Acta Veterinaria et Zootechnica Sinica

Cloning, Sequence Analysis and Chromosome Mapping of Porcine New NPM1 Gene

2007, 38(2): 105-109. doi:

Abstract ( 1441 )

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The cDNA and fourth intron sequences of porcine NPM1 gene were cloned, and verified by bioinformatics method. The cDNA fragment was 1 195 bp with an entire open reading frame （ORF）, encoding 294 amino acids. The intron fragment was 300 bp and splice junctions follow ‘GT/AG’ rule. The analysis of sequence revealed that the porcine NPM1 gene was highly homologous with that of other species such as human （95%）, mouse （91%） and rattus norvegicus （91%） in amino acid. The protein encoded by porcine NPM1 gene had a conserved domain ‘Nucleoplasmin＇. This gene was assigned to SSC16q21 region and closely linked to SW977 with the LOD of 9.16 by SCHP and IMpRH mapping, respectively.

Cloning and Sequence Analysis of SAA3 Gene Differentially Expressed in Mammary Gland at Two Lactation Stages of Xinong Saanen Goat

WU Hui-juan;LUO Jun;ZHANG Li-juan;HAN Xue-feng;YANG Bao-jin;WANG Hai-bin;SHAN Cui-yan;ZHANG Ning;YU Gang

2007, 38(2): 110-114. doi:

Abstract ( 1215 )

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To discover genes differently expressed between lactation periods, suppression subtractive hybridization method was employed to construct a cDNA library expressed in mammary gland of 60^th but not or little in 28^th. Serum amyloid A protein was one of the positive clones which was verified by Real-time PCR. Moreover the gene was cloned by RT-PCR and the sequence was analyzed. Results showed that a differently expressed library was successfully created in mammary gland of 60^th lactation. The goat mammary gland SAA3 complementary DNA has an open reading frame of 396 nucleotides encoding a putative protein of 131 amino acids, the GenBank number was DQ839400. The homologies of nucleotide and peptide sequence of Xinong Saanen goat mammary gland SAA3 with bovine（NM_181016）, rabbit （M64696. 1）, human（BCA）20795） and mouse＇s（NM_011315） are 95%, 84. 3%, 81.3% and 81.9% and 93%, 76%, 72% and 72% respectively. The 27 nucleotides in goat SAA3 261-287 were deleted in that of human, rabbit, and mouse. There were six conserved domains longer than 5 amino acids in the amino acids sequences among them. Goat SAA3 had three additional potential binding motifs compared to human＇s.

Research on Polymorphism of BoLA-DRB3 Gene in Holstein and the Relationship between Different Genotypes and Resistance to Dairy Mastitis

Zhang Fu-qian;Shi Yuangang;Zheng Xiaomin;Tang Dawei;Zhao Peng

2007, 38(2): 115-119. doi:

Abstract ( 1598 )

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In this study, the restriction polymorphism of BoLA-DRB3 loci in Holstein was investigated by PCR-RFLP，and the effect of year-season of calving and different genotypes of BoLA-DRB3 loci respectively digested by RsaI and HaeIII was analysed. The results suggested that SCS in individuals of RsaⅠAD genotype were significantly higher than that in individuals of RsaⅠEG genotype（P<0.05）; and that season of calving and different genotypes digested by RsaⅠhad significant effects on protein and fat percentage at level of P<0.05 ; and that protein percentage in individuals of Hae III AB genotype were significantly higher than that in individuals of Hae III AA genotype（P<0.05）.

Genetic Polymorphism of TLR4 Gene and Correlation with Mastitis in Bovine

2007, 38(2): 120-124. doi:

Abstract ( 1385 )

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Toll-like receptors 4 performed two functions: recognition of pathogen ligands and signaling to initiate innate and adaptive immune responses. In this experiment, a 243bp segment of the exon3 of TLR4 gene of Chinese Holstein, Sanhe cattle and Chinese Simmental was amplified by Created Restriction Site PCR. The genetic polymorphisms of three populations were detected by digestion with restriction endomuclease HinfⅠ. After sequencing, a polymorphic site in amplified production was identified of having either a C and a T at position 27bp, which induced that Thr changed into Ile. Statistical results of χ2 test indicated that the polymorphism sites locus in Sanhe cattle did not fit Hardy-Weinberg equilibrium(P<0.05). Meanwhile the effect of polymorphism of TLR4 gene on somatic cell score was analyzed,The results showed that: the somatic cell score of individual with genotype AA were lowest significantly than that of other genotype（P<0.05）. In a word, the allele A might play an important role in mastitis resistance in bovine.

