Acta Veterinaria et Zootechnica Sinica

Genetic Diversity of Five Native Pig Breeds of Chongqing Based on Microsatellite DNA Markers

BAI Xiao-qing;FAN Shou-jun;WANG Jin-yong;LIU Wen;GUO Zong-yi;HE Dao-ling;CHEN Si-qing;WANG Ke-tian;FU Wen-gui;CHEN Lei;ZHAO Jiu-gang;LAN Jing;ZHANG Liang

2010, 41(12): 1515-1522. doi:

Abstract ( 1029 )

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The study was conducted to analyze the genetic diversity and phylogenetics of five Chongqing indigenous pig breeds in Chongqing. Using 27 microsatellite markers recommended by the International Society for Animal Genetics(ISAG)and Food and Agriculture Organization(FAO),by effective number of alleles, expected heterozygosity， polymorphism information content and other indexs，the genetic variability within pig breeds were estimated．By Fstatistic, Nei’s genetic distance, Nei’s standard genetic distance， dendrogram analysis with the methods of UPGMA(unweighted pair group method with arithmetic averaging) and NJ(neighbor jioning )and principal component analysis, the genetic differentiation and genetic relationship among five Chongqing indigenous pig breeds were estimated. 542 alleles were observed at 27 loci, and 43 alleles of them belonged to certain pig breed. Abundant genetic diversity lied in five Chongqing indigenous pig breeds: effective number of alleles of pig breeds varied from 9.493 3 to 11.028 0, expected heterozygosity from 0.889 7 to 0.909 1, polymorphism information content from 0.868 9 to 0.892 6． F-statistic analysis implied that the genetic differentiation of Chongqing indigenous pig breeds was significant（P<0.001）and different degree inbreeding existed in every pig breed. Dendrogram analysis showed that the NJ methods was more reliable than UPGMA, and five Chongqing indigenous pig breeds pigs were clustered into two groups. Rongchang, Quxi and Hechuan pigs were clustered into one group, Luopanshan and Penzhoushandi pigs were clustered into the other one. Correlation analysis showed that there were no significant correlation between genetic distances and geographic distances（P>0.05）. The results could provide basic molecular data for the conservation and utilization of Chongqing indigenous pig breeds．

Activation of Wnt/β-catenin Signaling Pathway Inhibits the Adipogenic Differentiation of Porcine Adipose-derived Mesenchymal Stem Cells

LI Hui-xia;;LUO Xiao;LIU Rong-xin;YANG Ying-juan;YANG Gong-she

2010, 41(12): 1523-1528. doi:

Abstract ( 683 )

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To investigate the effects and molecular mechanisms of Wnt/ β-catenin signaling pathway in the adipogenic differentiation of adipose-derived mesenchymal stem cells (AMSCs), the porcine AMSCs was established, and the Wnt/β-catenin signaling pathway was activated by regulation of the pathway’s second messenger β-catenin. The expression level of CD44 and CD105 of porcine AMSCs cells was evaluated by cellular immunochemistry technique. The adipogenic differentiation of AMSCs was analyzed by Oil red O staining. The expression of Wnt/β-catenin signaling pathway factors and the adipogenic transcription factors were checked by semi-quantitative RT-PCR and Western blot analysis. The results showed that the porcine AMSCs expressed of CD44 and CD105, and they have multiple differentiation potential. Wnt/β-catenin signaling pathway was activated by stabilization of cytoplasmic β-catenin with continuous treatment AMSCs by 25 mmol·L^-1 LiCl. The number of adipocytes, and the content of triglyceride decreased during adipogenic induction differentiation of porcine AMSCs. In addition, the adipogenic transcription factors of CCAAT/enhancer-binding protein-α (C/EBPα) and peroxisome proliferator-activated receptor-γ (PPARγ) were repressed in the presence of LiCl. Taken together, the result indicated that activation of Wnt/β-catenin signaling pathway inhibits the adipogenic differentiation of porcine AMSCs, and its inhibition may due to reduce the expression of C/EBPα and PPARγ.

The Distribution of SCC and Its Correlation with Milk Production Traits in Chinese Holsteins

MA Pei-pei;YU Ying;ZHANG Yuan;ZHANG Qin;WANG Ya-chun;SUN Dong-xiao;ZHANG Yi

2010, 41(12): 1529-1535. doi:

Abstract ( 680 )

