Acta Veterinaria et Zootechnica Sinica

Investigation on Genetic Diversity and Systematic Evolution in Chinese Domestic Fowls and Red Jungle Fowls by Analyzing the mtDNA Control Region

BAO Wen-bin;SHU Jing-ting;WANG Cun-bo;ZHANG Hong-xia;Steffen Weigend;CHEN Guo-hong

2008, 39(11): 1449-1459. doi:

Abstract ( 1423 )

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Investigation of the genetic diversity and systematic evolution in Chinese domestic fowls and red jungle fowls was carried out by the structure and polymorphism of mtDNA D-loop region. A 560 bp DNA fragment of control region of mtDNA from 256 individuals of 14 Chinese domestic chicken breeds and two red jungle was determined, the percentage of nucleotide A, C, G and T was 25.00％, 37.40％, 4.40％ and 33.20％, respectively. There were 44 polymorphic sites accounting for 7.86％ of total analyzed sites. Only transition and transversion but no insertion/deletion were found in this region, the ratio of transition and transversion in this study was 0.13．32 haplotypes were defined, in which 9 haplotypes were shared among some chicken populations. The diversity of haplotypes was ranged from 0 to 0964, the average diversity of haplotypes was 0.909±0.014. The average number of nucleotide divergence (K) and average nucleotide diversity (Pi) were 7.276 and 1.851％, respectively. Inter-population nucleotide divergence (Dxy) in 16 chicken populations was ranged from 0.747％ to 3.125％, whereas inter-population net nucleotide divergence (Da) in 16 chicken populations was ranged from 0.015％ to 2.633％. The results indicated that the genetic diversity of 14 Chinese domestic chicken breeds and two subspecies of red jungle fowl was very abundant and the genetic divergence was significant among populations．Analysis of genetic diversity and phylogenetic relationships among Chinese domestic fowls and red jungle fowls indicated that some Chinese domestic fowls such as Gushi chickens and Xianju chickens derived from Gallus gallus gallus, other Chinese domestic fowls such as Chahua chickens and Tibetan chickens derived from Gallus gallus spadiceus. The genetic contribution of these two subspecies of red jungle fowl can be detected in some Chinese chicken populations. The results indicated that the Chinese domestic fowls don′t just derive from red jungle fowl in Thailand or just from red jungle fowl in China.

Compared Analysis of Genetic Structure among Chinese Indigenous Chicken Breeds Using Distance-ased and Model-ased Methods

LI Hui-ang;CHEN Kuan-wei;HAN Wei;ZHANG Xue-yu;GAO Yu-shi;CHEN Guo-hong;ZHU Yun-fen;WANG Qiang

2008, 39(11): 1460-1465. doi:

Abstract ( 840 )

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The Nei’s improved genetic distance (DA) and gene flow (Nm) were measured using 16 microsatellite markers. Then two dendograms of clustering based on DA genetic distance using neighbor joining (NJ) method and Structure program were constructed to analyze the genetic structure and relationship among ten Chinese indigenous chicken breeds. The results showed, in dendograms of clustering based on DA genetic distance using NJ method, ten Chinese indigenous chicken breeds were divided into two main clusters, one was light weighted (including CHA, TIB, XIA, GUS and BAI), the other was heavy weighted (including LAN, DAG, YOU, LUY and XIS). In light weighted cluster, TIB and CHA, XIA and GUS were respectively clustered into one branch. In heavy weighted cluster, XIS and LUY were clustered into one branch, but LAN, DAG and YOU were clustered into independent branches. This result was consistent with Nm among ten chicken breeds. Structure program properly inferred the presence of genetic structure at the condition of not defining the origin of individuals. The genetic cluster inferred by Structure program was probably consistent with DA distance cluster method. However, Structure program determined the migrants and admixed individuals in ten chicken populations, which could not be found through DA distance cluster method.

Preparation of Antiserums against Chicken Liver-type Fatty Acid Binding Protein (L-FABP) and Tissue Expression Analyses of L-FABP

SHI Hui;WANG Qi-gui;WANG Yu-xiang;WANG Ning;LI Hui

2008, 39(11): 1466-1469. doi:

Abstract ( 842 )

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The aim of this study was to prepare the antiserums against chicken livertype fatty acid binding protein (L-FABP) and analyze expression characteristics of L-FABP. Specific primers were designed to amplify the gene of chicken L-FABP by RT-PCR. The L-FABP gene was then inserted into pGEX4T-1 vector and expressed in Escherichia coli BL21 (DE3). The protein was purified by Glutathione Sepharose 4B affinity chromatography and the antiserums against L-FABP was produced by immunizing rabbits. The tissue expression characteristics of L-FABP gene were determined by Western blot analysis. The results showed that a 40 ku (14 ku L-FABP + 26 ku GST) fusion protein was induced; furthermore, high titer antiserums against L-FABP was obtained. The tissue expression analysis showed that L-FABP was highly expressed in liver and small intestine but no hybridization signal was detected in heart, fat, muscle, muscle stomach, spleen, lung and kidney.

