Acta Veterinaria et Zootechnica Sinica

Research Progress of Spermatogonial Stem Cells Transplantation Technology

LIU Ling, ZHU Hua-bin, WANG Chen, HAO Hai-sheng, ZHAO Xue-ming, FENG Rong, DU Wei-hua, QIN Tong, LIU Yan, WANG Dong

2012, 43(11): 1677-1682. doi:

Abstract ( 378 )

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The culture and transplantation of spermatogonial stem cells(SSCs) and related researches will be significant in some fields, such as male infertility, spermatogenesis mechanism research and set-up of transgenic technology. Biological characteristics of mouse SSCs had been studied clearly. The technology system of isolating, purifying and cultivating these stem cells had been built, as well as recipient preparation and transplantation technology. However, the efficiency of these technologies is not high. Reviewing all the above technologies, not only the optimum preparation time of the stem cells can be determined, but also the technique of recipient preparation and the techniques of purification, cultivation and transplantation of SSCs will be improved. And these will also provide guidance for the future study on farm animals in this field.

Advance on Biological Functions of Structural and Non-structural Proteins of Porcine Reproductive and Respiratory Syndrome Virus

ZHANG Song-lin, SHEN Zhi-qiang, LIU Lei, MA Yong-biao, LIU Ji-shan

2012, 43(11): 1683-1696. doi:

Abstract ( 368 )

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Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a threat, and causes huge economic losses in the swine industry. Commercial vaccines are still ineffective as they suffer from both PRRSV-induced immune evasion strategies and the antigenic heterogeneity of the virus. In this paper, the latest research progress of structural and nonstructural proteins of PRRSV are summarized, such as biological functions, which seems to be effective in PRSSV prevention and control, design of new vaccines.

Association of SNPs in Erythropoietin Receptor Gene with Blood Parameters

SONG Xin, CAO Sui-zhong, ZHANG Long-chao,et al

2012, 43(11): 1697-1702. doi:

Abstract ( 308 )

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The aim of present study was to investigate the effect of porcine erythropoietin receptor (EPOR) gene on blood parameters. Six blood parameters, hematocrit (HCT), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean cell volume (MCV), red blood cell count (RBC) and red blood cell distribution width (RDW), were measured in Large White × Minzhu intercross porcine population. EPOR was genotyped using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF). The associations of g.705G>T in intron 1 and g.2373C>T in intron 4 with the six blood parameters were analyzed. The result showed that the SNP at g.705G>T was not significantly associated with any trait. Two genotypes (CC and CT) of SNP at g.2373C>T were found in this population. The HCT of individuals with CT genotype was higher than that of individuals with CC genotype (P<0.05), while this SNP was not significant associated with other five traits. These results indicate that the mutation of EPOR gene can affect the HCT and this gene can be a potential candidate gene for immunity relate traits (e.g. HCT) for further study.

The Expression of Beijing-You Chicken AMPK Gene and Its Effects on the Adipogenesis in the Muscle and Adipocyte

YANG Ye, SONG Jiao, FU Rui-qi, LI Ying-ying, GOU Zhong-yong, SUI Yan-fa, ZHAO Gui-ping, WEN Jie

2012, 43(11): 1703-1709. doi:

Abstract ( 387 )

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The objective of the study was to explore the expression of adenosine monophosphateactivated protein kinase (AMPK) in the BJY chicken thigh muscle and its effects on the intramuscular fat(IMF). The BJY chickens were divided randomly into 6 treatments and 15 chickens each treatment. The chickens were fed under the same feeding standards and management condition. The chickens were slaughtered on the 1, 56, 112 days old and the expression of AMPK and lipid metabolic gene and IMF contents of thigh muscle were measured. The difference of gene expression was calculated by the method of of 2^{- ΔΔCT} value. The results showed that the expression levels of AMPKα2 and AMPKγ2 genes on the 56 days old in BJY chickens were lower than that on 1 (P>0.05) and 112 days old (P<0.05), which had the same trend with the gene expression of peroxisome proliferator activated receptor（PPAR）α and γ, Fatty Acid Translocase CD36 (FAT/CD36), Fatty acid synthase (FAS), Calciumsensing receptor (CASR). But the expression of AMPKβ1, AMPKβ2 and AMPKγ1 on the 56 days old were higher than that on 1 (P<0.05) and 112 days old (P>0.05), which had inverse trend with the expression of lipid metabolic genes (PPARα, PPARγ, FAT, FAS, CASR). The results of the study on cell incubation showed that the activation of AMPK by AICAR inhibited the expression of PPARα and PPARγ and reduced the lipid accumulation in preadipocyte. But inhibition of AMPK by compound C enhanced the expression of PPARα and PPARγ and promoted the lipid accumulation in preadipocyte. In conclusion, these data show that AMPK, especially AMPKβ1, AMPKβ2 and AMPKγ1, may play a negative regulation role in the process of adipogenesis.

