Acta Veterinaria et Zootechnica Sinica

Dynamic Expression of Cdc25B Gene in Early Chicken Embryo

ZHANG Hui;QIN Jun-hui;LIU Jin-xiong;FENG Ya-mei;CHEN Qiu-sheng

2011, 42(2): 157-162. doi:

Abstract ( 708 )

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This experiment was conducted to study the spatio-temporal expression patterns of the Cdc25B gene in early chicken embryos. The Cdc25B gene fragment was gained by molecular cloning. RNA probes of the Cdc25B gene were produced by in vitro transcription and Cdc25B transcripts were detected by whole mount in situ hybridization in the various developmental stages of the early embryo. The results showed that mRNA of Cdc25B was confined to the central nervous system (CNS), somites and tip of limb bud. The hybridization signal was weak in the caudal neural plate. In early stages, distribution of Cdc25B mRNA was asymmetrical in Hensen's node. As embryonic development progressed, the hybridization signal of Cdc25B in the CNS and somites became progressively stronger. Cdc25B transcripts displayed a ventro-dorsal decreasing gradient in the closing neural tube However, the notochord was negative in the various development stages The results suggest that chicken Cdc25B expression patterns support different specific roles of this regulator in development of the CNS, somites and tip of limb bud in early and later stages. These different roles depend on concentration of Cdc25B. In addition, asymmetrical development of inner organs was regulated by Cdc25B and other genes. The mechanisms which Cdc25B regulates CNS development may act through involvement of Cdc25B in progress of the cell cycle, and even in regulation of cell proliferation and differentiation events.

Pedigree Verification of Holstein Cows and Analysis of Factors Affecting Incorrect Paternity Based on Microsatellite Markers

CHU Qin;ZHANG Yi;SUN Dong-xiao;YU Ying;WANG Ya-chun;ZHANG Yuan

2011, 42(2): 163-168. doi:

Abstract ( 995 )

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To verify the pedigree data of Chinese Holstein cows,17 microsatellites on BTA3 were genotyped in 15 Chinese Holstein sires and their 2 220 daughters, which came from 21 herds in Beijing. The paternity with conflicts between the putative sire and daughter in at least 3 markers was considered to be erroneous. The paternity error rate was 16.62% in the Chinese Holstein population. Factors affecting incorrect paternity were further analyzed and results showed that the paternity error rate varied extremely significantly among herds. Since the daughters of sires were uneven distributed in different farms, the effect of sire was also significant. These findings indicate that it’s urgent to carry out paternity testing in the Chinese Holstein population.

Simulation Study on Paternity Identification in Dairy Cattle with Microsatelliteand SNP Markers

ZHOU Lei;CHU Qin;LIU Lin;LIU Jian-feng;WANG Ya-chun;ZHANG Yuan

2011, 42(2): 169-176. doi:

Abstract ( 1082 )

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The objective of this study was to compare the efficiency of microsatellites and single nucleotide polymorphism (SNP) markers in paternity identification Microsatellites or SNP genotypes of 10 000 pseudo-parent-child relationship individuals were randomly simulated, exclusion approach was employed in paternity identification, and this procedure was repeated 100 times. The results showed that exclusion probability was mostly influenced by the polymorphism of markers. To achieve the same exclusion probability, the needed number of low polymorphic markers was greatly higher than that of high polymorphic markers. When the genotypes of the actual mother were available, the efficiency of paternity inference could be greatly improved. No matter to high or low polymorphic markers, more SNPs were required than microsatellites in each inferential scenario, to achieve the same exclusion probability. When the average heterozygosity of SNPs and microsatellites were 0.484 and 0.875, the needed number of SNPs was 5～6 times more than that of microsatellites. When the average heterozygosity of SNPs and microsatellites were 0.363 and 0.566, the needed number of SNPs was about 2 times more than that of microsatellites. When at least 2 conflicts were required in inference, 0.45 times more markers were necessary, to achieve the same exclusion probability (> 0.99), comparing to that required at least 1 conflict, for both two kinds of markers. With the dropping of the genotyping cost, and other characteristics of SNPs, such as lower genotyping error rate, higher repeatability and amenable to high-throughput automated analysis, it is very promising that SNPs would replace microsatellites for dairy paternity identification in the future.

Preparation of Goat PTHrP Polyclonal Antibody and Analysis of PTHrPExpression Map in Tissues

ZHENG Hui-ling;YANG Zhen-yu;ZHU Zhen-zhen;AN Jun-hui;XING Rui-fang

2011, 42(2): 177-181. doi:

Abstract ( 703 )

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To further understand the biological function of goat PTHrP gene, the PTHrP polyclonal antibody was prepared and the tissue expression profile of PTHrP was analyzed in this study. Prokaryotic expression vector was constructed and expressed in E-coli BL21(DE3) host cells. Fusion protein was purified and used to immunize rabbit. Western blot was performed to assay the tissue expression profile. Restriction enzyme mapping and sequencing showed that prokaryotic expression vector was constructed successfully. SDS-PAGE showed that fusion protein (about 36.5 ku) was purified. The anti-PTHrP polyclonal antibody showed high sensitivity(1︰51 200) and specificity. The PTHrP protein was detected in mammary gland, kidney, pituitary, brain, heart, liver, bone, muscle and spleen.The result indicated that goat anti-PTHrP polyclonal antibody was obtained. The PTHrP expressed widely in goat tissues.

