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20 September 2015, Volume 42 Issue 9
Cloning of Buffalo BMP1 Gene and its Expression Pattern Investigation in Different Tissues
LEI Xiao-can, CUI Kui-qing, SU Jie, LI Zhi-peng, ZHAO Xian-hong, LIU Qing-you, SHI De-shun
2015, 42(9):  2215-2223.  doi:10.16431/j.cnki.1671-7236.2015.09.001
Abstract ( 230 )  
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Buffalo BMP1 gene was cloned in the present study, the BMP1 sequence was systemically analysed by bioinformatics techniques, and the expression level of BMP1 gene in different tissues were also assayed with Real-time fluorescence quantitative PCR (QRT-PCR).The results showed that with RT-PCR a 3 195 bp buffalo BMP1 gene was cloned and sequenced, including the whole ORF of 2 967 bp (coding 988 amino acid).The sequence multialigned results showed that buffalo BMP1 gene shared 99%, 96%, 96%, 96% and 95% of similar amino acid sequence with Bos taurus, Sus scrofa, Equus, Homa sapiens and Mus musclus, respectively.It was predicted that buffalo BMP1 protein contained a signal peptide domain, a preregion, a metalloproteinases domain, five complement-Uegf-BMP-1 domain (CUB domain) and two epitheloid growth factor-like domain (EGF-like domain).In addition, we also analyzed the expression level in different tissues through QRT-PCR, the results showed that BMP1 gene mRNA existed in all nine tissues with the most abundant expression in heart, followed by testis, ovary, genital ridge, while with lower amount of bone and other tissues, the minimal expression in liver was observed.
SNPs Screening and Bioinformatics Analysis of SIM1 Gene in Congjiang Pigs
LI Jun, ZHANG Yong, YANG Hong, ZHANG Xiong
2015, 42(9):  2224-2232.  doi:10.16431/j.cnki.1671-7236.2015.09.002
Abstract ( 267 )  
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In this study, Congjiang pig was served as a research object, crossbred pigs (Congjiang pig×boar), outside crossbred pigs (Duroc×Landrace×Yorkshire) as controls.Using DNA pools and direct sequencing detected polymorphism of 7 exons, part of introns and 3'untranslated region (3'-UTR) sequences of SIM1 gene for three groups.Bioinformatics software predicted what impact polymorphic loci had to mRNA secondary structure and protein primary, secondary structure of SIM1 gene.The results showed that 12 SNPs were screened in SIM1 gene of three groups, C77T located in exon 5, T29186C and A29195C located in intron 9, C63T and C225T located in exon 10, and C107T, A426G, T583C, A586C, A605C and A615C located in exon 11, G267T located in 3'-UTR. C77T, T29186C, A29195C, C63T, C225T, C107T and G267T were silent mutations, A426G, T583C, A586C, A605C and A615C were missense mutations.The five missense mutations respectively led to isoleucine (Ile) into valine (Val), leucine (Leu) into proline (Pro), glutamate (Glu) into alanine (Ala), glutamic (Glu) into aspartic (Asp), asparagine (Asn) histidine (His), and according to online software forecast, mRNA secondary structure and protein primary, secondary structure of SIM1 gene changed in mutations before and after.
Analysis of Codon Bias on Lysozyme Gene of Phage vB_EcoM-Bp7
LU Guo-min, ZHANG Can, ZOU Ling, LIU Wen-hua, WEN Jian-xin, REN Hui-ying
2015, 42(9):  2233-2239.  doi:10.16431/j.cnki.1671-7236.2015.09.003
Abstract ( 227 )  
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Bioinformatics softwares were used to analyze the codon bias of phage vB_EcoM-Bp7.Using Mobyle cusp of CodonW 1.4.4 and the program calculated the codon bias indicators, and calculated codon usage patterns of lysozyme gene of 7 phages in total GC content, the codon in the first and second position GC content and the codon in the third position GC content.Using MegAlign software analysis the amino acid sequence of 7 phage lyase, phylogenetic tree.The results manifested that codon usage patterns of lysozyme gene of Bp7 were basically consistent with those of other five phage lysozyme genes except phi68.Bp7 lysozyme preferred to use the A or U ending codon.Twelve kinds of high-frequency codons were identified besides in addition to AUG (Met) and UGG (Trp).Bias comparison of lysozyme codon among related phages showed that Bp7 was most similar with IME08, JS98 came second.Both phylogenetic relationship and cluster analysis demonstrated that Bp7 lysozyme was the closest with IME08 lysozyme.The results would provide further guidance for the evolution research of phage Bp7.
Development and Application of a Duplex PCR Assay for Detecting Mycoplasma ovipneumiae and Mycoplasma arginini
ZHENG Jia-qi, HUANG Hai-bi, WANG Xiao-hui, LI Zhen-ya, XING Meng-en, HAO Yong-qing
2015, 42(9):  2240-2245.  doi:10.16431/j.cnki.1671-7236.2015.09.004
Abstract ( 288 )  
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In order to establish a duplex PCR method for simultaneous detection of Mycoplasma ovipneumiae and Mycoplasma arginini, specific primers of Mycoplasma ovipneumiae and Mycoplasma arginini were designed, and evaluated its sensitivity and specificity after optimizing the reaction conditions of PCR.Then, a total of 40 nasal swabs were tested by duplex PCR.The assay could specifically amplify PCR fragments of 545 and 806 bp from Mycoplasma ovipneumiae and Mycoplasma arginini, respectively.While no PCR products were detected for other pathogens.The detection limits of the assay were determined to be 100 pg/μL for Mycoplasma ovipneumiae and 10 pg/μL for Mycoplasma arginini.The duplex PCR could detect Mycoplasma ovipneumiae and Mycoplasma arginini, and the coincidence rate could reach as high as 92.5% with enrichment culture about the 40 nasal swabs.The results suggested that the duplex PCR could be useful for clinical detection of Mycoplasma ovipneumiae and Mycoplasma arginini.
Prokaryotic Expression and Antigenic Characterization Identification of G Gene for Bovine Ephemeral Fever Virus
LI Zhi, ZHENG Fu-ying, GAO Shan-dian, WANG Su-yan, YUE Cheng, YIN Hong
2015, 42(9):  2246-2253.  doi:10.16431/j.cnki.1671-7236.2015.09.005
Abstract ( 267 )  
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In order to explore specific candidate gene of antigens and diagnostics development for the bovine ephemeral fever virus (BEFV), the target gene sequences were derived from G gene by PCR amplification using two specific primers.The PCR products were digested by Xho Ⅰ and Nde Ⅰ and cloned into pET-30 vector, and the recombinant plasmids (480-pET-30, 1107-pET-30) were transformed into E.coil BL21 (DE3) cells, the D600 nm of positive strains were 0.6 to 1.0.The recombinant strains were induced by IPTG (1.0 mmol/L) at 37 ℃.The characteristics of the target proteins were analyzed using SDS-PAGE and the immunogenicity was analyzed through Western blotting.At the same time, the target proteins were used as coating antigens to do ELISA and all rabbits were inoculated with recombinant proteins.The results showed that the expression feature of 1 107 bp gene fragment of G gene for the first time was segmented in vitro as well as has nicer biological activity and specificity, and it more be suitable as a candidate gene for molecular vaccine and diagnostics development.This study provided the theoretical foundation of the establishment of diagnostics technique and vaccine for BEFV.
Genetic Variation Analysis of NSP2 and ORF5 Genes of PRRSV Isolates in Jiangxi Province from 2013 to 2014
LI Hai-qin, LIN Juan, LIU Lei, JI Hua-yuan
2015, 42(9):  2254-2261.  doi:10.16431/j.cnki.1671-7236.2015.09.006
Abstract ( 266 )  
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To study the genetic varations of ORF5 and NSP2 genes of porcine reproductive and respiratory syndrome virus (PRRSV) isolates in Jiangxi province from 2013 to 2014, the complete ORF5 gene and subsequence of NSP2 gene of PRRSV were amplified by RT-PCR from lung samples of swine in Jiangxi.Sequence comparison and genetic variation analysis were conducted by DNAStar and Mega 6.0.The results showed that the homologies of ORF5 genes among the 12 PRRSV isolates were 83.7% to 99.8% and 82.1% to 99.5% in nucleotide and amino acid sequences, respectively.Compared to the reference strains JXA1, VR-2332 and LV, the homologies were 84.9% to 99.7%, 85.2% to 91.0% and 62.4% to 64.8%, respectively.Nsp2 sequences were amplified from positive samples and compared with the other published sequences, it indicated that 12 PRRSV isolates from Jiangxi belonged to Northern American genotype, 12 isolates had 30 amino acids discontinuous deletions in NSP2 protein which showed a typical structure as previously reported for highly pathogenic PRRSV.The phylogenetic tree of ORF5 of 12 isolates indicated that 10 PRRSV isolates were in the same branch with the highly pathogenic PRRSV, which showed that the highly pathogenic PRRSV had become the dominant PRRSV epidemic strain in Jiangxi.
