Acta Veterinaria et Zootechnica Sinica

Effect of Interleukin6 on Lipolysis in Porcine Adipocytes

YANG Yongqing;YANG Gongshe

2009, 40(8): 1131-1138. doi:

Abstract ( 780 )

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Abstract:The purpose of the study was to explore the effect of interleukin (IL)6 on lipolysis in porcine adipocytes and the underlying molecular regulation mechanism. Porcine primary adipocytes were cultured and incubated with different concentrations of IL6 (0, 25, 50, 75 and 100 ng·mL1) for 24 or 48 h. Then, the glycerol released into the media was measured as an indicator of the lipolysis. Lipid accumulation morphology was visualized by phasecontrast microscopy. Further, gene expressions of perilipin A, peroxisome proliferatoractivated receptorgamma2 (PPAR γ2), hormonesensitive lipase (HSL) and adipose triglyceride lipase (ATGL) were analyzed with semiquantitative RTPCR, and their protein level were determined with Western blot. The results showed that IL6 stimulated lipolysis in differentiated porcine adipocytes in a dose and timedependent manner. Meanwhile, the mRNA and protein level of PPAR γ2 and perilipin A were significantly reduced. Although the expression abundance of HSL mRNA was significantly elevated, its protein level had no marked changes. The mRNA and protein level of ATGL were not affected. These findings imply that chronic high dose of IL6 directly stimulates lipolysis in porcine adipocytes through repressing PPAR γ2 and its target gene perilipin A.

Study on the Nucleus Materials of Porcine First Polar Body Participating in Fertilization of Oocyte and Embryonic Development

WANG Gongjin;TAN Xiaodong; ZHOU Xiaolong; YU Jianning; XU Xiaobo

2009, 40(8): 1139-1144. doi:

Abstract ( 772 )

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The aim of the present study was to explore whether the nuclei materials of first polar body (FPB）in porcine oocyte could participate in fertilization and further developed into normal porcine embryo in vitro. The FPBs in MⅡoocytes were obtained by in vitro maturation of the Cumulus oocyte complexes (COCs) from ovary follicles. Live FPB was injected into an enucleated oocyte recipient by micromanipulation after it was isolated from mature oocyte. The oocytes reconstructed by FPB were fertilized by routine in vitro fertilization (IVF) or intracytoplasm sperm injection (ICSI), the recombinant zygotes derived from FPBs were cultured in vitro to observe their ability of further developing in vitro. The results showed that successful rate of recombinant oocytes was as high as about 80%, only about 20% recombined oocytes were degenerated or disaggregated during the process of short culture and fertilization in vitro. Some of recombinant oocytes were successfully developed into twocell, fourcell and eightcell stage embryos, the cleavage rates of twocell stage embryos were 12.9% and 29.8% after routine fertilization and ICSI, respectively. The conclusion was that the nuclei materials of porcine FPB could be able to participate in fertilization and further embryonic development. This might imply the possibility of reproducing normal porcine offspring by using FPBs in future.

Tandem Inhibin Gene Immunization to Induce Sheep Twinning

GUO Xian;YUE Yaojing;JIAO Shuo;CAO Xueliang;FENG Ruilin;ZHAO Xingxu

2009, 40(8): 1145-1149. doi:

Abstract ( 743 )

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To study the effect of tandem inhibin gene immunization on sheep twinning, the recombinant plasmid of tandem inhibin were constructed with inhibin αsubunit (1～32) of pig and complement 3d of sheep by RTPCR, which were used to immunize the sheep. The results showed that the recombinant plasmid, pcDNADPPISSDINH and pcDNADPPISSDINHsC3d3 were constructed successfully. After BHK21 cells were transfected with the recombinant plasmid pcDNADPPISSDINH and pcDNADPPISSDINHsC3d3, secreted expression was obtained. After immunization, the twinning rate of sheep was 12.5% and 25.0%, respectively, which was significantly higher (P＜0.05) than that in the control group. The construction of recombinant plasmid of tandem inhibin gene made the theoretical and technical basis for developing the inhibin gene as vaccine for sheep.

