Acta Veterinaria et Zootechnica Sinica

Association of Twelve Polymorphisms of the VIPR-1 Gene with Chicken Early Egg Production Traits

ZHOU Min;;LIANG Fei-fei;RAO You-sheng;;ZENG Hua;ZHANG De-xiang;ZHANG Xi-quan

2008, 39(9): 1147-1152. doi:

Abstract ( 955 )

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In this study，vasoactive intestinal peptide receptor-1 (VIPR-1) gene was used as a candidate gene to analyze the association of 12 polymorphisms sites with early egg production traits in 644 individuals in Ningdu Sanhuang population. Association analyses showed that the A-284G in the 5′ regulatory region was significantly associated with the age of first egg (P＜0.000 1) and total number of oafish egg from 90 to 300 d of age (P＜0.05). Allele G is positive for number of egg. The C+43 327T in intron 6 was significantly associated with total number of eggs and total number of qualified eggs from 90 to 300 d of age (P <0.05). CT was predominant genotype. Significant association was also found between C+53 327T in intron 8 and the age of first egg (P＜0.01). The allele C was positive for less age of first egg.

Genetic Variation of TYR Gene in Different Chicken Breeds

ZHANG Jian-qin;CHEN Hong;SUN Zhao-jun;LIU Xiao-lin;QIANG Ba-yang-zong;GU Yu-lan

2008, 39(9): 1153-1158. doi:

Abstract ( 1536 )

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In order to study genetic variation of TYR gene in four chicken breeds selected from special district including Guyuan， Wenchang， Tibetan and Hisex chicken， two sites of the gene exon1 and one site of 5′ flanking region were analyzed by PCRSSCP and DNA sequencing. The result indicated: there was no polymorphism at P2 site， while at P1 site， there were three genotypes (AA,BB,AB) in three Chinese chicken breeds respectively. They were all in equilibrium at P1 site. At P3 site， there were three genotypes (AA， BB and AB) in Chinese local chicken breeds (Guyuan， Wenchang and Tibetan chicken) and two genotypes (AA and AB) in Hisex chicken breed. Guyuan and Wenchang breeds were in equilibrium at the site， but Tibetan and Hisex breeds were extremely in unbalance. It is concluded that there was abundant variation of TYR gene in Chinese local chicken breeds. DNA sequencing of PCR products for different genotypes showed that there were two mutation sites: C to T at P1 site and G to A at P3 site, respectively， which didn’t cause amino acid variation at P1 site. The chisquare analysis revealed that there was significant statistical difference in distribution of genotype and allele frequencies at the two TYR sites among the analyzed four chicken populations (P<0.05 and P<0.01). It revealed that there was significant association between genotype and chicken breeds. This study provided original information for elucidating the possible roles of TYR gene of exon1 and 5′ flanking region in different chicken populations.

Analysis on Conserved Effect of Small Population in Jinding Duck Using Microsatellite Markers

DUAN Xiu-jun;WANG Li-hua;GONG Dao-qing;DONG Biao;SUN Guo-bo;CHEN Zhang-yan;WANG Lin-lin;BIAN You-qing

2008, 39(9): 1159-1164. doi:

Abstract ( 918 )

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The study was based on Jinding duck which was conserved in National Waterfowl Germplasm Resource Pool. The conserved effect was detected through comparing the difference of genetic diversity which was detected by 12 microsatellite markers in small populatin of three generations. The results suggested that 66 alleles were detected at 12 microsatellite loci in conserved population of three generations in Jinding duck. 60 alleles of the total were all detected in three generations， and 2 alleles were detected only in the first generation. The number of average alleles at each locus was more than 5. The mean heterozygosity of conserved population of three generations ranged from 0.712 9 to 0.729 8， and the mean polymorphism information content ranged from 0.665 8 to 0.687 1. The genetic diversity of Jinding duck was rich. The tendency was decrease following the raise of generation. The study showed that the small population conserved method in Jinding duck was feasible.

