Acta Veterinaria et Zootechnica Sinica

Comparative Genomic Analysis of Thyroid Hormone Responsive Spot14 Alpha Gene (THRSPα) in Ducks

2007, 38(6): 521-525. doi:

Abstract ( 1767 )

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To explore the mutation characteristic of the duck thyroid hormone responsive spot 14 alpha (THRSPα) gene cloned by the authors, the various PCR products covering the coding or intron regions of this gene were sequenced directly. The results showed that there was 23 single nucleotide polymorphisms (SNPs) per 1 000 nucleotides in the coding region compared to 28 SNPs per 1 000 nucleotides in intron region. The variations in the coding region contained three nonsynonymous mutations and thirteen synonymous mutations. Furthermore, the 81th Aspartate (Asp81) was substituted to Valine near the N terminus of the leucine zipper domain of the duck THRSPα peptide, in contrast with the insertion/ deletion (indel) of the Aspartate in the same site of the chicken THRSPα. The study implicated that the less selective pressure made the duck THRSPα gene keep more frequent and balanced variations than that of the chicken counterpart during the course of bird domestication. The deeper exploration was necessary whether the nonsynonymous substitution of the Asp81 locus for THRSPα gene was associated with fatness traits of the ducks.

Study of Polymorphysisms in Adipocyte Fatty Acid Binding Protein Gene and Their Relationship with Fatness Related Traits in Chick

2007, 38(6): 526-532. doi:

Abstract ( 1067 )

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PCRSSCP was used to detect polymorphic sites in chick adipocyte fatty acid binding protein gene (AFABP). Six Chinese local breeds, Beijing Fatty chick, Dwarf chick, Taihe Silky Fowl chick, Chongrenma chick, Xiayan chick, Luyuan chick and an imported breed, Arbor Acre broil, were used as tested populations. Three PCRSSCP loci were detected. The frequencies of genotype and allele were significantly different in different populations. Sequence analysis revealed that C→T, G→A, and C→T transitions were responsible for the polymorphisms. Some traits related fatness such as body weight, content of intramuscular fat (IMF) and percentage of abdominal fat weight to body weight (AFP) were measured in Beijing Fatty chick and Dwarf chick. We found that meat quality traits were significantly related to different genotypes in the populations tested.

Studies on Polymorphism of Prolactin Gene Exon2 in Wanxi White Geese

2007, 38(6): 533-536. doi:

Abstract ( 936 )

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Polymorphism in Exon2 of PRL gene of Wanxi White geese have been analysized by PCRSSCP. One SNP site has been detected. The result showed that allele frequency of A is 0423 7 and B is 0.576 3. Chi-square test showed that three genotypes frequencies significantly differed（P<0.01） in the population.

Demecolcine Assisted Enucleation of Bovine Oocyte

2007, 38(6): 537-541. doi:

Abstract ( 816 )

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Enucleating is one of the most important protocols for successive nuclear transfer. In this experiment the location of bovine oocyte chromosome was displayed with Demecolcine（DM）to enucleate .It focuses on the influence of the concertration of DM handling oocytes,the time of oocytes incubating in DM, and oocytes maturation time on displaying effect.The result showed that ①Matured bovine oocytes were activated with ionomycin for 5 minutes,and incubated in DM for 2 h to enucleate completely,but no oocyte was completely enucleated. ②Matured bovine oocytes with first polar incubating in DM(0.5 μg/mL) for 2 h,the displaying rate is 76.54%.③Bovine oocytes matured for 18 h,the displaying rate is 73.86%.④The displaying efficiency would be better(8082％) while adding DM to cumulus cellsoocyte,and development rate of blastula(18.60％) was higher than that of other groups.It indicates that adding DM to cumulus celloocyte is good to show the position of chromosome,and good to blastula development. Displaying chromosome position with DM can not only enucleat fast and effectively,but avoid fluorescence illumination on oocyte while using traditional enucleating methods.