Cloning and Sequence Analysis of gE and gI Genes of Pseudorabies Virus NP Isolate
LIN Qun-qun, ZHENG Xiao-xiang, CHEN Peng-qiang, CHEN Qiu-yong, ZENG Xian-cheng, HU Chong-wei, XIU Jin-sheng
2015, 42(9):  2262-2269.  doi:10.16431/j.cnki.1671-7236.2015.09.007
Abstract ( 347 )  
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According to published gE and gI gene sequences of pseudorabies virus (PRV) in GenBank, we designed two pairs of primers for PCR amplification of gE and gI genes of PRV NP isolate, after PCR products recycling, cloning and sequencing, the sequencing results were consistent with expectations of PRV gE and gI genes.Homology comparison analysis results revealed that compared with the domestic PRV strains, the homologies of gE and gI amino acids of PRV NP isolate were 95.7% to 99.8% and 89.9% to 99.5%, respectively.Phyogenetic tree analysis and amino acid sequence alignment results found that the gE amino acid sequence site changes of PRV NP isolate were the same with the PRV strains which were isolated from domestic in 2012, thus we could speculate that PRV NP isolate had mutanted.This study had laid the foundation for the epidemiological investigation and analysis of PRV, also provided the scientific basis for the development of scientific, effective and new pseudorabies vaccine.
Cloning and Sequence Analysis of P97 Gene R1 Region of Mycoplasma hyopneumoniae in Guangxi Luchuan Pig
LI Ke-yu, ZHAO Wu, LI Bin, QIN Yi-bin, LU Bing-xia, LIANG Jia-xing, HE Ying, ZHOU Ying-ning, WEI Ying-yi
2015, 42(9):  2270-2277.  doi:10.16431/j.cnki.1671-7236.2015.09.008
Abstract ( 241 )  
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Through in-depth understanding of sequence characteristics, structure and genetic variation of adhesion factor P97 gene R1 region of Mycoplasma hyopneumoniae (Mhp) epidemic strains in Guangxi Luchuan pig, the assay was aimed to provide theoretical basis for mycoplasmal pneumonia of swine (MPS) integrated effective prevention and control measures of Guangxi local variety Luchuan pig, and lay a solid foundation for further study the characteristics of Mhp epidemic strains in Guangxi Luchuan pig.A pair of primers was designed to amplify P97 gene R1 region according to Mhp genome sequence, the MPS positive disease materials of Guangxi Luchuan pig from 2011 to 2014 as the object of study, after genomic DNA extraction and PCR amplification of P97 gene R1 region, the PCR products were sequenced.The base composition and deduced amino acid sequence of P97 gene R1 region of Mhp epidemic strains of Guangxi Luchuan pig were compared.The analysis of DNAStar showed that there were multiple base mutations in P97 gene R1 region of 15 strains of Guangxi Luchuan pig Mhp strains (GXLC-1, GXLC-2, GXLC-3, GXLC-4, GXLC-5, GXLC-6, GXLC-7, GXLC-8, GXLC-9, GXLC-10, GXLC-11, GXLC-12, GXLC-13, GXLC-14 and GXLC-15) and 3 strains of Guangxi other breeds of pigs Mhp strains (GX227, GX595 and GX674).The statistical repeat number of five amino acid (AAKPV/E) of P97 R1 region were 9 to 18, the average value was 12, TN repeats were 1 to 4.We found that Guangxi Luchuan pig Mhp strains mutation made its virulence and adhesion ability enhancement, and respiratory of Luchuan pig with the special structure of short and narrow, which could more easily lead to the occurrence of Guangxi Luchuan pig MPS.
Establishment of Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry Determination Method of Amantadine in Chicken and Preparation of Positive Samples
CHANG Ping-ping, XU Chun-nan, ZHANG Li-xin, HAO Xiao-mei, GAO Xing, CHEN Chao
2015, 42(9):  2278-2285.  doi:10.16431/j.cnki.1671-7236.2015.09.009
Abstract ( 233 )  
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An ultra performance liquid chromatography (UPLC)-tandem mass spectrometry analysis method was established in this experiment for determination of amantadine in chicken.1% trichloroacetic acid with methanol as extraction liquid, the ultrasonic assisted solvent extraction was used to extract amantadine in chicken, extract liquid was purified and enriched in MCX solid-phase extraction column.Using C18 chromatographic column as separation column, in the positive ion mode, we detected the samples by electrospray ionization tandem mass spectrometry.Mass spectrometry and chromatographic conditions, the type of extract liquid, ultrasonic extraction time, the types of solid-phase extraction column and eluent were optimized.Under optimal conditions, a good linearity (r=0.9998) in the ranges of 0.1 to 20.0 ng/mL was obtained, the recoveries were 89.7% to 101.4% in the additive levels of 5.0, 10.0 and 20.0 μg/kg, the limit of detection (LOD) of amantadine was 0.07 μg/kg, the limit of quantiation (LOQ) of amantadine was 0.23 μg/kg, and the relative standard deviations (RSD) were below 7.0%.This novel approach had high sensitivity and accuracy, and was successfully applied to rapid and high sensitive detection of amantadine residues in chicken.The positive samples of amantadine in chicken were successfully prepared with concentrations of 11.16 and 7.18 mg/kg, which could be used for the research of ELISA kits of amantadine.
Cloning and Prokaryotic Expression of L1 Gene of Bovine Papillomavirus Genotype 13
ZHAO Tian-jing, JIA Xiao-xiao, SHI Qiao-yun, GUO Shi-yu, PANG Feng, ZHU Hua-pei, XU Kai-lian, LI Ya-ying, PENG Dong-mei, LI Guo-hua, WANG Feng-yang
2015, 42(9):  2286-2291.  doi:10.16431/j.cnki.1671-7236.2015.09.010
Abstract ( 243 )  
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This experiment was conducted to clone and express L1 gene of bovine papillomavirus genotype 13 (BPV13).Specific primers were designed according to the published sequences of BPV13, and 1 494 bp L1 gene fragment was amplified by PCR.After digestion by BamHⅠand Hind Ⅲ, the fragment was inserted into the prokaryotic expression vector pET28a(+) to construct the recombinant plasmid pET28a-L1.The recombinant plasmid pET28a-L1 was identified by double enzyme digestion and nucleotide sequencing, and was transformed into E.coli BL21(DE3).The expression of pET28a-L1 was induced by IPTG after selecting the best concentration and time.The results showed that L1 gene was exactly inserted into the prokaryotic expression vector pET28a(+), after induction, the target protein containing His-tag was successfully expressed by expression bacteria which included recombinant plasmid pET28a-L1;SDS-PAGE result showed that the molecular mass of the protein was 60 ku which was consistent with the expected size;After ultrasonic treatment, SDS-PAGE result showed that the protein existed in the precipitation;The target protein was a fusion protein with His-tag verified by Western blotting.This study laid a solid foundation for the research of function of BPV13 L1 gene and the development of effective DNA vaccine of BPV13.
Development of Nucleic Acid Lateral Flow Immunoassay Detection Method for Foot-and-mouth Disease Virus
GONG Zhen-li, JIANG Tao, QI Shu-yun, CHEN Guo-dong, LIU Yan-hong, LI Yong
2015, 42(9):  2292-2297.  doi:10.16431/j.cnki.1671-7236.2015.09.011
Abstract ( 273 )  
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In this article, through the combination of nucleic acid probes and immune chromatography, a simple, sensitive and specific detection system——nucleic acid lateral flow immunoassay (NALFIA) for amplifing foot-and-mouth disease virus (FMDV) 3D RT-PCR products was established.An ultrasensitive nucleic acid biosensor (NAB) based on streptavidin-labeled gold nanoparticles dual labels and lateral flow strip biosensor (LFSB) were used in this system.The biotinylated goat anti-rabbit IgG was marked to the NC membrane as the alleged strip and the anti-digoxin antibody was labeled to the NC membrane to capture the digoxin probe.After assemblying gold-labeled strip and detecting RT-PCR products, the detection limit of NALFIA was 0.3×10-3 to 3×10-3 μg/μL.The NALFIA was compared with agar gel electrophoresis analysis, the results showed that the sensitivity of NALFIA was higher than agar gel electrophoresis.There was an excellent agreement between the two methods.NALFIA was a method with high sensitive, low cost and short time.In conclusion, this method provided a good alternative to detect FMDV.