Study on Phylogenesis of Chinese Southern and Northern Cattle and Yak Based on the Polymorphisms of GH Gene

LI Yong-hong;CHANG Hong;GENG Rong-qing;CHANG Guo-bin;SONG Guang-ming;CHANG Chun-fang;JI De-jun

2008, 39(9): 1165-1170. doi:

Abstract ( 1443 )

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Applying simple after randomly sampling 18 Leiqiong cattle，19 Bayingole Menggu cattle and 18 Bayingole yak from typical colony in the central area of their habitats， the exon sequences (654 bp) of GH gene were analyzed by direct sequencing of PCR product. Together with homologous sequences of Bos taurus，Bos indicus，and Bos grunniens，using the sequence of Bubalus bubalis as the outgroup，the phylogenetic trees were constructed by NJ and MP methods respectively. The aim was to explore variation characteristics of GH gene，the relationships between southern and northern cattle of China and yak，and provide some considerable data for the evolution characteristics on Bovidae research and evaluate the relationship between adaptive evolution and molecular evolution research.The results showed that three mutations and five different haplotypes， two mutations and four different haplotypes，two mutations and three different haplotypes were detected in Leiqiong cattle，Bayingole Menggu cattle and Bayingole yak， respectively. Both three results supported almost the same topology，which indicated that GH gene differentiation happened before Bubalus bubalis differentiated from Bos indicus，Bos taurus and Bos grunniens. As to GH gene，the difference between yak and southern，northern cattle of China was not significant.

Comparison between 3 Individual Identification Methods Using Microsatellite Markers

MAO Yong-jiang;CHANG Hong;YANG Zhang-ping;CHANG Guo-bin;LIU Gui-qiong

2008, 39(9): 1171-1175. doi:

Abstract ( 1431 )

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30 Luxi and 30 Bohai Black individuals were genotyped with 12 microsatellites. The efficiencies of individual identification among phylogenetic tree analysis， maximum likelihood and Bayes approach were compared. The results showed that the individual identification accuracy rates were 100% for 3 methods using 12 microsatellites and 6 low heterozygosity microsatellites. By using 6 high heterozygosity microsatellites，the accuracy rates were 83.33%，76.67% and 71.67% for Bayes approach，maximum likelihood and phylogenetic tree analysis，respectively. All of these indicated that Bayes approach maybe the most accuracy and should be the first choice in individual identification research，and the maximum likelihood and phylogenetic tree analysis could be the complementary methods.

Sequence Analysis of the Deletion Region in PIS Goats

LI Dong-feng;ZHOU Rong-yan;LI Xiang-long;LI Lan-hui;FENG Fu-jun;XU Hou-gang

2008, 39(9): 1176-1182. doi:

Abstract ( 1536 )

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A 11.7 kb deletion region triggers intersexuality and polledness in goats， termed as Polled Intersex Syndrome (PIS). Through the sequence analysis of four repeated sequences (1-3 120 bp，3 988-4 565 bp，4 989-7 088 bp，7 875-9 135 bp) in goat PIS deletion region (AF404302)， we found that the 7 875-9 135 bp segment belonged to L1M3 subfamily of LINE-1 and the others were members of BDDF subfamily of RTE. Depending on the results of tblastn program searching using the fulllength protein sequences of RT domain in PIS region as query sequences against the bovine genome database，56 consensus sequences were constructed. Using the consensus sequences we deduced the formation time of PIS region at （22.5-30） million years， just the divergence time of Bovidae from Artiodactyla. Combined with other evidence， the PIS region might only exist in Bovidae， and another evidence of the role of PIS region and the non-LTR competition doctrine was provided in this paper.

Cloning，Sequence Characteristics and Expression Analysis of PGAM2 Gene

WU Xiao-xiong;TANG Zhong-lin;LI Yong;YANG Shu-lin;CHU Ming-xing;MA Yue-hui;LI Kui

2008, 39(9): 1183-1189. doi:

Abstract ( 1459 )