Effects of IUGR during Late Pregnancy on the Fetal Thymus Development of Mongolia Ovine

ZHANG Yuan, GAO Feng, LIU Ying-chun, AO-LI Ge-ri-ma

2012, 43(11): 1710-1715. doi:

Abstract ( 282 )

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This study investigated the effects of IUGR (Intrauterine growth retardation) during late pregnancy on the fetal thymus and the T cell lymphocyte development of Mongolia ovine. 42 Mongolia ewes mated at a synchronized estrus were selected and among them 6 ewes were slaughtered at the beginning of the experiment and the remaining 36 animals were allocated to three different groups: restricted group1 (RG1, 0.175 MJ·kgW^-0.75·d^-1, n=14), restricted group2 (RG2, 0.33 MJ·kgW^-0.75·d^-1, n=12) and control group (CG, ad libitum, 0.67 MJ·kgW^-0.75·d^-1, n=10). At 140 d of gestation, 6 ewes in each group were slaughtered and their fetuses were removed. The results showed that the fetal weight in both RG1 and RG2 groups were significantly reduced(P<0.01) by maternal under-nutrition during late pregnancy, which led to fetal thymus weight, thymus cortical thickness, thymus DNA contents in RG1(P<0.01) and RG2(P<0.05) groups, and thymus cortical/medullary ratio, protein contents in RG1(P<0.01) were decreased compared to that in CG group. The activities of SOD(P<0.05) and the contents of T-AOC(P<0.01) of fetal thymus in RG1 group were decreased, while the activities of GSH-PX and the contents of MDA were increased compared to CG group（P<0.05）. Additionally, the CD4⁺CD8⁺ T lymphocyte subsets of fetal thymus were lower in both RG2 and RG1 groups than that in CG group(P<0.05). These results indicate that the fetal thymus and T lymphocyte are affected seriously by IUGR during late pregnancy, which may influence fetal immunity capability and postnatal health.

Molecular Cloning and Differential Expression of clock and cry1 Genes in Goat Brain and Pituitary

ZHAN Si-yuan，LUO Wan-wei，CHENG Bo，JIANG Jing，LI Li，WANG Lin-jie，ZHANG Hong-ping，WANG Yong，GONG Hua-bin，DENG Zhong-bao

2012, 43(11): 1716-1722. doi:

Abstract ( 331 )

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The objective of the present study was to investigate the differential expression of clock and cry1 genes in the brain and pituitary of postnatal goats. The samples of cerebral cortex and pituitary were collected from a total of 24 (12 males and 12 females) Nanjiang Yellow goats at 30，60，90 and 120 days of age after birth. The mRNA levels of clock and cry1 genes were detected by real-time fluorescence quantitative PCR. The partial CDS of clock and cry1 genes were obtained by RT-PCR, containing exon 1 to exon 8 in clock gene and exon 6 to exon 15 in cry1 gene, and the lengths were 1 479 and 993 bp，respectively. The nucleotide sequence of CDS shared 99%, 90%, 94% and 92% consistency with the clock gene of sheep, cattle, human and mouse, respectively. Whereas the nucleotide sequence of CDS shared 99%, 98%, 94% and 87% consistency with the cry1 gene of sheep, cattle, human and mouse. The mRNA levels of clock and cry1 genes in pituitary were significantly higher than that in brain（P<0.01）, however there was no significant difference（P>0.05） between sexes. In pituitary, the mRNA levels of clock and cry1 genes were upregulated from 30 to 60 days, and then decreased. In addition, the expression of clock gene showed no significant difference at four development stages of brain, but the cry1 gene was upregulated slightly from 30 to 120 days. These results indicate that clock and cry1 genes are highly conservative, and play important roles in the brain and pituitary in goats. The mRNAs express developmentally in a tissue-specific manner.

Genetic Diversity and Evolution Relationship of Tibet Yaks Inferred from mtDNA cytb

JI Qiu-mei, TANG Yi-ting, ZHANG Cheng-fu, CHAI Zhi-xin, ZHAO Shang-juan, XIN Jin-wei, ZHONG Jin-cheng

2012, 43(11): 1723-1732. doi:

Abstract ( 389 )

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In order to explore the classification status and genetic diversity of Tibet yak at the molecular level, the cytb gene sequences of Tibet yaks were determined; its sequence polymorphism was analyzed; and the molecular phylogenetic tree was constructed for 110 yaks from 11 groups (Jiali, Sangsang, Sangri, Gongbujiangda, Sibu, Pali, Kangbu, Jiangda, Leiwuqi, Dingqing, Baqing yak). The results showed that the size of cytochrome b gene sequences of 110 yaks are 1 140 bp. 53 haplotypes were identified, 49 haplotypes were newly discovered, 14 SNPs loci among 110 sequences were detected, nucleotide mutation types included conversion and transversion, no insertions and deletions, the main mutations was synonymous mutations, which indicated that Tibetan yaks had abundant genetic diversity. 11 species or groups of Tibet yak can be divided into 5 strains:Pali,Jiangda,Baqing,Sangri,Leiwuqi yak.