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The objective of this study was to analyze the variation trends of the somatic cell counts (SCC) and its correlation with milk production traits in Chinese Holsteins in Beijing, then to conclude the criteria for sub-mastitis prediction. The dairy herd improvement (DHI) data came from 76 Holstein herds including 63 510 cows were tided up in this study. Twelve subsets and eight cumulative subsets were divided according to certain conditions using SAS 8.2 software. First, the curves of SCC and milk production traits of the twelve subsets were ploted with Excel. Then the SCC curves of the 12 subsets and eight cumulative subsets were ploted using the Spline function of Matlab, respectively. Except fat percentage, the averages of the other four milk production traits were highly correlated with the mean of the SCC for the 12 subsets (P<0.001). Milk yield, milk protein yield and milk fat yield decreased following the increase of SCC, while the protein percentage increased. The curves of the cumulated subsets indicated that there was significant difference between average of SCC<100 000 subset and of SCC<200 000 subset. But the difference between average of SCC<400 000 subset and of SCC<500 000 subset was not significant. Our results indicated that the criterion of subclinical mastitis in Chinese Holsteins should choose 100 000 to 500 000 SCC per milliliter milk.

Effects of Protease Inhibitor MG-132 on Nuclear Remodeling of Bovine Nuclear Transfer Reconstructed Embryos

LIANG Su-li;LI Xiang-chen;JIN Ya-ping;WANG Yi-peng;GUAN Wei-jun;MA Yue-hui

2010, 41(12): 1536-1542. doi:

Abstract ( 647 )

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To investigate the effect of MG-132 on the nuclear remodeling of bovine reconstructed embryos. Different concentrations MG-132 (0, 3, 5, 7, 10 μmol·mL^-1) were added at MI period oocytes, then mature oocytes were activated by Ionomycin. The appropriate concentration of MG-132 was obtained through the development of embryos. The role of MG-132 was studied by examining the developmental potential of reconstructed embryos. The entire process of nuclear transfer was acting by adding optimum concentration MG-132. The results was verified by immunofluorescence. The results showed that the cleavage and blastocyst rate in 5 and 7 μmol·mL^-1 MG-132 groups were significantly lower than that in the control and 2 μmol·mL^-1 MG-132 group (P<0.05), but there was no significant difference between the two groups (P>0.05). The 10 μmol·mL^-1 MG-132 group was the lowest (P<0.05). The cleavage and blastocyst rate had no significant difference among the three groups that the mature oocytes treated in the 5 μmol·mL^-1 MG-132 for 0, 2, 4 h , respectively (P>0.05), but 6 h group were significantly lower (P<0.05). The cleavage rate of adding MG-132 group in nuclear transfer operation was no significant difference compared with control group(P>0.05), while the morula and blastocyst rates were significantly higher than that of the control group (P<0.05). In the control group, the reconstructed embryos after fusion 1 h showed minimal or no change in donor cells, and after 4 h existing strong lamin staining of the control group. The test group after fusion 4 h showed patchy nuclear membrance attaining and significant chromatin condensation. The results indicate that 5 μmol·mL^-1 MG-132 can promote the reprogramming of reconstructed embryos, but the effect of implantation and pregnancy after transplantation remains to further study.

Influence of Different Factors on the Efficiency of Bovine Sorted Sperm Injection

ZHU Hua-bin;PANG Yun-wei;DU Wei-hua;HAO Hai-sheng;LI Hai-yan;SHEN Yan-jun;ZHAO Xue-ming;WANG Dong;WANG Zong-li

2010, 41(12): 1543-1549. doi:

Abstract ( 572 )

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This study was conducted to investigate the effects of different combined activation protocols,sperm preparing methods and injection methods on the efficiency of bovine ICSI. By the assistance of a micromanipulation system, bovine sorted X sperm, prepared by different pretreatments, was directly injected into an oocyte which was matured in vitro for 22-24 h, with or without performing cytoplasmic aspiration, then activated by three different combined activation protocols. The results showed that the cleavage and blastocyst rate of ICSI embryos activated by CR1aa + Ionomycin + 6DMAP were higher than those activated by A23187 + CHX and 7%ET + CHX groups（63.74% vs 61.09%, 53.75%；28.54% vs 17.68%, 22.00%，P<0.05）. After treating sperms with percoll density gradient centrifugation, the cleavage（77.10% vs 62.19%，P<0.05）and blastocyst rate（26.95% vs 22.82%，P>0.05）were both higher than that of the swim-up treatment group.The cleavage and blastocyst rate with performing cytoplasmic aspiration were both significantly higher than that of control during sorted sperm injection（63.10%，25.35% vs 41.56%，19.40%，P<0.05）. The results indicated that treating sperms with percoll density gradient centrifugation,performing cytoplasmic aspiration and activating ICSI embryos by CR1aa + Ionomycin + 6-DMAP,can significantly improved the efficiency of bovine sorted sperm injection.