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The cDNA sequences of porcine PGAM2 gene were cloned， being analyzed by bioinformatics method. The fulllength cDNA was 913 bp with an entire open reading frame (ORF)， encoding 253 amino acids. The analysis of sequence revealed that the porcine PGAM2 gene was highly homologous with that of other species， such as canis familiaris (97.6%)， Homo sapiens (96%)， Pan troglodytes (96%)， Rattus norvegicus (94.5%) and Mus musculus (94.1%) in amino acid. The protein encoded by porcine PGAM2 gene had ‘ ATHR’ and ‘QGKA’ motif at N- and C-terminatus sites， respectively. There were a cut site of signal peptide sequence at 14-15 AA and a typical conserved domain ‘carbohydrate transport and metabolism (GPMA)’. The PGAM2 gene was differently expressed during fetus skeletal muscle development with different expression pattern between Tongcheng and Landrace pigs. In Tongcheng pigs， it was differently expressed with wave pattern at selected stages， and expressed most abundantly at 65 days post coitus (dpc). In Landrace， PGAM2 gene was expressed with ‘upregulation’ expression pattern and expressed abundantly at 65 and 90 dpc more than that at 33 dpc. There was higher expression in Landrace pigs than that in Tongcheng pigs at 90 dpc， but no significant difference at 33 and 65 dpc between the two breeds.

Effects of Progesterone on Progesterone Receptor Expression in Cultured Bovine Endometrial Cells (bEC)

ZHANG Ming;GAN Xiao;ZHENG Jie;LAI Song-jia;ZHANG Hua;WANG Xiong-qing;ZHU Qing

2008, 39(9): 1190-1195. doi:

Abstract ( 832 )

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In the studies， bovine uterinal endometrium cells were cultured in medium including progesterone at 50， 100， 200 and 400 ng/mL respectively and collected after incubation for 1， 3， 6， 12 and 24 hours. Semiquantitative revervedtranscript polymerase chain reaction (RT-PCR) analysis was used to observe the effects of progesterone on progesterone receptor (PR) expression. Results of the present study indicate that progesterone inhibit the expression of PR in vitro.

Effect of Culture in vitro of Spermatogonial Stem Cells by Different Culture Systems in Chicken

WU Xin-sheng;SUN Si-yu;WU Hong;WEI Cai-xia;SUN Guo-bo;YU Fei;ZHAO Wen-ming;XU Qi;CHEN Guo-hong;LI Bi-chun

2008, 39(9): 1196-1203. doi:

Abstract ( 1420 )

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Spermatogonial stem cells (SSCs) were isolated from chicken embryo testis on 16^th hatching days. Growth of SSCs which were cultured in vitro in three culture solutions with or without feeder layer were compared， and the characteristic of SSCs was identified. The results showed that, without feeder layer of fibroblast cells， survival times of SSCs in DMEM， TCM199 and RPMI1640 were 6.5， 6.0 and 3.5 d, respectively， and there were extremely significant differences among in DMEM， TCM199 and in RPMI1640 (P<0.01). With feeder layer of fibroblast cells， survival times of SSCs in DMEM， TCM199 and RPMI1640 were 45.5， 38.0 and 14.0 d, respectively， and there were extremely significant differences among in three culture solutions (P<0.01). Survival time and rate of SSCs cloning in culture system Ⅳ were （45.5±3.2）d and 0.31±0.46, respectively， and those in culture system Ⅳ were extremely significant higher than those in other five cultures(P<0.01). SSCs were subcultured in the culture system Ⅳ to 3rd passage， and rates of cloning of systems at passage 1， 2 and 3 were 31.6%， 20% and 18%, respectively. SSCs formed cell clonies and proliferated， and cell clonies of SSCs were positive to alkaline phosphatase (AKP) and stage specific embryonic antigen-1 (SSEA-1). The 3^rd passage SSCs that had been cultured in vitro not only maintained the undifferentiated state and normal diploid karyotype (2n=78)， but also kept the necessary characteristics of SSCs， which was subcultured in culture system Ⅳ with DMEM and feeder layer of fibroblast cells.