Genetic Structure and Differentiation of three chinese Indigenous Cattle Populations

2007, 38(2): 125-132. doi:

Abstract ( 779 )

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Using the information from 12 microsatellite to estimate Level of genetic differentiation、gene flow and genetic structure of three indigenous cattle populations (Luxi, Bohai and Minnan) and two reference cattle populations (chinese Holstein and Qinhai yak) in China. The global heterozygote deficit across of all populations (Fis) amounted to 58.5% (P<0.001). The overall significant (P<0.001) deficit of heterozygotes because of inbreeding within breeds amounted to 43.2%. The five cattle populations were highly differentiated (Fst= 26.9%, p<0.001) with all loci. The heterozygote deficit within population (Fis) was highest in Luxi cattle and lowest in Yak. The average number of effective migrants exchanged per generation (Nem) was highest (1.149) between Luxi and Holstein, and lowest (0.509) between Luxi and Yak. With the application of prior population information, cluster analysis achieved posterior probabilities from 91.4% to 98.5% of correctly assigning individuals to their rightful populations. Combining the information of cluster analysis, gene flow and STRUCTURE analysis, five cattle populations belong to three genetic clusters, a taurine (Luxi and chinese Holstein), a zebu (Bohai and Minnan) and a yak cluster. This indicate that Bohai black have more blood of the Bos indicus than Luxi cattle. The evolution and development of three indigenous cattle populations were discussed in this article.

Analysis on the Molecular Phylogeograhpy in Domestic Ducks along the Changjiang-Huai River

2007, 38(2): 133-138. doi:

Abstract ( 709 )

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In order to analyze the distribution pattern and biogeographic evolution in genetic diversity of domestic ducks of this marker, the mitochondrial D-loop of 106 samples collected from 9 breeds of domestic ducks were sequenced, and 31 haplotypes were identified. The results showed the genetic diversity of mitochondrial DNA in breeds at middle reaches of Changjiang River was bigger than that at the lower reaches of Changjiang River. A unconspicuous negative correlation was observed between the estimated level of gene flow(Nm) and the geographic distance among breeds. The mismatch distribution of the haplotypes and neutrality tests of the Tajima’s D both showed that there might not be a population expansion and the population size was steady. Phylogenetic tree analysis demonstrated that no geographic clustering was observed, haplotypes were shared among geographic populations and an interwoven distribution pattern was presented.

Estimating Covariance Functions for Daily Milk Yields of Holstein Dairy Cows in Hunan Using a Random Regression Model

2007, 38(2): 139-143. doi:

Abstract ( 1163 )

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Data of 507 Holstein dairy cows were used to estimate covariance functions by the random regression model for test-day records of the first lactation as function-valued traits. The model with Legendre polynomials of age as independent variances was used restricted maximum likelihood employing the average information algorithm (AIREML). After estimating the covariance functions of the meta-meter, the 3-D graphs were made by the Excel. The result showed that covariance functions could reflect the change of genetic and permanent variation for test day records in continuous scale of age. When the order of polynomial fit M6-5-10, the changes for yields were described adequately and the 3-D graphs were smooth. What was more, a reduced order fit involved less parameters and smoothed out differences in estimates of covariance.

Culture and Cytochemical Analysis of Like-ES Cells from Neonatal Calf Male Germ Stem Cells

2007, 38(2): 144-148. doi:

Abstract ( 732 )

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Neonatal calf male germ stem cells (mGSCs) and calf sertoli cells(CSCS) were separated and purified through the method of adhering velocity difference, and in vitro cultivation of mGSCs was studied on feeder layer cells of CSCS. The feeder layer cells of CSCs survived time did not exceed 8 d. Like-ES clusters formed when mGSCs was cultured on CSCS till 5~6 d. The method of mechanical dispersion could favor formation and presence of clusters. At lest in first 2 passages, this clusters existed. Cells of part clusters center were AKP positive staining and cells of clusters margin were AKP light positive or negative staining. Immunohistochemical analysis of typical clusters showed SSEA1 strongly positive, SSEA3 light positive and Oct-4 positive. The results suggested neonatal calf mGSCss can form ES cells.