Study on Induction, in vivo Pick-up and in vitro Development of PrepubertalLambs Oocytes

HU Peng-fei;ZHANG Gui-xue;YU Guo-qing;WU Zeng-hua;LI Xiang-chen;GUAN Wei-jun

2011, 42(2): 182-189. doi:

Abstract ( 674 )

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This experiment was conducted to study the effect of Polyvinyl pyrrolidone （PVP) on superovulation of prepubertal lambs, and the effects of factors such as breed, age, hormone dose and frequency of ovum pick-up on number of oocytes obtained were also studied. One hundred and twenty 4.12 weekold Poll Dorset and Suffolk lambs were assigned to three FSH treatments with forty lambs in each group. 120 IU FSH was diluted by PVP with percentage 0, 15% and 30%, respectively. Meanwhile, 30% PVP treatment was subgrouped based on breed, age, hormone dose and frequency of ovum pickup, superovulation and ovum pick-up were taken in each group. In addition, in vitro fertilization and embryo transplantation of lamb oocytes were conducted compared with oocytes from slaughter house. The results showed that： 1) There were no significant differences between 30% PVP group and routine method （P>0.05), whereas 15% PVP group was lowest in each index （P<0.05); 2) There were no significant differences between Poll Dorset and Suffolk lamb in number of follicles picked per lamb and available oocytes gained per lamb （P>0.05), number of oocytes picked-up from 4, 6 and 8 week-old lambs were higher than that of 12 week-old lambs （P<0.05), when 40 IU FSH was injected, number of oocytes picked-up was lowest （P<0.05), there were no significant differences between 120 and 240 IU groups （P>0.05), efficiency of superovulation decreased significantly in second time ovum pick-up （P<0.05); 3) In vitro maturation rate, cleavage rate, morula rate and blastula rate of oocytes from lambs were lower than that of oocytes from slaughter house （P<0.05), but there was no difference in fertilization rate （P>0.05); 4) 95 and 140 blastulas from lambs and slaughter house were cryopreserved, survival rate after thawed were 95.8% and 94.3%, respectively, there were no significant differences in pregnancy rate after transplantation （P>0.05), 2 and 7 lambs were born, respectively. These results indicated that routine superovulation could be substituted by Polyvinyl pyrrolidone mediated superovulation in lambs. Number of oocytes picked-up and in vitro developmental capacity were affected by age, hormone dose and frequency of ovum pick-up, max number of available oocytes were gained when 4 week-old Poll Dorset lambs were superovulated by 120 IU FSH diluted by 30%PVP, offspring could be produced by ovum pick-up from prepubertal lambs, indicating that ovum pick-up oocytes have the same developmental capacity as oocytes from slaughter house.

Molecular Cloning and Analysis of Duck Follistatin Gene CDS and Its Expression in Escherichia coli

LIU He-he;JIN Hai-bo;WANG Ji-wen;CAO Wei;HAN Chun-chun;LI Liang;XU Feng

2011, 42(2): 190-195. doi:

Abstract ( 959 )

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In order to gain the duck FST exogenous protein and to elucidate about its role in muscle development and reproduction, the total RNA was extracted from adult female duck and the FST coding domain sequence was cloned using RT-PCR method. The duck FST mature peptide cDNA was inserted into pET-32a (+) vector, and transformed into Escherichia coli Rosetta, and then it was induced by IPTG, after that, the recombinant expression products were detected by SDS-PAGE and confirmed by Western blotting analysis. The results of sequence analysis showed that the full duck FST coding domain sequence was 1 032 bp, encoding 343 AA, and the deduced CDS amino acid sequence contains a 28 AA putative signal peptide. The FST amino acid sequence of duck was composed of four domains, including the N-domain, Domain Ⅰ, Domain Ⅱ and Domain Ⅲ. The follistatin Domain Ⅰ in FST was conservative between the duck and mouse, whereas there were many variations between DomainⅡand Domain Ⅲ. The results of SDS-PAGE showed that the molecule weight of induced protein was about 52 ku and display a positive reaction by Western blotting analysis. These results indicated that the three follistatin domains in duck have different roles, and it can be concluded that Domain Ⅰ is mainly related to muscle development, and the Domain Ⅱ and Domain Ⅲ are important for the animal reproduction. The duck FST protein has been gained successfully and these results will be benefit for further researching the effects of FST on promoting muscle growth and egg production of duck in the future.