Effect of Different Culture Condition on Bovine Spermatogonial Stem Cells Proliferation and Characteristic Identification

2007, 38(6): 542-547. doi:

Abstract ( 832 )

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The aim of this paper was to study the effects of serum, SCF, LIF and GDNF on proliferation of bovine spermatogonial stem cells(SSC) in vitro, the identification of SSC was also conducted. Experiment 1: Adding 0%, 2.5%, 5% and 10% fetal bovine serum(FBS), respectively, to the basal culture medium DMEM (Dulbecco minimal essential medium) with high glucose.The results showed that DMEM with 2.5% FBS was mostly proper for the proliferation of bovine SSC in vitro.Experiment 2:Adding SCF,LIF or GDNF to the medium could increase the number of SSCs. However, only the concentrations of SCF,LIF and GDNF 20 ,80 ， 10 μg/L, respectively,could significantly increase the number of SSCs compared to the control group(P<0.05). Alkaline phosphatase (AP) staining , immunohistochemical staining and RTPCR were conducted to identify bovine SSC. The results showed that AP staining was weakly positive ; Integrin β1 and c-kit staining were both positive. The sequences of Gfrα1 and ckit were also obtained by RT-PCR, the sizes(120 bp and 280 bp) were identical to the expected sizes. Therefore, DMEM with 2.5% FBS ,adding 20 μg/L SCF，80 ng/mL GDNF and 10 μg/L LIF,respectively, was appropriate for bovine SSCs culture in vitro.

Production of Transgenic Mice Expressing GFP by Intracytoplasmic Sperm Injection(ICSI)

2007, 38(6): 548-551. doi:

Abstract ( 1545 )

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Intracytoplasmic sperm injection (ICSI) is the process in which a high powered microscope is used to inject specially prepared single spermatozoa into a mature oocyte in order to effect fertilization.ICSI is now considered to be a useful procedure, not only for producing live offspring from nonmotile sperm cells but also for generating transgenic animals through spermmediated gene transfer.Mouse ICSI was conducted using mouse sperm or sperm which were incubated with GFP gene for 30 minutes.Genomic DNA was extracted from the offspring of the founder mice for PCR and Southern blotting analysis.PCR and Southern blotting analysis showed 3 of 11 mice were transgenic. These results indicated that the high efficiency of transgenic mouse could be produced by ICSI.

Priliminary Purification and Properties of the β-N-Acetyl-D-glucosaminidase from Cairina moschata

2007, 38(6): 552-557. doi:

Abstract ( 1268 )

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A β-N-Acetyl-D-glucosaminidase (EC3.2.1.30) was purified from the testes of Cairina moschata by ammonium sulfate fractionation,chromatography on DEAE32 and Sephadex G100 as well as. The specific activity of the enzyme was determined to be 19.18 U/mg. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p.nitrophenyl.N.acetyl.β.D.glucosaminide (pNP.NAG) were found to be at pH 5.6 and at 55 ℃, respectively. The enzyme is stable in the pH range from 3.0 to 7.6 and at temperatures below 45 ℃. The kinetic behavior of the enzyme in the hydrolysis of pNP.NAG followed Michaelis Menten kinetics with Km of 0.215 mmol/L and Vmax of 5.54 mol/L·min at pH 5.6 and 37 ℃, and the activation energy was determined to be 69.05 kJ/mol . The effects of metal ions on the enzyme were studied. Mg2+activated the enzyme, while, Na+,K+, Cu2+, Pb2+,Zn2+, Ca2+,Ba2+and Al3+ showed various degrees of inhibitory effects on the enzyme.

Drive of Incurrent Proton Gradient on Transport of Gly-Pro in Small Intestine of Piglets

2007, 38(6): 558-562. doi:

Abstract ( 2130 )

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In order to study the effects of incurrent proton gradient on transport of Gly-Pro in small intestine of piglets, radioactive isotope trace and in vitro incubation technique were used to observe the transmembrane transport of GlyPro in small intestinal brush border membrane vesicles (BBMV). The results showed that: When internal pH of BBMV remained 7.5,transport of Gly-Pro was optimized at an external pH 4.5-5.5; Gly-Pro transport (during 1.20 min period) was greater at an external pH 5.0 than that at 7.5;Presence of an inwardly directed proton gradient stimulated Gly-Pro transport;while in the absence of an inwardly directed proton gradient, Gly-Pro transport was not different at an external pH 5.0 as compared to a pH 7.5. It was suggested that transport of Gly-Pro into small intestinal BBMV of piglets should be driven by transmembrane inward proton gradient.