Effects of Liquid DLMethionine Hydroxy Analogue on Growth Performance and Immune Responses in Broiler Chickens

ZHANG Li-bin;GUO Yu-ming

2008, 39(9): 1204-1211. doi:

Abstract ( 860 )

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An experiment was conducted to determine the effects of different doses of Liquid DL-Methionine Hydroxy Analogue (LMA) on growth performance and immune response in broiler chickens. In a single factorial arrangement with 4 levels of LMA to meet 80%， 100%， 120%， 140% of methionine requirements of broiler recommended by Feeding Standard of China (2004)， 256 one-day-old Arbor Acres male broiler chickens were randomly divided into 4 treatments with 8 replicates of 8 birds each. The growth performance， cellular and hormonal immunity were respectively determined. The results showed that， with the LMA level increased， there were no significant changes (P＞0.05) in body weight gain and feed intake among the treatments， the ratio of feed to gain was linearly decreased and it was significantly highest (P＜0.05) in the group fed with 80% of methionine requirement; the relative weight of the spleen on day 21 were higher significantly (P＜0.05) in the group fed with 120% of methionine requirement than that in the 80% group; the serum globulin on day 21 and 42 were linearly increased significantly (P＜0.05); the phagocytosis of neutral red of peripheral blood lymphocyte was quadratic and the lowest was in the control group (P＜0.05); the proliferation of peripheral blood lymphocyte in response to LPS and those of the 120% group on day 21 and of the 100% group on day 42 were significantly higher than the others (P＜0.05); the antibody titers to NDV on day 4 post first inoculation of the vaccine was quadratically increased， and the anti-BSA antibody production on day 13 post second immunization was quadratic and the hormonal immunity were highest in the groups fed with 100% or 120% of methionine requirement. In conclusion， dietary LMA supplementation improves the feed utilization and humoral and non-specific immuno-competence of broiler chickens.

The Factors of Affecting Phytase Stabilities in Broiler Gastro-intestine

ZHANG Tie-ying;LIU Zhi-wei;SUN Jie;HUANG Yu-ting;CHEN Zhi-wei

2008, 39(9): 1212-1216. doi:

Abstract ( 1261 )

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The effects of pH value and protease in broiler gastro-intestine on Aspergillus niger phytase activity and the ability of phytase to survive in broiler proventriculus, gizzard and small intestine were studied in vitro. The results show that the phytase optimum pH value is 5.5 and is susceptible to broiler gastrointestinal pH and protease. When the phytase incubation at the pH which is higher or lower than its optimum pH5.5， the phytase activities decreased rapidly and most of phytase activity disappeared during 30 min. Following incubation at pH3.0 and pH6.0 in the presence of pepsin and pancreatin respectively， more phytase activity disappeared. Phytase is more susceptible to pancreatin than that to pepsin. 42%， 40% and 49% of phytase were survived on 14， 28 and 42 days broiler proventriculus and gizzard respectively and about 19％of phytase survived in small intestine of broiler in different ages in vitro. The phytase is more stable in proventriculus and gizzard than that in small intestine， where pH and pancreatin is not favor to phytase. The results suggest that phytase is susceptible to pH and protease in broiler gastrointestinal tract， which may be the main reason why only a small part of inorganic P could be replaced by phytase in broiler diet.

Effect of Flavones of Sea Buckthorn on Carcass Characteristics and Meat Quality of Arbor Acres Broilers

LI Yao;FU Jing;WANG Bao-dong;WANG Yan-bo;SHAN An-shan

2008, 39(9): 1217-1223. doi:

Abstract ( 966 )

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To study the effects of flavones of sea buckthorn on carcass characteristics and meat quality of AA broilers， a total of 360 1-day-old health male Arbor Acre broilers were randomly used for 3 dietary treatment. Each treatment was replicated four times with thirty broilers each. The three dietary treatments were basal diet，basal diet+0.1% flavones of sea buckthorn and basal diet+0.2% flavones of sea buckthorn. Apparent digestibility of nutrients (crude protein， ether extract， calcium， phosphorus)， carcass characteristics， meat quality and some blood biochemical index were determined. The results showed that apparent digestibility of dietary protein inclined to increase (P=0.054)， apparent digestibility of dietary calcium was significantly increased (P<0.01)， diameter of thigh muscle fiber tended to decrease (P=0.080) at the dose of 0.1% flavones of sea buckthorn. At the dose of 0.2% flavones of sea buckthorn， carcass percentage was significantly increased(P<0.01)， abdominal fat percentage was significantly reduced(P<0.01); meat color of breast and thigh muscle， crude protein of thigh muscle were significantly increased(P<0.01)， drip loss of breast and thigh muscle， ether extract of thigh muscle and serum triglyceride were significantly decreased(P<0.05). Apparent digestibility of dietary crude protein and calcium were increased by the diet supplemented with 0.1% and 0.2% flavones of sea buckthorn， and carcass characteristics and meat quality were improved by the diet supplemented with flavones of sea buckthorn. Moreover， flavones of sea buckthorn were nontoxic and harmless.

