Acta Veterinaria et Zootechnica Sinica

Polymorphism Analysis of the Horse GH Gene by PCR-SSCP

2007, 38(3): 209-213. doi:

Abstract ( 757 )

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The genetic polymorphism of 5＇-flanking region, 1st intron, 2nd exon and 5th exon of GH gene were investigated using PCR-SSCP methods in 281 horses from four local Mongolia breeds, one Chinese cultivating breed and one importing breed. The results showed that only the 5th exon had polymorphism that generated AA, BB and AB three genotypes; the rest loci were undetectable. Analysis of population genetics suggested that the frequency of A allele was dominant among the studied breeds except thoroughbred horse.The results of Hardy-Weinberg equilibrium test indicated that Wushen horse, Wuzhumuqin horse and Baerhu horse reached the equilibrium (p>0.05). All of the breeds had intermediate polymorphism information contents(0.25<PIC<0.5).These researches made an essential foundation for molecular breeding of horse in the future.

Study on DGAT1 Polymorphisms in Laiwu Pigs

2007, 38(3): 214-218. doi:

Abstract ( 1197 )

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Diacylglycerol acyltransferase 1 (DGAT1) is a microsomal enzyme that catalyses the final and only committed step in the formation of triglycerides. The Laiwu pigs are capable of depositing more fat than western pigs. It is possible that they have a higher DGAT1 enzyme activity. In this study, exon six through exon eight of Laiwu DGAT1 was amplified and sequenced (GenBank accession number DQ289596). Two single nucleotide polymorphisms (SNPs) were identified at 27bp of exon six and 56 bp of exon eight, respectively, both of which are silent mutations. A fragment of 737bp of Laiwu DGAT1 promoter was as well cloned and sequenced. A deletion of adenosine (A) at -241 (relative to initiation codon ATG) was identified in Laiwu pigs. The association of the mutation with fat deposition remains to be elucidated.

Study on mtDNA Genetic Diversity and Phylogeny Evolution of 5 Indigenous Sheep Populations in Southwest China

2007, 38(3): 219-224. doi:

Abstract ( 1426 )

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The genetic diversity and Phylogeny evolution of 232 individuals of 5 indigenous sheep populations in Southwest China was analyzed with PCR-SSCP technology and mtDNA D-loop sequenced. The five indigenous populations included Tengchong, Zhaotong, and Ninglang sheep from Yunan and Duoma and Jiangzi sheep from Tibet. PCR-SSCP analyses revealed that three genotypes A, B, and C of mitochondrial Cyt b and ND2 genes were detected in Duoma and Jiangzi sheep from Tibet and the rate of genotype C is higher than that of B；whereas only genotypes A and B found in Tengchong, Zhaotong, and Ninglang sheep from Yunan. mtDNA D-loop sequences of 39 individuals chosen from the five according to different genotypes were sequenced. Phylogenetic analysis indicated that three mtDNA lineages A, B, and C were found in Tibetan sheep and only lineages A and B in Yunnan sheep. These results from PCR-SSCP and D-loop sequences consistently indicated that Tibet sheep derived from three maternal origins, While Yunan sheep derived from two maternal origins. Polymorphism analyses showed that values of nucleotide diversity and average number of nucleotide differences in Tibetan sheep were higher than those of Yunnan sheep. This suggested that the level of genetic diversity in Tibetan sheep is higher than that in Yunnan sheep.