Effect of Undernutrition during Late Pregnancy on the Levels of GH,IGF-I,INS,T3, T4 Concentrations in Ewes

2007, 38(6): 563-567. doi:

Abstract ( 1034 )

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100 Mongolia ewes, which were synchronized for oestrus and mated at the same day, were selected and divided into three groups that offered 0.175 MJ/(kgw0.75·d) (restricted group1, RG1), 0.33 MJ/(kgw0.75·d) (restricted group2; RG2) and voluntary intake (control group, CG) during their late pregnancy (90 days) respectively. The concentrations of growth hormone(GH), insulinlike growth factor(IGF-I), insulin(INS), T3, T4 were determined to study the effect of different undernutrition. The results indicated that the GH concentration of ewes in RG2 and RG1 were increased, whereas the IGF-I, T3,T4 and INS concentration of ewes were decreased， and the difference were not significant (P>0.05) compared to CG with the nutrients supply increasing after parturition 10 days; The parameters of different groups changed differently after parturition 30 days, the concentrations of GH was decreasing and the concentrations of IGFI,INS,T 3 ,T 4 were increasing slowly; After parturition 150 days, the concentrations of GH,IGFI,INS,T 3,T 4 reached the levels of control group. And during the physiological compensatory, the RG2 group changed significantly.

Effects of Porcine Reproductive and Respiratory Syndrome Virus Infection on the Molecules Associated with Processing and Presentation Function of Porcine Pulmonary Alveolar Macrophages for Exogenous Antigen and Costimulatory Molecul

2007, 38(6): 568-573. doi:

Abstract ( 1516 )

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A quantitative competitive PCR (qcPCR) assay was used to detect mRNA transcription levels of SLA-DR, invariant chain (Ii), SLA-DM and costimulatory molecules CD40, CD80 and CD86 of porcine pulmonary alveolar macrophages (PAMs) on different phases after porcine reproductive and respiratory syndrome virus (PRRSV) infection in vivo. The mRNA transcription level of SLA-DR was found to be downregulated and lower than that of the control at day 3, 7, 14 post-infection (PI). The mRNA expression of Ii chain, SLA-DM and CD40 declined significantly at day 3 PI (P<0.01). CD80 and CD86 mRNA expression decreased markedly at day 3 PI (P<0.05). By using flow cytometry (FCM), it was showed that the percentage of SLADR + PAMs increased transiently at day 3 PI, declined quickly at day 7 PI and remained low level thereafter. Our results showed that PRRSV infection affected markedly processing and presentation of PAMs for exogenous antigen in early stage of infection, resulting in a decrease of antigen presentation function of PAMs for exogenous antigen and onset of impairment for humoral immune responses of swine.

Isolation, Identification of Porcine Reovirus from Diarrhea Feces of Pigs

2007, 38(6): 574-580. doi:

Abstract ( 1397 )

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Using Vero cell line and adding the trypsin into the viral culture MEM, reovirus SC-A strain was isolated form the pigs' diarrhea feces. The stable CPE characters of the reovirus were as follows: cell granulations increasing, cellular swelling and shedding. The virions of approximately 70 nm diameter, which shows typical lattice arrangement in cytoplasm of infected cells and the cytoplasmic inclusion bodies and the distinctive microbulelike structure occured. The S2 gene （DQ396805）of reovirus SC-A, obtained by RT-PCR, was 1 331 bp in length and encodes the σ2 protein, which with relative molecular weight of 47.14 ku. Sequence analysis of nucleotides indicated that the reoviurs SC-A S2 gene shared 86.9%, 76.7% and 85.4% similarity with standard strains T1L, T2J and T3D, respectively. And the similarity of deduced amino acid sequence of reoviurs SC-A S2 gene were 94.6%, 93.3% and 98.3% with standard strains T1L, T2J and T3D, respectively. Phylogenetic trees based on σ2 shows standard strains T1L, T2J and T3D were divided into branch A, B and C, respectively. Except T3C31 field isolated strain, serotype 3 of the other field isolate strains, SAC strain, all of serotype 1 strains were located in branch C of phylogenetic tree.

Molecular Epidemiology of Infectious Bursal Disease Viruses Isolated from Some Provinces of China

2007, 38(6): 581-588. doi:

Abstract ( 1942 )