Construction and Expression Study of Escherichia coli Phytase (appA2) Combined with Human MxA Gene Eukaryotic Expression Vector

JU Hui-ming;BAI Li-jing;MU Yu-lian;YANG Shu-lin;LIU Zong-ping;LI Kui

2010, 41(12): 1550-1555. doi:

Abstract ( 688 )

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The aims of this study were to explore the expression efficiency in double gene eukaryotic expression vector based on construction of Escherichia coli phytase gene (appA2) combined with human MxA gene Eukaryotic expression vector. CMV-appA2BGH pA fragment was amplified by PCR from pcDNAappA2 vector, and then ligated to the Mlu I site of the vector pcDNAMxA. The recombinant vector pcDNA-appA-MxA（AMP）was constructed. The pcDNA3.1（+）, pcDNA-MxA, pcDNA-appA2, and AMP vector DNA was transfected into porcine PK15 cells. Quantitative fluorogenic real-time PCR assay was performed for determining appA2 and MxA mRNA expression level. MxA protein in pcDNA3.1（+）, pcDNAMxA, pcDNA-appA2 and AMP cells groups was analyzed with Western blot method. The appA2 enzyme activity from pcDNA3.1（+）, pcDNA-appA2 and AMP cells groups was dectected with modified Molybdate colorimetric method. The results of sequencing and restriction showed that the AMP vector was constructed successfully. QRTPCR results showed that MxA mRNA expressiong level in AMP cells group was 1.118 times than that in pcDNAMxA cells group，appA2 mRNA expression level in AMP cells group was 1.134 times than that in pcDNA- appA2 cells group, and the difference was significant（P＜0.05）; Results of gray intensity analysis showed that MxA protein relative expression level in AMP cells group was 1.07 times than that in pcDNAMxA cells group. Phytase detection results showed that enzyme activity of phytase in AMP, pcDNA-appA2 and control groups were 0.294, 0.235 and 0.082 FTU·μL^-1, respectively, and there were no obvious difference in the two experimental groups, and the difference in the two experimental groups and control group was extremely significant（P＜0.01）. The results showed that the vector constructed had relatively higher expression efficiency compared with single gene expression vector. This study establish a foundation for preparation transgenic animal co-expression multiple foreign gene.

Comparison of Microbial Communities Adherent to Three Kinds of Feeds in Dairy Cows by PCR-DGGE

FENG Wei;WANG Jia-qi;LIU Kai-lang;BU Deng-pan;LI Dan;ZHAO Sheng-guo;YANG Kai-lun

2010, 41(12): 1556-1562. doi:

Abstract ( 621 )

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In order to get the information of microbes involved in coarse degradation, microbial communities adherent to alfalfa hay, corn silage and Chinese wild hay in the rumen of Holstein dairy cows were analyzed. Three healthy Chinese Holstein dairy cows with permanent rumen cannula and similar weight were selected, three kinds of feeds were put in nylon bags, respectively, then incubated in the rumen for 24 h, the microbial communities adherent to feeds were eluted with PBS method and compared by DGGE. Some different and the same bands were selected and sequenced. Phylogenetic tree was built by Mega 4.1. DGGE profiles showed the number and intensity of bands from different samples were different; The identity between alfalfa hay and corn silage, Chinese wild hay was low, but the identity between corn silage and Chinese wild hay was high; Simpson index of different samples were different significantly (P<0.05), Shannon-Wiener index was not different significantly (P>0.05); The phylogenetic tree derived from 16S rDNA sequence was found that the closest relatives belonged to Butyrivibrio sp., Fibrobacter sp., Prevotella sp., Succiniclasticum sp., Pseudobutyrivibrio sp.. The results showed that the microbial communities adherent to three feeds were different.

Preliminary Study on the Antigenic Relation of vIL8 Encoded by Marek’s Disease Virus and Chicken IL8 (cIL8)

GAO Wei;QIN Ai-jian;JIN Wen-jie;SHAO Hong-xia

2010, 41(12): 1563-1567. doi:

Abstract ( 591 )

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vIL8 is a chemokine encoded by Marek’s disease virus. cIL8 is an important chemokine of chickens which is useful in the antiviral activity. Studies on the relationship of vIL8 and cIL8 help to know the function of vIL8. In this study, the amino acid sequences of them were compared and analyzed firstly; then the cIL8 was expressed in E. coli. After induction with IPTG, the cIL8 fusion protein was obtained. The protein was separated by SDSPAGE and the band of interest was excised and minced for mouse immunization. The immunofluorescence test was carried out between the antiserum against cIL8 and vIL8 expressed by insect cells. The results showed that both vIL8 and cIL8 are typical CXC chemokines in structure, and they have similar key amino acids receptor binding. The result of the immunofluorescence test showed that the cIL8 antibody could react with vIL8 expressed by insect cells. The results suggest that vIL8 shares common antigenic determinants with cIL8; and they may have a common receptor. The feature of vIL8 suggests it may participate in the immune evasion of the virus.