Comparative Analysis on Partial Sequence of Follicle- Stimulating Hormone Beta Gene between 20 Goat Breeds and 8 Other Species

2007, 38(3): 225-231. doi:

Abstract ( 658 )

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Based on the FSHβ gene sequences of bovine and sheep, the primers were designed and the partial sequences of FSHβ gene from 20 goat breeds(16 indigenous breeds and 4 introduced breeds) were amplified and sequenced (GenBank accession number, AY838283 and AY853276). It contained the complete first exon (1～61bp), first intron (62～702bp) and partial sequence of exon2（703～731bp）in the 731base pairs fragment of FSHβ gene. The content of A+T (66.07%) was higher than that of G+C (33.93%) and it coded 7 amino acids. Three mutation loci [78(-/A), 132(G/A) and 343(G/A)]were found and they all exited in the sequence of intron1, while the correlation between mutation loci and the birth rate could not be confirmed. The phylogenetic trees constructed by the FSHβ gene intron1 of goat and other 8 species from GenBank were not completely accorded with factual taxonomy, which may be caused by the fact that most species’ gene length were not longer than 700bp and that the species relationships were beyond the congeneric limit.

Cloning and Analysis of Polymorphism of Fatty Acid Binding Protein Gene in Ducks

2007, 38(3): 232-236. doi:

Abstract ( 1504 )

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A pairs of primers were designed according to the sequence of chicken adipocyte fatty acid of binding protein (A-FABP) gene, and then PCR amplified to duck genome. The product of 475bp length of PCR was cloned and sequenced, it includes partial exon 2, exon 3 and complete intron2. Homologous comparison was done between duck and chicken, goose and porcine A-FABP gene and the homology are 84%, 94% and 77% respectively. Then 3 pairs of primers were designed on the base of the cloned gene, the SNPs was detected by the technique of single strand conformation polymorphism (SSCP) and then confirmed by sequencing. Four nucleotides mutation were found, A-T at –241bp, T-C at –243bp, T-C at–258bp and C-T at–299bp respectively. Three genotypes were detected and significant different genotypes frequencies among duck breeds were found.

Correlation Analysis Between Single Nucleotide Polymorphism of the ADSL Exon 9 and inosine monophosphate acid Content in five Chickens

2007, 38(3): 237-240. doi:

Abstract ( 735 )

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This study was designed to investigate the single nucleotide polymorphism(SNP) of ADSL gene in various breeds, including Recessive White chickens, Silkies chickens,Xiaoshan chickens, Baier chickens and Tibetan chickens. Primers for exon 9 in ADSL gene were designed from the database of chicken genomic sequence, Polymorphisms were detected by PCR-SSCP and DNA sequencing. Three genotypes were found among individuals within 5 chicken breeds and a mutation C→A at 9 839 nucleotide was found. It was a nonsynonymous mutation, resulted in amino acid sequence at 273 change accordingly from Pro to Thr . The results X2 showed that the frequency of genotype and the frequency of gene were significant different among breeds. However, The results from analysis of variation suggested that there was no significant relationship between this SNP site and IMP content in chickens.

Shadow Bands in PCR Amplification of Dinucleotide Microsatellites and Their Origin

2007, 38(3): 241-246. doi:

Abstract ( 1157 )

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The PCR products of two ovine dinucleotide microsatellites BM143 and BMS2508 were separated on nondenaturing polyacrylamide gel electrophoresis (nPAGE). Shadow bands were seen 2 bp below the main bands. Actually the main band was composed of two components: specifically a PCR-amplified high-density band that indicates the correct size of the microsatellite (specific band) and nonspecifically a PCR-amplified low-density band that is 2 bp shorter than the specific band (nonspecific band). The results of this study preliminarily demonstrated that the shadow band was heteroduplex DNA formed by a specific band and a nonspecific band. Appearance of shadow bands did not change the normal mobility of specific bands on nPAGE.