SNPs of IGF-ⅡGene and Its Correlations to Production Performancein Jinghai Yellow Chicken

YANG Feng-ping;LI Qi-song;DAI Guo-jun;XIE Kai-zhou;SHI Hui-qiang;WANG Jin-yu

2008, 39(11): 1470-1475. doi:

Abstract ( 1604 )

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Three SNP sites of IGF-Ⅱgene in Jinghai Yellow chicken was detected by PCR-SSCP method, its genetic effects on production performance were also analyzed. And the results showed that the mutations of C→G，G→C，G→A in P1，P2，P3 sites were identified, respectively. There were three genotypes in each site, and three genotypes in P1 site deviated the Hardy-Weinberg equilibrium(P＜0.05). The Least Square Means analysis showed the difference of the egg number in 300 days between AA and BB genotypes were significant(P＜0.05), and A was an enhancing allele, B was a leaky one; CD has higher 12-week-weight than CC(P＜0.05); EE and EF have higher average-egg-weight than FF(P＜0.05), and E was an enhancing allele, F was a leaky one. The com-genotypes AA/CC/EF has higher average-egg-weight than AB/CD/EE and AB/CD/EF(P＜0.05); AB/CD/EF has higher 300-day-weight than AA/CC/EF and BB/DD/EE(P＜0.05).

tudy on the Expression Rule of Krox20 Gene in Tissue and Adipocyte

ZHEN Xin;RUAN Jing-ling;ZHOU Jia-bo;LIU Di;

2008, 39(11): 1476-1481. doi:

Abstract ( 836 )

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The expression of Krox20 gene of Yorkshire in 1，6 months and Wild boar in 1 month were analyzed by using semi-quantitative RT-PCR method and detected in adipocyte and adipose tissue. The results showed that Krox20 gene was expressed in heart, liver, spleen, lung, kidney, stomach, large intestine, small intestine, fat, ovary, uterus, muscle; The expression level of Krox20 gene was different in the detected tissues, it was highly expressed in heart, liver, fat; The expression mode of 1 month Yorkshire and Wild boar were similar, there was little species-specificity; With the age increasing, the expression of Krox20 gene was gradually decreased in adipose tissue,it had the highest expression level at 1 month, and reached the least at 9 month; In the research of adipocyte, the expression of Krox20 gene was not detected in preadipocyte, but detected in adipocyte after induced for 5 hours.

Associations between Hae Ⅲ Polymorphism of PLTP Gene and Growth Traits of Pigs

WEI Lin;LIU Sheng-gui;SHI Xian-wei

2008, 39(11): 1482-1486. doi:

Abstract ( 852 )

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The polymorphism of intron 4 in phospholipid transfer protein(PLTP) gene was detected by PCR-RFLP. A Hae Ⅲ RFLP was detected in intron 4 and the polymorphic locus is common in all detected groups. Associations between the founded Hae Ⅲ RFLP in intron 4 and birth weight, body weight at 45 days, body weight at 60 days, body weight at 4 months, body weight at 6 months, body height at 6 months, body length at 6 months and circumference at 6 months were tested by least square analysis. No significant difference between Hae Ⅲ RFLP and body weight at 45 days, body weight at 60 days, body weight at 6 months, body length at 6 months and circumference at 6 months were founded in this study.The results showed that the body weight at 4 months with aa genotype was higher than that with AA genotype(P<0.01), the body height at 6 months with aa genotype was higher than that with AA genotype(P<0.05), the birth weight with Aa genotype was higher than that with AA genotype(P<0.05). Therefore, it could be seen that porcine PLTP genotypes had significant influence on growth traits, this SNP might be considered as a potential genetic marker for selection.

Effects of in vitro Developmental Competence of Bovine Somatic Cell Nuclear Transfer Embryos in Defined Culture Medium (IVD101 and G1/G2)

LI Rong;;LIU Ying;ZHAO Xing-bo;WANG Li-li;WANG Hai-ping;DING Fang-rong;LI Jing;LI Song;GAO Feng-lei;DAI Yun-ping

2008, 39(11): 1487-1492. doi:

Abstract ( 1201 )

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The purpose of the study was to investigate in vitro development, quality and frozen tolerance of bovine somatic cell nuclear transfer (SCNT) embryos in two defined culture media (IVD101 and G1/G2). The SCNT embryos were cultured in three media: IVD101, G1/G2, and CR1aa+5%FBS (control group). The results indicated that the development rates of SCNT embryos cultured in IVD101 and G1/G2 were lower than that in CR1aa+5%FBS, but had no significant difference among three groups (41.2%±9.1%, 42.2%±10.8% and 48.0%±9.2%, respectively,P>0.05). The result of differential staining showed that the ratio of ICM/Total of blastocysts cultured in IVD101 and G1/G2 were lower than that in the group of CR1aa+5% FBS, whereas there was no significant difference among them (31.8%±10.5%, 29.5%±11.9% and 33.0%±14.8%, respectively, P>0.05). No significant difference was observed in the survival rate among three groups (84.8%, 80.4% and 77.3%, respectively, P>0.05). Our study demonstrated that defined cultured media (IVD101 and G1/G2) could support in vitro development of bovine SCNT embryos, and freezing tolerance of them were similar with the culture medium with FBS.

