Acta Veterinaria et Zootechnica Sinica

Analyzing on Mitochondrial DNA D-loop Sequences Genetic Polymorphism of Partial Chinese Goat Breeds

2007, 38(4): 313-320. doi:

Abstract ( 815 )

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This study analyzed the sequences of mitochondrial DNA D-loop region of partial Chinese native goat breeds (200 individuals in 18 breeds) as well as 25 individuals in 4 introduced goat breeds, combined the completely D-loop sequences that deposited in Genbank from other regions in the world (77 individuals among 15 breeds and 2 wild goat). The results showed that there were 1 212 bp length and 228 mutation sites and 203 haplotypes defined in D-loop region for all the 304 individuals, Haplotype diversity was 0.993±0.001 and Nucleotide diversity was 0.018±0.001, respectively. The Chinese goats were belonging to Clades A and B type while the Pakistan goat contained all the clades (A, B, C and D). It suggested that the Chinese goat breeds had higher genetic diversity and high frequently gene flow among breeds when compared with the Chinese cattle breeds.

Cloning and Bioinformaton Analysis of cDNA Encoding Cattle Smad4 Gene

2007, 38(4): 321-325. doi:

Abstract ( 727 )

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Cattle Smad4 gene cDNA was cloned by RT-PCR, 3' RACE and 5' RACE and got a 3 503bp full-long cDNA sequence. The cloned cattle Smad4 cDNA sequence had been send to GenBank and got a accession number: DQ494856. Cattle Smad4 gene consists of 12 exons and codes 553 amino acids. Cattle Smad4 cDNA shares 99％, 96％, 95％, 91％, 91％ similarity in nucleic acid sequences and 99％, 98％, 98％, 99％, 98％ similarity in amino acid sequences with sheep, pig, human, rat and mouse respectively. Smad4 cDNA was found in testis, pancreas, liver, small intestine, ovary, lymph, cardiac muscle, skeleton muscle and thymus gland, this indicated Smad4 is broad expressed in cattle.

SNPs Detection of STAT5A Gene and Association with Milk Production Traits in Holstein Cattle

2007, 38(4): 326-331. doi:

Abstract ( 863 )

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Based on the primer sequences of two SNPs (AJ237937 and AF079568) of STAT5A gene from the GenBank database, polymorphisms on the two SNPs were detected in 758 Chinese Holstein cattles with PCR-SSCP and association between the polymorphisms and 5 milk production traits were analyzed. We found that the SNP1 was A/G (9501) and the SNP2 were T/C (12440) and CCT insert/delete (12550). Statistical results indicated that the SNP1 was significantly associated protein percentage (p<0.05), the SNP2 was significantly associated with milk yield (p<0.01)，fat yield (p<0.05) and protein yield (p<0.05), and the diplotype was associated with milk yield (p<0.01), protein yield (p<0.01) and fat yield (p<0.05). Duncan’s multiple-range test showed that the cows with genotype AB of SNP2 had significantly higher milk yield (p<0.01)，fat yield (p<0.05) and protein yield (p<0.05) than the cows with genotype AA. In addition, the least square mean for milk yield, fat yield, and protein yield of diplotype H1H2 was significantly higher than those of other four diplotypes (p<0.05 or p<0.01), respectively, and the least square mean for milk yield and protein yield of diplotype H3H4 was significantly higher than those of other four diplotypes (p<0.01). Our finding implied that STAT5A gene would be useful candidate gene in selection program on milk production traits in Holstein Cattle.

Polymorphism of Bovine TLR2 Gene and Its Associations with Somatic Cell Score

2007, 38(4): 332-336. doi:

Abstract ( 1093 )

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Three bovine breeds,including Chinese holstein, Sanhe cattle and Chinese simmental were used as experimental examples.T/G，G/A and G/A polymorphic sites which can be cut by EcoRⅤ，HaeⅢ and HhaⅠrestriction enzymes were genotyped by PCR-RFLP and CRS-RFLP analysis，T/G polymorphic sites cause the D/E AA variation.A chi-square analysis suggested that the three bovine breeds reached Hardy-Weinberg equilibrium in the three polymorphic sites ( P > 0.05). Chinese simmental has the highest polymorphism in the three bovine breeds. The associations were observed between genotypes and SCS traits at HaeⅢ and HhaⅠrestriction enzymes polymorphic sites in three bovine breeds ( P > 0.05),meanwhile EcoRⅤ restriction enzyme polymorphic site( P< 0.05). The results of least squares means showed that a significant different effect between genotype BB and AB or AA was found in EcoRⅤ restriction enzymes polymorphic site ( P< 0.05) ,The BB genotype of the EcoRⅤ restriction enzyme polymorphic site would be the best genotype to resist mastitis.