Difference of Cationic Amino Acid Transporters mRNA Expression in Different Intestinal Segments of Chicken

2007, 38(3): 247-252. doi:

Abstract ( 1365 )

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The objective of this study was to investigate the difference of mRNA expression of cationic amino acid transporter rBAT(system b0,+), y+LAT2(system y+L), CAT1(system y+)and CAT4(system y+) in different intestinal segments of chicken by relative quantitative RT-PCR. Yellow cover chickens at age of 30 days were used and the different intestinal segments were collected. The result showed: rBAT and y+LAT2 mRNA abundance in colorectum were very significant lower than that of duodenum, jejunum and ileum（P<0.01）. rBAT and y+LAT2 mRNA expression in ileum were higher than in duodenum and jejunum but had no significant difference between them (P>0.05); The expression of CAT1 mRNA in colorectum was very significantly higher than that of duodenum, jejunum and ileum （P<0.01）. Expression abundance in ileum was higher than in jejunum significant (P<0.01) and higher than duodenum by 27.9 %（P=0.111）; Same as CAT1 mRNA, the expression of CAT4 mRNA in colorectum was higher very significantly than that of duodenum, jejunum and ileum (P<0.01)；There was no difference between duodenum, jejunum and ileum (P>0.05).The result indicates that the tissue-specific of transporter mRNA expression of cationic amino acid transporter system b0,+ and system y+LAT2 are similar and discriminated with that of system y+ .

The Developmental Changes of LPL mRNA Expression in Muscle and Their Association with Intramuscular Fat for Pigs

2007, 38(3): 253-257. doi:

Abstract ( 825 )

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To investigate the developmental changes of lipoprotein lipase (LPL) mRNA expression in pig muscle and their association with intramuscular fat (IMF), relative quantitative RT-PCR with β-actin mRNA as internal standard was used in this study. Seventy-two Laiwu Black (LW) and Lulai Black (LL) castrated boars were fed in groups and slaughtered at weight of 40~90 kg (n=6). Results indicated that the developmental tendencies of muscle LPL mRNA in LW and LL pigs were similar. With gaining of body weight, the LPL mRNA abundance decreased from 50 kg to 70 kg (P<0.05), but increased and presented a peak at 80 kg phase for LW. The LPL mRNA abundance decreased gently, and significant difference was found between 40~60 kg and 70~90 kg phases for LL. On the whole, the expression level of LPL mRNA in muscle of LW tended to be higher than that of LL (P>0.05). Correlation analysis showed that the expression of LPL mRNA in muscle were positively correlated with the ratio of IMF to bodyfat for both LW (P<0.05) and LL pigs (P<0.01). The results suggest that LPL in muscle is one of the most important actors involved in IMF deposition, also the LPL gene expression is affected by body weight development, and has a certain extent effect on IMF content.

Detection the Immunity of the Recombinant virus Expressing Canine Distemper Virus H Gene Based on Canine Adenovirus Type-2

2007, 38(3): 258-262. doi:

Abstract ( 718 )

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In order to detect the immunity of the recombinant virus expressing canine distemper virus H Gene based on canine adenovirus type-2, twenty five 45~60 day-old dogs (CDV SN<1:2,CAV-2 HI<1:2) were divided into five groups randomly, the first group was inoculated with CAV-2/CDVLPH, the second group with pVAXLPH, the third group with pVAXLPH and CAV-2/CDVLPH, and the group with attenuated CDV vaccine as positive control ,the group with empty vector DNA pVAX1 as negative control. Indirect ELISA and neutralization assays were used to detect CDV ELISA antibody and CDV SN antibody of the inoculated dogs, respectively. CAV-2 HI assay was used to detect CAV HI antibody of the inoculated dogs. And lymphocyte transformation test was used to detect the proliferation of T-lymphocyte cell in peripheral blood lymphocytes of the inoculated dogs. The humoral and cellular immune responses against CDV and CAV of the CAV-2/CDVLPH were evaluated. As a result, CDV serum ELISA antibody and CDV SN antibody both were detected in the immunized dog, and the anti-CAV-2 HI antibody was detected in the dogs of the first group and the second group. And the PBMCs against ConA, CDV or CAV-2 stimulation of the immunized dogs obviously proliferated. The immune reaction against CDV induced by the second group was weaker than that induced by the third group, and that induced by the first group was stronger than that induced by the third group , but weaker than that induced by the CDV modified vaccine. It demonstrated that the dogs inoculated with CAV-2/CDVLPH developed humoral and cellular immune responses against CDV and CAV. The immune reaction against CDV induced by CAV-2/CDVLPH were stronger than that induced by pVAXLPH, but weaker than that induced by the CDV modified vaccine.