Cellular Reprogramming by in vitro Transcribed mRNA Cocktail of Oct4, Sox2 and SV40 T-antigen

PAN Chuan-ying, CHEN Hong, BISHOP E.Colin

2012, 43(11): 1733-1739. doi:

Abstract ( 322 )

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The aim of this study was to reprogram fibroblasts cell by in vitro transcribed mRNA cocktail of Oct4, Sox2 and SV40 T-antigen, and provide a safe, nonintegrating strategy for somatic cell reprogramming. The recombinated mRNA transcription vectors including the above three induced facotrs genes were constructed and transcripted, respectively, then 5′UTR and 3′ UTR of β-globin gene were used for stabilizing in vitro transcribed mRNA following in vitro capping and poly(A) tailing. After mRNA transfection of 293 and IMR90 cells, immunocytochemistry, immunoflurescence and Real-Time PCR methods were used to detect gene expression, protein location and endogenous gene expression related to pluripotence. The results showed that target gene expressions could be detected in 293 and IMR90 cells and all expressed protein were localized properly in the nucleus. The specific expression of Oct4 and Nanog were increased in transfection cells. Endogenous Nanog expression was induced by mRNA cock-tail transfection. These findings indicate that mRNA of Oct4, Sox2 and SV40 T in vitro can coorperate to initiate cellular reprogramming.

Effect of Glutamine on Growth Performance, Meat Quality and Expression Level of H-FABP of AA Broilers

ZHONG Jin-feng, HU Yong-ling, YANG Yong-sheng, LUO Jia-jie

2012, 43(11): 1740-1746. doi:

Abstract ( 398 )

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The experiment was conducted to determine the effect of exogenous glutamine on the growth performance, carcass traits, meat quality, the content of inosine monophosphate and the expression level of H-FABP in breast and thigh muscle of AA male broilers. A total of 240 AA male broilers were randomly divided into 4 groups (A, B, C and D) which were fed with basic diet supplemented with Glu at 0, 0.2%, 0.4% and 0.8% level, respectively, the average daily gain(ADG), average daily feed intake(ADFI) and feed/gain ratio(F/G) were determined at 21^st and 42^nd day, and the experiment lasted for 42 days. The shear force, drip loss and chromal(L^*, a^*and b^*) were determined and the dressing percentage, eviscerated yield, half eviscerated yield, percentage of breast and thigh muscle were calculated at 42^nd day. The content of inosine monophosphate was detected by HPLC, and RT-PCR was used to determine the expression level of H-FABP mRNA in breast and thigh muscle. The average daily gain, average daily intake, the content of inosine monophosphate and the expression level of H-FABP mRNA of broilers were improved significantly in group B(P<0.05). The eviscerated yield, percentage of thigh muscle, the breadth of subcutaneous fat, shear force of breast were increased and percentage of abdominal fat were decreased significantly in group C (P<0.05). The shear force of thigh, the L^* and a^*value of breast and thigh muscle were enhanced significantly in group D (P<0.05). The result indicate that Glu supplement at 0.2%, 0.4% and 0.8% levels could improve the growth performance, carcass traits and meat quality of broilers effectively, and the contents of inosine monophosphate and expression level of H-FABP mRNA in breast and thigh muscle could be increased to some extent. The 0.2% level is the recommended dosage in the production.

Effect of Dietary Riboflavin（Vitamin B₂）Levels on Growth Performance, Antioxidant Capacity and Hormone Secretion of Peking Ducks from 1 to 21 Days of Age

TANG Jing, XIE Ming, HOU Shui-sheng, HUANG Wei, WEN Zhi-guo, ZHU Yong-wen

2012, 43(11): 1747-1753. doi:

Abstract ( 354 )

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This experiment was conducted to evaluate the effects of dietary riboflavin levels on growth performance, antioxidant capacity and hormone secretion of Peking ducks from 1 to 21 days of age. Using a one-factor completely randomize design, a total of 512 one-day-old male Peking ducks were randomly divided into 8 groups with 8 replicates in each group and 8 ducks in each replicate. Eight groups were fed eight dietary riboflavin levels (1.69, 2.39, 3.09, 3.79, 4.49, 5.19, 8.69, 15.69 mg·kg^-1) diet, respectively. The growth performance were determined. The results showed that dietary riboflavin levels significant affected average daily weight gain, average daily feed intake and feed/gain (P<0.05); dietary riboflavin levels significant affected MDA and SOD in plasma and liver(P<0.05); dietary riboflavin levels significant affected plasma T₃ and GH content (P<0.05). Riboflavin was essential for growth of Peking duck from 1 to 21 days of age.