Effect of Different Concentrations of Neuropeptide P on Expression of BMP2 and BMP4 Genes in Skin Stem Cells of Mongolia Cashmere Goat
WANG Ya-juan, GAO Feng, LIU Ying-chun, ZHOU Huan-min
2015, 42(9):  2298-2302.  doi:10.16431/j.cnki.1671-7236.2015.09.012
Abstract ( 262 )  
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This study was aimed to explore the expression of BMP2 and BMP4 genes in skin stem cells of Mongolia Cashmere goat with the influence of different concentrations of neuropeptide P (SP).We set five different SP concentrations (0, 1×10-6, 1×10-7, 1×10-8 and 1×10-9 mol/L) to stimulate the skin stem cells, then identified the expression quantities of BMP2 and BMP4 at days of 0, 7, 14 and 21 by Real-time quantitative PCR with the purpose of determining the optimum SP concentration for the differentiation of skin stem cells.The results showed that SP with the concentrations of 1×10-6 and 1 ×10-8 mol/L promoted the expression of BMP2 and BMP4;The expression of BMP2 at 7 and 14 d in SP 1×10-8 mol/L group was significantly higher than that of the SP 1×10-6 mol/L group (P<0.05);The expression of BMP4 in SP 1×10-6 mol/L group had no significant difference with the SP 1×10-8 mol/L group (P>0.05);At 21 d, the expression of BMP2 and BMP4 significantly decreased in SP 1×10-8 and the SP 1×10-6 mol/L groups, and the expression of BMP2 in the SP 1×10-8 mol/L group was significantly higher than that of the 1×10-6 mol/L group (P<0.05), while the expression of BMP4 in the SP 1×10-6 mol/L group had no significant difference with that of SP 1×10-8 mol/L group (P>0.05).The results indicated that the concentrations of 1×10-6 and 1×10-8 mol/L not only might affect the expression of BMP2 and BMP4 of skin stem cells, but also provided a theoretical basis for the directional differentiation of skin stem cells.
Cloning and Sequence Analysis of the VP0 Gene of Porcine Kobuvirus
CHEN Ru-jing, XIU Jin-sheng, CHEN Xiao-luo, WU Xue-min, CHE Yong-liang, WANG Chen-yan, YAN Shan, WANG Long-bai, ZHOU Lun-jiang
2015, 42(9):  2303-2307.  doi:10.16431/j.cnki.1671-7236.2015.09.013
Abstract ( 267 )  
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In this study, specific primers were designed according to VP0 gene of porcine kobuvirus (PKV), and the full-length coding region of VP0 gene was amplified by RT-PCR method and sequenced, then bioinformatics analysis were conducted to investigate the structure and function of the porcine kubovirus VP0 gene.The results showed that the porcine kobuvirus VP0 gene was 1 098 bp in length, coding an open reading frame (ORF) with 366 amino acids.The isoelectric point, molecular weight and instability index of porcine kubovirus VP0 were 6.71, 38.489 ku, 34.65, respectively.The maximum and minimum hydrophobicity were 2.622 and —2.122, respectively.Compared with the porcine kubovirus VP0 genes published previously in GenBank, the sequenced gene shared the highest homology with HNXX-4 strain, which was 89.1%;And shared the lowest homology with S-1-HUN strain, which was 81.1%.The porcine kubovirus VP0 gene shared two different phylogenetic genotype branches, and the Chinese porcine kubovirus isolates distributed in the two phylogenetic genotype branches.
Isolation,Identification and Sequence Analysis of Leukotoxin Gene lktA1 of Fusobacterium necrophorum of Cow Footrot
GAO Jing, LI Chun-qiu, YAO Shuang, ZHANG Li-chun, WANG Xin-yu, WANG Zhi-hui, ZHAO Xi-wen, WEI Shan, WANG En-yu, SUN Dong-bo, WU Rui, GUO Dong-hua
2015, 42(9):  2308-2312.  doi:10.16431/j.cnki.1671-7236.2015.09.014
Abstract ( 248 )  
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For separation and purification of Fusobacterium necrophorum of cow footrot, and analysis of genetic relationship with other strains, the hoof ministry swab samples were detected by PCR based on specific primers of leukotoxin gene, and genomic DNA were isolated from PCR positive samples of Fusobacterium necrophorum culturing in anaerobic medium.The genes of leukotoxin were cloned and sequenced.The results showed that nine of hoof ministry swab samples were all PCR positive samples, and we obtained Fusobacterium necrophorum pure culture from one of the samples which named bFR13-1.The gene sequencing results indicated that the homologies of leukotoxin gene nucleotide sequence of bFR13-1 strain compared with H05, A25 and B35 strains from GenBank were 98.40%, 98.35% and 90.79%, respectively, and the homologies of deduced amino acid sequence were 97.7%, 97.6% and 89.0%, respectively.Phylogenetic tree analysis results showed that leukotoxin gene of Fusobacterium necrophorum bFR13-1 and H05 had high homology and bFR13-1, H05 and A25 showed a close genetic relationship.The result indicated that leukotoxin showed variability between different Fusobacterium necrophorum isolated strains, and it was worth to study whether this change and pathogenicity of Fusobacterium necrophorum were related.
Effects of Estrogen and Progesterone on Canine Endometrial Stromal Cells Proliferation and Progesterone Receptor Expression
ZHANG Meng-xiao, QIU Yu-hua, MA Wei-ming
2015, 42(9):  2313-2318.  doi:10.16431/j.cnki.1671-7236.2015.09.015
Abstract ( 254 )  
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In this study different digestion and isolation methods were applied to obtain canine endometrial stromal cells (ESCs), different concentration levels were set for estrogen (E2) and progesterone (P4), then MTT method was used to measure their effect on cell proliferation in vitro, also cell immunohistochemistry was used for cell identification and measurement of effect on progesterone receptors (PR) expression.The results indicated that E2 (15, 30 and 100 pg/mL) showed no significant regulations on both cell proliferation and PR expression, P4 (15 and 30 ng/mL) had significant promoting effect on proliferation of ESCs (P<0.05), P4 (3, 15 and 30 ng/mL) showed significant inhibitory effect on PR expression (P<0.05), the regulation level was related to concentration and acting time.
Preparation and Identification of Mycoplasma hyrhinis Antiserum
LI Xin-ping, HE Sun, HOU Feng, WANG Gang, CAO Jian, LI Yan-tao
2015, 42(9):  2319-2323.  doi:10.16431/j.cnki.1671-7236.2015.09.016
Abstract ( 343 )  
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Mycoplasma hyrhinis (Mhr) was a common inhabitant in the nasal cavity of pigs.To prepare antiserum of Mhr, we immuned New Zealand White rabbit with Mhr which was emulsified with freund's adjuvant.Western blotting analysis showed that antiserum had the immuneoreactivity to Mhr.The antibody titer was more than 1:160 000 detected by established indirect ELISA.The growth inhibition test confirmed that antiserum could significantly inhibit the growth of Mhr, but the effect on the growth of Mycoplasma pneumoniae (Mhp) was weak.The above results would provide means for identification and detection of Mhr, and also provide references for the separation of Mhp.
Effect of Dietary Energy Level on Growth Performance,Onset of Puberty and Serum Hormone Contents of Wuzhishan Sows
CAO Ting, XUN Wen-juan, SHI Li-guang, ZHOU Xiong, ZHOU Han-lin, HOU Guan-yu
2015, 42(9):  2324-2329.  doi:10.16431/j.cnki.1671-7236.2015.09.017
Abstract ( 307 )  
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This study was conducted to investigate the effect of dietary energy level on growth performance, onset of puberty and serum hormone contents of Wuzhishan sows.Twenty Wuzhishan sows which had the similar body weight (2.5±0.6)kg and age (30±2)d were randomly allocated to 2 treatments named NRC group (Standard group:Feed the basal diet to Wuzhishan sows with the amount of the normal standard) and 0.7 NRC group (Limited energy intake group:Feed the basal diet to Wuzhishan sows with the 70% amount of the normal standard), there were 10 replicates in each group and 1 pig per replicate.The trial lasted for 10 weeks.The results showed that the body weight in NRC group was significantly higher than the 0.7 NRC group 6 to 10 weeks after weaning (P<0.05).The average daily gain weight in NRC group was significantly higher than the 0.7 NRC group (P<0.05).Dietary energy level had significant effect on the age of puberty (P<0.05), and the age of puberty in 0.7 NRC group was later than the NRC group.The thickness of back fat in the age of puberty of 0.7 NRC group was significantly lower than the NRC group (P<0.05), and decreased by 39.7%.In the age of puberty, the contents of FSH, LH, E2 and Leptin in serum of NRC group were significantly higher than that of 0.7 NRC group (P<0.05).In conclusion, higher feeding energy level had a better body condition, which stimulated the puberty and estrous symptom in Wuzhishan sows.