Effects of Dietary Structural and Nonstructural Carbohydrate Ratio on RuminalFermentation for Sheep

WU Qiu-jue;HAO Zheng-li;LI Fa-di;YE De-he;ZHENG Chen;ZHANG Xiao-qing;LI Yong

2011, 42(2): 196-202. doi:

Abstract ( 644 )

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This study was conducted to investigate the effects of ratios of structural to nonstructural carbohydrates(SC/NSC=1.57（Ⅰ）, 1.95（Ⅱ）, 2.29（Ⅲ）)in the three diets on ruminal fermentation parameters（pH, VFA, total nitrogen, NH₃-N, UreaN，protein nitrogen）for sheep. Six Gansu high mountain fine wethers fitted with permanent ruminal cannulae, about one and half year old, and weighing 25.30 kg , were used according to a double 3×3 Latin square design with the three feeding cycles of 19 days each (10 days for adapting and 9 days for sampling). The results showed that the average of pH in rumen fluid of wethers for treatment Ⅰwere lower significantly than those for treatment Ⅱ（P<0.01） and Ⅲ（P<0.05） the pH range of all the rumen fluid were from 6.2 to 7.0 except the pH(5.86) of the treatmentⅠ was lower after 3 h of feeding in this trial. There were the tendencies that averages of TVFA concentration in rumen fluid was higher forⅠand Ⅱ than that for treatment Ⅲ（P=0.09） no significant difference on the molar ratio of acetic, propionic and other acid as well as acetic/propionic ratio among the three treatments were observed (P>0.05), the molar ratio of butyric were higher for Ⅰthan that for Ⅲ( P<0.05). As the SC/NSC being increase, the averages of total- and protein nitrogen in rumen fluid were reducing（P<0.01or P<0.05）; but no significant difference on the levels of NH₃-N and Urea-N among the three treatments occur（P>0.05）. These results indicated that as the rising of the value of SC/NSC, the pH of rumen fluid were increasing, the averages of TVFA, total and protein N concentrations were decreasing, but there were no significant influence on the levels of NH₃-N and Urea-N under portions of forage in all the three diets being higher more than 60%.

Continuously Expressing HIV-1 Gag and EGFP by Retroviral Vector InducedSP2/0 Cell Lines

WANG Jing;LI Chang;LI Lin-xi;HU Bo;CONG Yan-zhao;REN Da-yong;WANG Zhuo-yue;DU Shou-wen;JIN Ning-yi;

2011, 42(2): 203-209. doi:

Abstract ( 996 )

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To construct a retroviral vector mediated mammalian cell expression cell line of the human immunodeficiency virus (HIV-1) core protein, the recombinant retroviral vector pFB-gag-EGFP was constructed by inserting core protein gene gag of HIV-1 and enhanced green fluorescent protein gene EGFP into pFB-neo by turns. The recombinant retroviral vector, the pVPack-GP plasmid containing the helper virus gag-pol gene and pVPack-10A1 plasmid containing env gene were cotransfected into packaging cell line HEK-293T with liposome-mediated transduction, and then the recombinant retrovirus was collected and infected SP2/0 cells. The green fluorescent protein was observed under fluorescence microscope, and the expression of core protein Gag of HIV-1 was verified. The resistance cells were screened by G418. The core protein Gag of HIV-1 and green fluorescent protein could express in SP2/0 cells, and the mammalian cell line of the core protein gene gag of HIV1 had been constructed successfully, which laid a mouse cell model of activity assay of anti.AIDS genetic engineering therapeutic agents and targeted drugs.

Construction and Virus Rescue of an Infectious Clone of Type Asia 1 Foot-and-Mouth Disease Virus Containing RSD Receptor Recognition Site

LI Ping-hua;BAI Xing-wen;SUN Pu;LU Zeng-jun;CAO Wei-jun;XIE Bao-xia;WU Run;YIN Hong;LIU Zai-xin

2011, 42(2): 210-216. doi:

Abstract ( 724 )

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Foot-and-Mouth Disease Virus (FMDV) has the high mutation rates and exhibits an important potential for adaptations. The new variants will be generated upon environment change to adapt new environmental conditions, these variants contained one or several amino acid replacements within the capsid protein, including the RGD cell receptor-binding motif. Following serial passages of FMDV Asia1/JS/China/05 isolates under different host condition, the viruses which contained an RSD and RDD sequence in the cell receptorbinding site were generated. To study the effect of single amino acid replacements within RDD receptor-binding motif on production of infectious virus particle, we constructed type Asia 1 FMDV full-length cDNA clone pFMDV-RSD by using over-lap PCR, based on FMDV Asia1/JS/China/05 full-length infectious cDNA clone- BHK-21 cells were cotransfected with linearized recombinant plasmids and plasmids expressing T7 RNA polymerase Apparent CPE were observed after 56 h incubation. Furthermore, the rescued FMDV were verified by RT-PCR, IFA, electron microscope and sulk mice pathogenicity analysis. These results suggested that the infectious FMDV were acquired, and one amino acid replacement in the cell receptorbinding did not affect the virus viability. This study lays a foundation for further study of biology characteristic of rescued virus with RSD receptor recognition site.