Correlation between Phenotype and Genotype of Resistance to Aminoglycoside in 98 Strains of Pathogenic E. coli from Ducks

YU Xue-hui;HUANG Lan;YANG Xiao-nong;LIU Qun

2010, 41(12): 1568-1575. doi:

Abstract ( 1049 )

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The objective of this paper is to investigate aminoglycoside resistance genes in E. coli from ducks and to study the correlation between the phenotypes and the genotypes of resistance. By using the K-B method and choosing aminoglycoside antibiotics including streptomycin (STR), neomycin (NEO), gentamycin (GEN), kanamycin (KAN), amikacin (AMK) and spectinomycin (SPT), the drug sensitivity tests were conducted for 98 strains of pathogenic E. coli from ducks. All strains of E. coli were detected by multiplex PCR method based on the main genes of AMEs to aminoglycoside, the resistance genes including ant(3″)-Ⅰa, aac(3)-Ⅱa and aph(3′)-Ⅱa randomly selected from 3 E. coli strains with positive amplification were cloned and sequenced, then the results obtained from drug sensitivity tests were compared with that of PCR detection. The results showed that 67 strains out of 98 strains are resistant to one or several antibiotics with the resistance rate of 68.4%(67/98), and AMEs genes were amplified in 49 strain of the bacteria with the positive rate of 50%(49/98), in which the detection rates of ant(3″)-Ⅰa, aac(3)-Ⅱa, aph(3′)-Ⅱa, ant(3″)-Ⅰa＋aac(3)-Ⅱa and aac(3)-Ⅱa＋aph(3′)-Ⅱa were 30.6%(30/98), 13.3% (13/98), 3.1% (3/98), 2.0% (2/98) and 1.0% (1/98) respectively, and aac(6′)-Ⅰb wasn’t be discovered. The sequences analysis indicates that the amplified products shared high homology (over 99%) with the corresponding sequences in GenBank. The results of comparison show that the correlation rate between phenotype and genotype of resistance gene AMEs is 67%(45/67), in which the correlation rates of SPT, GEN, STR, KAN, NEO and AMK were 60% (3/5), 55% (11/20), 33.3% (22/66), 19% (4/21), 12.5% (1/8) and 0% (0/3), respectively. In addition, 4 strains carrying resistance genes showed the sensitive phenotype, while 22 strains with resistance phenotype didn’t carrying the relevant genes. The main resistance genes of pathogenic E. coli from ducks to aminoglycoside are ant(3″)-Ⅰa and aac(3)-Ⅱa, and the drug resistance is positively correlative to the detection rate of relevant resistance genes.

Research on Class I Integrons of rmtB Positive Isolated E.coli and Their Transconjugants from Pig Farms

CHEN Lin;LIU Jian-hua;CHEN Zhang-liu;ZENG Zhen-ling;ZHANG Jun-feng;HUANG Dong-zhang;WEI Dong-xia

2010, 41(12): 1576-1583. doi:

Abstract ( 611 )

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The objective of this study was to analyze the prevalence and characteristics of class I integrons in 48 isolated rmtB positive E. coli and their transconjugants. The Polymerase Chain Reaction and DNA sequencing were used to analyze the detection rate and the conformation characteristics of class I integrons in 48 rmtB positive E. coli and their 45 transconjugants.The microdilution method were applied to analyze the resistance of 48 rmtB positive E. coli isolates and their transconjugants against 20 antibacterial agents. And statistical method was used to compare the resistant ratio between cassette-positive and cassette-negative isolated E.coli. Of all 48 rmtB positive E.coli, 100%, 77.1% and 58.3% strains were positive for 5′conserved segment, 3′-conserved segment and the gene cassettes, respectively. And 84.4%, 62.2%, 42.2% of 45 transconjugants were positive for 5′-conserved segment, 3′-conserved segment and the gene cassettes, respectively. Missing, acquisition and replacement of gene cassettes were happened in the procedure of transconjugation. Indistinctive deviation of the resistant ratio between gene cassettepositive and gene cassettenegative isolated E. coli to 20 antibacterial agents were demonstrated except for spectinomycin and apramycin. The results indicated that class I integrons were widely prevalent in rmtB positive E. coli and their transconjugants. And the detection rates for 5′-conserved segment, 3′-conserved segment and gene cassettes were different. Gene capture and recombination were taken place in the procedure of transconjugantion. The ratios of gene cassette were unrelated to the multi-drug resistance of rmtB positive E. coli and their transconjugants.