Study on the Nutritional-ecologic Environment of Fe,Cu,Mn,Zn and Se in Yili Summer Pasture of Xinjiang (Ⅱ)
YANG Ju-qing, LUO Qiu-jiang, YANG Kai-lun, ZHANG Guo-qing, WANG Xiao, XIE Peng-gui, ZHANG Ji-rong
2015, 42(9):  2330-2336.  doi:10.16431/j.cnki.1671-7236.2015.09.018
Abstract ( 246 )  
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This experiment objective was to study the Fe, Cu, Mn, Zn, Se five kinds of trace element contents and change rule in the blood and wool of sheep pastured of different age and different gender, provide the basis for summer grazing sheep scientific supplementary feeding trace elements.At the altitudinal belt of 1 400 to 2 999 m of the summer pasture of the Karajon grassland (South slope) and Tangbula grassland (North slope) of Yili, Xinjiang, Chinese merino (Xinjiang type) sheep wool and sheep blood were collected, to determine their contents of Fe, Cu, Mn, Zn and Se for evaluating the nutritional-ecologic environment of trace elements of summer pasture of Yili.The results showed that the contents of Fe, Cu, Mn, Zn and Se in wool of grazing sheep wool were 520.70, 3.89, 30.16, 102.64 mg/kg and 41.18 μg/kg, respectively, in which the contents of the Cu and Se were lower than normal value by 48.3% and 79.9%, respectively, and that of Fe was higher by 603.4%.The contents of Fe, Cu, Mn, Zn and Se in blood were 357.05, 4.18, 0.25, 3.08 mg/L and 45.23 μg/L, respectively, in which the contents of the Cu and Se were lower than normal value by 29.9% and 73.9%, respectively, and that of Fe was higher by 95.5%.The contents of Fe, Cu, Mn, Zn and Se in the wool and blood were increased with the age.The blood contents of Zn and Se between male and female were extremely different.
Effects of Lactic Acid Bacteria Cultures on Growth Performance and Immunity Function of Broilers
FU Li, ZHAO Wei, LI Li-jia, WANG Xiao-dong, DU Chao, ZHEN Yu-guo
2015, 42(9):  2337-2344.  doi:10.16431/j.cnki.1671-7236.2015.09.019
Abstract ( 329 )  
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This experiment was conducted to study the effects of inactivated lactic acid bacteria cultures on growth performance and immunity function of broilers.A total of 300 one-day-old AA broilers with similar weight were randomly assigned to 5 groups with 6 replicates per group and 10 broilers per replicate (half male and half female).The control group was fed a basal diet;The antibiotic group was fed the basal diet supplemented with 0.01% chlortetracycline;The lactic acid bacteria cultures groups (L1, L2, L3) were fed the basal diets supplemented with 0.16% lactic acid bacteria cultures L1, L2, L3, respectively.The whole experiment period was 42 days, including two phase of days 1 to 21 (starter period) and days 22 to 42 (finisher period).The results showed as follows:From 1 to 21 days of age, ADFI of broilers in lactic acid bacteria cultures group L1 was extremely significant higher than that in control and antibiotic groups (P<0.01), the serum content of IL-2 was significantly lower than that in control group (P<0.05), the indexes of spleen, thymus and bursa of fabricius were significantly higher than that in control group (P<0.05).The indexes of spleen and thymus of broilers in lactic acid bacteria cultures group L2 were significantly higher than that in control group (P<0.05);ADG of broilers in lactic acid bacteria cultures group L3 was extremely significant higher than that in control and antibiotic groups (P<0.01);The serum content of IFN-γ of broilers in antibiotic group was significantly higher than that in lactic acid bacteria cultures groups (P<0.05).From 22 to 42 days of age, ADG of broilers in lactic acid bacteria cultures group L1 and group L2 were extremely significant higher than that in control and antibiotic groups (P<0.01);The serum content of IgG was significantly higher than that in antibiotic group (P<0.05), Newcastle disease antibody titers in serum and the indexes of spleen, thymus and bursa of fabricius of broilers were significantly higher than that in control group (P<0.05);The serum content of IgA and the thymus index of broilers in lactic acid bacteria cultures group L3 were significantly higher than that in control group (P<0.05).Therefore, supplementing inactivated lactic acid bacteria cultures in a diet could improve growth performance and immunity function of broilers to some extent.Among them, adding L3 was better during starter period for improving growth performance of broilers, while add L1 and L2 during finisher period.Adding L1 was better during starter period for improving immunity function of broilers, while add L2 during finisher period.
Effect of Alfalfa Flavonoids on Growth Performance and Serum Indexes of Sheep
WANG Meng-zhu, LIU Yan-feng, WANG Wen-qi, NIE Cun-xi, TANG Shu-zhen, ZHANG Wen-ju
2015, 42(9):  2345-2351.  doi:10.16431/j.cnki.1671-7236.2015.09.020
Abstract ( 298 )  
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This experiment was conducted to evaluate the effect of alfalfa flavonoids (Medicago sativa) on growth performance and serum indexes of sheep.This experiment was performed with hybrid F1 sheep of Suffolk sheep×Small-tailed Han sheep.Twenty-eight sheep with the body weight of (27.02±3.03) kg were randomly divided into 4 groups with 7 replicates in each group and 1 sheep per replicate.Sheep in different groups were fed diets with 0 (group Ⅰ), 0.1% (group Ⅱ), 0.2%(group Ⅲ) and 0.4% (group Ⅳ) alfalfa flavonoids, respectively.The experiment was cosisted of 7-day-preliminary trial and 41-day-formal trial.The results showed as follows:① Dietary supplementation of alfalfa flavonoids had no significant effects on growth performance of sheep(P>0.05).② Dietary supplementation of alfalfa flavonoids had significant effects on the contents of GLB, ASP, BUN, HDL and P in serum (P<0.05).Serum GLB content at the 1st day in group Ⅲ was significantly lower than that in groupsⅠand Ⅱ(P<0.05), serum ASP contents at the 1st and 21th days in group Ⅳ were significantly lower than those in group Ⅰ(P<0.05), serum BUN content at the 1st day in group Ⅳ was significantly lower than that in group Ⅰ(P<0.05), serum HDL content at the 21th day in group Ⅱ was significantly lower than that in groups Ⅰ and Ⅲ (P<0.05), serum P content in group Ⅳ at the 21th day was significantly lower than that in groups Ⅱ and Ⅲ (P<0.05).In conclusion, dietary supplementation of alfalfa flavonoids might modulate lipid metabolism, improve nitrogen utilization, maintain energy metabolic and balance Ca and P contents, and have the potential function for promoting growth and no adverse effect on liver.The results suggested that the appropriate supplemental level of alfalfa flavonoids for sheep was 0.2%.
Analysis of Regular Nutritional Components and Amino Acid Contents of Mongolia Bullock Muscle Grazed in Kubuqi Desert
ZHAO Chen-he, Aorigele, WANG Chun-jie, LIU Wen-cai, JIANG Jing, ZHANG Yan
2015, 42(9):  2352-2357.  doi:10.16431/j.cnki.1671-7236.2015.09.021
Abstract ( 296 )  
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In order to evaluate the Mongolia muscle characteristics and nutritional value, six Mongolia bullocks aged 4 years grazed in Kubuqi desert were chosen to analyze the regular nutritional components and amino acid contents of Mongolia muscle.The results showed that the contents of water, crude protein (CP), crude fat (EE), crude ash (ash), calcium and phosphorus of Mongolia muscle (fresh) were 68.21%, 22.60%, 6.49%, 2.69%, 0.58% and 0.55%, respectively.A total amino acid (TAA) content of beef was 24.297 g/100 g, essential amino acid content was 10.265 g/100 g, the proportion of essential amino acid justified and exceeded the FAO/WHO standard.The ratio of essential amino acid and total amino acid contents (EAA/TAA) was 42.25%, the ratio of essential amino acid and the non-essential amino acid (EAA/NEAA) was 73.15%.In functional amino acid, the content of palatable taste amino acid and sweet flavor amino acid in total amino acid was 46.891%.In addition to valine, essential amino acid contents of Mongolia muscle were superior to ideal protein of FAO/WHO standard, the percentage of valine reached 83.972% in ideal protein.These results indicated that Mongolia muscle belonged to the comprehensive nutrition and high quality protein food on the evaluation of the regular nutrient content and the amino acid content of beef.