Isolation and Characterization of One H11N2 SubtypeAvian Influenza Virus A/duck/Jiangxi/k0701/2009

ZHAO Kun-kun;ZHANG Yan;DUAN Zhi-qiang;ZHONG Lei;ZHU Yan-mei;ZHAO Guo;LIU Xiao-wen;LIU Wen-bo;LIU Xiu-fan

2011, 42(2): 217-223. doi:

Abstract ( 1075 )

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To explore the evolutionary patterns of avian influenza virus (AIV) in poultry in Eastern China, epidemiological surveillance of AIVs was carried out in Eastern China, and one H11N2 subtype avian influenza virus, A/duck/Jiangxi/k0701/2009, was isolated from domestic duck in a live bird market. The analysis of complete genome sequences and genetic evolution showed that all eight segments of A/duck/Jiangxi/k0701/2009(H11N2) belong to Eurasian lineage, but genetic recombination was observed between A/duck/Jiangxi/k0701/2009(H11N2) and AIVs in some regions in Asia and Europe in varying degrees and extensively. Moreover, the PB2 and NS gene may be originated from A/mallard/Korea/GH170/2007(H7N7), NA gene may have the same origin with NA of HPAIV A/duck/Hebei/0710/2009(H5N2), and NP, PA, PB1 genes may have the same origin with A/Muscovy duck/Thailand/CU-LM1984/2009(H4N9). Our results indicated that A/duck/Jiangxi/k0701/2009 may be a multi-origin reassortant virus, and may play an important role in the evolution of HPAIVs.

Evaluation on ALV Infection in Fertilized Eggs from A Wan-nan Yellow-feather Parent Broiler Breeder Flock

WANG Bo;LI Qing-yuan;LIU Shao-qiong;ZHANG Yong-guang;CUI Zhi-zhong;;SUN Shu-hong;

2011, 42(2): 224-227. doi:

Abstract ( 684 )

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In the present study, the infection of avian leukosis virus (ALV) in parent breeder flock was evaluated by the inspection of fertilized eggs. 120 eggs were obtained for a flock of Wan-nan yellow-feather parent layers. 40 eggs were hatching for 9 to 11 days for the preparation of CEF, which were cultured for 10 days and the supernatants were collected for the measurement of p27 antigen by ELISA. The egg white was obtained from the other 80 eggs and used for the direct measurement of p27 antigen or indirect measurement after inoculation with CEF (DF1) and cultured for 10 days.The results showed that ALV positive rate (10/36) was higher in the measurement with cultured CEF than that in the determination with egg white (17/80) or egg white inoculated with CEF (7/80). The ALV-J positive rate in DF1 positive samples was 6/7 by the measurement of IFA with JE9, a monoclonal antibody to ALV-J. The positive rates of ALV-J and ALV-AB in egg yolk antibody were 18/80 and 1/80, respectively. The results showed that the parent flock was infected with ALV-J. The result suggests that the measurement of ALV antigen from eggs is an available method for the detection of ALV infection.

Expression of Streptococcus suis Type 2 Secreted Nuclease A (SsnA), Development of the SsnAELISA and Its Application in the Differential Diagnosis

ZHU Jian;KANG Chao;ZHANG An-ding;JIN Mei-lin

2011, 42(2): 228-235. doi:

Abstract ( 663 )

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Streptococcus suis serotype 2 (SS2) is a zoonotic infectious pathogen associated with a wide range of diseases in human health and pig industry This article aims to study the role of SsnA gene as a molecular diagnosis marker in the differential diagnosis of SS2-SsnA gene was amplified using SS2-LT genome DNA as template and the sequence was then inserted into expression vector pET-28a(+). The plasmid was introduced into E- coli BL21(DE3) for expression and 46 kD recombinant protein was obtained, which had been proved to be immunoreactive by SDS-PAGE and Western blotting. An SsnA-ELISA diagnostic method was developed by using the purified SsnA fusion protein as coating antigen. The detected results of immune serum and infected serum were compared by using SsnA-ELISA Kit, and the significant difference （P<0.05） presented indicates that the established SsnA-ELISA owns excellent detecting effect. 1 446 serum samples collected from 8 provinces of China were then tested by SsnAELISA Kit and the result show that the positive rate ranged from 0%-14.77% and the average positive rate was 5.39%. Our study show that SsnA of Streptococcus suis serotype 2 could be used as a good molecular diagnosis marker in distinguishing infected or immunized animals; in addition, our clinical data indicates that there exists an evident relationship between the infection and epidemicity of SS2 and its climatic environment.