Cloning of SLA-2 and 2m from Bama Miniature Pig and Construction of the Protein Complex

2007, 38(3): 263-270. doi:

Abstract ( 872 )

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The SLA-2 and β2m genes were cloned from a Bama miniature pig. Then the extracellular part of SLA-2 was linked to the mature peptide of β2m gene via (G4S)3, a linker encoding a 15-amino acid glycine-rich sequence, using splicing overlap extension-PCR (SOE-PCR). The reconstructed gene SLA-2-linker-β2m was inserted to pMAL-p2X and expressed in E. coli TB1 system. The fusion protein was processed followed by Western-blot, purifying and cleaving by Factor Xa and then to separate the monomer protein from MBP. The secondary structure of the monomer and fusion protein was determined by circular dichroism (CD) spectrum. The results indicated that the MBP-SLA-2-(G4S)3-β2m was 84.1ku, and the monomer protein SLA-2-(G4S)3-β2m was 41.6 ku. The a-helix, b-sheet, turn, and random coil of the fusion protein and the monomer protein shared high homology ratio at 100%，97.3%，97.1% and 97.9%, respectively, detected by CD estimation. The results indicated that the complex protein SLA-2-(G4S)3-β2m had correct secondary structure and could be used to have further research of peptide binding in vitro.

ImmunoAffinity Chromatography - GC/MS for Determination of Salbutamol and Clenbuterol in swine liver

2007, 38(3): 271-275. doi:

Abstract ( 855 )

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An IAC column was developed by coupling the purified specific IgG against salbutamol to CNBr-activated Sephrose 4B with the dynamic capacity of 400ng/mL gel for salbutamol and 416ng/mL gel for clenbuterol. After extraction using dilute HCl, the extracts of swine liver homogenization was loaded on the IAC column for purification and determined by GC/MS. The limit of detection for salbutamol and clenbuterol were 0.5ng/g and 0.8ng/g, and the limit of quantification for salbutamol and clenbuterol were 2.0ng/g and 2.5ng/g respectively. Recoveries ranged 78.4%-106.9% for salbutamol and 77.1%-102.9% for clenbuterol at fortified levels of 1,5,10,20ng/g in blank swine liver. It was the first time that the IAC-GC/MS for detection salbutamel and clenbuterol in swine liver was reported in China.

Proliferation of Pulmonary Artery Smooth Muscle Cells in the Development of Ascites Syndrome Induced by Low Ambient Temperature in Broilers

2007, 38(3): 276-281. doi:

Abstract ( 674 )

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Pulmonary vascular remodeling and proliferation of vascular smooth muscle cells (VSMCs) of pulmonary arteries of broilers in the development of ascites syndrome (AS) induced by low ambient temperature were dynamically analyzed to study the mechanism of pulmonary vascular remodeling. Broilers in control group (22±1.5℃) and low temperature group (11±2℃) were sampled every week from 15 to 50 days of age. Mean medial thickness (mMTPA) and the ration of vessel wall area to vessel total area (WA:TA), the indexes of pulmonary vascular remodeling, were examined by computer-image analyzing system. Proliferative indexes (PI) of VSMCs in pulmonary arteries were assessed with proliferating cell nuclear antigen (PCNA). The higher incidence (18.75%) of AS was induced, and pulmonary vascular remodeling were significantly elevated in low temperature group from 36 days of age (P <0.05). PI in pulmonary arteries with 20-50um and 50-150um diameter in low temperature group were significantly higher than those in the control group from 22 and 36 days of age separately(P <0.05). Our study demonstrated that VSMCs proliferated and pulmonary artery remodeled progressively in the development of AS, which suggest that proliferation of VSMCs facilitate pulmonary vascular remodeling and have a pivotal role in the development of AS in broilers.