Comparison of the Ovine Sperm Capacitation and Fertilization in vitro among the Different Concentrations of Serum Collected from Their Different Estrous Stages

LIU Lieqin;LIU Yunhai;GUO Yong;YANG Feige;NI Hemin

2009, 40(8): 1150-1156. doi:

Abstract ( 744 )

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Abstract:This study was aimed to provide some experimental references for improving the in vitro fertilization system of sheep. The effect of different ovine serum concentrations during their different estrous stages on their sperm capacitation, enduring durations and fertilization were analyzed. After the basic sperm capacitation medium(Sheep Oviduct Fluid, SOF medium) was supplied respectively with 0%, 2%, 5%, 10% and 20% ovine serum collected during their different estrous stages (the first day of estrus, the last day of estrus, and diestrus ), the effects of their sperm capacitation, enduring durations were compared and analyzed reasonably by CTC（chlortetracycline）staining and appropriate fertilization in vitro．The results showed as following:①While the SOF medium was supplied with the ovine serum collected on the first day of their estrus, its best effect was initiated by treating for 45 min with 10% and 20% serum. Their sperm rates (45.28％ and 44.52%) by capacitation but without acrosome reaction(AR) were significantly higher than those among the other groups treated with 0%, 2% and 5% serum, respectively (30.14％, 28.71％ and 35.49％，P<0.05); ②While the SOF medium was supplied with the ovine serum collected on the last day of estrus and diestrus, there were no significant differences of the ovine sperm capacitation percentage among all the groups treated for 45 minutes（P>0.05）; ③While the SOF medium was supplied with 20% ovine serum collected on the first day of their estrus, enduring duration of sperm (18 h)was much longer than that of the other two groups with 20% serum collected on their last day of estrus or diestrus respectively(11 h, 11 h); ④Following their appropriate fertilization in vitro, the cleavage and blastocyst rates（62.5％ and 26.25％）in the group treated with the serum collected on the first day of their estrus, were significantly higher than those in the other two groups treated with the serum collected on the last day of estrus（42.22％ and 11.11％，P< 0.05）or diestrus（37.36％ and 15.38％，P< 0.05）,respectively. It can be recommended that the serum be applicable for improving ovine IVF system, while the sheep sperm is treated for 45 minutes by SOF medium with 20% ovine estrous serum collected on the first day of their estrus.

Cloning and Expression of Porcine MEF2a Gene

GAO ZhiSheng;;YANG Long;ZHANG DongJie;LIU Di

2009, 40(8): 1157-1163. doi:

Abstract ( 1111 )

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In order to understand the structure and function of porcine MEF2a gene,porcine MEF2a gene was cloned by bioinformatics and RTPCR,and two variant sequences were obtained. Variant 1 (1 524 bp, EF576923) encodes 505 amino acids which is 54.481 ku. Variant 2 (1 416 bp, EF576922) encodes 471 amino acids which is 50.846 ku. Amino acid sequence homology between MEF2a variants is 86.79%. It is completely identical in 1st–77th and 132th -470th amino acids, and is hypervariable in 78th-131th region. Variant 2 has 34 amino acids (102 bp) deletion in 224th-258th compared with variant 1. MEF2a mRNA is a broadspectrum expression gene. It was detected in heart, liver, stomach, spleen, kidney, lung, large intestine, small intestine, dorsal muscle and leg muscle, especially superior expression in leg muscle by Realtime PCR. It was showed that in phylogentic tree, for variant 1,horse and house mouse was clustered one group and for variant 2,cattle and human was clustered another group. So it was presumed that MEF2a gene undergoes apparent variation during evolutionary process.

Study on Association of Single Nucleotide Polymorphism of MEF2A Gene with Carcass Traits in Chicken

ZHOU Yan;LIU Yiping;JIANG Xiaosong;DU Huarui ;ZHU Qing

2009, 40(8): 1164-1170. doi:

Abstract ( 779 )

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To understand the relationship between SNPs of MEF2A gene and carcass traits in chicken, three SNPs loci in MEF2A gene were detected by PCRSSCP method in 240 Daheng quality meat chicken and its genetic effects on 9 carcass traits were also analyzed. Three polymorphic sites were C/T mutation at nt 46 023, G/A mutation at nt 72 626 and G/T mutation at nt 89 232, respectively. Statistical analysis showed that the three SNPs and diplotypes significantly associated with partial carcass traits (P<0.05 or P<0.01). The multiplerange test indicated that A2A2 chicken of SNP1 had higher live weight, carcass weight, breast muscle weight, breast muscle percentage (P<0.01), abdominal fat weight (P<0.05) than that of A1A1 chicken; B1B1 chicken of SNP2 had higher live weight, carcass weight, breast muscle weight (P<0.01), leg muscle weight and abdominal fat weight (P<0.05) than that of B2B2 chicken; C1C1 chicken of SNP3 had significantly higher semieviscerated percent than that of C2C2 chicken. The LSM of live weight, carcass weight, breast muscle weight and leg muscle weight of diplotype H1H2 were higher than that of all the other diplotypes, while the H5H5 diplotype had the lowest LSM of live weight, carcass weight and breast muscle weight in all the diplotypes. It was concluded that MEF2A gene can be regarded as a useful candidate gene for carcass traits of quantity meat chicken.