Isolation of Vancomycin Resistant Enterococci and Detection of Its Phenotype,Genotype from Pig Farms

YU Dao-jin;YIN Bing;YI Xiu-li;MA Yu-fang;LI Jian;HUANG Yi-fan

2011, 42(2): 236-242. doi:

Abstract ( 596 )

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This experiment was conducted to investigate the isolation ratio， the phenotype and the genotype of Vancomycin-resistant Enterococci (VRE) from 3 different pig farms and provide basic data for the epidemiology of VRE. A broth dilution method recommended by American Society of Clinical and Laboratory Standards Institute (CLSI) was used to determinate the resistant phenotypic, a PCR method was used to identify Enterococci and detect Vancomycin-resistant gene at the same time. The results were as follows: 30 strains of VRE were isolated from 250 samples of 3 different pig farms. The isolation ratio of VRE was highest in the sucking piglets，followed in weaned pig, milking sow, farrowing sow, and hog, and lowest in boar- Sensitivity test results of VRE showed that the resistance rate of oxytetracycline was highest (93.3%), and the sensitive rate of ampicillin was highest (80%). Resistance gene of 30 VRE strains were detected by PCR method and there were two VanA-positive，12 VanB-positive，7 VanC1-positive，5 VanC2/3-positive in all the VRE, though there were no Van gene amplified from 4 strains of VRE. The isolation ratio of VRE from 3 pig farms was related to the extent of antibiotic usage. The VRE had different resistant rate to 6 kinds of antibiotics. There was highly agreement between phenotypes and genotypes in 30 strains of VRE．

Interactive Effects of Dietary Zearalenone and Soybean Isoflavoneon Development of Reproductive Organs and Transcription ofEstrogen Receptors Gene in Replacement Gilts

WANG Ding-fa;PENG Yun-zhi;ZHANG Ni-ya;QI De-sheng

2011, 42(2): 243-250. doi:

Abstract ( 664 )

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To study the interactive effects of zearalenone (ZEA) and soybean isoflavone (ISO) on growth performance, development of reproductive organs and transcription of estrogen receptor genes in replacement gilts, ninety 75-day-old female crossed-bred pigs (Duroc × Landrace × Yorkshire) were randomly allocated to nine diet treatments with 5 replicates in each treatment of 2 pigs per replicate during the 21-day research. The experiment was a 3×3 factorial design using a none-soybean meal diets with the level of ZEA at 0, 0.5 or 2.0 mg·kg^-1 and the addition of ISO at 0, 300 or 600 mg·kg^-1. The results showed that interaction of ZEA and ISO in diets has no significant influences on the average daily gain (ADG) and average daily feed intake (ADFI) in the replacement gilts (P>0.05). ISO could enhance the increased weight of the reproductive organs and the width of vulva induced by ZEA with 0.5 mg·kg^-1 in diets. However, ISO with 300-600 mg·kg^-1 in diets could reduce the increased weight of reproductive organs and edema of vulva induced by ZEA with 2 mg·kg^-1 in diets of replacement gilts（P<0.05）.Simultaneous provision of ISO and 2 mg·kg^-1. ZEA to replacement gilts increased the mRNA transcription of ERα, and reduced the mRNA transcription of ERβ in uterus and vagina compared with the pigs offered the diets containing 2 mg·kg^-1 ZEA alone. The results suggested interaction of ZEA and ISO in diets has no significant influences on ADG and ADFI in the replacement gilts. The estrogenic effect of ZEA would be reinforced when coexistence of ISO and low dosage of ZEA (0.5 mg·kg^-1) in the diets. While ISO could offset the estrogenic effect of ZEA when the diets containing the high dosage of ZEA (2 mg·kg^-1). The effects of ZEA and ISO on the development of reproductive organs in replacement gilts were mediated through the dual estrogen function of ISO and changing the mRNA transcription of ERs in the reproductive organs.

Studies on Alany-glutamine on Antioxidant Ability and Protects of Small Intestine for Early Weanling Piglets

CHEN Heng-can;MA Li;CHEN Ke-lin;TAN Li-qin;YUAN Xue-bo;GUO Rong-fu

2011, 42(2): 251-259. doi:

Abstract ( 1025 )