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Tissue samples of Fabricius′ bursal, spleen and bone marrow collected from the provinces of Gangxi, Anhui, Jiangshu, Zhejiang and Hainan during the years of 20002006, were detected by a developed reverse transcriptase polymerase chain reaction (RT-PCR) technique for IBDV. Viral isolations were performed on the positive samples by chicken embryo inoculation via chorioallantoic membrane (CAM) or cell culture with chicken embryo fibroblast (CEF) and 23 isolates of IBDV were obtained. A set of primer was designed to amplify the vVP2 of 23 isolates and 5 commercial vaccine strains with intermediate and intermediateplus virulence by RT-PCR. The PCR products were analyzed by restriction enzyme analysis (REA) and sequenced respectively. The sequences of all the isolates and reference viruses were analyzed and compared, and their phylogenetic tree was made based on the nucleotide sequences. The results indicated that isolate BH09, BH11, JS1, JS7, YY1, YY6, YL051, 050222, TSC-2(9) ,TZ(3) and HN0602 were classified to be very virulent IBDV (vvIBDV), and have close phylogenetic relationship with other Chinese isolates, Japanese isolate OKYM and European isolate DV86 and UK66, while isolate 040124, YL052, 020180, YLZF2, 040131, BH15, YY2, 050045, 050057, 050258, TSC-1(3) and A038 were classified to be classical IBDV (cIBDV). Isolate 040124 and YL052 were further identified as intermediate and intermediateplus virulent vaccine strains, and the rest 10 isolates were identified as attenuated vaccine strains. The results of the study demonstrated that the viruses prevailing in chickens in these 5 provinces during 20002006 are vvIBDV and there is no significant evident divergence in vVP2 of all the isolates on both time and geology.

Determination and Residue Depletion of Ractopamine Hydrochloric in 4 Porcine Tissues by HPLC

2007, 38(6): 589-594. doi:

Abstract ( 2116 )

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A high performance liquid chromatography (HPLC) method was developed to determine ractopamine hydrochloric in porcine tissues. The limit of detection (LOD) was 1 ng/g and the limit of quantification (LOQ) was 2 ng/g. The average recovery rate in liver was 73.4%-83.2% with coefficients of variation (CV) of 2.1%-6.6%; in kidney was 73.1%-95.5% with CV of 3.8-4.0%; in muscle was 80.7%-82.5% with CV of 4.9%-9.1%; and in fat was 73.3%-78.8% with CV of 2.9%-6.7%. Sixty healthy pigs were administered ractopamine hydrochloric at a dosage of 18 mg/kg in feeds for 28 days, then fed without the drug for 14 days. Six pigs were slaughtered at day 7, 14, 28 since the first feeding and with withdrawal periods of day 1, 2, 3, 7, 9 and 14. Tissues including kidney,liver, muscle and fat of each pig were collected and determined by HPLC. Results showed that the concentration of ractopamine hydrochloric in kidney was the highest, and lower in liver. Residues were significantly lower after the last feeding with the drug. Residues were declined below the LOQ at day 14 withdrawal period in kidney, and at day 9 of withdrawal period in liver. The concentration of ractopamine hydrochloric in muscle and fat were much lower than that in liver and kidney, residues were declined below the LOQ at day 28, and dropped to LOD at day 1 of withdrawal period.

The Change of Ca2+ and Ca2+-ATPase of Right Ventricular Myocardium in Ascites Syndrome of Broiler Chickens

2007, 38(6): 595-600. doi:

Abstract ( 870 )

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Right ventricular hypertrophy and failure is an important step in the development of ascites syndrome. Cytoplasmic calcium concentration is a major regulator of cardiac contractile function and various physiological processes in cardiac muscle cells. The purpose of this study was to measure the right ventricular pressure and investigate the precise ultrastructural location of Ca2+ and Ca2+-ATPase in the right ventricular myocardium of chickens with ascites syndrome induced by low ambient temperature. The results showed that the right ventricular diastolic pressure (RVDP) of ascitic broilers was significantly higher than that of control broilers (P<0.01), and the maximum change ratio of right intraventricular pressure (RV dp/dtmax) of ascitic broilers was significantly lower than that of the controls (P<0.01). Extensively increased calcium deposits were observed in the right ventricular myocardium of ascitic broilers, whereas in the agematched control broilers, calcium deposits were much less. The Ca2+-ATPase reactive grains were obviously found on the sarcoplasmic reticulum and mitochondrial membrane of the control right ventricular myocardium, but rarely observed in the ascitic broilers. The data suggested that in ascitic broilers there was the right ventricular diastolic dysfunction, in which the overload of intracellular calcium and inhibition of Ca2+-ATPase might be the important factors.