Comparative Proteomics Analysis of Changes of the Milk Protein during Thermal Treatment

ZANG Chang-jiang, WANG Jia-qi, YANG Yong-xin, BU Deng-pan, YANG Jin-hui, YUAN Ting-jie, ZHOU Ling-yun, SUN Peng, LI Fa-di

2012, 43(11): 1754-1759. doi:

Abstract ( 306 )

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To investigate the changes of milk protein after treated with 75 ℃/15 s, 125 ℃/4 s, 135 ℃/4 s and 145 ℃/4 s, two-dimensional gel electrophoresis was used to separate proteins samples and mass spectrometry was used to identify differential protein spots. These results showed that four protein spots were down-regulated after treatment with 135 ℃/4 s and 145 ℃/4 s compared with raw milk and milk treated with 75 ℃/15 s in which protein profiles had no difference. These protein spots were identified as α-lactoalbumin, beta-casein variant, κ-casein and Ig γ chain. Content of IgG from raw milk and thermal treated milk was detected using ELISA method and in consistent with data of two-dimensional gel electrophoresis coupled with mass spectrometry. These finding indicated that milk protein contents were not affected by treatment with 75 ℃/15 s and affected by treatment with 135 ℃/4 s and 145 ℃/4 s.

Development of Duplex PCR Assay for Mycoplasma hypopneumoniae and Porcine Circovirus 2 Detection and Its Application

HU Jun-yong, ZHANG Qian, WANG Dan-dan, TU Zhi-qin, HU Rui-ming, TANG Xi-biao, WU Bin

2012, 43(11): 1760-1766. doi:

Abstract ( 341 )

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A PCV2/Mhp duplex PCR detection assay was established in this paper. Specificity was tested by amplifying the genome DNA of PRV, PPV, Salmonella, E. coli, S. suis, Hps, Pm, B. bronchiseptica and none detectable amplification was found. Sensitivity of the duplex PCR assay proved that the minimal detectable template concentration were 130 and 180 fg·mL^-1 for PCV2 and Mhp respectively. By applying both single and duplex PCR assays in PRDC cases in Hubei Province, 174 samples were tested, and the coincident rate between single and duplex PCR are 100% and 98.28% for PCV2 and Mhp respectively. By applying the duplex PCR assay, the infection dynamic of both PCV2 and Mhp were investigated in different herds, and the results suggested that the infection of both PCV2 and Mhp started in suckling stage with various proportions, and most infection took place in nursery and growing stages. Actually, different herds showed different infection dymanic. In this study, we established a duplex PCR assay and showed excellent specificity and sensitivity, which provides support for clinical diagnosis and epidemic investigation.

Epidemiological Investigation and Antigen Phylogeny Analyses in Guangdong for Porcine Transfusion Transmitted Virus

CHEN Yi-wen, LI Zhong-sheng, BAI Ai-quan, CHEN Shan-zhen, LI Qi-chang, YAO Huo-chun, WANG Gui-ping

2012, 43(11): 1767-1772. doi:

Abstract ( 294 )

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The objective of this study was to investigate the prevalence of porcine transfusion transmitted virus (TTV) species (TTSuV1 and TTSuV2). The study was conducted in 7 different intensive pig farms of Guangdong province in China. A total of 159 swine sera were collected, and then the virus were tested using Nest-PCR. The results demonstrated that the TTSuV1 and TTSuV2 infection rates were 54.72% and 34.22% respectively, and the co-infection rate was 29.56%. We designed PCR primers based upon the conserved region of the two viruses for amplifying the two virogenes. By blast analysis with our cloned TTsuVs sequences in NCBI database, we found that our isolated TTsuV1 ORF1and TTsuV2 ORF1, have high homologousto to those known TTSuVs sequences,72%-75% and 88%-97% respectively; while have big gaps with the TTVs which infecting other species like feils, canis, tupaia, tamarin, douroucouli and homo in the nucleotide sequence. To sum up, TTSuVs had been widely distributed in Guangdong’s pig populations. This study laid the foundation for further research of the prevalence situation, serologic diagnostic methods and to prepare for safe and valid vaccine.