Polymorphism of Luteinizing Hormone Betasubunit Gene and Its Relationship with Litter Size of Jining Grey Goats

DI Ran ;LIANGG Chen;CHU Mingxing;LIU Wenzhong;FANG Li;MA Yuehui;LI Kui

2009, 40(8): 1171-1178. doi:

Abstract ( 779 )

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In present study, the luteinizing hormone betasubunit (LHβ) gene was selected as a candidate gene for the high prolificacy in Jining Grey goats. According to the sequence of ovine LHβ gene, six pairs of primers were designed to detect single nucleotide polymorphisms in the 5′ regulatory region, exon 1, exon 2 and exon 3 of ovine LHβ gene in high prolificacy breed (Jining Grey goat), medium prolificacy breed (Boer goat) and low prolificacy breeds (Liaoning Cashmere and Angora goats) by PCRSSCP method. The results showed that the similarity in nucleotide sequence of goat and ovine LHβ gene was 98%, and only the products amplified by primers P1 and P5 displayed polymorphisms. For primer P1, three genotypes (AA, AB and BB) were detected in all four goat breeds. Sequencing revealed that two nucleotide substitutions (202C→A and 210C→T) at the 5′regulatory region of LHβ gene in the BB genotype compared with the AA genotype. The differences of the litter size between different genotypes were nonsignificant in Jining Grey goats（P>0.05）. For primer P5, three genotypes (CC, CD and DD) were detected in Jining Grey goats, two genotypes (CC and CD) were detected in Liaoning Cashmere goats, and only one genotype CC was detected in Boer and Angora goats. Sequencing revealed one single nucleotide mutation (1124C→T) at exon 2 of LHβ gene in the DD genotype compared with the CC genotype. Genotype frequency of CC, CD and DD was 0.38, 0.44 and 0.18 in Jining Grey goats, respectively. The Jining Grey does with genotype DD or CD had 0.99 (P<0.01) or 0.87 (P<0.01) kids more than those with genotype CC. The fitness tests showed that the Jining Grey goat population was in HardyWeinberg equilibrium (P1 locus:χ2=1.66, P=0.437; P5 locus:χ2=1.22, P=0.544).

Genetic Diversity and Origin of Gansu Indigenous Sheep Breeds Determined by Mitochondrial DNA Dloop Sequence

CHENG Shuru;;LUO Yuzhu;;HAN Jianlin;;OLIVIER Hanotte

2009, 40(8): 1179-1185. doi:

Abstract ( 819 )

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To explore the genetic diversity and origin of Gansu indigenous sheep breeds determined by mitochondrial DNA Dloop sequence, one pair of primers was designed to amplify the mtDNA Dloop sequence of sheep. By sequencing we got the basic sequencing data. Then the haplotype, genetic diversity, phylogenetics, and Network analysis were carried out. The results showed that 3 sequences were 573 bp length, 117 sequences are 648 bp length. 77 haplotypes are identified in 117 sequences. Through analysis, it is found that haplotype proportion, haplotype diversity, nucleotide diversity and average number of nucleotide difference of Lanzhou Fattailed sheep and Minxian Black Fur sheeps were very low, but it was very high in Mongolian sheep. The phylogenetic and network analysis demonstrated that Gansu 6 sheep breeds were divided into 3 clusters. So we concluded that 4 repeat units was the characteristic of Gansu sheep mtDNA Dloop sequence. The genetic diversity in Mongolian sheep is the highest, and it is the lowest in Lanzhou Fattailed sheep and Minxian Black Fur sheep. Phylogenetic and network analysis of the 77 haplotypes identified in Gansu 6 sheep breeds supported that there are 3 maternal origins in Gansu local sheep breeds.

Association between the Polymorphism of GHR and Milk Production Traits in Chinese Holstein Cows

MA Yan;JIA Jin;ZHANG Yi;SUN Dongxiao;ZHANG Yuan

2009, 40(8): 1186-1190. doi:

Abstract ( 793 )

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PrimerIntroduced Restriction AnalysisPolymerase Chain Reaction （PIRAPCR） and Restriction Fragment Length Polymorphism（RFLP）were developed to identify the F279Y mutation within the exon8 of GHR gene. Further, the association between F279Y and five milk production traits were investigated in a population of 1 145 Chinese Holstein cows. Statistical results indicated that 305-day milk yield and protein percentage（P<0.01）and fat percentage（P<0.05）was significantly associated with the F279Y mutation of GHR, respectively. The results of Bonferroni ttest showed that the difference between genotypes TT and AT were significant for 305day milk yield and fat percentage（P< 0.05）. Also, the difference between genotypes TT and AT was significant（P<0.01）for protein percentage and the difference of protein percentage between genotypes TT and AA was significant（P<0.05）. The additive effect of A allele for protein percentage was -0.025 8%（P< 0.01） and dominance effects were -214.005 （P<0.01）and -5.515 kg（P<0.05）for 305day milk yield and protein yield. The substitution effects for fat percentage and protein percentage were -0.047 5% （P<0.05） and -0.029% （P<0.01）, respectively. Therefore, allele T is the dominant allele for milk composition, while allele A is the dominant allele for milk yield. Our findings in the present study implied that F279Y mutation of GHR gene could be of considerable in marker-assisted selection programs of milk production traits in Chinese Holstein cows.