The Immunogenicity Analysis of Expression Product of the ClfA Gene of Staphylococcus aureus in Mice

YANG Ming-feng;CHEN Chuang-fu;CAO Xu-dong;LIU Jun;WANG Zheng-rong;LI Zhen;QIAO Jun;

2010, 41(12): 1584-1591. doi:

Abstract ( 683 )

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This experiment was conducted to study the immunogenicity of partial ClfA gene product of Staphylococcus aureus in mice. The region of clumping factor A (ClfA) gene was amplified by PCR from Staphylococcus aureus causing mastitis in dairy cows, and sequenced. The epitopes were analyzed by DNAman software. Then the fragment was inserted into the vector pET-28a(+) constructed the recombinant plasmid ClfA-pET-28a+. The plasmid was transformed into the host cell BL21(DE3), and the protein was expressed in BL21(DE3) after being induced by IPTG. The target protein was purified by His-bind chromatography. Mice were immunized with the purified protein emulsified with Freund’s adjuvant. Challenge test was performed by injecting S. aureus via the mammal glands to mice after parturition. And the immunogenicity was evaluated by detecting the pathological change，the concentration of TNF-α，the amounts of S. aureus in per-gram mammal gland tissues and the antibody levels. The results showed that the expressed protein was ClfA protein of S. aureus, and the protein had significant effects on improving the pathological change，reducing the concentration of TNFα and the amounts of S. aureus in mammal tissues in mice. These demonstrated that the prokaryotic expressed protein of ClfA have a better immunoprotection effect against S. aureus， and provide a reference for the study on cows.

Sequence and Phylogenetic Analysis of Mitochondrial cox1 and nad1 Genes for Ascaridia galli Isolates from Hunan Province

WU Hui-lan;TAN Mei-ying;LIU Guo-hua;LI Fen;XU Ping-yuan;LUO Dong-sheng;LIU Yi

2010, 41(12): 1592-1597. doi:

Abstract ( 995 )

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The objectives of the present study were to examine sequence variation in the mitochondrial cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes among Ascaridia galli isolates from Hunan province, and to study the phylogenetic relationship among roundworms of the other family using cox1 and nad1 sequences. The partial cox1 (pcox1) and nad1（pnad1）were amplified from each Ascaridia galli sample, and pcox1 and pnad1 sequences were aligned using the ClustalX 1.81. MP and NJ trees were constructed by using the software Phylip 3.67 version 4.0 and Mega version 4.0, and ML tree was also constructed using Puzzle version 5.2. The results were as follows: the lengths of all the pcox1 and pnad1 sequences were 394 and 408 bp, respectively. The phylogenetic tree showed that the Hunan isolates were clustered in the same clade. There is no significant variation in pcox1 and pcox1 sequences within Ascaridia galli, while inter-species difference is obvious. It is concluded that pcox1 and pcox1 sequences can be used as genetic marker for population genetic studies of roundworms. It was the first time that the pcox1 and pcox1 sequence of Ascaridia galli were reported．The results of this study lay the foundation for further study on molecular epidemiology and diagnostics of Ascaridia galli.

Effect of High Molybdenum on the Pathologic Lesion and Antioxidative Function of Kidney in Broilers

XIAO Jie;CUI Heng-min;YANG Fan;PENG Xi;CUI Wei;CHENG An-chun;CHEN Tao;BAI Cai-min

2010, 41(12): 1598-1604. doi:

Abstract ( 639 )

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300 one-day-old Avian chicks were randomly divided into four groups and feed on diets as follows: control diet(Mo 13 mg·kg^-1) and high molybdenum(Mo) diets(Mo 500 mg·kg^-1, high molybdenum group Ⅰ; Mo 1 000 mg·kg^-1, high molybdenum group Ⅱ; Mo 1 500 mg·kg^-1, high molybdenum group Ⅲ) for 42 days, examining the effect of dietary high molybdenum on pathologic lesions in kidney of broilers. Compared with those of control group, the renal tubule epithelial cells showed swelling, granular degeneration and vacuolar degeneration in high Mo groups. Ultrastructurally, endoplasmic reticulum and nuclear membrane expanded, mitochondrial were swelling or loss of volume and increases in electron density, and myelin-like structure in the microvilli were appeared in renal tubule epithelial cells in high Mo groups. The renal glutathion peroxidase (GSH-Px) and superoxide dismutase (SOD) activities were lower (P<0.01), and the malondialdehyde(MDA) contents were higher(P<0.01) in high Mo groups Ⅱand Ⅲ than that in control group. Changes of the above mentioned enzymes activities and MDA contents in serum were consistent with those of kindey. At the same time, the serum creatinine and serum uric acid contents were much higher in high Mo groups Ⅱand Ⅲ than that in control group(P＜0.05 or P<0.01).The results showed that dietary molybdenum in excess of 1 000 mg·kg^-1 caused renal pathological injury and decreased antioxidant enzymes activities. The renal function was finally injuried.