Genomic Characterization and Evolvement Analysis of a Porcine Reproductive and Respiratory Syndrome Virus Isolate with Deletion in GP5

FU Xiang-jing, WANG Dan-yang, WANG Xing-long, ZHANG Wei-dong, SONG Xiang-jun, LIU Yang-kun, TANG Wen-ya, MU Guo-hui, LIU Hai-jin, HE Sheng-fang, DU En-qi, YANG Zeng-qi

2012, 43(11): 1773-1779. doi:

Abstract ( 441 )

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In order to understand the variation of porcine reproductive and respiratory syndrome virus (PRRSV) in Shaanxi province, the virus was detected from swine clinical samples, NSP2 and ORF5 genes were amplified, cloned and sequenced for analyzing the genetic variation of PRRSV. The result showed that the strain belonged to the North American genotype, named HZ1007. It had discontinuous 30 amino acids deletion in the aa position of 481 and 533-561 of NSP2, which was the same deletions with the highly pathogenic PRRSV. Moreover, the ORF5 gene of HZ1007 strain contained a continuous 11 amino acids deletion in the aa positon of 85-95. PRRSV HZ1007 was highly homologous to HUN4 and SY0608 strains. Phylogenetic tree analysis revealed that HZ1007 had closer relationship with the highly pathogenic PRRSV than classical strains, it may be a new variant of the highly pathogenic strain. The athogenic force and biological characteristics needed further research and analysis.

Development of an Indirect ELISA of Infectious Bronchitis Virus by Using Tandem Epitopes of S1

SUN Luo-mei, YI Lin, ZOU Nian-li, LIU Ping, HUANG Yong

2012, 43(11): 1780-1787. doi:

Abstract ( 317 )

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The aim of this study was to establish an indirect ELISA for detection of antibodies against avian infectious bronchitis (IB). The published amino acid sequences of S1 gene of avian Infectious bronchitis virus (IBV) strain ZY3 were analyzed by bioinformatics software, and four dominant epitopes named as F1, F2, F3 and F4 were selected and ligated together as a chimeric gene F. Bioinformatic analysis showed that this protein if highly antigenic and flexible. Then the chimeric gene was then inserted into expression vector pET-32a（+）for the expression of target gene and a 42 kD recombinant protein was obtained. The result of westernblot showed that the chimeric protein could react specifically with anti-IBV positive serum. An indirect ELISA was then developed using purified protein as coating antigen.175 sera samples were examined by this ELISA and commercial kit, the results showed that the positive coincidence rate could reached 90.2%, negative coincidence rate could reached 85.7%, and the total coincidence rate reached 89.7%. The results indicated that the indirect ELISA was sensitive and specific, and no cross-reaction with positive sera of other chicken diseases. The indirect ELISA for detection of chicken antibodies against Infectious bronchitis were successfully developed.

Protective Effect Analysis of Inactivated Vaccine of Subgroup B Avian Leukosis Viruses in Chicken Breeder Farms

LI Xue, LI Wei-hua, ZHAO Peng, CUI Zhi-zhong, WANG Xin, DONG Xuan

2012, 43(11): 1788-1794. doi:

Abstract ( 387 )

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To develop a inactivated vaccine against subgroup B avian leukosis viruses (ALV-B) and determine if vaccination of chicken breeders could protect young chicks from ALV-B horizontal infection at early stage and accelerate eradication progress. Chicken embryo fibroblast (CEF) cells were inoculated with SDAU09C2 strain of ALV-B and ALV-CEF was inactivated for preparation of oil-adjuvant vaccine. Eggs were collected from un-vaccinated and 9 vaccinated great parent female chickens for incubation. One-day-old chicks were bled for testing their maternal antibodies to ALV-A/B and then inoculated with ALV-B. Viremia and cloaca p27 detection dynamics were tested and compared between chick groups with or without maternal antibody to ALV. In 3 weeks after the 3rd vaccination with the inactivated vaccine, all 9 vaccinated breeders developed high antibody titers against ALV-A/B with ELISA read values of 1.69-1.89 (the positive base line was 0.4) and kept at the high titers for at least another 4 weeks. Maternal antibody was detected in 70% (12/17) of chicks from breeders with high antibody titers to ALV-A/B. Only 4 of 12 chickens with maternal antibodies developed temporary viremia and no viremia was detected in the left 8 maternal antibody positive chickens during the whole 14 weeks after inoculation of ALV-B at 1 day of age. But the persistant viremia was detected in 2-8 weeks in all 9 maternal antibody negative chickens and the viremia persisted in the whole tested period of 14 weeks after inoculation of ALV-B. The inactivated ALV-B vaccine could induce high titer antibody reaction to ALV-B, it could provide matarnal antibodies to 1-day-old chickens and protect chickens from early infection of ALV-B.