Effects of Enzyme Predigested Feed on Growth Performance and Nutrients Apparent Digestibility in Weaned Piglets

ZHANG Shiyuan;YU Lu;WANG Yaqian;ZHU Peiji;XIE Yuehua;LI Haibin;WANG Dong

2009, 40(8): 1191-1195. doi:

Abstract ( 786 )

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To study the effects of enzyme predigested feed on growth performance and apparent digestibility of nutrients in weaned piglets, eighty (Duroc×Landrace×Yorkshine) 28 old age of weaned piglets were randomly divided into control (group Ⅰ), no enzyme addition predigesting group(group Ⅱ), common enzyme addition group(group Ⅲ), enzyme addition predigesting group(group Ⅳ) with 2 duplicates in each group which had 10 piglets each. The experimental period included 7 days of preliminary trial and 28 days of test trial. The results showed that daily gain and feed conservation rate was increased in group Ⅳ compared with groupⅠ and Ⅱ (P<0.05); Pigs fed enzyme predigested diets have significantly lower diarrhea rate than that of groupⅠ, Ⅱ and Ⅲ(P<0.01). Apparent digestibility of energy and calcium was also higher in group Ⅳ than that in groupⅠ, Ⅱ(P<0.05) and group Ⅲ (P>0.05). The apparent digestibility of phosphorus was increased significantly in group Ⅳ compared with groupⅠ, Ⅱ(P<0.01) and Ⅲ (P<0.05), there was no difference for apparent digestibility of crude protein among all groups. It was suggested that the enzyme predigested feed was effective to improve digestibility performance of nutrients and growth of weaned piglets.

Effect of γ-Aminobutyric Acid on Growth Performance, Antioxidation and Hormones of HPA, HPT Axis in Growthfinishing Swine in Hot Summer

ZOU Xiaoting;HU Jiacheng; CAO Derui;DONG Jinge

2009, 40(8): 1196-1201. doi:

Abstract ( 835 )

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A study was conducted to determine the effects of feeding γaminobutyric acid (GABA) on the growth performance, antioxidation and hormones of HPA, HPT axis in growthfinishing swine in the hot summer. 96 (Duroc×Landrace×Yorkshine) growthfinishing pigs with an average initial body weight (BW) of (43.07±5.76) kg, were randomly allotted into 4 treatment groups with 3 replications of 8 pigs per replicate pen for each treatment in 48 days trial. The treatments were designed in a single factorial arrangement, and were as follows: basal diet without GABA, basal diet with 10 mg·kg1 GABA, basal diet with 20 mg·kg1 GABA, and basal diet with 40 mg·kg1 GABA. The results showed that growth performance of growthfinishing swine was affected in all treatments. 10 mg·kg1 was the best dosage which significantly increased the average daily gain (ADG), feed/gain (F/G) by 13.08% and 7.81% ,respectively (P<0.01). However, average daily feed intake (ADFI) was not affected in every treatment. In addition, 10 mg·kg1 GABA increased the activity of GSHPx (P<0.01), the activity of SOD was increased and MDA concentration was decreased (P>0.05). The concentration of serum cortisol, corticosterone and ACTH were all decreased too (P<0.05). 10 mg·kg1 GABA obviously promoted the secretion of T3 and FT3 (P<0.05) but had no obvious effect on the secretion of T4 and FT4 (P>0.05). In conclusion, GABA can effectively improve the growth performance of growthfinishing swine in the hot summer when they were added into diet, 10 mg·kg1 was the optimal dosage under this condition. 10 mg·kg1 GABA also mitigated stress induced by high temperature in the summer and enhanced antioxidation performance and thyroid function.

Localization of a Highly Pathogenic Avian Influenza Virus，A/duck/Guangdong/220/2004（H5N1）in Experimentally Infected Ducks

LI Yugu;YE Yuanlan;ZHOU Quanhe;CUI Congying;ZHANG Yuan;MA Yongjiang;LI Chuxuan

2009, 40(8): 1202-1208. doi:

Abstract ( 805 )

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Using streptavidinbiotin complex immunoperoxidase staining, the tissue localization and distribution of a highly pathogenic avian influenza virus, A/duck/Guangdong/220/2004(H5N1) were investigated in ducks inoculated by intravenous and ocularnosaloralcloacal routes. The results showed that the viral nucleoprotein antigen was most often detected in the cardiac muscle cells, hepatocytes, Kupffer cells, pancreatic acinar epithelial cells, renal tubular epithelial cells, vascular endothelial cells and phagocytic leucocytes, and occasionally in the mucosa epithelium of trachea, bronchia and intestinal tract, encephalon, and macrophages, histiocytes and necrotic cellular debris of thymus, bursa of Fabricius, spleen and lung. But no viral nucleoprotein antigen was detected in the oesophagus, stomach, testis, ovary, thyroid glands, adrenal glands, Harderian glands and skeletal muscles.