Effect of Different Selenium Sources on Selenium Contents andthe Antioxidant Capacity of Hen Tissues

PAN Cui-ling;HUANG Ke-he;ZHAO Yu-xin;QIN Shun-yi;CHEN Fu

2010, 41(12): 1605-1613. doi:

Abstract ( 909 )

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This experiment was conducted to compare the effects of Seleniumenriched probiotics(SP) and sodium selenite(SS) on selenium contents and the antioxidant capacity of hen tissues. Both Se sources were added into basal diet at 0.2, 0.5 and 1.0 mg·kg^-1 of Se to make six types of experiment diets. At the same time, probiotics was added into basal diet as the probiotics control, and basal diet served as the blank control. Eight hundred healthy Rohman laying hens were randomly and equably allocated into eight groups and were investigated for 28 days. At the end of the experiment, hens were slaughtered, and liver, kidney and cardiac muscle were sampled for Se content and GSH-Px, CAT, and SOD activity and T-AOC value assay. The results showed that supplemental Se significantly increased Se contents and the activity of GSHPx, CAT and SOD and T-AOC value in liver, kidney and cardiac muscle of hens. With supplemental Se level increase, Se contents and the activity of GSH-Px in liver, kidney and cardiac muscle all were significantly increased. Supplemental SP had more ability than SS to increase Se content in liver and cardiac muscle, and to increase the activity of GSH-Px and CAT in liver, kidney and cardiac muscle, and the activity of SOD and T-AOC value in kidney. It was concluded that supplementation of Se can increase Se contents and the antioxidant capacity of hen tissues. Especially, advocating to supplement SP at a lower or medium Se level, 0.2 or 0.5 mg·kg^-1.

Effects of Melamine and Cyanuric Acid on Renal Injury and Its Mechanism in Mice

CHEN Xi;YUAN Hui;XIE Zhi-hui;GUO Cheng-zhi;ZHU Li;DENG Si-jun;LU Yin;WEI Qiang;YI Jin-e

2010, 41(12): 1614-1621. doi:

Abstract ( 648 )

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The aim of the study was to investigate the effects of melamine and cyanuric acid on renal injury. 120 male SD mice were randomly divided into six groups : control group, melamine group (50 mg·kg^-1bw), cyanuric acid group (50 mg·kg^-1bw), melamine + cyanuric acid group (equal amount of melamine and cyanuric acid mixed) at the dose of 50,10 and 2 mg·kg^-1bw, respectively. Mice were administrated drugs for 30 days at 48 h intervals, and the follow researches were taken: determination of the level of blood urea nitrogen (BUN) and creatinine (CRE) in serum, activity of superoxide dismutase (SOD) and content of malondialdehyde (MDA) in kidney homogenization, observation of renal tissue morphological; apoptosis of renal cell by TUNEL, expression of Bcl-2, Bax and Cyt-c gene by RT-PCR, and expression of the related proteins by immunohistochemistry. The results showed that the mixture of melamine and cyanuric acid could significantly induce renal injury in a dose- dependent manner. Such as the level of BUN and CRE in serum were increased, the activity of SOD was decresed and the content of MDA was enhanced, and renal tubular injury were obviously observed, apoptotic rate of renal cell in melamine + cyanuric acid group at the medium and high doseage was markedly higher than that in the control group (P<0.01). At the same time, some changes of genes expression were also found, a markedly increase in the expression of Bax and Cyt-c gene and a markedly decrease in the expression of Bcl-2 gene were observed. Renal injury melamine- and cyanuric acid-induced caused by lipid peroxidation and mitochondriamediated apoptotsis.

Study on Nutrition Condition of E.tenella Cultivation in Cecum Epithelial Cells from Chicken Embryo in Vitro

GU Shao-peng;HOU Jin-huan;CHEN Zhao-ying;LI Bao-jun;ZHENG Ming-xue

2010, 41(12): 1622-1628. doi:

Abstract ( 619 )