Effects of Different Substrates on Fermentation Parameters
LIU Xiao-ming, YANG Zai-bin, LI Zhao-yong, WANG Xiao-ming, WANG Zhi-heng
2015, 42(9):  2358-2363.  doi:10.16431/j.cnki.1671-7236.2015.09.022
Abstract ( 271 )  
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Fermenting different raw materials (corn, soybean meal and cottonseed meal) with three kinds of strains (Lactobacillus, yeast and Bacillus subtilis) were employed to optimize the fermentation parameter with sensory evaluation, pH and amount of probiotics retention.The results showed that the time to reach stable pH of materials with different substrates was different.Cottonseed meal had the longest time to reach stable pH followed by soybean meal, while corn had the shortest time under the condition of same water content.Different substrates had no effect on initial pH of corn, soybean meal and cottonseed meal.The count of the probiotics was also influenced by different substrates.When pH reached steady state, corn had the largest number of the probiotics followed by soybean meal and cottonseed meal.The color and smell of three materials after fermented had distinctive features.But cottonseed meal had the highest viscosity followed by soybean meal and corn.Because of shorter time and more microorganisms, corn was better than soybean meal and cottonseed meal to be fermenting medium.
Effect of RH-3 Fusion Hybrid on the Immune Function and Intestinal Microlora of Three Buff Chickens
ZHAO Chun-miao, XU Chun-hou, ZHONG Ri-cong
2015, 42(9):  2364-2370.  doi:10.16431/j.cnki.1671-7236.2015.09.023
Abstract ( 211 )  
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The purpose of this experiment was to study the effect of RH-3 fusion hybrid of Bacillus subtilis and Lactobacillus rhamnosus on the immune function and intestinal microflora of three buff chickens.96 one-day-old chickens were randomly divided into 4 groups with 3 replicates per group and 8 chickens per replicate.Chickens in control group were drinking water that had not added the RH-3 fusion hybrid culture;Chickens in the experimental group were drinking water that supplemented with 0.5%, 1.0% and 1.5% RH-3 fusion hybrid culture, respectively.The whole experiment period was 42 days, including two phases of days 1 to 21 and days 22 to 42.The results showed that compared with the control group, the supplementation of RH-3 fusion hybrid increased the Newcastle disease HI antibody titer, SOD activity, lymphocyte transformation rate in serum and the immune organ index of three buff chickens at days 21 and 42, and the difference was significant of 0.5% level (P<0.05).Compared with the control group, the supplementation of RH-3 fusion hybrid reduced the number of Salmonella and E.coli and increased the number of the Bifidobacterium and Lactobacillus in jejunum, ileum and cecum of three buff chickens at days 21 and 42, and the difference was significant of 0.5% level (P<0.05).In summary, RH-3 fusion hybrid could improve the immune function and regulate the intestinal microflora of three buff chickens, promote the growth of beneficial bacteria and inhibit the growth of harmful bacteria, but the effects showed a downward trend with the increasing of the amount of RH-3 fusion hybrid.So the adding amount of RH-3 fusion hybrid in the drinking water of three buff chickens was suggested as 0.5% under this experimental condition.
Effect of Probiotics-Chinese Herbal Compound on Oviduct, Production Performance and Egg Quality of Hy-Line Brown Hens
XIE Quan-xi, GU Ai-min, YANG Jia-mei, ZHANG Jian-mei, YU Jia-min, GU Wei
2015, 42(9):  2371-2376.  doi:10.16431/j.cnki.1671-7236.2015.09.024
Abstract ( 298 )  
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This experiment was conducted to investigate the effect of probiotics and chinese herbal compound on oviduct, production performance and egg quality of Hy-Line Brown hens.Seven hundreds and twenty 45-week-old Hy-Line Brown hens with similar body weight and laying rate were divided into 4 groups with 3 replicates in each group and 60 hens per replicate.The hens in control group were fed a basal diet and those in the experimental groups (Ⅰ, Ⅱ, Ⅲ) were fed the basal diets supplemented with 1.5‰ probiotics and 1‰ the Chinese herbal medicine one (patrinia, dandelion, radix, forsythia, etc.), 1.5‰ probiotics and 1‰ the Chinese herbal medicine two (honeysuckle, astragalus, epimedium, motherwort, etc.), 1.5‰ probiotics and 1‰ the Chinese herbal medicine three (folium, treats, skullcap, gardenia, etc.), respectively.The preliminary trial lasted for 7 days, and the experimental period lasted for 28 days.The results showed as follows:① Probiotics-Chinese herbal medicine could repair damaged fallopian tubes, reduce tubal adhesions with the surrounding tissue and keep tubal patency, compared with control group, the contents of TNF-α and EGFR of group Ⅱ were significantly decreased by 40.85% and 17.79%(P<0.05), respectively;② Compared with control group, the laying rate of group Ⅱ was significantly increased (P<0.05), meanwhile the feed/egg and soft-broken egg rate were significantly reduced (P<0.05);③At the end of 48 weeks and 50 weeks, egg shape index and yolk ratio of group Ⅱ were significantly increased compared with control group (P<0.05).Therefore, dietary supplementation of probiotics-Chinese herbal medicine could reduce the oviduct inflammatory reaction of hens in a certain extent, improve the laying rate and egg quality.The better combination was group Ⅱ, including probiotics and honeysuckle, astragalus, epimedium, motherwort, etc.
Isolation,Culture and Multiple Differentiation Induction of Nanyang Bovine Bone Marrow Mesenchymal Stem Cells
ZHU Xue-min, WEI Lan, ZHANG Zi-qiang, DENG Wen, SUN Yu, ZHAO Qian-ning, WANG Meng-fan
2015, 42(9):  2377-2383.  doi:10.16431/j.cnki.1671-7236.2015.09.025
Abstract ( 238 )  
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The study aimed to build the method of Nanyang bovine bone marrow-derived mesenchymal stem cells (BMSCs) separation culture in vitro, and study its bionomics and the ability of multiple differentiation induction on this basis. Using bone marrow puncture to take rib bone marrow of 3 months calf, isolation and culture BMSCs, subcultured and determined its curve of growth, detected Oct4, Nanog, Sox2 genes expression by RT-PCR technique, then took P3 BMSCs into neural and fat cells to carry out induction differentiation, and took the use of histology staining technique and RT-PCR technique to detect. The results of the BMSCs were uniform sindleshaped in appearance; RT-PCR could dectect the expression of stem cell factor Oct4, Nanog, Sox2. The results showed that an "S" shape growth curve of cell at different generations times, it entered exponential growth stage at the 3rd day generally, and entered plateau after then the 7th day. Into nerve after induction, stained with toluidine blue was apparent throughout the structure of Nissl substance, ENO2 and GFAP genes showed a positive expression. After adipogenic differentiation of ASCs was assessed by oil Red O staining which showed a large number of lipiddroplet, RT-PCR dectected Leptin and PPAR genes showing a positive expression. The tests showed that had successfully isolated from Nanyang bovine BMSCs and it had the potential of multidirectional differentiation.
Effects of Fusarium Toxins on Serum Enzymes Activity,Liver Antioxidant Function and Histopathology of Weaned Gilts
CHEN Ning-bo, ZHANG Chong-yu, JIANG Shu-zhen, Huang Li-bo, CHEN Xiang-xing, SUN Jian-li, YANG Wei-ren
2015, 42(9):  2384-2390.  doi:10.16431/j.cnki.1671-7236.2015.09.026
Abstract ( 298 )  
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The objective of the present study was to investigate the effects of Fusarium toxins contaminated diet on serum enzymes, liver antioxidant function and histopathology of weaned gilts.Forty healthy weaned gilts (Duroc×Landrace×Yorkshire) aged 35 d with body weight of (8.45±0.94) kg were randomly allocated into 2 groups with 20 replicates per group and 1 pigs per replicate.Gilts in the control group were fed with basal diet, and those in the experiment group were fed with Fusarium toxins contaminated diet made by replacing 50% of corn and corn gluten meal in the basal diet with the naturally contaminated corn and corn gluten meal (0.90 mg/kg ZEN, 1.43 mg/kg DON, 5.85 mg/kg FUM).Gilts were fed individually in a metabolic cage for 35 d after 7 d adaptation.The results showed as follows:① Compared with control group, the serum activities of AST, ALT and ALP in experimental group were significantly increased (P<0.05), and the liver index of experiment group was significantly decreased (P<0.05).②Compared with control group, the activities of GSH-Px and T-SOD in serum and liver of experimental group were significantly decreased (P<0.05), and the MDA content in serum and liver were significantly increased (P<0.05).③ Histological examination of liver in experiment group revealed a certain degree of damage, with obvious inflammatory cell infiltration partly at the margin of hepatic lobule and vacuoles degeneration of local hepatic cells.The results indicated that Fusarium toxins contaminated diets (0.90 mg/kg ZEN, 1.43 mg/kg DON, 5.85 mg/kg FUM) could change serum enzymes activity and impair liver antioxidant function, with a certain degree of pathological injury of liver in the present study, which had a negative impact on the normal liver function in weaned gilts.