Complete Genome Sequencing and Variations Analysis of Seven Isolated Strains and One Vaccine Strain of Duck Hepatitis Virus

MA Xiuli;YU Kexiang;WU Jing;SONG Minxun;LIAO Ming;XIN Chaoan

2009, 40(8): 1209-1214. doi:

Abstract ( 742 )

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This experiment was conducted to study the genetic variations and phylogenetic analysis of Duck Hepatitis Virus（DHV） in China. The complete nucleotide sequences of 7 isolated strains and 1 vaccine strain of DHV1 had been determined and comparative genome analysis with other DHV strains available in GenBank was performed. The results showed that the VP1 was the most variant gene and hypervariable regions were found in 180194aa and 213219aa of VP1 protein. Variant NLinked glycosylation sites were found in different strains. Phylogenetic and evolutionary analysis showed that our sequencing strains belonged to genotype A, which were on different branches from genotype B and C. Strain JYZ02 had a very close relationship with strain R85952 which suggested that strain JYZ02 was evolved from strain R85952. The results of amino acid sequence identity and virus neutralization test proved that there was no antigenic variation occurred in the seven strains we determined. Phylogenetic analysis revealed that the dominant serotype of epidemic DHV in China was type 1 at present.

Mixed Infection of ALVJ and MDV in a Flock of Shandong Free Range Chickens

ZHANG Honghai;LIU Qing;QIU Bo;LIU Gongzhen;CHENG Ziqiang

2009, 40(8): 1215-1221. doi:

Abstract ( 726 )

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An outbreak of tumors diseases occurred in Shandong free range chickens on a commercial farm of Shandong Province in China during September 2008. The flock containing 6 000 chickens, and the symptoms were first seen at 80dayold. The mortality reached 15% at 120dayold. The main symptoms were characterized by emaciation, ruffled feathers, paralysis and visceral tumors. Some birds showed unilateral limb paralysis and skin tumors. 66 serum and feather samples were collected from the sick chickens to evaluate the presence of antibodies to MDV, ALVJ and REV using agar diffusion reaction and enzymelinked immunosorbent assay (ELISA) technique. The results of agar diffusion reaction showed that the MDV antigen positive rate was 59.09%, and MDV antibodies positive rate was 65.16%, antigenantibodypositive rate was 37.88%. The overall seroprevalence of ALVJ antibodies was 7.58%, while the antibodies to REV were negative. Numerous similar nodular lesions and resembling tumors in one or more tissues were seen at necropsy. Some organs showed intumescence. Histopathological findings revealed large islands of leukomonocytes or myelocytes in tissues containing tumors. Immunostaining for MDV, ALVJ and REV monoclonal antibody revealed a diffuse presence of MDV and ALVJ antigen. PCR analysis revealed the presence of MDV and ALVJ proviral sequences in DNA of affected tissues. All the findings observed in this case were considered to be related with the MDV and ALVJ mixed infection. Prevention and decontamination to these diseases in local free range chickens should be noticed.

Development of a Rapid Multiplex PCR for Simultaneous Detection of Five Major Pathogenic Bacteria of Respiratory Tract Diseases in Swine

XUE Yun;ZHAO Zhanqin;CHEN Yang;ZOU Haoyong;HE Qigai;LI Wenjie;XIONG Jinfeng;CHEN Huanchun

2009, 40(8): 1222-1228. doi:

Abstract ( 1197 )

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Actinobacillus pleuropneumoniae (App), Haemophilus parasuis (Hps), Bordetella bronchiseptica (Bb), Toxigenic Pasteurella multocida (T+Pm) and Mycoplasma hyopneumoniae (Mhp) are five kinds of the most common pathogenic bacteria which can cause a series of respiratory tract diseases in swine. Five pairs of specific oligonucleotide primers were designed according to the DNA sequences of App apxIV, Hps 16S rRNA, Bb fla, toxigenic T+Pm toxA and Mhp ldh reported in NCBI website. A multiple PCR detection method was established according to improved systems and programmes on the basis of five single PCR methods. No cross reaction was detected by the multiplex PCR method between these five bacteria templates and other common bacteria templates. The sensitivity of multiple PCR was lower than 160 pg genome template. Using this multiple PCR method, bacteria could be detected from lung tissues of challenged mice with App, Hps, Bb and T+Pm respectively after being cultured for 8 h in Tryptic Soy Broth (TSB) supplemented with 5% calf serum and NAD+ factor. Simultaneous detection of pathogens in 269 clinical samples from the central area of China revealed the universal existence of App, Hps, Bb, T+Pm and Mhp. In conclusion, the multiple PCR assay showed excellent specificity, sensitivity and reduplication and could detect five pathogenic bacteria of respiratory tract in swine. We expected that this multiple PCR assay could be useful extensively for epidemiological survey and clinical differential diagnosis of five common respiratory diseases in swine.