Effect of Trichostatin A on in vitro Maturation of Porcine Oocyte and Development of Parthenogenetic Embryo

LIU Xiao;PAN Deng-ke;CHEN Kou-kou;FENG Chong;ZHANG Wei-hong;ZHENG Mao-en;LONG Chuan;FENG Shu-tang;YANG Bo-hui

2008, 39(11): 1493-1498. doi:

Abstract ( 814 )

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Trichostatin A (TSA) is an inhibitor of histone deacetylase.It is reported that treatment of mouse somatic cell nuclear-transferred oocytes with TSA significantly increased the blastocyst rate.The present study was designed to examine the effect of TSA on the maturation of porcine oocytes and development of parthenogenetic embryos in vitro.We evaluated the concentration of TSA to oocyte maturation, the concentration of TSA to parthenogenetic embryo, treatment duration of TSA to parthenogenetic embryo in vitro. The maturation oocytes to the metaphase II (M II) stage cultured with 5 nmol/L TSA was not significantly different from the TSA-free treatment, meanwhile 5 nmol/L TSA treatment supported a higher cleavage and blastocyst development rate (P<0.05); The parthenogenetic embryo exposured in 50 nmol/L TSA supported a higher cleavage and blastocyst development rate (P<0.05).The parthenogenetic embryo exposured in 50 nmol/L TSA for 24 h supported a higher cleavage and blastocyst development rate than the others (P<0.05, 82.1±2.6% and 37.4 3.1% ).The data demonstrated that treatment of porcine oocytes and parthenogenetic embryos with TSA significantly improved the in vitro blastocyst production.5 nmol/L TSA treatment enhanced the oocytes maturation in vitro, and 50 nmol/L TSA-treatment for 24 h following oocyte activation resulted in more efficient development of parthenogenetic embryo.

Effect of Dietary Protein Levels on Growth Performance and Carcass Composition in Wujin Pig

GE Chang-rong;ZHAO Su-mei;ZHANG Xi;LAI Hua;LI Chang-qiang;GAO Shi-zheng

2008, 39(11): 1499-1509. doi:

Abstract ( 1562 )

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The aim of this study was to investigate the effect of dietary protein levels on growth performance and carcass composition so as to investigate the suitable dietary protein level according to the best lean meat growth and carcass composition by Fuzzy Comprehensive Judgment Model. Ninety pigs were randomly allotted into five groups fed diet containing different protein levels. There are eighteen pigs in each group with three replicates. Six pigs were used in per replicates. The experiment was designed by single factor random arrangement. The dietary protein levels were high high protein（HHP, 18%, 16% and 14%）, high medium protein（HMP, 17%, 15% and 13%），medium medium protein（MMP, 16%, 14% and 12%）, medium low protein（MLP, 15%, 13% and 11%）and low low protein（LLP, 14%, 12% and 10%）respectively during 15.30, 30-60 and 60-100 kg body weight. The daily gain and feed efficiency were calculated. The carcass composition was measured when the pigs were slaughtered at 30, 60 and 100 kg body weight. Fuzzy Comprehensive Judgment Model was analyzed to make sure the suitable dietary protein level to attain the best growth performance and carcass composition in Wujin pigs during different growth stages. The results showed that dietary protein levels affected significantly the growth performance and carcass composition during different growth stages. With dietary protein level decreased, the daily gain reduced but the ratio of feed to weight increased gradually. The difference was significant among high group, medium group and low groups during 30-60 and 60-100 kg body weight. The time reaching 100 kg body weight of five groups were the days of 163, 168, 173, 179 and 185, respectively. The carcass composition except for suet weight and loin eye area were no significant difference among each groups at 30 kg body weight. Lean meat weight, lean meat percentage and loin eye area decreased, but fat meat weight, fat meat percentage and backfat thickness increased with dietary protein levels reducing at 60 and 100 kg body weight. Moreover, the differences were significant among high, medium and low groups. Fuzzy Comprehensive Judgment Model was analyzed basing on the lean meat weight. The suitable dietary protein levels were 15.95%, 14.30% and 11.81% during 15-30, 30-60 and 60-100 kg body weight respectively from the fitting curve. Fuzzy Comprehensive Judgment Model was draw depending on the lean meat percentage, backfat thickness and loin eye area which represented the carcass composition. The suitable dietary protein levels were 16.47%, 13.71% and 10.72% during 15-30, 30-60 and 60-100 kg body weight, respectively. It was revealed that the suitable dietary protein levels were 14.98%, 11.34% and 11.71% combined lean meat growth with carcass characteristics by Fuzzy Comprehensive Judgment Model. This result could provide scientific basis for the reasonable feeding of Wujin pigs.

Effect of Different Protein Levels on the Growth, Nutrient Utilization and Amino Acids Digestibility of Dairy Calves

LI Hui;DIAO Qi-yu;ZHANG Nai-feng

2008, 39(11): 1510-1516. doi:

Abstract ( 851 )

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This study was designed to examine the effects of different dietary protein levels on growth performance, nutrients utilization, and amino acid (AA) digestibility of early-weaning dairy calves. Fifteen healthy new born calves were randomly allotted into three experimental groups and fed three kinds of milk replacers with different protein levels (18%, 22% and 26%) respectively, labeled as LP, MP and HP group. The results showed that the body weight (BW) gain of MP group was higher than that of other groups (P<0.05), the ADG of three groups were 598.10, 829.52 and 628.57 g/d, respectively. The apparent digestibility of DM, EE and Ash were no difference among three groups (P>0.05), however, the apparent digestibility of CP in MP group was higher (P<0.05). Intake N and Fecal N increased as dietary protein content increasing (P<0.05), however, Urine N was unaffected by dietary CP levels significantly (P>0.05). The retained Ca, P and expressed as a percentage of intake N in MP group were all higher than that in other groups (P<0.05). The average digestibility of the total AA were 82.73%, 83.41% and 79.45% in LP, MP and HP groups, respectively, and the digestibility of amino acids（except for Ala） were no effected by CP levels (P>0.05). It was concluded that milk replacer with 22% CP produced higher body weight gain and better utilization of nutrients in comparison with lower protein and higher protein contents milk replacer.