Isolation,Identification and Biological Characteristics Analysis of Bovine Viral Diarrhoea-mucosal Disease Virus
BAO Zhen-zhong, LEI Cheng-hong, CAI Yuan-qing, GAO Dou, BIAN Sai-sai, GE Ting
2015, 42(9):  2391-2398.  doi:10.16431/j.cnki.1671-7236.2015.09.027
Abstract ( 254 )  
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In order to isolate and identify bovine viral diarrhea-mucosal virus (BVDV) strain of Xinjiang, grasp the biological characteristics of the strain, we gathered feces which were suspected as bovine viral diarrhea-mucosal disease infected feces in the northern parts of Xinjiang.The strain was separated and identified by RT-PCR, cell isolation and culture, indirect immunofluorescence antibody test, immune electron microscopy and serum neutralization test.After determining TCID50 of the strain, ether sensitivity test, chloroform sensitivity test, trypsin sensitivity test, acid and alkali sensitivity test, temperature sensitivity test and nucleic acid typing test were used to detect the physical and chemical properties of the strain.The samples appeared fragment at 286 bp by RT-PCR, and then inoculated the positive samples into a density of about 80% monolayer MDBK cells, and appeared cytopathic effect, passaged to the 5th generation caused the typical cytopathic effect.The F5 cell cultures were detected by indirect immunofluorescent antibody, the result showed that a yellow-green fluorescence appeared same as C24V.A lot of spherical BVDV particles with the size of 20 to 40 nm were observed by immune electron microscopy.Serum neutralization test had no cytopathic effect, viruses were completely neutralized by positive antibody serum.The isolated strain was determined to be BVDV based on the methods above.The TCID50 of the isolated strain was 10-4.5/0.1 mL.Physicochemical assays showed that the isolated strain was sensitive to chloroform, ether, trypsin and temperature, sensitive to acid and resistant to alkali in acid and alkali sensitivity test, and was completely inactivated by 54 ℃ for 1 h.The virus genome was RNA.The study successfully isolated a BVDV strain in Xinjiang and grasped the biological characteristics of the strain, which laid a foundation for future diagnosis and prevention of the disease.
Detection of Selenium Content in Beef by Hydride Atomic Fluorescence Spectrometry
LIU Li-na, XU Fa-ting, ZHAO Fang-hong, QIN Shun-yi, YAO Wan, LIU Jia, LI Yu-zhu
2015, 42(9):  2399-2404.  doi:10.16431/j.cnki.1671-7236.2015.09.028
Abstract ( 233 )  
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In this assay, we detected the selenium content in beef by hydride atomic fluorescence spectrometry.The conditions of the test were optimized by the selections of clearing up solution, pre-reducing agent, medium acid and so on, the limits of detection, precision and the standard addition recovery were determined.The results showed that 2:1 mixed acid as the clearing up solution, 6 mol/L hydrochloric acid as pre-reducing agent and the volume ratio of 10% hydrochloric acid as acid medium were chosen, the calibration curve was linear in the range from 0 to 8.0 μg/L with correlation coefficient was 0.9999.The limit of detection (LOD) was 0.01 ng/mL.At three adding levels of 20, 30 and 40 μg/L for selenium, the recovery of beef was 94% to 104% with RSD of 3.80%.This method had the characteristics of simple and effective, it had important referential significance for the detection of selenium in meat.
Research Progress on Hair Cortisol as Evaluation Indicators of Chronic Stress
DOU Jin-huan, LI Zhong-shu, XU Qing, WANG Ya-chun
2015, 42(9):  2405-2410.  doi:10.16431/j.cnki.1671-7236.2015.09.029
Abstract ( 250 )  
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Making hair cortisol as the gold standard of reflecting chronic stress level is prospective and innovative.Hair cortisol not only can be used in detecting and estimating the chronic stress level of animals, but also can be benefit for understanding the body's physiological and pathological process and making effective health management strategy in time.Blood, saliva, urine and milk cortisol concentrations can be regarded as matrices for reflecting acute stress level as well, they have been used in detecting and establishing the diurnal profile of circulating cortisol studying.But hair which is a good supplementary matric can reflect the chronic stress level retrospectively.Hair cortisol as a reflection of the biochemical markers of chronic stress has the advantages of no invasive, relatively sensitive detection and convenient sampling stress, and hair cortisol is a heritable trait (h2=0.31).The utility of hair cortisol as a maker for HPA activity, and a useful tool for identifying genetic variation influences on long term individual differences in HPA activity.This article summarized the relationship between the cortisol and animal's healthy, the regulating mechanism and genetic adaptation change under chronic stress and the research progress of evaluation indicators during chronic stress, the research progress of hair as a matric of cortisol, the limitations and challenge of regarding hair cortisol as the biochemical marker to reflect the chronic stress level in the future.
Research Progress on the Development of Mammary Glands
AN Pei-pei, GUO Qian-qian, LI Chun-yi
2015, 42(9):  2411-2417.  doi:10.16431/j.cnki.1671-7236.2015.09.030
Abstract ( 266 )  
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Mammary gland is a kind of exocrine glands and a dynamic organ.Structure of mammary gland changes throughout the female reproductive cycles.Development of mammary gland occurs in the defined stages that are closely linted to sexual development and reproduction, namely embryonic, prepubertal and pubertal stages, pregnancy, lactation and involution.In this review, the roles of epithelial-stromal (mesenchymal) interactions in embryonic and postnatal mammary development were summarized, elaborated the regulations of different kinds of hormones on the mammary development after pubertal stage, and described the discovery of mammary stem cell populations (MaSC) and selection markers of mice and human.The objective of this work was to learn the basic processes and potential regulation mechanisms of the mammary development to provide some help for the researchers in the field of mammary gland.
Distribution Difference of Microsatellite in 7 Domestic Animals Genomes
WANG Yue-yue, LIU Xue-xue, DONG Kun-zhe, CHEN Xiao-fei, YE Shao-hui, MA Yue-hui
2015, 42(9):  2418-2426.  doi:10.16431/j.cnki.1671-7236.2015.09.031
Abstract ( 315 )  
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To investigate whether difference in SSRs distribution of 7 domestic animals genomes, the microsatellite sequence were searched by using MSDB (Microsatellite Search and Building Database) and analyzed in pig, horse, cow, goat, sheep, chicken and dog genomes.Preliminary results showed that SRRs number, frequency and density were found to be largely variable among these animals.The most abundant distribution was observed in dog genome with 1 436 242 identified SSRs loci.The minimum SSRs (276 564) number was found in chicken genome.Yet, the minimum SSRs (430 760) frequency and density were found in horse genome.Among these SSRs, mononucleotide repeat type motif was the most abundant and pentanucleotide repeat type motif was the rarest in all animals.Meanwhile, all the dominant SSRs for different repeat types were abundant in nucleotide A and T.In addition, there was variability among 7 animals of SSRs distribution.In general, SSRs distribution was the most similar in cow, goat and sheep genomes, and chicken was the most different from other animals.The results also showed that the length of microsatellite ranged mainly from 12 to 20 bp in all animals, which might be due to selection pressure of convergence.In short, these results revealed difference as well as conservation in SSRs distribution among different domestic animals genomes.In particular, genetically close animals tended to have similar SSR distribution patters.This would provide a basis for subsequent research of SSRs function.