Study on Pharmacokinetics of Two Effective Components from Andrographis Paniculata Ultrafine Powder in Chickens

LIU Kaiyong;HUANG Xianhui;GAO Hai;;HE Limin;JIANG Zhigang;ZENG Zhenling

2009, 40(8): 1229-1234. doi:

Abstract ( 741 )

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The pharmacokinetic features of andrographolide and dehydroandrographolide, two effective components of Andrographis paniculata, were investigated in chickens administrated with ultrafine powder of A. paniculata in the paper. The two compounds in chicken plasma were determined simultaneously by HPLC method following oral administration of Andrographis paniculata ultrafine powder at a single dose of 5 g·kg1 B.W., the pharmacokinetic process were evaluated by Pharmacokinetics Program 3P97. The results showed that the plasma concentrationtime data of andrographolide and dehydroandrographolide in chicken plasma could be described by two compartment model with firstorder absorption, with t1/2α of (47.4±3.5 )and (41.9±3.9) min，t1/2β of (104.8±12.8 ) and (143.0±12.8) min，t1/2Ka of (23.2±2.6 )and (9.77±1.22) min，AUC of (329.3±28.8) and (329.1±25.5) (μg·mL1)·min，tmax of (51.5±3.5) and (37.2±2.8) min，Cmax of (1.60±0.32 )and (1.44±0.09) μg·mL1，MRT of (175.2±15.9) and (219±18 )min for them respectively. The pharmacokinetic rule of the ultrafine powder could be reflected by kinetic process of the two constituents in chickens, between which significant or extremely significant difference exited in absorbtion and elimination stage, and the study could provide references for the clinical application of the powder preparation of Andrographis paniculata, also do for the pharmacokinetic study on multiconstituents from Chinese traditional medicine.

Effect of Dietary High Fluorine on the Morphologic Structure, Cell Cycle and Apoptosis of bursa of Fabricius in Broilers

CHEN Tao;GONG Tao;BAI Caimin;PENG Xi;CUI Hengmin

2009, 40(8): 1235-1243. doi:

Abstract ( 791 )

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The experiment was conducted to study the effects of high fluorine on the bursa of Fabricius in chickens by the methods of experimental pathology and flow cytometry (FCM). 300 onedayold Avian broilers were divided into four groups and fed with Control diet(F 23 mg·kg1) or High fluorine diets(F 400 mg·kg1, High fluorine group Ⅰ; F 800 mg·kg1, High fluorine group Ⅱ; F 1 200 mg·kg1, High fluorine group Ⅲ) for 6 weeks. The results showed that the weight and the relative weight of bursa of Fabricius were decreased（P<0.01 or P<0.05）, and lymphocytes were histopathologically decreased in number in high fluorine group Ⅱ and high fluorine group Ⅲ. The G0/G1 phase of high fluorine groups Ⅱ and Ⅲ rised significantly（P<0.05）, while the G2+M phase and the PI(Proliferating index) were decreased at 14 days of age. The S phase and apoptosisrates of high fluorine groups Ⅱ and Ⅲ were abnormally increased（P<0.01） at 28 days of age. The G0/G1 phase was significantly increased（P<0.01） and S phase,G2+M phase and PI decreased（P<0.01） in high fluorine groups Ⅱ and Ⅲ at 42 days of age. Also, the percentage of cellular apoptosis was higher in high fluorine groups Ⅱ and Ⅲ than in control group at 42 days of age. Typical morphological changes of apoptotic cells and some swelled mitochondria of the bursa of Fabricius in high fluorine group Ⅱ and high fluorine group Ⅲ were ultrastructurally observed. It is concluded that fluorine exceed 800 and 1 200 mg·kg1 in diet can cause differentiation and proliferation retardation of lymphocyte in the bursa of Fabricius and increased apoptotic cells, which leads to inhibition on the development of the bursa of Fabricius and impaired the function of humoral immunity in chickens.