Development and Primary Application of an Immunochromatographic Colloidal Gold Test Strip for Rabbit Hemorrhagic Disease Virus

CAI Shao-ping, WANG Fang, JIA Hua-min, ZHANG Hai-bin, LI Chao-mei, FAN Zhi-yu, HU Bo, XIONG Fu-qiang, WEI Hou-Jun

2012, 43(11): 1795-1801. doi:

Abstract ( 315 )

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To establish a rapid, simple assay for rabbit hemorrhagic disease virus (RHDV), purified polyclone antibody against RHDV was labeled with colloidal gold particle. Monoclonal antibody (McAb) A3C against RHDV was prepared with recombinant RHDV VP60 protein expressed by baculovirus expression system. The detection zone and quality control zone of the nitrocellulose membrane was made by A3C and goat anti-rabbit IgG. The immune colloidal gold test strip was developed by optimizing the experiment conditions. The sensitive test showed that the test strip can detect the RHDV which erythrocyte agglutination test (HA) titer was 1∶10, which is negative result if the RHDV was detected by HA. It means that the test strip is more sensitive than erythrocyte agglutination test. The crossing test showed that the test strip could not react with other rabbit common pathogens. 127 clinical liver samples of rabbit were detected by the test strip, while a parallel experiment was conducted using HA, and the positive consistent rate of both was 100%. All of the results showed that the test strip is a rapid, sensitive and specific method for detecting RHDV infection. The method has provided a effective tool for diagnosing RHDV, particularly in clinical diagnosis.

Construction of a gE and TK Double Genes Deletion Mutant of Pseudorabies Virus by Cre-loxP System

LIANG Yuan-yan，HU Yan-fen，ZHANG Xiao-rong，CHEN Su-juan，PENG Da-xin，LIU Xiu-fan

2012, 43(11): 1802-1809. doi:

Abstract ( 327 )

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In order to construct a gE and TK double genes deletion mutant without a reporter gene, the recombinant virus rPRV-gE^-/GFP with loxP sites was first constructed using PRV JSZ as a parental strain and an enhanced green fluorescent protein (EGFP) gene as a reporter gene. The recombinant virus rPRV-gE^- was obtained by treating the genomic DNA of rPRV-gE^-/GFP with Cre recombinase and transfecting PK15 cells. Then, the recombinant virus rPRV-gE^-/TK^- was constructed using rPRV-gE^- as a parental strain. The results of fluorescence detection and PCR amplification showed that a gE gene deletion mutant and a gE/TK double genes deletion mutant were constructed successfully. The characteristics of the viruses revealed that rPRV-gE^- had a similar growth curve in PK15 cells when compared with its wild type strain, but rPRV-gE^-/TK^-had a relatively slower growth speed. The mouse LD₅₀ of rPRV-gE^-/TK^- was more than 1×10⁵ TCID₅₀, which was significantly higher than that of rPRV-gE^- and wild type strain. The mice immunized with the rPRV-gE^-/TK^- provided an 80% protection against wild type virus challenge. These results indicated that a multiple gene deletion vaccine against PRV can be constructed by repeat use of Cre/loxP system, and it will provide a platform for the development of the recombinant attenuated PRV vaccine and its vectored vaccine.

Deep Sequenceing-based Early Transcriptome Analysis of the RAW264.7 Cell with the Brucella Infection

LIU Qian-hong, HAN Wen-yu

2012, 43(11): 1810-1817. doi:

Abstract ( 306 )

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This experiment was designed to screen the genes of host involved Brucella intracellular infection and lay a foundation for elaborating the pathogenic mechanism. The murine macrophages were infected with Brucella melitensis 16M strains, and then the differently expressed genes of macrophages were screened with the digital gene expression profiling technology. The genes differently expressed were verified with the quantitative real-time PCR. Then the genes were analyzed with the GO Term and KEGG to screen the signals significantly enriched. There were 3 576 genes expressed significantly difference 4 hours post infection, approximately 58% genes were up-regulated. NOD-like receptor signaling pathway, lysosome pathway, Fc gamma R-mediated phagocytosis, p53 signaling pathway, apoptosis pathway and protein processing in the endoplasmic reticulum pathway were enriched. Transcriptomics profile of murine macrophages infected with Brucella melitensis 16M strain was successfully analyzed.

Effects of Adding Ipriflavone to Low-calcium Diet on Microstructure and Histomorphometry of Bone in Cage Layers

LV Wen-ting, MA Li-qin, YANG Yong-hong, WANG Peng, LI Kai

2012, 43(11): 1818-1824. doi:

Abstract ( 307 )