Phylogenetic Analysis and Detection of Positively Selected Site on the Coding Regions of TYRP1 Gene
LI Li-sha, LI Xiang-long, ZHOU Rong-yan, LI Lan-hui, ZHANG Yong-gai, MA Meng-yun
2015, 42(9):  2427-2435.  doi:10.16431/j.cnki.1671-7236.2015.09.032
Abstract ( 334 )  
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Tyrosinase-related protein 1 (TYRP1) is one of the tyrosinase family members, which was also the first successfully cloned pigment gene.The study aimed at exploring the selective pressure on TYRP1 gene in the process of biological evolution.The study used NCBI to download nucleotide and protein sequences of TYRP1 gene coding regions in different species, through the Clustal X to compare TYRP1 gene encoding protein sequences, using Pal2nal online tool to generate codon multiple sequence alignment, DAMBE software was used to determine the base substitution saturation for constructing phylogenetic trees, using maximum likelihood method and PAML software to analyse TYRP1 gene in different species phylogenetic relationships and selection pressure.The results showed as follows:TYRP1 gene evolution and evolutionary classification of mammals was most close, and the site specific model revealed that TYRP1 genes were mainly affected by purifying selection, and it had been subjected to positive selection in its adaptive evolution, the software found thirteen positive selection amino acid sites.The study analyzed the evolutionary pattern of the TYRP1 gene from the molecular evolutionary perspective, providing a new way for the study of the TYRP1 gene structure and function.
Genetic Diversity Analysis of Two Population of Captive Tianshan Red Deer Using Microsatellite Markers
Rahmutulla·Abdukerim, Zilajigvl·Ekram, NING Li-qun, Guzalnur·Zibibilla, Ezizjan·Nabi, Buzohra·Tursun, Shamshidin·Abduriyim, Mahmut·Halik
2015, 42(9):  2436-2443.  doi:10.16431/j.cnki.1671-7236.2015.09.033
Abstract ( 284 )  
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For scientific evaluation, protection and utilization of the valuable resources of 53 captive Tianshan Red deers (CJ, n=31;HM, n =22), the genetic polymorphisms of 8 microsatellites, analyzed the genetic diversity, genetic diversity and kept in captivity Tianshan Red deer was discussed.Microsatellite data indicated that 8 loci alleles were 3 to 7, 37 alleles were detected.The results showed that genetic diversity of Shenghua domesticated Red deer breeding base were A=4.625, He=0.6398, Ho=0.5887, PIC=0.5851, Ne=2.8357, Fis=0.0338, Hami Red deer farm field were A=4.375, He=0.6888, Ho=0.6818, PIC=0.6238, Ne=3.2078, Fis=-0.0175.The two population was not significantly deviated from Hardy-Weinberg equilibrium (HWE).It suggested that the genetic diversity of the captive Tianshan Red deer from two areas was still higher, however the date of F-statistics indicated that the captive Tianshan Red deer in Changji exchanged individuals resulting to inbreeding. Therefore, at the present, the focus of management of captive Red deer population should shift to avoiding the inbreeding and the loss of genetic diversity.
Polymorphism Analysis of 3 Quail Groups by Using Microsatellite Marker
BAI Jun-yan, PANG You-zhi, ZHANG Xiao-hui, YUN Yin-xian, ZHAO Shu-juan, QI Yan-xia
2015, 42(9):  2444-2452.  doi:10.16431/j.cnki.1671-7236.2015.09.034
Abstract ( 317 )  
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In order to reveal the genetic structure and genetic relationship of Korean quail, Chinese Yellow quail, Chinese Black quail, the 3 quail populations from the molecular level, to provide for the study of genetic resources of quail.This study used PCR amplification, polyacrylamide gel electrophoresis, cluster analysis and other methods of 3 quail populations were polymorphic analysis of microsatellite markers.The results showed that 12 microsatellite markers in 3 quails was detected in the observed number of alleles was between 4 and 7, the average polymorphism information content of Chinese Yellow quail, Chinese Black quail, Korean quail were 0.6853, 0.6401 and 0.6565, respectively;The average heterozygosity were 0.7333, 0.6957 and 0.7111, respectively, which Chinese Yellow quail population genetic diversity was the most abundant polymorphism.The cluster analysis showed that the Chinese Black quail and Korean quail smallest genetic distance was 0.0628, so the Chinese Black quail and Korean quail were clustered into one category, and then was Chinese Yellow quail together.This was further evidence of Chinese Black quail and Korean quail had a closer genetic relationship, and the genetic relationship of Chinese Black quail and Korean quail was more distant with Chinese Yellow quail.
The Association Analysis of Prolactin Receptor Gene Polymorphisms with Milk Yield in Yili Horse
YU Xi, ZANG Chang-jiang, LIU Ling-ling, MA Hai-yu, HE Mei-sheng, JIANG Xin, CAO Hang, LIU Wu-jun
2015, 42(9):  2453-2458.  doi:10.16431/j.cnki.1671-7236.2015.09.035
Abstract ( 285 )  
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This experiment was designed to analyze association of prolactin receptor (PRLR) gene polymorphism with milk yield of Yili horse.The number of 60 horse were selected as samples, detected genetic polymorphism of PRLR gene using PCR-SSCP technique and sequencing technology, and then analyzed the polymorphism with milk yield of Yili horse.The results showed that there were two polymorphism fragments of PRLR gene flanking region, there were three genotypes:AA, AB and BB.Two novel SNPs (g.29764513 G>A and g.30106804 C>A) were identified of PRLR gene flanking region by sequencing.The g.29764513 G>A SNP caused amino acid variations as p.33His>Arg, while the g.30106804 C>A SNP was synonymous mutation without causing amino acid changes.Statistical results indicated that the g.29764513 G>A and g.30106804 C>A SNPs were significantly associated with daily milk yield in Yili horse (P<0.05).These mutations might be as a crucial DNA genetic markersfor milk production traits selection in Yili horse.
Comparison Study on Effects of Three Chinese Herbal Medicine on Intestinal Morphology in Weaning Piglets
LI Hui-feng, LI Qing-hong, LI Hong-quan, HAO Rui-rong, CHEN Jian-hui, GU Jian-wen
2015, 42(9):  2459-2466.  doi:10.16431/j.cnki.1671-7236.2015.09.036
Abstract ( 193 )  
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The objective of this study was to compare the effect of three Chinese herbal medicine (medicated leaven, hawthorn, fructus hordei germinates) on histomorphology of different parts of small intestine in weaning piglets.40 28-day old ternary pigs (Duroc×Landrace×Yorkshire) with the body weight (8.13±1.32)kg were divided into four groups, control group, medicated leaven group, hawthorn group and fructus hordei germinates group, and feeding with 1% Chinese herbal medicine, there were two repeats in each group and each repeat had 5 piglets, slaughtering and sampling at 7th and 14th day morning, respectively, sections were used for HE staining.The staining tissues were taken pictures under a microscope camera (200×) and measured VH, CD, IWT and V/C in different parts of small intestine with Image-Pro Plus.The result showed that the IWH of ileum in medicated leaven group was significantly or extremely significantly higher than control group (P<0.05;P<0.01).The VH and IWT of duodenum and jejunum at 7th day in hawthorn group were extremely significantly lower than control group (P<0.01);The IWT of ileum and the VH of ileum at 14th day were extremely significantly higher than control group (P<0.01).In fructus hordei germinates group, the VH of duodenum and jejunum at 7th day and IWT of jejunum were extremely significantly lower than control group (P<0.01);The CD of ileum at 7th day, the IWT of duodenum and ileum at 7th day were extremely significantly higher than control group (P<0.01).The effects of different simple Chinese herbal medicine on histomorphology of different parts of small intestine in piglets were different, because of the different digestive functions of small intestine, the most effects of Chinese herbal medicine an duodenum, jejunum and ileum were fructus hordei germinates, medicated leaven and hawthorn, respectively.
Preparation of Adjuvant Diluent for Live Vaccine against Classical Swine Fever and Evaluation of its Immune Effects
LIANG Kong-xian, LV Xiao-jun, DING Dan, XU Qi, ZHOU Jie, LIU Wei
2015, 42(9):  2467-2473.  doi:10.16431/j.cnki.1671-7236.2015.09.037
Abstract ( 339 )  
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The aim of this study was to develop a novel oil-in-water (O/W) emulsion as adjuvant diluents (AD) for live vaccine against classical swine fever (CSF) that could effectively enhance the immune effect of vaccine.The AD was prepared by high-pressure homogenization technique.Formulations and preparation parameters were optimized with response surface design.Its stability, particle size, polydispersity (PDI) and Zeta potential were characterized.The humoral immune response and cellular immune response of the AD were evaluated with BALB/c mice by intramuscular injection.The particle size of the AD prepared by optimized formulation and parameters was 100.4 nm, PDI was 0.147, and Zeta potential was —28.7 mV.The experiment results showed that the AD had good stability.The AD was inoculated combined with live vaccine against CSF into BALB/c mice by intramuscular injection.The results showed that the live vaccine against CSF specific immune responses could be evoked in mice by co-inoculation with AD and vaccine.The cellular immune response levels in co-inoculated groups were significantly higher than control group (P<0.05), with obvious phenomena of higher levels of IFN-γ, IL-6 and IL-4 in serum.The result revealed that cellular immune capability significantly improved with the AD.The results strongly revealed that cellular immune capability significantly improved by introducing AD for effective immune-adjuvant for live vaccine against CSF.