Effects of the Combined Anaesthetic for Miniature Pigs on Hemodynamics and ReninAngiotensinAldosteroneSystem in Miniature Pigs

FAN Honggang;LU Dezhang;HU kui;ZHANG Jiantao;LI Jing;WANG Hongbin

2009, 40(8): 1244-1248. doi:

Abstract ( 819 )

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In order to investigate the effects and mechanisms of the Combined Anaesthetic for Miniature Pigs(CAMP) on the hemodynamics in miniature pigs, fourteen Chinese laboratory miniature pigs were injected with the CAMP (0.15 mL·kg1). Precaval vein blood was collected at 0, 5, 10, 30, 45, 60, 80, 100 and 120 min before and after administration, and noninvasive blood pressure（NIBP）and heart rhythm（HR）were monitored at the same time. Plasma renin activity（PRA），angiotensin Ⅱ（AⅡ）and aldosterone（ALD）were determined by radioimmunoassay. Results showed that NIBP and HR were significantly changed at 5—10 min and 80 min（P<0.01 or P<0.05）. Changes of PRA, AⅡand ALD were similar to that of SBP, DBP, MAP and HR, and correlation among them was found, especially in SBP, DBP and MAP. It was concluded that PRA, AⅡand ALD participated in changing of hemodynamics caused by the CAMP, and ReninAngiotensinAldosteroneSystem might be one of the main factors to change hemodynamics.

Effect of the Various Concentration of Azone and Menthol on the in vitro Percutaneous Penetration of Terbinafine through Excised Canine Epidermis

CAI Wenhe;LIN Degui

2009, 40(8): 1249-1252. doi:

Abstract ( 702 )

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This experiment was conducted to study permeationenhancing effect of various concentration of Azone and menthol on the in vitro percutaneous penetration of terbinafine(TBF) through canine epidermis. Modified Franz vertical glass diffusion cells were used and excised canine epidermis was chose as transdermal barrier. The in vitro permeation effects of every combination of TBF with various concentration enhancers were evaluated through calculating and comparing the cumulative penetration amount, steadystate flux, permeability coefficient and enhancement ratio. The results revealed that various concentration of enhancers had the permeationenhancing effect, the sequence of effect was 1%AZ**>5%MT**>3% AZ**>3% MT**>1%MT*>5%AZ*. The linear correlation between the effect of combinations of TBF with menthol and the concentration of menthol was positive, while the linear correlation between the effect of combinations of TBF with Azone and the concentration of Azone was negative. Though all combinations of TBF with enhancers had the enhancement effect, the effect of menthol was better than that of Azone. Besides, the menthol is a natural product with low toxicity and side effect, so it is a preferable enhancer for the external preparation.

Expression, Purification of Sheep Myostatin Cdomain in E.coli and Preparation of Its Polyclonal Antibody

MA Yi;SHU Jianhong;LI Qing;GUAN Hong;AN Xiaorong;CHEN Yongfu

2009, 40(8): 1253-1257. doi:

Abstract ( 750 )

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To further understand the biological functions of myostatin in sheep and its molecular mechanism, both of the recombinant myostatin protein and its polyclonal antibody are indispensable. The Cdomain of sheep myostatin gene was amplified and cloned into the expression vector pET30ac(+). After induced by IPTG, sheep myostatin was expressed in E.coli. The recombinant protein was further purified by Ni2+ affinity chromatography and its purity and content were detected by thinlayer scan analysis and Bradford assay, respectively. Thinlayer scan showed that the purity of recombinant protein reached at least 95%. Bradford assay showed the content was about 5 mg·mL1. Rabbits were immunolized with purified recombinant myostatin. The titer of polyclonal antibody was detected by indirect ELISA assay. The specificity of polyclonal antibody was evaluated not only by Western blot but also Cytochemical stain.The titer of antiserum was as high as 1250 000, with very high specificity proved by Western blot and Cytochemical stain. The expression of endogenous myostatin was detected in myoblast by the polyclonal antibody. These results showed that recombinant protein of sheep myostatin Cdomain with high purity and its specific polyclonal antibody were obtained, and this polyclonal antibody could be used in future research work.

The Tendency of Mitochondrial distribution during Maturation in vitro of Ovine Oocytes

QU Yangyan;LIU Yunhai;GUO Yong;LI Kai;ZHAO Yanwei;LI Xiaoyan;NI Hemin

2009, 40(8): 1258-1261. doi:

Abstract ( 752 )

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To investigate the distribution of mitochondria in ovine oocytes during maturation in vitro,ovine oocytes for the active analysis of mitochondria during their in vitro maturation were directly incubated with GENMED kit and then stained by PI for nuclear staining to observe their distribution tendency by confocal laser scanning microscope. The results showed that the distribution of mitochondria was gradually translocated from the cortex to the perinuclear area of oocytes, and then spread in the cytoplasm thoroughly, as well the chromatosis of mitochondria clusters compressed tightly. It can be concluded that the tendency of mitochondria distribution in ovine oocytes, seem to be a reliable reference for effectively judging the status about the cytoplasm maturation of ovine oocytes matured in vitro.