Effect of Corticosterone Treatment at Different Hatching Time on Embryo Development of Broiler Chickens

GAO Jing;SONG Zhi-gang;JIAO Hong-chao;LIN Hai

2008, 39(9): 1224-1229. doi:

Abstract ( 879 )

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The experiment was conducted to investigate the possible effect of corticosterone treatment at different hatching time on the development of chicken embryos. Seven hundreds of AA broiler eggs were divided into 7 groups and randomly subjected to one of the seven treatments:corticosterone treatment (200 ng) at E0， E7 and E14， three positive control groups that injected with corn oil at E0， E7 and E14， and one negative control group. The incubation time，hatching ability and posthatching body weight were recorded.The blood samples were collected and the organs weight were measured after hatching.The results indicated that corticosterone treatment at E0 significantly reduced hatching ability(P＜0.05) and increased mortality of embryo.Corticosterone treatment at E7 shortened the incubation time (P＜0.05) and tended to reduce the hatching ability.The development of heart and liver were restrained by corticosterone treatment(P＜0.05).The result showed that the effect of corticosterone on the development of chicken embryo was age dependent.Moreover，the effect of injection had an adverse effect on embryos as well.

Establishment of ELISA Based HA1 Protein for Detecting Antibodies against H3N2 Subtype Swine Influenza Virus

DING Xuan-ya;QIAO Chuan-ling;CHEN Yan;YANG Huan-liang;XIN Xiao-guang;HAN Qing-gong;CHEN Hua-lan

2008, 39(9): 1230-1234. doi:

Abstract ( 874 )

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The HA1 gene of H3N2 subtype swine influenza virus (SIV) was cloned into expression plasmid pET-30a. And the recombinant plasmid DNA was named as pET-HA1. Then it was transformed into E.coli BL21(DE3)， the recombinant protein was expressed in E.coli BL21(DE3)by induction of IPTG. The expressed HA protein was identified by SDSPAGE and Western blotting analysis. The results showed that HA1 protein was a 45 ku protein with immunogenicity. The purified HA protein was used to establish the indirect ELISA for detection of the antibodies against H3 subtype of SIV. The assay has excellent specificity， high sensitivity and excellent reproducibility. The total of 40 serum samples were randomly collected from field and evaluated by ELISA with recombinant HA1 and HI test， the coincidental rate between the two tests is 86.5%. These results showed that recombinant HA1 based ELISA is specific， sensitive and easy to perform for serologically diagnosis of SIV infection.

Expression of Major B-cell Epitopes within 2C Non-structural Protein of FMDV and Its Bioactivity

FU Yuan-fang;LU Zeng-jun;TIAN Mei-na;ZHANG Xiao-li;LIU Zai-xin;CAI Xue-peng

2008, 39(9): 1235-1239. doi:

Abstract ( 841 )

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A pair of primer was designed according to the 2C gene sequence of footandmouth disease virus (FMDV) for amplification of a fragment including 174 bp 5′-end and 279 bp 3′-end of 2C gene， which encoded abundant B-cell epitopes of 2C protein. The amplified fragment was inserted into pET-30a plasmid (Novagen) via two unique restriction sites of NcoⅠand SalⅠ. Open reading frame of target gene was confirmed correctly inserted into the positive recombinant plasmid by sequencing， the recombinant plasmid was transformed into the host strain bacteria BL21(DE3)pLys for protein expression. After inducing by IPTG at 37 ℃ for five hours， the expressed product was analyzed by SDS-PAGE and Western Blotting. The results revealed that the target gene fragment of 2C had been expressed successfully. The product is a 23 kD fusion protein and can react with sera derived from FMDV infected animal. It would provide an useful antigen for establishment of enzyme linked immunetransfer blot (EITB) diagnostic method， which can be used for differentiation of the FMDV infected animals from the vaccinated animals.