The Effect of High Copper on Lymphocyte Apoptosis of Lymphoid Organs in Chickens

2007, 38(6): 601-607. doi:

Abstract ( 190 )

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The experiment was conducted with the objectives of examining the effect of dietary high copper on lymphocyte apoptosis of the lymphoid organs in the chicken by the methods of ultrastructural pathology and Flow Cytometry (FCM). 420 onedayold Avian chickens were randomly divided into seven groups, and fed on diets as follows：control (Cu 11 mg/kg), and high copper （Cu 100 mg/kg，high copper group Ⅰ；Cu 200 mg/kg，high copper group Ⅱ；Cu 300 mg/kg，high copper group Ⅲ；Cu 400 mg/kg，high copper group Ⅳ；Cu 500 mg/kg， high copper group Ⅴ；Cu 600 mg/kg，high copper group Ⅵ）for six weeks. Ultrastructurally, the frequency of lymphocyte apoptosis was higher in high copper groups Ⅳ,Ⅴ and Ⅵ than in control group and high copper groups Ⅰ,Ⅱ,Ⅲ. The mitochondria of lymphocytes of lymphoid organs were swelled, and its crista were broken or/and disappeared. The apoptotic lymphocytes showed that the chromatin become condensed and marginated, and the shape of nuclei were crescent,Cshaped, dark round and petallike. Also, the statistical analyses by FCM indicated that the percentage of apoptotic cells of the lymphoid organs was higher in high copper groups Ⅳ, Ⅴ and Ⅵ than in control group and high copper groups Ⅰ,Ⅱ,Ⅲ. The aforementioned results demonstrated that dietary copper in excess of 300 mg/kg diet could induce apoptosis in lymphocytes of lymphoid organs.

Effects of Leptin Gene on Litter Size in Luchuan and Large White Pig

2007, 38(6): 608-613. doi:

Abstract ( 831 )

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Single nucleotide polymorphisms(SNPs) of the leptin gene were tested using PCR-SSCP in Luchuan and Large White pigs, and the effects of the leptin gene on litter size traits were also analyzed. Two pairs of primers for leptin gene were designed based on the known sequence in GenBank, and two SNPs were identified within the product amplified by PCR-SSCP and sequencing. The results showed that: (1) the polymorphisms were due to two synonymous single base mutation T to C and T to G at base position 3 469 and 3 714, respectively; (2)using least square analysis, it was seen that T3 714G SNP had significant effect on litter size in Luchuan pigs ( P<0.05), but no effect in Large White pigs (P>0.05); T3469C SNP had no significant effect on litter size in Luchuan and Large White pigs(P>0.05).

A Preliminary Study on the Maturation of Buffalo Denuded Oocytes in vitro

2007, 38(6): 614-619. doi:

Abstract ( 2260 )

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The feasibility of maturing denuded buffalo oocytes in vitro was explored in this study, so as to provide a model to investigate the mechanisms of oocytes maturation. Denuded buffalo oocytes were allocated randomly to culture in medium directly (M1), co-culture on cumulus cell monolayers (M2), coculture with surrounding cumulus cell pieces (M3), co-culture on expanding cumulus cell clumps (M4) and co-culture with surrounding ovarian tissue pieces (M5). At 24 h after maturation in vitro, rate of the first polar body (PB1) extruded was checkd, and the developmental competence of oocytes was evaluated by parthenogenetic activation using ionomycin and 6-dimethylaminopurine. The PB1 rate of oocytes matured using M4 was significantly higher than that of oocytes matured using M1 and M5 (P<0.05). Significantly more oocytes developed to blastocysts when they were matured using either M3 or M4 in comparison with them matured using M1 and M5 (P<0.05). In addition, the cleavage rate of oocytes matured using M4 and M1 was also significantly higher than that of oocytes matured using M5 (P<0.05). In conclusion, co-culture with either cumulus cell pieces or expanding cumulus cell clumps can promote the maturation of buffalo denuded oocytes in vitro, but coculture with cumulus cell monolayers does not work, and coculture with ovarian tissues may be unfavorable to the maturation of buffalo denuded oocytes in vitro.

The Preliminary Study on Differentiation of Chicken Spermatogonial Stem Cells to Osteoblasts in vitro

2007, 38(6): 620-624. doi:

Abstract ( 787 )

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Investigate the capability of spermatogonial stem cells(SSCs) differentiating to osteoblasts under different experimental conditions in vitro. In this study, the osteoblasts were induced from SSCs by desamethason, glycerol 2phosphate disodium salt hydrate and vitamin C and were examined with Von Kossa’s stain,cytochemistry stain and immunohistochemistry stain (Collagen Ⅰ).The results indicated that the osteoblasts formed Von Kossa stain positive nodules, dark granules were found in the cell, suggesting the deposition of mineralized materials; the osteoblasts showed brown or deep dark by alkaline phosphatasepositive stain and showed CollagenⅠpositive with immunohistochemistry stain. And so，we may know that the Spermatogonial stem cells（SSCs） have the capability to be differentiated to osteoblasts in vitro.

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