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This experiment was conducted to study the effects of adding Ipriflavone to low-calcium diet on microstructure and histomorphometry of bone in hyline brown laying hens. Two hundred 24-week-old hyline brown laying hens were randomly divided into four groups and fed on diets as follows: control group (containing calcium 3.55% in basic diet), low calcium group (containing calcium 1.27% in low-calcium diet), test groupⅠ(adding 8 mg·kg^－1 Ipriflavone to low-calcium diet) and test groupⅡ(adding 20 mg·kg^－1 Ipriflavone to low-calcium diet) for 60 days. At the end of experiment, 10 hens in each group were killed. Then, demineralized bone sections of the left middle tibias were made for observing the changes of bone microstructure by using OLYMPUS BX41 metalloscope, and some static parameters of bone histomorphometry were measured by Image-Pro Plus 6.0. Tibia microstructure was observed. Compared with control group, cortical bone of low calcium group had many resorption cavities, there were a large number of osteoclasts in the resorption cavities, and osteoclasts increased in the surface of medullary bone trabeculas, the effect of bone resorption was more than that of bone formation and led to osteoporosis. Cortical bone of test groupⅠ had many resorption cavities, there were more osteoclasts in the resorption cavities and the surface of medullary bone trabeculas, the effect of bone resorption was not inhibited. There were a large number of osteogenic cells and osteoblasts in the outer surface of cortical bone in test groupⅡ, the effect of bone formation was increased. And, Static parameters of bone histomorphometry were measured. Compared with low calcium group, Tb.Ar and %Tb.Ar in test group Ⅰ and Ⅱ were very significantly increased (P<0.01). Tb.Pm and Tb.N were higher in test group Ⅰ and Ⅱ than in low calcium group, but the differences were not significant (P>0.05). In test group Ⅱ Tb.Sp was significantly decreased (P<0.05) and Tb.Th was significantly increased (P<0.05). The results showed that adding 20 mg·kg^－1 Ipriflavone to low-calcium diet strengthened bone reconstruction, improved bone metabolism and relieved the occurrence of osteoporosis.

Detection of SNPs of Bone Morphogenetic Protein 15 Gene Exon1 and Its Association with Egg Production Traits in Female Line of Shaobo Chicken

LI Chun-miao, LI Shou-feng, ZHAO Zhen-hua, HUANG Hua-yun, XUE Long-gang

2012, 43(11): 1825-1832. doi:

Abstract ( 350 )

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The objective of the present study were to elucidate the relationship between polymorphisms of bone morphogenetic protein 15 (BMP15) gene and egg production traits in female line of Shaobo chicken and to provide a scientific basis for marker-assisted selection for reproductive traits of chicken. Polymorphisms of exon1 of BMP15 gene were detected in 261 female line of Shaobo chicken by PCR-RFLP. The relationship between polymorphisms of BMP15 gene and egg production traits in female line of Shaobo chicken was analyzed by least squares linear model. Three polymorphic sites C397T, A474G and C594T were found in exon1 of BMP15, and C397T mutation resulted in an amino acid change of leucine→phenylalanine. Three genotypes were found at all three mutation sites in 261 female line of Shaobo chicken by RFLP. Chi-square test showed that Shaobo chickens were in Hardy-Weinberg equilibrium. The relationship between three site polymorphisms of BMP15 gene and egg production traits in Shaobo chickens was analyzed by least squares linear model. The result showed that three genotypes (CC, CT and TT) were found at C397T mutation locus, and individuals with TT genotype had significant younger age at first egg than that for CT (P<0.05), TT genotype birds had also significant more the total number of eggs at the age of 300 days than that for CT (P<0.05). Three genotypes (AA, AG and GG) were found at A474G mutation site, and there was no significant effect on egg production traits at the locus. Three genotypes (CC, CT and TT) were found at C594T mutation locus, and CC genotype had significant younger age at first egg than that for CT and TT (P<0.05). The combined genotype TTAATT also had significant genetic effects on age at first egg (AFE), weight at first egg(WFE), body weight at first egg(BWFE) and the total number of eggs with 300 age(EN)（P<0.05）. The combined genotype TTAATT was the most favorable genotype. The result indicate that combined genotype TTAATT of BMP15 gene is a potential DNA marker for improving egg production traits in female line of Shaobo chicken.

The Study on Dietary Nitrogen Efficiency and Its Influential Factors for Milk Protein Synthesis in Lactating Cows

AI Jin-tao, SU Peng-cheng, LIN Xue-yan, WANG Yun, LIU Gui-mei, WANG Zhong-hua

2012, 43(11): 1833-1840. doi:

Abstract ( 311 )