Study on the production of chimeras derived from the transgenic embryonic stem cells by blastocysts injection

2007, 38(2): 149-154. doi:

Abstract ( 1443 )

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cell system, introduction of foreign genes into ES cells and production of germ line chimeras, also plays as a key step to obtain transgenic mouse through embryonic stem(ES) cell. The experiment used ES cells line(S8 cells) transfected by LacZ gene, which was cultured on mouse mbryonic feeder layers made by fibroblast of mouse embryo, and through blastocyst injection, the S8 cells that had been digested into single cell was injected into the blastocyst of the outbreed mouse straining Kunming albino females as the provider for recipient -embryo which had grown for 3.5 days after coitum(p.c), then transplanted the manipulated embryos into the uterus of 2.5 p.c. pseudopregant Kunming albino female recipients to allow the embryos to develop in vivo after recovering culture, which offsprings were identified one week after the chimera mouse being born according to the contribution of ES cells to tissues of chimeric mice by coat color. The experiment treated total 597 embryo with S8 cells of 8-13 generation through blastocyst injection, there were 585 embryo which got blastocyst cavity again after recovery culture of 1～3 hours, not only the figure of cells were clear, but also the juncture between the cells of feeder layers could be seen clearly, the surviving rate of embryo achieved 97%. All 37 liver-born offsprings were obtained after 17～19 days of gestation by using embryonic transfer(the fetiferous rate was 16%), among which 8 are chimeras (the generative rate of chimera was 21.6%)with coat color. The result showed that the chimeras mouse can be generated by blastocyst injection using the ES (S8) cells, and all of them occurred the sex-converted. This experiment was firstly shown that chimeras could be obtained in china at present by using S8 calls transfected LacZ gene.

Effects of Isovalerate on Rumen Fermentation and Purine Derivatives of Urine in Simmental Steer

2007, 38(2): 155-160. doi:

Abstract ( 754 )

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Four Simmental steers(average BW 420±8.6kg, aged 2.5) with permanent rumen cannula and consuming a corn straw diet were used in a 4×4 Latin square design and supplemented with four levels(0, 0.02, 0.04 and 0.06g/kgW) of supplemental isovalerate to evaluate the effects of isovalerate on ruminal pH, NH3-N, VFA, nutrients effective degradability and purine derivatives of urine. The results showed that ruminal pH in the steers given supplemental isovalerate 0.06g/kgW decreased (P<0.05). The NH3-N concentrations in the rumen of the animals in 0.04g/kgW and 0.06g/kgW group were lower than control and in 0.02g/kgW group significantly (P<0.05). Soybean DM, OM and CP effective degradability in the rumen of the animals given isovalerate supplementation with 0.04g/kgW and 0.06g/kgW were lower than control. The effective degradability of DM, OM, NDF and ADF of corn straw in the rumen of the animals given isovalerate supplementation with 0.04 g/kgW were increased significantly (P<0.05). Ruminal actate, butyrate, acetate/propionate, TVFA, and purine derivatives in urine of the animals given isovalerate supplementation with 0.04g/kgW were increased significantly (P<0.05) . These results indicated that the optimum dose of isovalerate supplementation was 0.04g/kgW.

Effect of Dietary Conjugated Linoleic Acid on the Growth and Lipid Metabolism of Wulong-geese

2007, 38(2): 161-168. doi:

Abstract ( 767 )

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One hundred and ninety two Wulong-geese of one day old were selected and divided into four groups at random. They were fed diets of different CLA levels respectively (0.0, 5.0, 15.0 and 25.0g/kg), the body weight gain (BWG), feed intake (FI), the ratio of feed and gain (F/G), the abdominal fat percentage (AFP) of different growing stages and a few serum biochemical indexes about lipid metabolism were measured to investigate the effects of different levels of dietary Conjugated linoleic acid(CLA) on the growth and lipid metabolism of Wulong geese. The results showed that, 5.0g/kg and 15.0g/kg dietary CLA had no significant effect on the BWG of Wulong geese (P > 0.05), while the FI and F/G were decreased significantly (P < 0.05), the AFP and the content of serum TG, TC, VLDL were decreased significantly or very significantly (P < 0.05 or P < 0.01) and the content of serum HDL-C and the ratio of HDL-C/LDL-C were increased significantly (P < 0.05); 25.0g/kg dietary CLA decreased FI and AFP very significantly (P < 0.01), and had no significant effect on serum TG、TC、HDL-C、VLDL and HDL-C/LDL-C (P > 0.05). Conclusions: Appropriate amounts of dietary CLA could better promote the growth and regulate the lipid metabolism of Wulong geese, that’s to say, on the whole growing stage of Wulong geese, when dietary CLA was no higher than 15.0g/kg, it could not only improve the feed utilization ratio of Wulong geese in the premise of no effects on BWG, but also prevent atherosclerosis by reducing fat deposition and regulating lipid metabolism; but superfluous supplementation of CLA would restrain the growth of Wulong geese.