Effect of the foreign matter on the number of mast cells and variation of histamine concentration in mammary gland tissues from different lactation phases

2007, 38(3): 282-287. doi:

Abstract ( 1246 )

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Rats in lactating and nonlactating stages were chose, and the number of mast cells in mammary gland, draning and undraning lymph nodes from control rats and those stimulated by horseradish peroxidase (HRP) was studied by modified toluidine blue (MTB) staining. At the same time, the histamine concentration of mammary gland in different stages was detected by fluorospectrophotometry. The results indicated that the number of mast cells from control lactation mammary glands and lymph nodes tested in the experiment was remarkably less than that during non-lactating period(P<0.01). The variation of histamine concentration had the same tendency. The mast cells of mammary glands and draining lymph nodes increased after injecting HRP during lactating period(P<0.01), but those of undraining lymph nodes decreased slightly(P>0.05). During non-lactating period, after injecting HRP in mammary gland or the sole of foot, the mast cells from mammary and undraining lymph nodes decreased significantly, but the mast cells from draining lymph nodes decreased after injecting HRP in the sole of foot and increased after injecting HRP in mammary. The dynamic changes of the number of mast cells in mammary glands during lactating and non-lactating periods resulted from the development of mammary gland, local immune state of mammary gland and endocrine regulation. Furthermore, the mast cells have different susceptibility to foreign matter during different phases.

Effect of selenium-enriched malt on dynamic variation of hypoglycemia and relative humoral regulation of hepatocarcinoma rats diethylnitrosamine-induced

2007, 38(3): 288-293. doi:

Abstract ( 1083 )

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193 SD rats weighting 100~120 g were divided randomly into 5 groups. The animals in Group I，II and III were fed with selenium as Se-enriched malt（SEM）supplemented diets (0.3，1.0 and 3.0 mg/kg). Animals in group IV and group V received 0.1 mg/kg selenium in sodium selenite only and were severed as the positive control and negative control respectively. Animals of groups I~IV were induced hepatoma by diethylnitrosamine resolved in sterilized water (100 mg/L) at the dosage of 10 mg/kg body weight every day for 16 weeks，then drunk with sterilized water for another 2 weeks. Eyeground blood samples of five animals selected randomly from each group were respectively collected in every 4 weeks. All the rats were denied diet for 12 hours before sampling. Then the values of plasma glucose at different sampling time were measured. Subsequently, the values of the hormones related to plasma glucose metabolism, including insulin, glucagon, and the ratios of insulin/glucose (IGR1), insulin/glucagon(IGR2)，glucagon/glucose(GGR) and insulin-like growth factors-II（IGF-II） were determined. At the same time, the correlation of the plasma glucose concentrations related to hormones was analyzed respectively. The results indicated that the values of plasma glucose and the relative hormones, including insulin, glucagon and GGR in the groups treated with DEN were decreased significantly compared with the negative control group, however, the values of IGF-II and IGR2 were increased significantly. SEM showed a significant effect in suppressing the decreasing of plasma glucose and glucagons, delaying the increasing of IGF-II and IGR2 in the DEN-induced hepatoma rats. The plasma glucose concentrations revealed a significant relation to the hormones. In conclusion, SEM can efficiently deaden the development of hypoglycemia in the DEN-induced hepatoma rats by regulating the mutifactors forenamed, including the levels of the relative hormones and balance among the hormones.