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This experiment was conducted to investigate the effects of Alanyglutamine (Ala-Gln) on antioxidant ability and protecting intestine of early weanling piglets. 48 piglets of weanling at 35 days (half male, half female), reproduced by Yunnan Saba sows with similar body weight, parity, milk yields and litter size, were designed randomly to 4 groups, each has 3 repeats with 4 piglets per repeat. Group A (control) was fed the basal diet, and experimental diets with adding 0.15%, 0.30% and 0.45% for group B, C and D, respectively. The duration of experiments was from 35 to 60 days. The results indicated that growth performance of piglets can be improved with Ala-Gln supplementation of 0.15％, 0.30％ and 0.45%, respectively. With the increase of dietary Ala-Gln supplementation，body weight and daily feed intake for piglets were increased (P<0.05) and feed:gain was decreased as a quadratic function (P<0.05). At the 49th day，the serum T-AOC and SOD concentrations were increased (P<0.05)，and the serum MDA contents was decreased as quadratic equation (P<0.01). The results of intestinal morphology determination showed that the length of duodenum and ileum villus for piglets increased as quadratic equation (P<0.01),and length of jejunum villus increased as a quadratic function (P<0.05), duodenal and jejunal crypt depth reduced as the quadratic function (P<0.05), ratio (V / C) of duodenum, jejunum and ileum villus length to crypt depth can be improved as the quadratic manner (P<0.01). It was concluded that the growth performance and antioxidant ability of Saba weanling piglets can be improved, and intestine structures and functional development can be protected with adding dietary Ala-Gln.

Effects of Denervation of SSN and ISN on Testicular Developmentand Spermatogenesis in Rat

HUO Shu-ying;MA Ai-jin;WU Xian-jun;LI Yu-rong

2011, 42(2): 260-266. doi:

Abstract ( 678 )

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The experiment was conducted to reveal whether the superior spermatic nerve (SSN) and the inferior spermatic nerve (ISN) were involved in regulating testicular development and spermatogenesis. The present study was thus designed to transect the SSN and ISN of rats on PD10 and then analyzed changes on PD25 and PD50. The results demonstrated that testicular denervation have no obvious influence to testis mass on PD25（P<0.05）, but significantly reduced testis mass, cauda epididymal sperm counts on PD50（P<0.01）. Proliferating cell nuclear antigen (PCNA) immunohistochemistry staining proved that the denervation had no influence on the proliferarion of germ cells on PD25, but obviously inhibited the proliferation on PD50. In addition, RT-PCR results showed that testis denervation significantly decreased testis 3β-hydroxy sterol dehydrogenase 1 (3β-HSD1) on PD25（P<0.05）and significantly decreased testis 3β-HSD1 and androgen binding protein (ABP) mRNA levels（P<0.01）, luteinizing hormone receptor (LHR) and follicle stimulating hormone receptor (FSHR) mRNA levels on PD50（P<0.05）. These results suggest that the testicular nerve supply plays an important role in supporting seminiferous tubule development and spermatogenesis after testis descend to scrotum.

Analysis the Contents of SOD in Blood Taken from the Male Sika Deer in Different Physiological Periods

WANG Xiao-song;TANG Chao;MA Ze-fang;LEI Yu-fa;LIU Yong-ju;JIANG Xiao-ming

2011, 42(2): 267-271. doi:

Abstract ( 603 )

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Study on content of SOD in whole blood of sika deer from different physiological period. The blood of male sika deer in different physiological periods was centrifugated, and then using pyrogallol method to detect the content of SOD in blood corpuscle, plasma, dissolve blood and cell membrane, lastly estimated content of SOD in whole blood. The results showed that the highest content of SOD ((1 007.07±11.03) U·mL^-1) appears during the time of developing antler, besides we found that there was a remarkable discrepancy between developing antler period and remaining two periods(P<0.01). But no significant difference there was between preantler period and service period(P>0.05). The research demonstrated that there were some certainly correlative bewteen the change of SOD content and being different physiological periods. The developing anlter period have the highest SOD content in deer blood, that could be to improve and increase the medicine value, especially the function of anti-aging. In addition, during various physiological stages, we found the dissolve blood should be the best raw material for producing deer blood against aging, because of the highest SOD content has had been happening in it.

The Relationship Between Liver Injury and Hydrogen Sulfide in Endotoxemiaand Analysis of Protection of Cation A

CHEN Chao;GAO Hong;YAN Yu-lin;CHEN Li-ping;GAO Li-bo;ZHAO Ru;CHEN Ling

2011, 42(2): 272-277. doi:

Abstract ( 624 )

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To explore the realationship between liver injury and hydrogen sulfide (H2S) in endotoxemia and protection of Cation A (CA) in rats. Seventy-two adult healthy SD rats were randomly divided into three groups: control group, ET group, CA group. Hepatic tissue and blood serum of three groups were collected for tests at the 3rd, 4th, 8th and 12th hour after treatment The concentration of ALT and AST in serum were detected. The production rate of H2S in liver were measured by spectrophotography. The transcriptional level of CSE mRNA were investigated by in situ hybridization and by image analysis. Compared with control group, ALT and AST level in serum of ET group were significantly higher. In CA group, ALT and AST level is significantly lower than that of ET group. The production rate of H2S in liver of ET group were significantly higher than that of control group, while these items in CA group were significantly lower compared with ET group. The transcriptional level of CSE mRNA and the production rate of H2S in liver were significantly up-regulated and the up-regulation were inhibited markedly by CA. These above results suggested the novel gaseous transmitter H2S mediate liver injure in endotoxemia, whereas CA have a significantly pretective effect on inflammatory injure through down-regulation of the CSE/H2S pathway in liver.