Immunological Adjuvant Effect of Recombinant Porcine IFN-γ and IL-4 Plasmid on Foot-and-mouth Disease Vaccine

MENG Xue-lian;FANG Yong-xiang;DOU Yong-xi;CHEN Guo-hua;JING Zhi-zhong;CAI Xue-peng

2008, 39(9): 1240-1244. doi:

Abstract ( 826 )

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In order to certify the immunological adjuvant effect of recombinant pcDNA3.1/IFN-γ and pcDNA3.1/IL-4 plasmid， we investigated the changes of antibody levels and the CD4⁺/CD8⁺ relative value induced by a vaccine against foot-and-month disease and the mentioned adjuvants. The results showed that the recombinant plasmids significantly increased the level of antibody against foot-and-mouth disease (P<0.01)， and the adjuvant effect of pcDNA3.1/IFN-γ was much higher than that of pcDNA3.1/IL-4 concerning the antibody level of Serotype AsiaⅠ(P<0.01)，whereas the adjuvant effect of pcDNA3.1/IL-4 was more obvious than that of pcDNA3.1/IFN-γ concerning the antibody level of Serotype O(P<0.01). Relative values of CD4⁺/CD8⁺in groups pcDNA3.1/IFN-γ and pcDNA3.1/IL-4 were statistically significantly higher than that of the other groups(P<0.01)， and that on 21 d post immunization was statistically significantly higher than both on 7 and 45 d(P<0.01).

Construction and Identification of Recombinant Porcine Parvoviruslike Particles Formed by the Hybrid VP2 Protein Carrying Immunoreactive Epitope of Porcine Circovirus Type 2

PAN Qun-xing;HE Kong-wang;WANG Yong-shan;WEN Li-bin;MAO Ai-hua;GUO Rong-li

2008, 39(9): 1245-1250. doi:

Abstract ( 817 )

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The gene encoding immunoreactive epitops, residues 165 to 200 from the PCV2 capsid protein, was cloned into VP2 gene from porcine parvovirus. Then the recombinant porcine parvovirus-like particles(PPV-VLPs) was acquired by adenovirus vector system. After transfection， recombined virus (rAd-PPV∶VP2-PCV2∶Cap) was developed. The specific 64 kD band was showed by analysis of Western-blotting and specific fluorescence was observed by IFA which indicated that chimeric PPV VP2 protein had expressed in HEK-293 cells. Moreover， PPV:VP2-PCV2:Cap protein was to be similar to PPV in haemagglutinating activity. It was noteworthy that many self-assembled virus-like particles \[PPV:VLP(PCV2)\] were found in VP2 crude extracts by electronmicroscope.

Cloning and Expression of floR Gene from Bovine Pathogenic E.coli and the Preparation of Its Polyclonal Antibody

WU Bei-bei;SHEN Jian-zhong

2008, 39(9): 1251-1255. doi:

Abstract ( 885 )

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For expressing the recombinant GST-FloR1 protein in prokaryote expression system, floR gene was cloned into pGEX-4T-2， and BL21(DE3) codon plus was chose as corresponding expression hosts. The floR1 gene could be successfully expressed in the systems， by optimizing the IPTG induction concentration， incubation temperature and time. The method of tritonurine with sonication lysis of cells was chose to get the relatively good washing effect. After the protein refolding of denaturant inclusion body following dialysis， we got the pure recombinant GST-FloR1 protein by GST affinity columns. A murine anti-FloR antibody was produced by using the recombinant protein of GST-FloR1 as antigen. The antibody lays the foundation for further research on location of FloR protein and inhibition of drug efllux function.