Simulation Study of Chinese Different Types of Pig Farms by Bio-economic Model: The Construction and Preliminary Results of the Model

2007, 38(4): 337-343. doi:

Abstract ( 1499 )

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Five typical types of pig farms were the abstraction of Chinese intensive pig production according to the current three-level hybrid pig production system and their bio-economic farm models including biological efficiency were constructed. The annual profit of the simulated commercial pig farm, sow farm, hybrid multiplying farm, Duroc nuclear farm and Landrace (Yorkshire) nuclear farm of 600 sows in the base situations is 0.560 1, 0.129 9, 1.005 3, 1.340 9 and 1.641 0 millions RMB, respectively. The efficiency in net energy utilization of the five farms is respectively 27.69, 32.98, 28.25, 28.32 and 28.48 MJ/kg. Model could also output the cost structures, income structures and production performance of different types of pig farms. The sensitivity analysis showed that the models were sensitive to different market situations, production levels and management measures.

Construction of Phage Display Library of Fab Antibodies Repertoies of Mouse Male Specific Antigen

2007, 38(4): 344-351. doi:

Abstract ( 709 )

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Isogenic C57BL/6 female mice at 6~7 weeks of age were inoculated with male spleen cells from the same strain. RNA was isolated from immunized female mice spleen cells using TRIZOL method. The first-strand cDNA was reverse transcribed from total RNA using an oligo(dT)18 primer, the κ light-chain and the Fd (Vh-Ch1) regions of heavy-chain products were amplified from the cDNA template by polymerase chain reaction (PCR) with specific primers. To generate the pComb3-κ and pComb3-κ-Fd library, κ light chain and Fd gene fragments were inserted into phagemid vector pComb3 respectively, then transformed into E. coli strain of XL1-Blue competent cells, the transformants were plated for a blue-white color screening. The recombination rates of κ light chains and Fd fragments were 80％which was confirmed by PCR and endonuclease digestions. A mouse combinatorial Fab library was constructed by the help phage
VCSM13 infection, which gave 1.5×107 colonies with the titer of 3.2×1011 pfu/ml. The presence of mouse Fab on the phage surface was determined by ELISA. 9 of 18 strains had specific binding capacity to male spleen cells，8 of 18 strains had specific binding capacity to testicle cells. The mouse Fab antibody library could be used for selection of H-Y antigen binding Fab phage. These new molecules are potentially valuable tools in the study for molecular characteristics and functional analysis of H-Y antigen and antibody, the application of embryo sexing in mammals.

The Inhibitory Effects of siRNA Expression Vector on the Expression of avUCP Gene

2007, 38(4): 352-355. doi:

Abstract ( 701 )

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To investigate the inhibitory effects of RNAi（RNA interference） expression vector on the expression of avUCP gene. Four siRNA expression vectors (A, B, C and D) were constructed to be aimed directly at avUCP gene. The recombinants were transfected into myofibroblast with liposomes. Expression of avUCP was detected with fluorescence quantitative PCR（FQ-PCR）and flow cytometry. Four kinds of expression vectors could reduce the expression of avUCP mRNA and protein all in myofibroblast. The expression of avUCP mRNA reduced to 14.6% of control group, and the protein inhibition ratio reached 93.7%. The RNAi expression vector can effectively inhibit the expression of avUCP gene.

Effects of Sunflower Seed Oil Supplementation on Rumen Digestion and Fatty Acids Composition in Goats

2007, 38(4): 356-361. doi:

Abstract ( 781 )

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This experiment was conducted to evaluate effects of sunflower seed oil supplementation on rumen digestion and fatty acid composition. 5 female adult Xuhuai White goats were surgically installed with ruminal fistula. The experiment was divided into two periods by self-control design. Each period lasted for 15 days. Goats were fed a low-concentrate diet in two periods (non sunflower seed oil or 7mL sunflower seed oil per day). Rumen liquid was taken from rumen fistula at the last day of each period. The results showed that the pH value of ruminal liquid was not affected by sunflower oil supplementation. Compared with control period, the concentration of ammonia N, total volatile fatty acid (TVFA), acetate and propionate in the oil supplementation period increased significantly (P<0.05). However, the contents of acetate, propionate and butyrate, the ratio of acetate plus butyrate to propionate ((C2 + C4)/ C3), microbial crude protein (MCP) concentration in ruminal liquid were not affected by the oil supplementation. The concentration of oleic acid (C18:1) and cis9, trans11-conjugated linoleic acid (c9,t11-CLA) in ruminal fluid increased remarkably by sunflower oil supplementation compared with control period (P<0.05). The results implicated that appropriate dose of sunflower oil supplementation could improve rumen digestion and manipulate long chain fatty acid composition in goats fed low-concentrate diet.