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To investigate the nutrition condition of E. tenella cultivation in cecum epithelial cells from chicken embryo in vitro and to observe the development process of E. tenella. E. tenella were cultivated in cecum epithelial cells of chicken embryo in vitro. The basal medium, the nutrient content and the serum concentration for sporozoite inoculation were optimized, and the development process of E.tenella under optimized nutrition condition was observed. The infection rate of E. tenella cutivated with MEM199 at 24, 48 and 72 h， respectively were obviously higher than that with DMEM(P<0.05 or P<0.01). The infection rate of different stages at 24, 48 and 72 h were 25.33%, 12.67% and 12.33%， respectively when folacin, VB6 and VB1 were added together with the same supplementation of 6 mg·L^-1，which were remarkably higher（P<0.05）than that of other groups. More mature schizonts were observed when glucose and trypsin were added at 3 000 and 0.40 mg·L^-1， respectively in the MEM199 medium. The later E. tenella development was promoted when medium was changed at 4, 48 and 96 h. Meanwhile, the serum concentration in medium was 2.5% at 0, 4 and 48 h, and then was 1% at 96 h. Under the optimized conditions, The E.tenella infection rate at different stages of 4, 24, 48, 72, 96 and 120 h after inoculation were 42%, 28%, 20%, 15%, 13.67% and 13.67%， respectively. In addition, the E. tenella could complete the whole life cycle in cecum epithelial cells of chicken embryo and develop into oocysts. The optimized nutrition condition for E. tenella cultivation in vitro in cecum epithelial cells from chicken embryo was obtained.

Therapeutics Research of Phacoemulsification in Canine Cataract Models

XIA Nan;YAO Hua;WANG Xue-qiao;YUN Shui;ZHANG Zhong-wen

2010, 41(12): 1629-1635. doi:

Abstract ( 614 )

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Canine cataract is a common disease in veterinary clinic, and oxidative damage is an important pathogenesis of the cases. Nowadays more effective therapy is phacoemulsification. This study was conducted to establish canine cataract model by H₂O₂ and explore therapeutics. Fifteen adult dogs were selected, 13 for modeling and 2 for control. Phacoemulsification was used to cure the left eyes in model dogs. We assessed the method about the model establishment and the curative effect of phacoemulsification by lens opacity ratio, intraocular pressure and Schirmer tear test. We investigated the morphology characteristics of lens by light microscope and transmission electron microscope. Results showed that 13 canine cataract models have established by detecting lens and mitochondrion vacuolization. After phacoemulsification, the models showed the incision healing and eyesight recovering well. The index of lens opacities ratio was proved to be more sensitive marker for the canine cataract modeling. Phacoemulsification could be used to cure canine cataract cases and it would be adopt in veterinary clinic.

Construction of Recombinant Retroviral Vector with Bovine Klf4 andEstablishment of a Virus Producing Cell Strain

XIN Xiao-ling;LV Chang-rong;CHEN Dong-mei;DOU Zhong-ying

2010, 41(12): 1636-1641. doi:

Abstract ( 690 )

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In order to construct recombinant retroviral vector containing bovine Klf4 gene cDNA and get a stable virus producing cell strain, the primers with restriction enzyme sites were designed and the ORF(open reading frame) sequence of bovine Klf4 gene was amplified by RT-PCR from MDBK cells. Subsequently bovine Klf4 gene cDNA was inserted into the restriction sites of retroviral vector pMSCVneo to construt recombinant retroviral expression vector pMSCV- Klf4. The pMSCV- Klf4 was transfected into packaging cell line(PT67) by LipofectamineTM2000 to gain infectious retroviral particles which can be found under electron microscope. Then NIH 3T3 cells infected with infectious retroviral particles was used to test viral titer. The results showed that the ORF sequence was highly homologous to the published sequence(NM-001105385). RNA from the recombinant retroviral vector pMSCV- Klf4 could be packaged into infectious retroviral particles in PT67 cell line . Then we successfully got a stable virus producing cell strain whose viral titer was up to 7.16×107 CFU·mL^-1. The recombinant retroviral vector pMSCV- Klf4 and the stable virus producing cell strain were gained in this study, which would be helpful to the further work about bovine pluripotent stem cells induced by defined factors.

Studies on Subcellular Localization and Anti-viral Activity of the Mx-EGFP Fusion Protein

TIAN Zhi-quan;NI Li-gang;WU Xiao-wei;REN Li-wei;YANG Hai-yan;SUN Min;DING Ai-jun;LI Bi-chun

2010, 41(12): 1642-1648. doi:

Abstract ( 600 )

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This experiment was conducted to study the expression location of chicken Mx gene in cell and the anti-viral activity of the Mx-EGFP fusion protein. The EGFP encoding sequences were subcloned and constructed into the eukaryotic expression vector pcDNA3.0-MMx. Then, NIH-3T3 cells were transfected with the recombinant vector-pcDNA3.0/EGFP-Mx and control vector to observe the subcellular localization of EGFP-Mx. In addition，the fusion protein Mx- EGFP anti-viral activity were studied by anti-VSV virus experiment. The recombinant expression plasmid was ensured after restriction enzyme digestion.Then the recombinant plasmid was transfected into 3T3 cells.Observed under Inverted fluorescence microscope, most of the fusion protein were expressed in the cytoplasm and partly in the nucleus Attacked with VSV, the cells transfected with only plasmid pcDNA3.0/EGFP-Mx were not pathological changed. But the negative control group transfected with pcDNA3.0 of the NIH-3T3 cells were pathological changed in 48 h These results indicated that fusion protein was expressed in the cytoplasm mostly and could slow down the time for virus infection lesions.

