in order to ensure the sperm had complete functions during the fertilization process.Four stain methods of SYBR-14/PI (Propidium Iodide),YO-PRO-1/PI,PNA/PI,Mito tracker/YO-PRO-1 were used to detect the plasma membrane integrity,apoptosis,top body ntegrity and mitochondrial function of Rongchang pig sperm.SYBR-14+/PI- expressed live sperm with complete plasma membrane,YO-PRO-1-/PI- expressed live sperm,PNA-/PI- expressed live sperm had no top body reaction,and Mito Tracker+/YO-PRO-1-expressed live sperm with high mitochondrial activity.The functional integrity of the tested sperm was evaluated according to the above comprehensive results.Through the study of Rongchang pig sperm quality detection by flow cytometry,it suggested that the method could provide theoretical basis for clinical diagnosis,and was very important for swine infertility treatments.