Construction and Characterization of UL14-null Recombinant Pseudorabies Virus

2007, 38(4): 369-375. doi:

Abstract ( 743 )

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UL14 is a highly conserved protein among alphaherpesvirus. However, until now, little is known about the function of pseudorabies virus (PRV) UL14. In this study, an UL14-negative PRV mutant (PRV-UL14D) was constructed by inserting an EGFP expression cassette into the UL14 coding sequence. In noncomplementing cells, the mutant was able to replicate, but exhibited an extended growth cycle and significantly reduced plaque sizes, when compared to the field PRV-Ea. These deficiencies were corrected in UL14-expressing cells. In vivo, the average death time of BALB/c mice inoculated with PRV-UL14D was obviously delayed, compared with the same dose of PRV-Ea. From the above results it was therefore concluded that the PRV UL14 product is not essential for virus replication in vitro but delay virus replication and is involved in cell-to-cell spread.

Isolation, Identification and Biological Characterization of Streptococcus from Swine in China

2007, 38(4): 376-381. doi:

Abstract ( 844 )

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Studies on drug susceptibility, serotype and molecular epidemiology of Streptococcus from swine were investigated through biochemical reaction test, drug susceptibility test, PCR typing, PCR test of virulence factor genes and animal test assays. 97 strains of Streptococcus were isolated from heart blood, liver, lymph node, brain and joint of swine in China. Drug susceptibility test for 97 strains showed their resistance patterns were different, but their drug resistance rate to cephalosporin V and ciprofloxacin is lower than 5℅ respectively. PCR typing assay indicated that 26 strains were Streptococcus suis including 1 serotype 1 strain, 16 serotype 2 strains, 4 serotype 7 strains and 5 other serotype strains. The genotypes of their mrp, epf and sly were identified by PCR method as well. Result of mouse virulence test showed that the genotypes of streptococcus, which were lethal to all of the mice, were mrp+, epf+ and sly+ serotype 2 strains. One serotype 2 strain and one serotype 7 strain could reproduce typical swine streptococcosis.

Effects of PRRSV and PCV2 Co-infection in vivo on the Transcriptions of IL-10, IL-12p40 and IFN-γ mRNA in Porcine Pulmonary Alveolar Macrophages

2007, 38(4): 382-387. doi:

Abstract ( 898 )

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Forty-day-old healthy piglets were co-infected with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2). The changes of mRNAs of cytokines in porcine alveolar macrophages (PAM) of the inoculated piglets were analyzed using a real-time fluorescent quantitative PCR (real-time FQ-PCR). The results showed that the transcriptions of IFN-γ mRNAs exhibited two transcription bottoms at day 3 and 28 post-inoculation (PI) and a peak at 7DPI and 42DPI respectively, with significant differences at 3DPI and 7DPI (P<0.05). The transcriptions of IL-12p40 mRNAs reached the highest level at 7DPI, with significant differences at 3 DPI and 14DPI (P<0.05), and then downregulated at 28 and 42DPI with no significant differences. In addition, the mRNAs transcriptions of IL-10 peaked at 14DPI with significant differences at 3DPI (P<0.05), 7DPI (P<0.05) and 14DPI (P<0.01), then declined gradually to the level of the control at 42DPI. Accordingly, the co-infection of PRRSV and PCV2 could significantly activate the transcriptions of IL-12p40 and IL-10 mRNAs early during the co-infection, while the transcription of IFN-γ mRNAs witnessed more kinetic changes, an indication of the impairments of cellular immune response, immune regulatory and anti-infection function of PAM in dually infected piglets.

Porcine CD58: cDNA cloning, expression and prediction of the structure and function

2007, 38(4): 388-394. doi:

Abstract ( 1391 )

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CD58 plays an important role in mammal immune system. In this study, a pair of primers was designed according to human and sheep CD58 sequence and used to clone porcine CD58 gene by RT-PCR method. In addition, the protein structure of porcine CD58 were predicted through bioinformatics methods and compared with human and sheep CD58. The results showed that an 800 bp fragment of porcine CD58 cDNA with an ORF of 735bp was obtained. Then, this cDNA was ligated into pGEX-4T-1 and transformed to E.coli BL21 cells. The recombinant transformant was induced with IPTG and the expressed product could be recognized by anti-CD58 serum. The prediction of the protein structure indicated that the porcine, sheep and human CD58 have high similarity, especially in V region which was the molecular foundation of xeno-adhesion between lymphocyte and RBC. This research will help us to develop new adjuvant and/or immune-regulated drugs, and laid a solid foundation for further study of structure of CD58 molecule and the mechanism of CD2-CD58 complex triggering immune system.