Effects of Pentoxifylline and Platelet Activating Factor on Giant Panda (Ailuropoda melanoleuca) Post-thaw Spermatozoa Fertilizing Capability

2007, 38(3): 294-300. doi:

Abstract ( 1266 )

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Post-thaw Giant Panda sperm was incubated in Ham’s F-10 medium with different dosage pentoxifylline (PF) or platelet activated factor (PAF) under 37℃. The effects of PF or PAF on giant panda post-thawed sperm fertilizing capability were evaluated by spermatozoa motility, survival time, acrosome reaction ratio, membrane integrity ratio and heterogenetic penetration-egg ratio. The results showed: PF and PAF affected giant panda post-thaw sperm fertility in vitro. In the experiment, 1mg/mL PF was best for improving giant panda post-thaw sperm in vitro fertilizing capability. When giant panda post-thaw sperm was treated with 1mg/mL PF under 37℃, the survival time of sperm was 15.33±4.73h, the heterogenetic penetration-egg ratio was 51.44% after incubating for 4 hours, the heterogenetic penetration-egg ratio was still 7.49% after incubating for 6 hours. The results were significant higher than control group（P＜0.01(), and better than other treated groups; When giant panda post-thaw sperm was treated with PAF, sperm quality in 50ng/mL group was better than that in 100ng/mL group, but the sperm quality would obviously decreased when incubation time exceeded 2 hours.

Effect of Vitrification on Chicken PGCs

2007, 38(3): 301-306. doi:

Abstract ( 1861 )

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Six kinds of vitrification freezing medias were used to cryopreserve isolated primordial germ cells (PGCs) from gonads at stage 19 and stage 28 by ficoll density-gradient centrifugation. The six kinds of vitrification freezing medias were following: I：10%DMSO+10%EG+10%PVP，II：20%EG+10%PVP，III：20%DMSO+10%PVP，IV：10%DMSO+10%EG+0.5mol/LSurcose，V：20%EG+0.5mol/LSurcose，VI：20%DMSO+0.5mol/LSurcose. The viabilities of PGCs were performed after thawing by way of staining with trypan blue. The results showed: For the viability of the frozen-thawed PGCs at stage 19, it showed significant difference（P <0.05）between the viability under freezing media VI and the viability under freezing media V, and showed insignificant difference （P >0.05）between the viability under freezing media II and the viability under freezing media IV. But it showed very significant difference（P<0.01）among the viabilities under the other freezing medias. For the viability of the frozen-thawed PGCs at stage 28, it showed significant difference（P<0.05）between the viability under freezing media IV and the viability under freezing media V, and showed insignificant difference （P >0.05）among the viability under freezing media III 、IVand V, and so were the viabilities under freezing media II、III and IV（P >0.05）. But it showed very significant difference（P<0.01）among the viabilities under the other freezing medias. The second passage PGCs cultured after thawing were positive to PAS staining and AKP staining and still kept intact karyotypes. The purpose of this experiment was to conquer the disadvantage of needing long time equilibrium and bringing down the temperature gradually during the slow speed cryopreservation, and set up the quick method of vitrification. So it could supply another way to conserve the diversity of genetic resources of avian, and supply adequate PGCs for manipulation in vitro.

Expression of Mature Equine Interleukin 18 in Escherichia coli and its purification

2007, 38(3): 307-312. doi:

Abstract ( 734 )

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Using the total RNA extracted from ConA stimulated equine peripheral blood mononuclear cells (PBMC) as template, the cDNA of interleukin l8 was amplified by RT-PCR. The cDNA was subsequently cloned into the vector pCR2.1-TOPO and sequenced. Then the gene encoding mature equine interleukin 18 was amplified from the recombinant plasmid named pCR2.1-pEIL-18 by polymerase chain reaction (PCR) and subcloned into pET-28a(+). The recombinant plasmid pET-mEIL-18 was transformed into E. coli BL21 (DE3) after sequence analysis, and a fusion protein was then expressed and purified. Results showed that the fragment of mEIL-18 gene was 474 bp in length, which contained an open reading frame and encoded the protein of 157 aa, and the expressed product existed in soluble portion and inclusion bodies. The SDS-PAGE and Western-blotting analysis indicated that the fusion protein was 20 ku in molecular weight and had immunological activity. The mEIL-18 was expressed successfully in E. coli BL21 (DE3), and purified efficiently using Ni+ column chromatography in non-denatured and denatured conditions, which lays the foundation for investigating the structure and biological effects of EIL-18.

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