Differentiation of Porcine TLR4 Gene mRNA Expression between Resistantand Sensitive Resource Populations to ETEC F18

.Key Laboratory for Animal Genetics;Breeding;Reproduction and Molecular Design of Jiangsu Province;College of Animal Science and Technology;Yangzhou University;Yangzhou 00;China;. College of Veterinary Medicine;Yangzhou University;Yangzhou 00;China;. Jiangsu Engineering Research Centre for Molecular Breeding of Breeder Pig;Changzhou;China;. Jiangsu（Yangzhou）Public Technical Service Centre for Efficient and Healthy Culture Technology in Largescale Swine Farms;Yangzhou 00;China

2011, 42(2): 278-283. doi:

Abstract ( 1029 )

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This study was conducted to detect the expression of TLR4 gene in various tissues of pig, and then to analyze the expression difference between resistant and sensitive resource populations to ETEC F18, which aimed to discuss the role that TLR4 gene played in immunity and resistance to ETEC F18. Porcine TLR4 mRNA was amplified by SYBR-GreenⅠ semi-quantitative RT-PCR, total RNA was extracted from 11 organizations of sixteen 35-day-old piglets(eight ETEC F18 resistant piglets and eight F18 sensitive piglets) including heart, liver, spleen,and so on. Using GAPDH as an inner control gene, the relative TLR4 gene mRNA expression level in various tissues and then in resistant and sensitive resource populations to ETEC F18 was calculated according to the fluorescence threshold (Ct value). The results showed that TLR4 gene was expressed in all tissues and higher expression levels were detected in immune organs like lung, lymph node, kidney, spleen and thymus gland. The mRNA expression of TLR4 gene in sensitive individuals were higher than that in resistant individuals generally, the ratio of sensitive individuals and resistant individuals in all tissues apart from stomach ranged from 1.083 to 2.980. In tissues of lung, lymph node, kidney and spleen, the mRNA expression of TLR4 gene in sensitive individuals were significant higher than that in resistant individuals (P<0.05). The results speculated that TLR4 gene function may be related to innate immune recognition, particularly have a certain impact on porcine Gram-negative bacteria such as ETEC F18 etc The resistance to ETEC F18 in pig is related to down regulation of mRNA expression of TLR4 gene to some degree.

Polymorphism of KAP3.2 Gene and Its Effect on Partial Economic Traitsin Tibetan Sheep

WANG Zhi-you;CHEN Yu-lin;XU Qiu-liang;MA Zhong-ren;QI Quan-qing

2011, 42(2): 284-288. doi:

Abstract ( 697 )

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PCR-SSCP and DNA sequencing techniques were used to analyze the polymorphism of partial sequence of KAP3.2 gene in 254 Tibetan sheep including three types (Plateau, Oula and Qiaoke). Based on the data, the correlation between the polymorphisms and production traits (body weight, wool length and wool yield) was studied using least square linear model The results showed that three genotypes (AA, AB and BB) were detected at KAP3.2 gene locus. Allele A was predominant in Plateau type, and allele B was predominant in Oula and Qiaoke types. Frequencies of genotype deviated from Hardy-Weinberg equilibrium in three types. The polymorphism was medium in Plateau and Oula types, while low in Qiaoke type. There was one mutation (C to T) at 271 bp on KAP3.2 gene. For the body weight, there was no significant difference among various genotypes in three types of Tibetan sheep. For the wool length, individuals with genotype AA was significantly higher (P＜0.05, P＜0.01) than those with BB genotype, and there was no significant difference between AA and AB genotypes in three types of Tibetan sheep. For the wool yield, individuals with AA genotype was significantly higher(P＜0.05) than those with AB and BB genotypes in three types of Tibetan sheep. The results indicated that the KAP3.2 could be regarded as marker for wool length and wool yield traits in Tibetan sheep.