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The aim of the study was to determine the dietary nitrogen conversion efficiency of dietary nitrogen, and analyze its influential factors and provide the basis for improving the dietary nitrogen conversion efficiency in lactating cows. Dietary nitrogen conversion efficiencies were measured in 18 lactating Holstein herds distributed in 7 farms. The factors influencing the dietary nitrogen conversion efficiencies were identified by principal component analysis (PCA). The results showed that the average daily milk yield of the 18 herds ranged from 6.00 to 28.40 kg·d^-1 and dietary CP (crude protein) ranged from 11.47% to 18.34%. The nitrogen balance analysis results indicated that CP nitrogen intake ranged from 185.72 to 653.00 g per cow per day, CP in feces comprised 25.58%-42.73% of dietary CP, that in urine comprised 25.04%-74.65% of dietary CP, that in the milk comprised 12.26%-27.99% of dietary CP. The true protein intake ranged from 160.06 to 582.20 g·d^-1 per cow, and 24.10%-43.18% were secreted in feces, 29.17%-75.54% were excreted in urine, and 10.77%-30.98% were used for milk production. The percentage of nitrogen in feces and urine was negatively correlated with milk yield, and that in milk was positively correlated with milk yield. Dietary CP level, DMI and milk yield were the major factors affecting dietary CP nitrogen conversion efficiency for milk protein synthesis. The multiple regression equation was MNE_CP(%) = 36.96-1.45 CP (%)-0.83 DMI （kg·d^-1）+ 1.11milk yield（kg·d^-1），R²=0.87. The equation was verified using 21 sets of data collected from published paper, and the difference between the measured and predicted efficiencies was non-significant (P>0.05). The results indicated that the crude protein conversion efficiency was 12.26%-27.99% in lactating dairy cows, true protein conversion efficiency was 10.77%-30.98%. Dietary protein conversion efficiency was mainly affected by lactation milk yield, dietary CP content and DMI, which was positively correlated with milk yield, but negatively correlated with DMI and CP content. The results will provide the basis for the application of technologies to improve the dietary nitrogen conversion efficiency for milk protein synthesis in lactating cows.

Molecular Characteristics of 3′ Untranslated Regions of Avian Leukosis Virus Subgroup J in Layer Flocks

GAO Yu-long, YUN Bing-ling, QIN Li-ting, PAN Wei, WANG Yong-qiang, GAO Hong-lei, QI Xiao-le, WANG Xiao-mei

2012, 43(11): 1841-1846. doi:

Abstract ( 324 )

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Avian leukosis virus subgroup J (ALV-J) was first isolated from meat-type chickens in 1988. No field cases of ALV-J infection and tumors in layer chickens were observed worldwide until 2004. However, layer flocks in China have experienced outbreaks of this virus in recent years. The molecular epidemiology of 3′UTR of ALV-J strains isolated from layer flocks was investigated. A total of 205 nucleotides were deleted from the 3′ untranslated region of 89.5% (17/19) of the ALV-J layer isolates. Approximately 94.7% (16/17) of the layer isolates contained a complete E element of 146-149 residues. The U3 sequences of 84.2% (16/19) of the ALV-J layer isolates displayed less than 92.5% sequence homology to the ALV-J broiler isolates, although the transcriptional regulatory elements that are typical of avian retroviruses were highly conserved. Several unique nucleotide substitutions in the U3 region and the E element of most of the ALV-J layer isolates were detected. These results suggested that the E element and U3 region in the ALV-J layer isolates have evolved rapidly and were significantly different from those of the ALV-J broiler isolates. These findings will contribute to a better understanding of the pathogenic mechanism of layer tumor diseases induced by ALV-J.

Isolation and Rapid Detection of Avian Borna Virus by a Reverse Transcription Loop-mediated Isothermal Amplification Assay for Outbreaks in Psittacine Birds

TIAN Chun-jian, WANG Hong, LUO Qiong，et al

2012, 43(11): 1847-1854. doi:

Abstract ( 396 )

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In this study an avian bornavirus（ABV） strain was isolated from sick parrots with proventricular dilatation disease(PDD). The virus grew in swine testicular (ST) cell monolayer with granulating, shrinking, rounding and falling off although classical Borna disease virus strains replicate very efficiently in cultured mammalian cells in which persistent, noncytolytic infections was readily established. Viruses were successfully isolated and demonstrated by reverse transcription-PCR analysis from the proventricular glands of parrot “glass 363” and “color” with confirmed PDD. The 351 bp product of the expected size bands of matrix protein (M) gene was cloned, the sequence and phylogenetic tree analysis showed that the isolated virus belonging to genotype ABV5. Five sets of M gene RT-LAMP primers ID1, ID6, ID19, ID30 and ID37 were designed using DNAStar and PrimerExplorer V5.0 (network) and later three set reactions showed positive color reaction with specific electrophoretic bands. The amplification curves of of real-time RT-LAMP using fluorescent indicator calcein were shown in 36 (ID30), 38 (ID37) and 49 (ID19) minutes, respectively and an amplification peak in 60 minutes. Meanwhile the three amplification curves of turbidimetric determination were shown in 56, 58 and 65 minutes. According to the results of clinical and related samples detected by RT-LAMP and RT-PCR, proventriculus were the ABV positive while large intestine, small intestine, duodenum, heart, liver, spleen, lung, kidney, gizzard, proventriculus contents , pancreas, crop, brain, and muscle were negative. The virus was positive in 10^-1 to 10^-5 of ST cell culture material by RT-LAMP detection while it was negative after 10^-3 by RT-PCR detection. There was also no RT-LAMP positive response to Newcastle disease, avian flu, bursal disease, leukemia subgroup J, encephalomyelitis. The findings provide new technological tools for the PDD control of parrots and BDV public health research.

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