Effect of Daidzein and Genistein on Proliferation and Antioxidation of Mammary Epithelial Cell of Dairy Cow in vitro

LIU Chun-long;;LI Zhong-qiu;ZHANG Fan;JIANG Wen-bo;XU Yan;SHAN An-shan

2008, 39(11): 1517-1522. doi:

Abstract ( 861 )

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Effect of Daidzein and Genistein on the proliferation and antioxidation of mammary epithelial cell of dairy cow were determined in vitro. The experiments were assigned into 9 groups(blank,1,10,100,1 000 ng/mL Daidzein and Genistein).According to MMT,the proliferation experiment results showed that 10,100 ng/mL Genistein and 100,1 000 ng/mL Daidzein significantly improved dairy cow mammary epithelial cell proliferation(P＜005),but all weaker than 10 ng/mL E₂group (P＜0.05) after 72 h cultured. In the other experiment,the mammary epithelial cell during the logarithm growing period were collected after 24 h cultured to measured the TSOD,GSH-PX activity,MDA,NO content.Results showed that 100,1 000 ng/mL Daidzein and 100,1 000 ng/mL Genistein significantly increased the T-SOD activites and decreased MDA concentration (P＜0.05). 100,1 000 ng/mL Daidzein and 10,100, 1 000 ng/mL Genistein significantly increased NO concentration(P＜0.05).1 000 ng/mL Daidzein and 100,1 000 ng/mL Genistein significantly increased the GSH-PX activites (P＜0.05).It was proved that proper levels of Daidzein and Genistein can advance the proliferation and antioxidation of mammary epithelial cell of dairy cow.

Effect of Selenium Source and Level in Diets on Spleen Selenium Content and Immune Function of Laying Hen

PAN Cui-ling;HUANG Ke-he;ZHAO Yu-xin;CHEN Fu;JIAO Miao;RAO Jing-jing

2008, 39(11): 1523-1529. doi:

Abstract ( 1153 )

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Se sources (Se-enriched probiotics, SP and sodium selenite, SS）were added into basal diet at 0.2, 0.5 and 1.0 mg/kg of Se. Feeding test lasted for 35 days. The results showed that the addition of either SS or SP significantly increased Se content in spleen. With an increase of the supplemental Se level, the Se content in spleen had a significant increase. Supplemental SP had a more significant action to increase Se content in spleen than SS. On days 14, supplemental Se from either SS or SP significantly promoted the T lymphocyte transformation in hen peripheral blood. With supplemental Se level increase, the T lymphocyte transformation were increased. On days 14 and 28, supplemental Se from either SS or SP significantly increased the plasma IL-2 level of hen. With supplemental Se level increase and the extension of experiment duration, the plasma IL-2 level also significantly increased. On days 28, there was a more significant increase in the plasma IL-2 level of hens fed by SP than that of hens fed by SS. The conclusion is that supplemental Se from either SS or SP significantly increased Se content in spleen, significantly promoted immune function of hen. With supplemental Se level increase, the effect was better. With the extension of experiment duration, supplemental SP had a better effect than SS.

Molecular Characteristics and Phylogenetic Relationships of cDNA Encoding αv Subunit as FMDV Receptor in Cloven-hoofed Domestic Animals

DU Jun-zheng;GAO Shan-dian;CHANG Hui-yun;ZHAO Jian-yong;CONG Guo-zheng;SHAO Jun-jun;LIN Tong;LIU Xiang-tao;CAI Xue-peng

2008, 39(11): 1530-1536. doi:

Abstract ( 816 )

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Foot-and-mouth disease virus（FMDV）has been showed at least to use four integrins, αvβ1, αvβ3, αvβ6 and αvβ8 as receptors to initiate infection. In this study, the integrin αv genes were cloned from the lung tissue of cattle and pig, both infected experimently with FMDV, healthy sheep and Bactrian camel by RT-PCR. The nucleotide and predicted amino acid sequences of the αv genes of sheep, cattle, pig, Bactrian camel, human, dog, horse, chimpanzee, rhesus monkey, chicken, and mouse were analyzed. The ORF of integrin αv cDNA of sheep, cattle, pig, Bactrian camel consists of 3 147 nt, 3 147 nt, 3 141 nt, 3 165 nt, encoding a polypeptide of 1 048 aa,1 048aa, 1 046 aa, 1 054 aa, respectively. The putative signal peptide, transmembrane domain and cytoplasmic domain consists of 30, 29, 32 residues, respectively. The ectodomain of integrin αv contains 957 aa, 957 aa, 955 aa, 963 aa in sheep, cattle, pig, Bactrian camel, respectively. The homology analysis showed that the signal peptides exhibit the lowest conservation, and the transmembrane domain and cytoplasmic domain exhibit the highest degree of conservation, and the degree of similarity was somewhat higher for ectodomain. Phylogenetic analysis revealed that integrin αvs of sheep, cattle, pig, Bactrian camel belonged to the same branch, suggesting that they had a close genetic relationship. It is possible that host and tissue tropism of FMDV may related to divergence in αv subunit among different species.