Study on Anti-viral Effect of Rhubarb on Porcine Epidemic Diarrhea Virus in vitro
CHEN Tian-tian, CHENG Fu-liang, ZHANG Jian-mei, NIE Zhao-jing, FANG Dong, FAN Mei-na, LI Fu-hui, CHENG He-gang
2015, 42(9):  2474-2480.  doi:10.16431/j.cnki.1671-7236.2015.09.038
Abstract ( 369 )  
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The inhibitory effects of rhubarb on cell lesion, which was caused by porcine epidemic diarrhea virus (PEDV), and its mechanism had been studied in this research.The maximum safe concentration of rhubarb solution applied to Vero cells was determined through morphological observation, and then the PEDV infected Vero cell model in vitro was established, by which the inhibitory effect of rhubarb on virus was studied.The experimental method had also been established based on this cell model.The result showed that the maximum safe concentration was 3.28 mg/mL.Both morphological observation and MTT assay were indicated that rhubarb could not only block the attachment of the virus to Vero cell and inhibit the synthesis and reproduction of the virus, but also could directly inactivate the virus.This research showed that rhubarb had an inhibitory effect on PEDV, and provided a theoretical basis for the prevention and treatment of clinical porcine epidemic diarrhea.
Pathogen Analysis of Bovine Respiratory Disease Complex
MA Chun-xia, LI Jun, QIN Yong, ZHONG Shu-hong, TAO Li, PENG Hao, LAN Mei-yi, YANG Wei, CHEN Ze-xiang, HU Shuai, LI Chang-ting, ZHAO Xiao-hong
2015, 42(9):  2481-2486.  doi:10.16431/j.cnki.1671-7236.2015.09.039
Abstract ( 377 )  
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To investigate the pathogen of bovine respiratory disease complex (BRDC) of a dairy farm in Guangxi, a strain of Mycoplasma and a strain of gram-negative pathogenic bacterium were isolated and identified by the means of field surveys, clinic observation, pathological examination, isolation studies and so on.Treatments were taken according to drug sensitivity test results.The Mycoplasma strain, growing on PPLO medium, formed typical "fried egg" colonies.A 448 bp of oppF fragment was amplified by PCR from the strain and had 98.4% nucleotide identity with Mycoplasma bovis reference isolate PG5 of USA.The biochemical features of the gram-negative bacterial isolate were same with Serratia marcescens.The PCR amplified 16S rDNA of the gram-negative pathogenic bacterium strain was 1 400 bp.It shared 99.0% nucleotide identity with other Serratia marcescens reference strains obtained from GenBank.Animal experiment showed that the gram-negative pathogenic bacterium isolate could cause the mice to die.The drug sensitivity tests showed all isolates were sensitive to spectinomycin, azithromycin, amikacin, gentamicin and neomycin.It was effective to treat with dexamethasone and spectinomycin.Pathogen analysis and drug treatment showed that the BRDC was caused by Mycoplasma bovis and Serratia marcescens.
Detection of Resistance and Resistance Genes of Escherichia coli to β-lactams Antimicrobial Agents Isolated from Waterfowl in Guangdong Province
ZHANG Ji-pei, TAN Hua-long, WEI Qing-lan, CHEN Jian-hong
2015, 42(9):  2487-2492.  doi:10.16431/j.cnki.1671-7236.2015.09.040
Abstract ( 227 )  
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In order to understand the resistance to β-lactams antimicrobial agents and the prevalence of resistance genes, 243 Escherichia coli (E.coli) were isolated and identified from waterfowl in Guangdong province during 2012 to 2014.Drug sensitivity of total 243 E.coli isolates to 4 kinds of β-lactams antimicrobial agents were determined by double broth dilution method.All β-lactams resistant isolates were detected for the presences of blaCTX-M, blasub>TEM and blaOXA genes by PCR.The results showed that the occurrence of ampicillin and amoxicillin were ranked the higher (80.00% to 100.00%), and were 9.09% to 50.00% for ceftazidime and ceftiofur.The blaCTX-M, blaTEM and blaOXA genes were detected in 88.46% to 90.78%, 67.21% to 88.52% and 51.28% to 59.02% of the β-lactams resistant isolates to the four kinds of antimicrobial agents, respectively.The results indicated that the resistance of E.coli isolates from waterfowl to β-lactams antimicrobial agents were widespread, and penicillin was particularly serious.The blaCTX-M, blaTEM and blaOXA genes were mainly carried β-lactams resistant genes in E.coli isolates from waterfowl in Guangdong province.It had a great relationship between the prevalence of resistance genes and growth of antimicrobial resistance.
Study on the Immunity Effect of Immune Complex Vaccine for Infectious Bursal Disease on One-day Old Low Maternal Antibody Chicken
ZHANG Zhen-hua, LI Lin, JING Xiao-dong, ZHANG Jian-wei, SHEN Jia, SHI Ai-hua, ZHENG Xiao-lan, HUANG Feng-jun, JIANG Bei-yu
2015, 42(9):  2493-2498.  doi:10.16431/j.cnki.1671-7236.2015.09.041
Abstract ( 288 )  
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Five kinds of infectious bursal disease (IBD) immune complex (IC) vaccines were prepared with infectious bursal disease virus (IBDV) BX strain mixed with IBDV hyperimmune serum according to a certain proportion (containing 32, 8, 4, 0.5 and 0.125 units IBDV neutralizing antibody, respectively).One-day old low maternal antibody chickens were vaccinated with IBD IC vaccines 1 to 5 and BX strain live vaccine, respectively, pathological changes of the bursa of fabricius in chickens were observed at the 9th day after immunization.On the day of 28 after immunization, blood samples were taken and the IBDV neutralizing antibody were tested, meanwhile all experimental chickens were challenged with high virulent IBDV, the protective rates of vaccines were calculated.The results showed that at the 9th day after immunization, the bursa of fabricius were normal in IC vaccine 1, 2 and 3 groups, however 2/10, 4/10 and 5/10 of pathological changes of the bursa of fabricius in IC vaccine 4, 5 groups and BX strain vaccine group, respectively.At the 28th day after immunization, the IBDV neutralizing antibodies in IC vaccine 1, 2, 3, 4, 5 groups and BX strain vaccine group were 8.34log2, 9.60log2, 9.21log2, 7.88log2, 9.50log2 and 9.12log2, while 90%, 100%, 100%, 80%, 90% and 80% protection rates were provided, respectively.The results showed that IBD IC vaccines 2 and 3 (containing 8, 4 units IBDV neutralizing antibody, respectively) had the best immunity effect on one-day old low maternal antibody chickens, protection rates were both 100%.
Research Status on the Development of Live Vector Vaccine and Biological Agents of Orf Virus
ZHANG Kai-zhao, YU Meng, NING Zhang-yong
2015, 42(9):  2499-2505.  doi:10.16431/j.cnki.1671-7236.2015.09.042
Abstract ( 272 )  
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Orf virus (ORFV) has special advantages as the live viral vaccination vectors due to its structure of genome, replication characteristics of encoding gene and quick activation of immunoreactions in host and non-host.In addition, ORFV and its coding proteins have huge potential as clinical biological agents for their function as dual immune modulators.Research statuses on recombinant vaccine of ORFV and biological agents were reviewed which would provide the reference for the development and clinical application of vaccination and biological agents.
Research Progress on Porcine Epidemic Diarrhea Virus
LIU Zheng-long, WANG Jin-liang, SHEN Zhi-qiang
2015, 42(9):  2506-2511.  doi:10.16431/j.cnki.1671-7236.2015.09.043
Abstract ( 385 )  
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Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) is a contagious acute gastrointestinal infection, which has brought great economic losses to the pig industry in recent years.The article reviewed advances in molecular biology, methods of detection, genetic variation and vaccine development to provide references for further study in preventing porcine epidemic diarrhea.