Screening of Sheep Fibroblast Cell Line with the Spider Dragline Silk Protein Gene

WANG Chunsheng;LIU Xin;YUAN Lu;AN Tiezhu

2009, 40(8): 1262-1265. doi:

Abstract ( 765 )

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According to spider dragline silk protein gene sequence in GenBank(AY555585 and AH015065)，tetrapolymerized silk protein gene(4S) was artificially synthesized. Eukaryotic expression vector was constructed by linking the 4S to pcDNA3.1K2.10(hair folliclespecific promoter). Sheep fibroblasts were transfected with the plasmid by cationic liposome method and filtrated by using G418. After identified, transgenic cell line with spider dragline silk protein gene was established. Results showed that sheep skin fibroblasts were isolated from fresh and were transfected with linearization vector. Using G418 to select cells, G418 resistance cells were gained. G418 resistance cells were identified by PCR method, the result showed that the vector constructed was integrated into sheep genome. At the same time, chromosome of G418 resistance cell was analysed, the result showed that chromosome of these cells were stable and there were no different between these cells and normal cells. The transgenic sheep fibroblast cell line with artificial synthesized spider dragline silk protein gene would be used in the future transgenic sheep study.

Study on SPAPiezoelectric Immunosensor for the Detection of H9 Serotype Avian Influenza Virus

ZHAN Aijun;WANG Xinwei;JIN Xin;CHEN Shukun;LU Tikang;HUA Qunyi;QIN Zhifeng;QIN Aijian

2009, 40(8): 1266-1270. doi:

Abstract ( 1101 )

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By using the high sensitivity of resonance of quartz crystal and specificity of immunoreaction, the gold membrane on quartz crystal electrode was coated with SPA, and the monoclonal antibodies against H9 serotype avian influenza virus(AIV) were coupled with the SPA, then the SPApiezoelectric immunosensor was established and applied for detecting H9 AIV. The detection results showed that the immunosensor displayed better specificity to H9 AIV, and didn’t respond to H5 AIV and NDV. The sensitivity test indicated the detecting limit for the H9 AIV could reach to 20 EID50. The accordance rate between the immunosensor and virus isolation by chicken eggs was 91%. The method provided a foundation for further research on immunosensor for detecting H9 AIV and it could be a new exploration to detect AIV quickly and quantitatively in practical poultry industry.

Variation Analysis of ORF5 Gene of Porcine Reproductive and Respiratory Syndrome Virus Isolates from Guizhou Province

TANG Deyuan; ZHAO Qizu;LI Chunyan ;LU Xia;ZHOU Xingqi;FAN Yunfeng;ZENG Zhiyong;ZHU Yongxing

2009, 40(8): 1271-1275. doi:

Abstract ( 1063 )

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To investigate variation of porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 gene, 153 suspected samples of PRRSV were collected from 16 largescale farms of Guizhou province. With RTPCR detecting and ORF5 gene sequencing,results show that among all these samples the homology of ORF5 gene was up to 98.7%—100%, and the homology of amino acids was up to 98.5%—100%;compared with VR2332，CH1a and LV，the homologies of gene sequence were up to 89.2%—90%, 94.8%—95.6%, 59.7%—60.2% respectively, and the deduced amino acid homologies were up to 88.8%—89.3%, 92.7%—94.2%, 54.9%—55.3% respectively. It is indicates that the epidemic strains are from the same origin and belong to Northern American genotype.The ORF5 gene from the variant can be separated into three clusters. The relationship is closed to the highly pathogenic PRRSV that separated at other parts of China,but alienate from PRRSV strain separated early.Our results indicated that the ORF5 gene of PRRSV existed variation.

Proliferation of Infectious Bursal Disease Virus in the Chicken Blymphocyte Cell Line DT40

LUO Jun;TENG Man;FAN Jianming;XING Guangxu;LI Qiaomu;YANG Yanyan;ZHANG Gaiping

2009, 40(8): 1276-1280. doi:

Abstract ( 733 )

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In the present study, the infectivity and propagation of infectious bursal disease virus (IBDV) in DT40 cells were investigated based on a systematic selection of the optimal maintenance medium for virus infection. The results showed that both of the two tested attenuated IBDV strains, TAD and HN3, could directly infect and proliferate in DT40 cells without adaptive passage. The IBDV particles could be rapidly propagated and continuously harvested at high titers for a long time, accompanying with the rapid growth of DT40 cells. Our data suggest that DT40 could be used as an ideal model for the research of the pathogenetic mechanisms of IBDV. Additionally, it could also serve as a potential system for the preparation of the commercial vaccines against IBDV.

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