Effects of Dietary Phosphorus on Phosphorus Metabolism and Regulation ofSodium-dependent Phosphate Transporter mRNA for Broilers

FANG Re-jun;HE Jia;CAO Man-hu;CHEN Juan;LI Mei-jun;FANG Chen-kun

2011, 42(2): 289-296. doi:

Abstract ( 633 )

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To study effects of dietary phosphorus on phosphorus metabolism and Na/Pi-Ⅱb mRNA expression in intestine of broilers. Forty 60-day-old broilers with an average initial weight of about 1.5 kg were allotted to four treatments randomly, each treatments had five replicates of two broilers. Levels of dietary total phosphorus were: group A:0.4%, group B:0.6%, group C:0.8% and group D:1.0%(all with calcium∶phosphorus rations of 2∶1), group B was as control group. The results showed that serum phosphorus level increased as dietary phosphorus increased while serum calcium level decreased. At the 0.8% level of dietary phosphorus, the activity of alkaline AKP tended to be decreased significantly, but the metabolic rate of Ca and Pi were the highest, respectively. Compared with control group, low Pi diet (TP 0.4%) increased expression of Na/Pi-Ⅱb mRNA in jejunum forepart (P<0.01) and jejunum tail (P>0.05) of 60 days broilers. Whereas, higher dietary Pi (TP 0.8% and TP 1.0%) decreased duodenal (P<0.01), jejunum forepart and jejunum tail (P>0.05) expression of Na/Pi-Ⅱb mRNA. Phosphorus uptakes from feedstuffs of broiler dissimilar small intestine were measured by everted gut sac. The result showed: the absorption rate of phosphorus in vitro of duodenum and jejunum forepart declined as dietary phosphorus increased, and the absorption rate of phosphorus in vitro of each intestinal segment respectively was highest at lower Pi diet (TP 0.4%). The result implies that increasing dietary phosphorus levels could increase expression of Na/Pi-Ⅱb mRNA and phosphorus absorption at low Pi diet, whereas the exorbitant dietary phosphorus levels inhibit its expression.

Sequence Analysis of Neuraminidase Genes of 63 H9N2 SubtypesAvian Influenza Viruses

WANG Shou-chun;YIN Yan-bo;WANG Xiao-hong;LI Hai-ying;WANG Jian-lin;ZHANG Yi;GUO Yan-yan

2011, 42(2): 297-305. doi:

Abstract ( 1090 )

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In order to explore the genetic mutations of the neuraminidase and the law of the molecular epidemiology of H9N2 subtype Avian Influenza virus (AIV) in mainland of China, 63 H9N2 AIV strains were isolated from some poultry yards in China, the whole NA cDNA fragments of them were amplified by reverse transcription polymerase chain reaction, with primers specific to NA gene. After cloning and sequencing, homology and heredity evolution of NA gene sequences were analyzed. The sequence analysis showed that the homology of NA gene in this study was between 87.4%-99.5% Neuraminidase of 52 strains had a deletion of 3 amino acid residues at positions 63-65, belong to Y280-like branch; the other 11 strains had no deletion in the stalk, belong to G9like branch. Amino acid residues at hemadsorbing (HB) sites and antigenic determinants of these isolates exist variation. The amino acid residues in the enzyme active sites of the isolates were highly conserved and showed no mutations associated with resistance to the sialidase inhibitors oseltamivir and zanamivir. 36 strains viruses sequenced in this study contained potential glycosylation sites at 264 amino acid residues 28 strains lost potential glycosylation sites at 402 amino acid residues. The results indicated that by now the NA gene of H9N2 AIV in mainland China belong to two branches, Y280-like and G9-like, and the most NA gene of the H9N2 isolates in China mainland belong to Y280-like.

Establishment and Application of the Triplex PCR for Detecting M. Bovis, M. mycoides subsp. mycoides Small Colony Type and M. agalactiae

LI Da-wei;HUANG Can-ping;ZHANG Yan-ming;XIE Jian-hua;RAN Zhi-guang;XIONG Zhong-liang;FAN Wei-xing

2011, 42(2): 306-310. doi:

Abstract ( 624 )

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The study was aimed at establishing a triplex PCR to differentiate M.bovis, M. mycoides subsp. mycoides Small Colony Type and M. agalactiae out of the same sample, for clinical diagnosis and epidemiological survey. Three pairs of primers were designed and synthesized according to the published literatures in GenBank. And a triple PCR was developed to amplify conservative regions of each bacterial genomes of M. Bovis, M. mycoides subsp- mycoides Small Colony Type and M. agalactiae, with the target sequences of 448, 549 and 375 bp, respectively. Sensitivity of this triple PCR was analyzed. Specificity was verified with template from M. gallisepticum, M. Swine, M. agalactiae and M. mycoides subsp- mycoides Small Colony. And reliability was tested by comparing the results from determinative bacteriology. Three specific fragments of 448, 549 and 375 bp, corresponding to M. Bovis, M. mycoides subsp- mycoides Small Colony Type and M. agalactiae, were amplified under optimized condition, without interference between templates and the primers. And results were in agreement with determinative bacteriology. No band was shown in targeting genomes from M. gallisepticum and M. Swine. Sensitivity was determined as 0.8 ng·μL^-1 These results suggest that the triplex PCR could differentiate M. Bovis, M. mycoides subsp- mycoides Small Colony Type and M. agalactiae based on one single sample, with high sensitivity, specificity and reliability，and can be applied in clinical diagnosis and epidemiological survey.

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