The Construction of GHRH Gene Expression Vector Based on RNA Replicon and Its Expression in 293 Cell

2007, 38(4): 395-399. doi:

Abstract ( 1253 )

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In the present study, GHRH gene was cloned into pCMV-Rep-lacZ which digested by BamHⅠin order to replace the LacZ gene with our gene of interest and dephosphorylated by shrimp alkaline phosphatase, creating pCMV-Rep-GHRH. pCMV-Rep-GHRH was transfected by Lipofectin to prepared 293 cells. Results of RT-PCR, Tricine-SDS-PAGE ,Western Blot and immunofluorescence antibody assay(IFA) showed the our interested gene was expressed efficiently in transfected 293 cell. In order to compare the expressed efficacy between RNA replicon vector(pCMV-Rep-GHRH) and ordinary vetor（pIRES-GHRH and pCDNA3-GHRH）, we also detected the concentration of GHRH from transfected cell supernatant by RIA in this study. The results showed that the concentration of GHRH from pCMV-Rep-GHRH transfected cell supernatant was 36.12%（P<0.05）and 67.94%（P<0.05）higher signifiantly than that of GHRH from pIRES-GHRH and pCDNA3-GHRH transfected cell supernatant respectively. We conclude that that GHRH can be expressed efficiently in eukaryotic expression vetor pCMV-Rep-GHRH transfected 293 cell. We can also draw another conclusion that the express level of RNA replicon vector was superior to ordinary vetor. It may be a potential way to increase animal productivity.

Study on the Chromosomal Aberrations of Liangshan Semifine-Wool Sheep and Tibetan Sheep

2007, 38(4): 400-406. doi:

Abstract ( 666 )

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Four Liangshan Semifine-Wool Sheep and four Tibetan Sheep were used to compare the karyotypes, which were employed on slides obtained from the peripheral blood cell culture. The result showed that: the karyotype of ram was 54, XY; and the ewe’s was 54, XX.In the two breeds,the frequency of numerical chromosome change were 12.00% and 17.60%, and the frequency of chromosomal structural change were 33.00% and 32.25%;the type of the chromosomal structural change includ chromosome break,chromatid break and centric change;Some break points of chromosomal structural change were correspondence with the loci of specific Ag-NORs; Some homologues had the different length in the two breeds, and this phenomenon in Liangshan Semifine-Wool sheep was more significant than in Tibetan sheep.

Elementary Study on Immortalization of Porcine Vascular Endothelial Cell by Telomerase Reverse Transcriptase Transfection

2007, 38(4): 407-411. doi:

Abstract ( 778 )

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To elongate the proliferation life-span of porcine arterial endothelial cell(PAEC) in vitro. The eukaryotic expression plasmid pCI-neo-hTERT was transfected into PAEC by lipofection. FactorⅧ related antigen and CD34 of PAEC were identified after transfection by immunohistochemical method; we also detected telomerase activity of PAEC after transfection by PCR-ELISA. The results showed that Factor Ⅷ related antigen and CD34 in PAEC is positive after transfection. The PAEC after transfection expressed the vascular endothelial cell-specific protein still; Telomerase activity in PAEC is higher by hTERT introduction, the cell proliferation life-span was elongated to 20 population doublings compared to control. Conclusion, Telomerase activity can be activated in PAEC, thus the cell proliferation life-span was elongated in vitro.

Clone and Prediction of the Secondary Structure and B cell Epitopes for the Env Protein of Porcine Endogenous Retrovirus

2007, 38(4): 412-416. doi:

Abstract ( 749 )

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Porcine Endogenous Retrovirus（PERV）is a kind of virus correlatively to the safety of pathogens during pig xenografts. The env gene encodes the Env protein, which plays an important role in virus subtyping, host ranging, cells tropism and mechanism of virus infect, as well as induction of neutralizing antibody secretion. In this research, the envelope gene of PERV was amplified by RT-PCR and sequenced from the peripheral blood lymphocyte of WZS minipigs, The Secondary Structure and B cell Epitopes of the Env Protein of PERV were analysed and predicted subsequently by the Bioinformatics software. The results showed that the PERV-Env protein was supposed to exist18 potential antigen epitopes and 7 potential glycosylated sites. These results indicated that it would be helpful not only to the PERV vaccine designing and the preparation of both of monoclonal antibody and diagnostic reagent, but also to research of Env Protein function and mechanism of PERV infecting human-derived cells.

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