Expression of GnRH in the Hypothalamic-Pituitary-Ovarian Axis of the Pregnant Cattle

XU Yuan-qing;WANG Jian-lin;SONG Guo-qiang;WANG Feng-ling;SHAO Bao-ping

2010, 41(12): 1649-1654. doi:

Abstract ( 692 )

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The experiment was conducted to study the expression of gonadotropin releasing hormone (GnRH) in the hypothalamic-pituitary-ovarian axis of the pregnant cattle, and to explore its function. The expression of GnRH in the hypothalamus, pituitary and ovary of the pregnant cattle and performed a detailed analysis were examined by immunohistochemical ABC and ImagePro Plus 6.0. The results showed as following: (1) GnRH immunopositive production were found in the anterior hypothalamic nucleus (AH), medial mammillary nucleus (MMN), ventromedial nucleus (VMN), arcuate nucleus (ARC) and periventricular nucleus (PVN). Sum area (S) and IOD of GnRH immunopositive production in the AH and MMN are larger in the hypothalamic nucleus (SAH > SMMN > SVMN > SARC > SPVN and IODAH > IODMMN > IODVMN > IODARC > IODPVN; P<0.001); (2) GnRHpositive cells are mainly distributed in the anterior pituitary and GnRH-positive nerve fibers were only observed in the neurohypophysis; (3) GnRH immunopositive production was found in the granulosa cells, stroma of ovary (stroma ovarii) and corpus luteum of pregnancy (corpus luteum verum) in the ovary. The results suggested that GnRH-positive neurons in AH and MMN may play a important role in the maintenance of pregnancy in cattle; in the pituitary and ovary, GnRH may have important roles in hormone homeostasis and regulation of pregnancy in cattle by the way of autocrine and paracrine.

Studies on the Pharmacokinetics of Melamine in Dairy Goat

WANGLiang;JIN Ming-wu;LIU Li-cheng;ZHAO Hong-bo;WEN Qi-nan;ZHANG Yong-gen

2010, 41(12): 1655-1658. doi:

Abstract ( 654 )

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This experiment was conducted to study the pharmacokinetics of melamine in dairy goat. A single intravenous administration of the melamine at the dose of 6.13 mg·kg^-1bw to 6 dairy goats. Blood samples were collected at different intervals after administration and concentrations of melamine were determined by HPLC method. The plasma concentrationtime data of melamine was analyzed by Pharmaceutical 3P97. The results showed that Two-compartment open model was the optimum one of melamine in the dairy goats .The main pharmacokinetic parameters were as follows :Vd was (0.03±0.01) L·kg^-1, t1/2β was (3.64±0.02) h, AUC was (41.96±0.52) μg·h·mL^-1, CL was (0.14±0.01) L·kg^-1·h^-1. The results indicated that the distribution of melamine in dairy goat was limit, and the elimination of melamine was rapid.

Cloning and Sequences Analysis of HA and NA Genes of H9N2 Avian Influenza Viruses Isolated from Tibet Autonomous Region of China

SOULANGSIZHU;ZHOU Wei;WANG Can;CHEN Huan-chun;JIN Mei-lin

2010, 41(12): 1659-1666. doi:

Abstract ( 1022 )

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To evaluate the molecular characterization and the evolutional situation of the H9N2 avian influenza viruses (AIV) spreading in Tibet, the HA and NA genes of three AIV strains isolated from different farms in 2009 were amplified by RT-PCR and completely sequenced. The homology of three Tibet isolates was analyzed and the phylogenic characterization was analyzed with the other H9N2 viruses available in GenBank database. The results showed little degree of variation among the three isolates and the homologies of HA and NA gene are up to 98%. And the three Tibet isolates were belonged to the branch of the Bj/94-like sub-branch. The nucleotide homologies of HA gene between the Bj/94 and Tibet isolates were from 88.7% to 88.8%, and the homologies of the deduced amino acids of HA protein were 93.4%-93.6%. The nucleotide homologies of NA gene between the Bj/94 and Tibet isolates were 92.7%-93.0%, and the deduced amino acids of NA protein were 93.4%-94.0%. These results indicate that the three Tibet isolates maybe evolute from the virus Bj/94. The HA gene of virus Tb/1 contains a novel potential glycosylation site at site 313 and the NA gene of virus Tb/4 lost a potential glycosylation site at site 146 because of point mutation. The mutation happened in the connecting peptide of HA protein and the receptor binding domain of NA protein as well. The mutation in HA and NA genes maybe associated with the adaption of the virus to special climate and environment of the high altitude and high altitude anoxia.

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