The Construction and Morphology Observation of Three-dimensional Uterine Tissue

CHEN Xiu-li;ZHAO Yong-zhen;YIN Bao-ying;ZHANG Yan-ming

2008, 39(9): 1256-1261. doi:

Abstract ( 792 )

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Three-dimensional uterine tissue was reconstructed in vitro，which was composed of 3 kinds of cells. Rabbit endometrial cells and smooth muscle cells were successfully isolated and cultured in vitro. Smooth muscle cells less than 3 generations ( 1×10⁷ cells/mL) were mixed with the mixture of 3 mg/mL collagen Ⅰ solution derived from mouse tail tendon and Matrigel gel（4∶1)， and were inoculated quickly into self-made moulds， the cellgel mixture solidified for 30 min in 5% CO₂ at 37 ℃. Endometrial stromal cells ( 1×10⁷cells/mL) were seeded in the mixture by the same ways， the suspension of endometrial epithelial cells was inoculated on the gel when it was solidified; The tissue were cultured in 5% CO₂ at 37 ℃.HE staining and immunohistochemical staining were used to analyze and identify the threedimensional uterine tissue. The results indicated that three-dimensional uterine tissue was successfully reconstructed in vitro， which was composed of three kinds of cells and whose structure was similar to native uterine tissue. HE staining analyses showed that reconstructed uterine tissue was stretched along the stretching direction on 10 d， cells moved freely in extracellular matrix; The epithelial layer showed curving. Immunohistochemical staining revealed that reconstructed uterine tissue distributed three layers of cells; Cells of coiled epithelial layer showed column aspect. Obviously， endometrial cells and smooth muscle cells successfully grow in extracellular matrix; The cultured system of three dimensional uterine was reconstructed in vitro， three-dimensional uterine tissue was composed of three kinds of cells and similar to native uterine tissue. The established model is feasible for research on the mechanism of embryo implantation.

Effects of 17-β-Estrogen on Expressions of Bcl-2 and Bax Protein in Pituitary of Ovariectomized Rats

LONG Min;ZHAO Hui-ying;CHEN Shu-lin;LU Yong-xin;YANG Jing;ZHOU Xu;PENG Zheng-wu

2008, 39(9): 1262-1266. doi:

Abstract ( 852 )

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In order to study the effects of estrogen on development and apoptosis of the pituitary cells， immunohistochemical SP method was used to detect the expressions of Bcl-2 and Bax in pituitary of rat that ovariectomized and treated with 17-β estrogen in different periods. The results showed that the expressions of Bcl-2 and Bax of the group sham operated (SHAM) in different periods had no remarkable change (P>0.05). The group ovariectomized (OVX) had significantly difference with the group SHAM and the group ovariectomized and treated with 17-β-E2 (OVX+E2)within the same stage (P<0.05). The expression of Bcl-2 in the sixth week after injection had a little increase than that before， and the Bax decreased. In terms of the eighth week， no remarkable difference was observed with the sixth (P>0.05). The results indicated that estrogen had certain regulative effect on the structure maintaining and function educing in pituitary.

Effect of Estrogen on Structure of Mucosal Epithelium and Number of Intraepithelial Lymphocytes and Goblet Cells in Small Intestine of Rat

YAN Jin-kun;CHEN Yao-xing;WANG Zi-xu

2008, 39(9): 1267-1271. doi:

Abstract ( 850 )

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To explore the effect of estrogen on the intestinal mucosal barrier function of the rat， eighteen female Wistar rats were induced oestrous synchronizotion and then were divided into three groups in the dioestrus: Control group (Shame operation)， OVX group (Ovariectomy)， and OVX+E₂ group (Ovariectomy and injecting with 100 μg/kg·d estradiol benzoate). After a week， the structure of mucosals epithelium and the number of intraepithelial lymphocytes and goblet cells were observed by light microscope. The results showed that in the OVX group， the villus was atrophic， the villus height and the ratio of villus height to the depth of crypt decreased， and the number of intraepithelial lymphocytes and goblet cells decreased too. There were significant differences between control group and OVX group(P＜0.05)， and between OVX group and OVX+E₂group(P＜0.05). There were also differences between control group and OVX+E₂group， but some indexes between them were not significant. This study indicated that the intestinal mucosal barrier function was greatly damaged in ovariectomy rat， and proper dosage of estradiol benzoate (100 μg/kg·d) would improve the small intestinal mucosal barrier function of ovariectomy rat to some degree.