Display of NP Gene of AIV on the Surface of Bacteriophage T4 and Development of Indirect ELISA on the Basis of the Expressed NP Protein

WANG Xin-wei;;BI Ying-zuo;HE Hong-xuan;WANG Xian-wen;ZHAN Ai-jun;MA Jing-yun

2008, 39(11): 1537-1543. doi:

Abstract ( 1594 )

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A 1 320 bp fragment of NP gene of avian influenza virus (AIV) was cloned and integrated into the genome of T4-z1 phage with SOC-deleted and lysozymedefected by homologous recombination. The recombinant phage was named as T4-z1-NP. The results of SDS-PAGE and Western blot showed that nucleoprotein of AIV was successfully displayed on the surface of the T4-z1-NP. Based on the purified T4-z1-NP, an indirect enzyme-linked immunosorbent assay (T4-NP-ELISA) was developed for detection of antibodies against AIV. Its sensitivity relative to AGP was 100% but the specificity was 87.62%. The detection accuracy rate of T4-NP-ELISA was 91.43%. It could detect antibodies to H5, H7 and H9 subtype AIV, but has no significant reaction to positive serum of IBD, IB, MD, ND and EDS. The sensitive test confirmed that when the positive serum of AI was diluted to 1∶640, the OD450 value was still positive, which suggested it had better sensitivity. The accordance rate between T4-NP-ELISA and commercial IDDEXX ELISA was 968%, when 192 serum samples were detected with them. These results indicated that T4-NP-ELISA could not only detect antibody to AIV, but also provide an alternative technique for diagnosis and control of AI.

Studies on Viral Characteristic of Myeloblastosis and Hemangioma Associated with ALV-J in Commercial Layer Chicken

MENG Xiang-kai;LIU Qing;WANG Hai-lun;ZHANG Hong-hai;QIU Bo;LIU Gong-zhen;CHENG Zi-qiang

2008, 39(11): 1544-1547. doi:

Abstract ( 1171 )

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Myeloblastosis and hemangioma cases associated with avian leukosis virus subgroup J (ALV-J) were first diagnosed in a layer chicken flock in Shandong province on July 2007. The CEFs inoculated by virus extracted from liver were cultured, and detected by indirect immunofluorescence with ALV-J monoclonal antibody G2-3, the results were intense positive. The four isolated strains were named as WS0701, WS0702, WS0703 and WS0704. A pair of primers P1 /P2 was designed according to the sequence of prototype ALV-J HPRS103. DNA extracted from the positive CEFs were selected as PCR template, and a 924 bp fragment was amplified. PCR products were sequenced and demonstrated differ from 95.0% to 97.5% identity with ALV-J HPRS-103. The identity ranged from 92.0% to 95.9% among the four strains and other Chinese field strains. Genetic and antigenic analysis showed the identity of ALV-J which characterized myeloblastic and angiomatous is being more close to HPRS-103 strain than other Chinese filed strains. It indicated that the replication of ALV-J has shown atavism in commercial layer chicken during the several years, and the disease caused by ALV-J showed novel oncology character.

Development and Application of the Recombinant ELISA Based on the Assembling and Expression of VP7 Gene of African Sickness Virus

GAO Zhi-qiang;ZHANG He-xiao;LAI Ping-an;GU Qiang;PU Jing;WANG Lin;QIAO Cai-xia;WU Dan;BAI Ya-duo;ZHANG Wei

2008, 39(11): 1548-1553. doi:

Abstract ( 831 )

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A DNA fragment, which encodes the most linear epitopes of VP7 of African sickness virus(AHSV), was assembled artificially, then cloned into pET-30a. Target protein expressed at very high level as inclusion body after the recombinant pET-30a-VP7 plasmid was transformed into BL21(DE3), and induced with 1.0 mmol/L IPTG. The indirect ELISA for detecting AHSV VP7 protein antibody was established after the reaction activity of the recombinant protein was determined by Dot-ELISA and ELISA. The results obtained indicated that the optimum antigen concentration was 0.25 μg/mL, and optimum serum dilution was 1∶40. The cut-off value was determined as 0.25. 184 serum samples were detected by this method and commercial ELISA kit and the results were all negative.

Development and Application of Double-PCR Method for Quick Detection of Bacteria and Yeast from Bovine Mastitis

GE Sheng-qiang;CHAI Tong-jie;MA Bao-chen;LI Xiao-xia;CAI Yu-mei LV- Jing;QIN Mei;WANG Bing-xiao

2008, 39(11): 1554-1561. doi:

Abstract ( 1373 )

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A double polymerase chain reaction (PCR) was developed for simultaneous detection and germ group classification of bacteria and yeasts of bovine mastitis. We developed a new method that extracts both bacteria and yeast DNA from milk using β-mercaptoethanol, which can decompose cell walls. At the same process we used lysozyme and snailase, which can assimilate cell walls, and quartz sand, which can further abrade the outside of cell. The conserved regions from 16S rRNA and 18S rRNA were selected as target sequences of bacteria and yeast respectively. We evaluated the technique for the detection and identification of which species it was in artificially infected milk and milk from cows with moderate or severe clinical mastitis. The performances of the double-PCR and traditional culture method were evaluated on 84 mastitis milk samples. The results indicated that the sensitivity of double-PCR in detecting bacteria and yeast was 102 CFU/mL and 103 CFU/mL, respectively. There was no significant difference as compared to the culture method in detecting bacteria (P>0.05), but doublePCR was more sensitive than the culture method in detecting yeasts (P<0.01).