Development and Application of an in-situ Hybridization for Detection of Porcine Circovirus Type 2

2007, 38(2): 169-174. doi:

Abstract ( 793 )

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A fragment of PCV2 BF strain with a size of 341 bp was amplified by PCR with a set of primer designed according to the genomic sequence of porcine circovirus type 2 available on GenBank. A nucleic acid probe labeled with digoxigenin-dUTP was prepared by random priming. The probe was not reactive to PCV1, porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV) and pseudorabies virus (PRV), and could detect 1.78 pg DNA of PCV2. Thirty clinical samples were detected with the developed in-situ Hybridization (ISH) and polymerase chain reaction (PCR). The results indicated that the ISH had a specificity of 100% and a sensitivity of 88.9%. Distribution of PCV2 in tissues of piglets inoculated with PCV2 was analyzed with ISH. The lymph nodes, thymus gland, tonsil, lung, spleen and nasal mucosa from the inoculated piglets exhibited positive signal at day 3 post-inoculation. The liver, kidney, pancreas and ileum were detected to be positive at day 21 post-inoculation. The heart, stomach and brain were positive at day 42 post-infection. The epiglottis cartilage, stomach, urinary bladder, skin, muscle and the tissues from un-inoculated piglets were negative during the experiment. The results suggested the in situ hybridization were sensitive and specific, and could be applied for clinical and laboratory detection of PCV2, as well as location of infected target cells of PCV2 .

Immune Efficacy of DNA Vaccine of Avian Infectious Laryngotracheitis Virus

2007, 38(2): 175-183. doi:

Abstract ( 1074 )

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The gB and gD gene of Infectious Laryngotracheitis virus (ILTV) Wang Gang (WG) strain were cloned into the eukaryotic expression vector pCAGGS and designated as pCAGG-gB, pCAGG-gD,respectively. Seventy-nine 30-day-old SPF white Leghorn chickens were randomly divided into seven groups ,five of them were immunized by four sites intramuscular injection with pCAGG-gB, pCAGG-gD, pCAGG-gB+pCAGG-gD，pCAGG-gB+rFPV-ILTVgB，rFPV-ILTVgB, respectively. Another two groups were injected with sodium chloride solution and pCAGGS as control groups. Two weeks after the second immunization, chickens were challenged intralaryngealy with 500EID50 of highly virulent ILTV WG strain. The sera antibodies detection result showed that all chickens immunized with pCAGG-gB, pCAGG-gD, pCAGG-gB+pCAGG-gD，pCAGG-gB+rFPV-ILTVgB，rFPV-ILTVgB developed ILTV-specific antibodies 2 weeks post-inoculation. The percentages of CD4+, CD8+ and TCRγδ+ T lymphocytes of these immunized groups were higher than control groups, and expression level of cytokine IFN-γ and IL-18 were higher in pCAGG-gB and pCAGG-gD groups than control groups. After challenge, chickens in control groups began to die at day 2 post-challenge. Chickens immunized with pCAGG-gB, pCAGG-gD had been partially protected (44%), the groups immunized with two plasmids (pCAGG-gB and pCAGG-gD) or plasmid and recombinant virus (pCAGG-gB+rFPV-ILTVgB) had higher protection (56%),the group immunized with recombinant virus (rFPV-ILTVgB) had the highest protection (69%). These results indicated that DNA immunization can induce immune response and partially protect chickens from homologous ILT virus challenge.

Study on PCR-Microtiter Plate Hybridization Assay for Detection of Eperythrozoon suis

2007, 38(2): 184-189. doi:

Abstract ( 1403 )

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Molecular cloning and characteristics of cDNA encoding bovine αv subunit for FMDV receptor

2007, 38(2): 190-195. doi:

Abstract ( 1415 )