Regulating Effect of Sulfated Total Saponin of Panax Ginseng on the Activity of Peripheral Lymphocyte of Chicken Infected with MDV

WANG Lu;WANG Chun-yuan;FU Ben-dong;CHU Xiu-ling;YI Peng-fei;SHEN Hai-qing;WEI Xu-bin

2008, 39(9): 1272-1277. doi:

Abstract ( 798 )

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Eight-day-old chickens were injected peritoneally with Marek’s disease virus (MDV) to establish MD model， and the effects of sulfated Total Saponin of Panax Ginseng(s-TSPG) on the number of leucocytes， proliferation of lymphocytes， and the expression level of IFN-γ mRNA in peripheral blood lymphocytes by semi-quantitative reverse transcription-PCR were evaluated besed on the Model at 15 days postinfection. The results showed that the percentage of lymphocytes in chicken infected by MDV was relatively increased (P＜0.01)， s-TSPG and TSPG did not change the increasing level (P＞0.05)， and there was no difference between s-TSPG and TSPG (P＞0.05). The lymphocyte proliferation were significantly depressed in chicken infected by MDV (P＜0.01)， and TSPG improved this suppression (P＜0.01)， while s-TSPG promoted this suppression (P＜0.01). The expression levels of IFN-γ mRNA from lymphocytes induced by MDV were enhanced by s-TSPG and TSPG， and the expression level of IFN-γ mRNA enhanced by s-TSPG was significantly higher than that of the control group (P＜0.01). These data suggested that the regulating effect of s-TSPG on the activity of peripheral lymphocyte of chicken infected by MDV is higher than that of TSPG.

Study on RAPD and SCAR Markers for Heat-tolerance in Holstein

SU Guang-hua;HU Xiong-gui;YAN Hai-feng;XIAO Bing-nan;ZHANG Yuan-yue;

2008, 39(9): 1278-1284. doi:

Abstract ( 1582 )

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The heat-tolerance of the Chinese Holstein (52 herds high yield and heattolerance, together with 41 herds high yield and heat-sensitivity) was studied using RAPD-PCR ( 200 RAPD primers). The results indicated that the 581 bp (primer S441) and 680 bp (primer S463) fragments were found in heat-tolerance group. The homology between 581 bp fragment and KCNN2 gene got to 89%, as well as the homology between 680 bp fragment and NW_001024067.1|BtUn_WGA9442_2 got to 99%. According to its function it seems that the KCNN2 gene was a candidate of heattolerance gene. The 581 and 680 bp fragments were submitted to GenBank and the accession numbers of EF123743 and EF123744 were got, respectively. The ORF was not found in 581 bp fragment, however, 4 ORFs （ORF1, coding 48AA; ORF2, coding 34AA; ORF3, coding 45AA; ORF4, coding 34 AA）in 680 bp gene fragment were found. Using the sequence of RAPD-S441 and RAPD-S463, together with the SCAR primer designed by Premier 5.0 software, the RAPD-S441 marker (581 bp) was converted to SCAR marker.

Detection of Bovine Leukocyte Adhesion Deficiency (BLAD) and Haplotype Analysis

LI Yan-hua;HAN Guang-wen;ZHANG Sheng-li;LI Ning;SUN Feng-jun

2008, 39(9): 1285-1288. doi:

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Bovine leukocyte adhesion deficiency （BLAD） is an autosomal recessive disease caused by A→G mutation in the CD18 gene. The disease is characterized by greatly reduced expression of the heterodimeric β2 integrin adhesion molecules on leukocytes， resulting in multiple defects in leukocyte function and significant deficits on performance traits in Holstein cattle. In this study， primers were designed to amplify the exon2 fragment of CD18 gene and BLAD was detected by PCRSSCP. There were four different haplotypes which were H1， H2， H3， and H4， respectively. Two SNPs were confirmed in the amplification fragment by DNA sequencing. C20T mutation of the exon2 is silent mutation which is novel and A55G is missense mutation which induces BLAD. Haplotype H1， H2， H3 are normal while haplotype H4 is BLAD carrier. Our aim is the identification of BLAD carriers， and therefore this study provides a more reliable and useful method for extensive screening of BLAD.

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