Experimental Study on Dogs Immunized with CPV DNA Vaccine, CPV Recombinant Vector Vaccine and CPV Modified Vaccine

XIE Zhi-jing;YANG Song-tao;XIA Xian-zhu;YAN Fang;ZHAO Zhong-peng;GAO Yu-wei;ZOU Xiao-huan;HUANG Geng

2008, 39(11): 1562-1566. doi:

Abstract ( 1232 )

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To test the immunogenicity of the different CPV vaccines, the dogs were respectively vaccinated with pVCPV-VP2, CAV2/CPV and CPV modified vaccine. The antibody titers against CPV were detected by CPV ELISA, CPV HI and CPV SN. And the proliferation of the PBMCs of the vaccinated dogs was detected by MTT. As a result, both antiCPV ELISA antibody and SN antibody were checked up on in the dogs vaccinated with CAV2/CPV or pVCPV-VP2. AntiCPV HI antibody was discovered in the dogs immunized with CAV2/CPV or CAV2/CPV combined with pVCPV-VP2, but not in the dogs vaccinated with pVCPV-VP2. And the PBMCs of the inoculated dogs obviously proliferated against both CPV and ConA stimulation, respectively. It demonstrated that CAV2/CPV and pVCPV-VP2 induced humoral and cellular immunity against CPV in dogs. The immune levels elicited by CAV2/CPV or CAV2/CPV combined with pVCPV-VP2 were higher than that by pVCPV-VP2, but weaker than that by the CPV modified vaccine. At the same time, CAV2/CPV can also induce specific anti-CAV-2 immunity.

Pathological Observation of Ovine Theileriosis Caused by Theileria uilenbergi

LI You-quan;LUO Jian-xun;GUAN Gui-quan;MA Mi-ling;GAO Jin-liang;LIU Zhi-jie;DANG Zhi-sheng;LIU Ai-hong;REN Qiao-yun;LIU Jun-long;LU Bing-yi;CHEN Huai-tao;BAI Qi;WEI Yong-hong;NIU Qing-li;TIAN Zhan-cheng;YIN Hong

2008, 39(11): 1567-1574. doi:

Abstract ( 803 )

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Sheep were infected experimentally with Theileria uilenbergi by subcutaneous injection, and the pathology of theileriosis was observed. The results showed that the principal microscopic founding was hyperaemia, hemorrhage and proliferation of reticuloendothelial cell in lymph nodes, proliferation of interstitial lymphocytes and reticular cells in lung, and thickened alveolar septum, proliferation of glomerular endothelial cells and vascular endothelial cells in kidney. Lymph reticulocytes were accumulated into a tubercle in kidney. Cardiac muscle cells showed apomorphosis and uneven dyeing. The tunica propria was obvious hyperaemia and hemorrhage in the intestinal tract. Additionally, the hepatocytes showed granular degeneration and necrosis. Various degrees of haemorrhage were seen in submucosa and muscle layer of rumen.

Recombinant Expression of Microneme Protein 5 Gene of Eimeria necatrix LZ Strain in E. coli and Antigenicity of Recombinant Protein

LIU Hong-xia;JIA Wan-zhong;GUO Ai-jiang;ZHANG Shao-hua;YAN Hong-bin;YUE Cheng;CAI Xue-peng

2008, 39(11): 1575-1580. doi:

Abstract ( 1508 )

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Based on the published nucleotide sequence of MIC5 gene of Eimeria tenella, a pair of primers was designed. The EnMIC5 gene was amplified by PCR from the genomic DNA of E. necatrix. The amplicons were cloned into pMD18-T simple. Sequence analysis showed that the PCR fragment of EnMIC-5 gene is 1 470 base pairs in length and encodes a partial protein of 490 amino acids. Then the gene was introduced into pET-28a(+) vector, and the recombinant plasmid pET-EnMIC-5 was expressed in the E. coli BL21. The results of SDSPAGE revealed that the fusion protein with molecular weight about 57-5 ku was over-expressed after induction of IPTG. Western-blot results demonstrated that the expressed recombinant protein was reacted with sera of chickens against E. necatrix infection. A high titer of antibody was detected at two weeks after the injection of the recombinant protein in Kunming mice suggesting that it should have good immunogenicity, and make mice produce high level of specific antibody.

Study on Lymphocyte Proliferation and Apoptosis in the Embryonic and Post Embryonic Development of the Duck Bursa of Fabricius

FANG Jing;CUI Hengmin;LIAO Tingbin;CUI Xue

2008, 39(11): 1581-1587. doi:

Abstract ( 817 )

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By proliferating cell nuclear antigen(PCNA) immunohistochemisty, terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling(TUNEL), light and electron microscopy techniques, the changing patterns of lymphocyte proliferation and apoptosis during the embryonic and post embryonic development of the Tianfu duck bursa of Fabricius were studied. In the embryonic period, the lymphocyte proliferation in the bursa was prominent. As the embryos grew, the proliferative index of lymphocytes in the follicle (PIF) increased gradually, and reached the peak at 26 embryonic days. After hatching, both proliferative indexes of lymphocyte in the cortex (PIC) and medullar (PIM) in each age group dropped progressively. PIM was higher than PIC in each group. During the embryonic development of the organ, the apoptosis index of lymphocyte in the follicle (AIF) increased gradually. During 0-3 weeks posthatching, the apoptosis index of lymphocytes in the medullar (AIM) rose continually, whereas the apoptosis index of lymphocytes in the cortex (AIC) kept unchanged. Both AIM and AIC decreased at 5 weeks and increased at 17 weeks, reaching their plateaus at 29 weeks. During 0-14 weeks after hatching, AIM in each group was higher than AIC, whereas AIM in each group was lower than AIC during 17-29 weeks. The nucleus of apoptotic lymphocytes with swollen mitochondria and broken cristae mitochondriales presented different futures. The results indicated that the proliferation and apoptosis of lymphocytes exist universally during the normal embryonic and post embryonic development of the duck bursa of Fabricius which undergoes obvious age-related changes, and then supervise and control the whole development，and regression of the bursa cooperatively.