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Foot-and-mouth disease virus（FMDV）has been showed to use four integrins, αvβ1, αvβ3, αvβ6 and αvβ8 as receptors to initiate infection. In this study, the bovine integrin αv gene was cloned from the lung tissue of healed cattle infected experimently with foot-and-mouth disease virus by RT-PCR, and compared the nucleotide and predicted amino acid sequence homology with the αv gene of other animals.The secondary structure of bovine αv protein was predicted by the biological softwares. The 3147bp cDNA of bovine integrin αv encodes a polypeptide of 1048 amino acids consisting of a 30-residue putative signal peptide, a 957-residue ectodomain, a 29-residue transmembrane domain, and a 32-residue cytoplasmic domain. The ectodomain contains 12 potential N-linked glycosylation sites（NXT/NXS）, 2 calcium binding domains(DX[D/N]XDGXXD), 18 cysteine residues, and a proteolytic cleavage site located between amino acid residue 890 and 891(KR-D). The nucleotide sequence similarity of integrin αv between cattle and rheses monkey, house mouse, dog, human,chicken is 91.0%,85.7%,90.1%,91.2%,73.1%, and the amino acid sequence similarity is 94.7%,91.6%,96.3%,95.0% and 81.6% respectively.The secondary structure of bovine αv contains few α-helix and many β-sheet and β-turn regions.The hydropathic analyses of the polypeptide revealed two hydrophobic domains,the signal peptide(1-30aa) and transmembrane domain(988-1017aa).

Comparative study on morphology and distribution of NOS-positive neurons in myenteric plexus of the goat small intestine

2007, 38(2): 196-201. doi:

Abstract ( 794 )

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Using NADPH-diaphorase histochemistry staining, the morphology and distribution of the nitric oxide synthase (NOS) positve neurons in myenteric plexus of goat small intestine were studied at 15 days,4 months and 12 months of age. The results showed that the NOS positive neurons in myenteric plexus of goat small intestine and NOS positive nerve fibers formed clearly a three-class reticular formation. The shapes of NOS positive neurons were different, and lots neurons gathered together, coming into some different size ganglion. With the goats growing, the density of NOS positive neurons in the plexus decreased about 27.07，20.80 and 16.18 neuron/mm2 respectively, but the total number increased about 1.86×106,4.06×106,4.41×106 respectively. The neurons density in the jejunum of different ages was the lower,but the number was the highest. The mean somatic area and nuclear area of the neurons enlarged firstly and then decreased late with goat growing, and the mean ratio between nuclear and plasmic area decreased with growing. There was a notable difference between 15 day-age and 4 month-age, 15 day-age and 12 month-age goat（P<0.05）. The mean somatic and nuclear of the neurons from the duodenum to the ileum showed differences, but the mean ratio between nuclear and plasmic area had no notable difference(P>0.05).

Accumulation dynamics of erythromycin in Streptococcus strains isolated from veterinary clinic

2007, 38(2): 202-205. doi:

Abstract ( 716 )

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To identify if there was the mechanism of active efflux in erythromycin resistant Streptococcus strains with mefA gene isolated from veterinary clinic，several erythromycin resistant strains with mefA gene, ermB gene and several susceptible strains were selected in this test. The uptake of 14C-erythromycin in all strains and the effects of carbonylcyanidem chlorophenylhydrazone (CCCP, a proton conductor,) on the erythromycin accumulation were assayed with intact cells. The results showed that increased active exocytosis and decreased accumulation of erythromycin (P<0.05) were occurred in strains with mefA gene, and CCCP led reverse results. The accumulation and the response to the addition of a proton conductor indicated that the erythromycin resistant strains with mefA gene could actively pump out 14C-erythromycin from the cell.

Immunohistochemical localization of ghrelin in the small intestine of adult WanXi white goose

2007, 38(2): 206-208. doi:

Abstract ( 779 )

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Objective To study the distribution pattern of ghrelin-producing cells in small intestine of WanXi white geese. Methods The immunohistochemical SABC method and DAB staining technology were used. Results Ghrelin immunopositive cells (ghrelin-ic) were mainly found in the mucosal layer of the small intestine, but not in the submucosa and tunica muscularis. These positive cells, the intensely immunoreactive stain was found in the basal cytoplasm, named ‘‘closed type cells’’, which were rounded or oblong in shape, were not in contact with the lumen. A few ghrelin-ic, which were cone in shape, showed an apical process that contacted the lumen and were the so-called ‘‘open-type cells’’. In the duodenum in particular the two types of ghrelin-ic, ‘‘closed’’ and ‘‘open’’cells, were still observed along villi intestinales. Ghrelin-ic were less numerous in the jejunum and ileum, and were generally of the ‘‘closed’’ type. Conclusion The results suggested that ghrelin-producing cells exist in the mucosal layer of the small intestine of the adult WanXi white goose, ghrelin may regulate the function of the small intestine.

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