Effects of Two Histamine Receptor Antagonists on Right Ventricle’s Function in Broiler Chickens with Pulmonary Hypertension

OU De-yuan;QIAO Jian;LIU Wen-ju;TIAN Xing-gui;GAO Ming-yu;LI Jing

2008, 39(11): 1588-1593. doi:

Abstract ( 1376 )

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The aim of the study was to evaluate the effect of histamine receptor antagonist on right ventricle’s function in broiler chickens with pulmonary hypertension induced by low ambient temperature. One hundred and sixty male 17-day-old broilers were divided randomly into four groups. There are forty birds in each group. (1) Normal temperature group (NT): The birds were reared at 22-23 ℃and normal saline was given twice by intramuscular injection each day. (2) Low ambitent temperature group (LT): The birds were reared at 9-11 ℃and normal saline was given twice each day. (3) Chlorphenamine maleate group in low ambient temperature (CMLT): The birds were reared as LT, and chlorphenamine maleate was given twice each day. (4) Cimetidine group in low ambient temperature (CLT): The birds were reared as LT, and cimetidine was given twice each day. At 24, 31, 38, 45 days of age, 7 birds were randomly taken from each group for measurement of right ventricular pressure (RVP) using right cardiac catheter. Mast cells (MC) was stained with Alicience blue. Histamine was shown using immunohistochemistry. The ascites heart index (AHI) were accounted. The results were as follows: (1) The number of mast cells and histamine positive cells were decreased by low ambient temperature, but it was no affected by chlorphenamine maleate and cimetidine. (2) The RVP of CMLT group was maintained at same levels, and the RVP of CLT group was kept at low levels. (3) The dp/dt of CMLT group was significantly (P<0. 05) different with that of LT group. (4) AHI of LT group was significantly higher than that of NT and CMLT group (P<0. 05). (5) The ratio of right ventricular hypertrophy and failure in CMLT group were lower than those in LT group. It was concluded that internal histamine can promote the development of pulmonary hypertension syndrome by dysfunction of right ventricle through H1 receptor in broilers.

Preparation of Polyclonal Antibody against Sulfonamide and Preliminary Study on ELISA Method

SUN Jun-ying;YU Hong-yi;HUANG Xian-hui;GAO Hai;YANG Gui-xiang;ZENG Zhen-ling

2008, 39(11): 1594-1598. doi:

Abstract ( 1510 )

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New Zealand White rabbits were immunized with three artificial immunizing antigens that modeling the common structure of sulfonamides, and the immune effect were detected by ELISA. A new Ci-ELISA method against sulfonamides and standard curves were established using two polyclonal antisera because of their good inhibitory effect. The IC10 of polyclonal antisera from group A against SMD, SMM, SMZ, SDM, SDM′ and SD was 0.010, 0.046, 0.010, 0.067, 0.019 μg/mL respectively, which was less than the MRLs(0.1 μg/mL). The IC10 of polyclonal antisera from group C against SMD, SMM, SMZ, SDM, SQ, SD, sulfachloropyridazine and sulfachaoropyrazine were 0.009 1, 0.051, 0.058, 0.088, 0.020, 0.010, 0.014, 0.011 μg/mL respectively, less than the MRLs. The recoveries of two polyclonal antibodies ranged 54.5%111.8%, 54.2%-130.3% with CV of 3.6%-10.9% and 3.7%-5.9%, respectively.

Effect of Tiletamine on the Activity of NOS, NO Production, and cGMP Content in Rat Different Brain Regions

WANG Hong-bin;FAN Hong-gang;LU De-zhang;HU Kui;ZHANG Jian-tao;LI jing

2008, 39(11): 1599-1605. doi:

Abstract ( 1430 )

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The effect of tiletamine on the activity of NOS, NO production, and cGMP content in different brain regions of rat were observed to investigate the modulation of NO/cGMP signal transduction in the molecular mechanisms of general anaesthesia mediated by tiletamine. 168 SD rats were randomly divided into conrol group, high dose tiletamine group(intraperitoneally tilelamine 60 mg/kg) and low dose tiletamine group(ip tilelamine 30 mg/kg )，and each dose group was divided into three sub-groups,eg anesthesia group, recovery groupⅠand recovery groupⅡ. The activity of NOS and production of NO were measured by spectrophotometric analysis, and the cGMP content were measured by radioimmunoassay (RIA) in different brain regions. After administering tiletamine, the activity of NOS in the cerebral cortex, the hippocampus and the thalamus in the anesthesia group were obviously inhibited, and the NO production and cGMP content in abovementioned regions were obviously decreased（compared with control group,P<0.05）. The activity of NOS, NO production and cGMP content in recovery groupⅠat the two dosage were recoverd in different degree, and they were significant recovered（compared with control group, P＞0.05）other than cGMP content in the thalamus (compared with control group, P<0.05) in the recovery groupⅡ. Different dose of tiletamine affected significantly the activity of NOS, NO production and cGMP content in the brain stem, and cerebellum. The anesthesia effects of tiletamine might relate to inhibition of NO/cGMP signal transduction in cerebral cortex, the thalamus, and the hippocampus.

Characterization of 5′-Regulatory Region and Expressional Distribution of Myostatin Gene in Goose

GU Zhi-liang;LU Xiang-yun;ZHU Da-hai;LI Hui

2008, 39(11): 1606-1611. doi:

Abstract ( 852 )

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The primers of 5′-regulatory region were designed according to chicken Myostatin gene. The fragment was amplified from goose genomic DNA, and cloned into pGEMT-Easy Vector and sequenced. A 1.3 kb long fragment of 5′regulatory region of goose Myostatin gene, which includes the putative initial transcriptional site, was acquired. The sequence homology analyses showed that the goose Myostatin 5′-regulatory region shared 94.8% with duck, 692% with chicken and 14.3% with mouse. Sequence analyses showed that there are a TATA box at －34 bp and a CCAAT box at －77 bp upstream of the putative transcriptional site. In addition, some muscle specific transcriptional factor binding sites including 5 E-boxes, 2 MEF2 binding sites, a MSX2 box and a muscle TATA box were determined. The Northern blot results in goose showed that Myostatin mRNA only expressed in breast muscle and thigh muscle and were not detectable in liver, heart, intestine, kidney, brain and lung.

Cloning, Expression and Polyclonal Antibody Preparation of Duck MHC Class Ⅱβ Chain Gene

ZHANG Shu-jie;GONG Yan-zhang;FENG Yan-ping;LI Shi-jun;YANG Qing-lei;HU Fu-li;YANG Huan;PENG Xiu-li

2008, 39(11): 1612-1615. doi:

Abstract ( 771 )

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According to mRNA gene sequence registered in GenBank, a pair of specific primers for the gene of duck MHCⅡβ chain was designed and synthesized. Using total RNA from duck spleen, the target gene fragment was obtained by RT-PCR, then cloned into the T cloning vector pMD18-T. After being identified by restriction digestion and DNA sequencing, the insert fragment was subcloned into the expression vector pGEX-KG. After transforming into E.coli BL21, the recombinant plasmid were induced to obtain the interest protein. Then the purified protein was immunize to mouse for preparing polyclonal antibody. The results showed that MHCⅡβ gene was cloned, the obtained 798 bp fragment has 92% identities to the previously identified duck MHCⅡat nucleotide level; The prokaryotic expression vector was successfully constructed and the protein was expressed efficiently. The purity of protein reached 95%. A high titer and specific antibody has been prepared by purified protein, it was proved by the methods of ELISA and Western blot. All this makes it possible to do further studies on duck MHCⅡgene.

Cloning and Analysis of Partial cDNA Sequence of Lmbr1 Gene in Pigs

WANG Jin-yong;BAI Xiao-qing;ZHAO Xian-zhi;FAN Shou-cheng;LI Qin;LIU Wen

2008, 39(11): 1616-1620. doi:

Abstract ( 1514 )

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In this study we cloned partial cDNA sequences of Lmbr1 gene from a normal pig and a polydactyly pig by RT-PCR and RACE and analyzed the sequences. The results showed that the cDNA sequence from the normal pig was 1 797 bp,including 1 178 bp in CDS and 619 bp in 3′UTR.The cDNA sequence from the polydactylous pig was 1 069 bp,including 768 bp in CDS and 301 bp in 3′UTR. the CDS sequences similarity of Lmbr1 gene was near 77% between the normal pig and the polydactyly pig, while the 3′UTR sequences similarity was below 20%.There were 13 mutations among 755-768 base pairs between the CDS sequences, including G755C, A756T, A757G, T758C, C759A, A760G, C761T, T762G, A763C, G764T, A765G, T767G and T768A.The former 11 mutations belonged to missense mutations，which resulted in 4 amino acids change, including G → A, I → A, T → V and R → L. The later 2 mutations belonged to nonsense mutations，which resulted in the termination of ORF in advance. It is presumed that the fundamental reason for the form of the polydactyly pig is the mutations of Lmbr1 gene, which could result in the exceptional expression of SHH.

Detection of Wild-type Porcine Pseudorabies Virus by Digoxigenin-labeled DNA Probe

LIU Zhi-jie;REN Hui-ying;WEN Jian-xin;LIU Wen-hua;ZOU Ling;HAN Xian-jie;WEI Xiao-xiao

2008, 39(11): 1621-1624. doi:

Abstract ( 859 )

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A 304 bp fragment of gE gene of PRV was amplified from recombinant plasmid pMD18-T-gE and labeled with digoxigenin as DNA Probe. Dot blot hybridization was developed for detection of PRV wild strains. The recombinant plasmid DNA, PRV Bartha-k61DNA, PPV DNA, PCV DNA,PRRSV cDNA and CSFV cDNA were tested by this method. The results showed that positive signal appeared only in recombinant plasmid, while PRV Bartha-k61 DNA, PPV DNA, PCV DNA, PRRSV cDNA and CSFV cDNA were all negative. As little as 4 pg of known positive target DNA (PCR product of gE) could be detected with the digoxigenin-labeled probe. 11 samples collected from cases with reproductive disorders were detected by this probe, and among which, 4 samples were positive. The results of dot blot hybridization were in accordance with that of PCR, it showed that the probe could be used in the clinical diagnosis of pseudorabies.

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