Acta Veterinaria et Zootechnica Sinica

Methodology of Genetic Analysis for Discrete Traits of Livestock

YIN Zong-jun;ZHANG Qin;DING Xiang-dong;WANG Chun-kao;ZHOU Jie

2008, 39(12): 1625-1630. doi:

Abstract ( 887 )

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The objective of this paper is to provide an introduction to generalized linear mixed models(GLMM), and to compare the efficiency of heritability estimate for discrete traits using three methods (LM, TM, GLMM). Binary response traits are simulated in this study, and the design is mixed family of full-half sib. The results showed that generalized linear mixed model have a great advantage in heritability estimation, and have nice accuracy for discrete traits. The results of estimation heritability using GLMM method are higher than those using LM method in all different parameter case. In addition, the accuracy of estimation depended on the true heritability and incidence of the trait directly.

Polymorphism and Sequence Analysis of Partial Fragment of BMP6 Gene in Five Sheep Breeds

XIAO Chao-ting;DI Ran;CHU Ming-xing;FU Yan;FANG Li;MA Yue-hui;LI Kui;XU Zuo-peng

2008, 39(12): 1631-1639. doi:

Abstract ( 1617 )

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Single nucleotide polymorphisms (SNPs) of exons 5, 6, 7 of bone morphogenetic protein 6 (BMP6) gene were detected in 301 individuals of five sheep breeds (Small-tailed Han, Hu, Dorset, Texel and Corriedale sheep) with three pairs of primers by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP). No polymorphisms were detected in the amplified regions of three pairs of primers in five sheep breeds tested, which may indicate that the sequences of exons 5, 6, 7 of BMP6 gene were rather conserved. On the other hand, partial sequences of BMP6 mRNA were cloned from Small-tailed Han sheep (695 bp) and Jining Grey goat (699 bp) respectively, and the sequences (785 bp) of intron between exon 3 and exon 4 with some of the two flanking exons were also sequenced in Small-tailed Han sheep. The two BMP6 sequences show high identity with 99.28% at nucleotide level and 98.1% at amino acid level, and besides an insert of alanine in goat, only 4 amino acid alterations were found between them (211 in total). Compared with the ovine nucleotide and amino acid sequence of BMP6 with that of cattle, human, mouse and rat, identities of 97.04%, 81.82%, 84.68%, 85.06% and identities of 99.05%, 91.43%, 90.48%, 92.38% were revealed respectively. The results suggested that although the nucleotide changes are a little big, the amino acid sequences of BMP6 are very conservative among species.

Analysis on Associations of SNPs of Leptin Gene with Growth Traits in Four Sheep Breeds

HUANG Dan-li;CHEN Ren-jin;YANG Zhang-ping;MAO Yong-jiang;LI Yun-long;TIAN Dai-jun;CHEN Liang;ZHAO Xiao-yong

2008, 39(12): 1640-1646. doi:

Abstract ( 1568 )

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PCR-SSCP was applied to detect single nucleotide polymorphism (SNP) of exon 2 and exon 3 of the Leptin gene for 358 sheep, including Poll Dorsets (PD), Suffolks (SF), Texels (TX) and Tan sheep (Tan). The polymorphisms were demonstrated, and exon 2 and exon 3 of leptin gene were sequenced. Seven SNPs were detected, five of which were new SNPs. There is no SNP in exon2, but four SNPs were detected in parts of intron 2. They are A99G, G115A, C150T and C171T. Three SNPs occurred in exon3, all of which resulted in amino acid changes. The association between some SNPs and growth traits were analyzed. The results showed that the average values of birth weight, daily gain and withers height of Aa were significantly higher than those of AA at A99G SNP(P<0.05), while the values of heart girth and rump width of Aa were very significantly higher than those of AA (P<0.01). The average values of daily gain, withers height, body length, heart girth and rump width of mutantions types were significantly higher than those of wide-types at C150T SNP (P<0.05). The results of A99G+C150T polymorphic site were the same to C150T SNP.

Genetic Analysis of IGFBP-3 Gene and Its Association with Economic Traits in Goats

LI Mei-yu;LI Lan;MIN Ling-jiang;WANG Jian-min

2008, 39(12): 1647-1653. doi:

Abstract ( 838 )

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501 goat individuals in seven populations were detected by PCR-RFLP. The results showed the polymorphisms of XspⅠ existed in every population and the number of the individuals with genotype AG was more than that with genotype AA while the number of genotype AA was more than that of GG in all the populations. At the same time, the results of χ2 test indicated that all the populations were at Hardy-weinberg equilibrium (P>0.05) and genotype distributions were not relative to populations (P>0.05). We also found that frequency of allele A was higher than that of allele G and the frequencies of allele A and G were nearly invariable in all populations. PCR products were sequenced and the result showed that the sequence contains partial exon2, complete intron2, exon3 and partial intron3. The homology of the gene sequences with and dairy goat, sheep, dairy cattle, were 99.1%, 98.1% and 94.1%, respectively. The correlations between genotypes at the gene locus and weaning weight, rib eye area, 10th month body length, 10th heart girth and 12th month body length were significant (P<0.05) and the associations were significant for 3rd month body length and 3rd heart girth (P<0.01). The genetic effects of genotype AG were significantly higher than those of genotype GG (P<0.05 for weaning weight and rib eye area while P<0.01 for above five physical measure traits). Further more, the effects of genotypes for above traits were AG>AA>GG.

Polymorphism of Exon 10 of Prolactin Receptor Gene and Its Relationship with Milk Performance of Xinong Saanen Dairy Goat

SUN Rui-ping;WANG Li-xin;ZHU Guang-qin;WANG Jian-gang;SONG Yu-xuan;LIANG Zhao-yi;CAO Bin-yun

2008, 39(12): 1654-1660. doi:

Abstract ( 1359 )

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Prolactin receptor (PRLR) gene was studied as a candidate gene for the milk performance of in Xinong Saanen dairy goat. Four pairs of primers were designed to detect single nucleotide polymorphisms of exon 10 of PRLR gene by PCR-SSCP. The results showed that only the products amplified by primer P1 didn’t display polymorphisms. For primer P2,two genotypes (AA and AB) were detected in Saanen dairy goat.Sequencing revealed two mutations of PRLR gene in the genotype AA in comparison to the genotype AB, The mutation C27T did not cause any amino acid change,The deletion of 189th result in two amino acid changes of His→Tyr and Thr.The difference of the least squares means (LSM) for milk yield between AA and AB was significant (P<0.05) in Saanen dairy goat, the third lactation milk yield of individuals with genotype AA was higher than that with genotype AB(P<0.05).For primer P3, three genotypes(CC,CD,DD) was detected in Saanen dairy goat.Sequencing revealed two mutations(C103A and T106C) of PRLR gene in the genotype CC in comparison to the genotype DD. The former mutation resulted in an amino acid change of Lys→Thr,and the later mutation resulted in an anino acid change Thr→Ile.The difference of LSM for milk yield among CC,CD and DD was non-significant(P>0.05).For primer P4,two genotypes(EE and EF) were detected in Saanen dairy goat. Sequencing revealed one mutation(A114G) of PRLR gene in the EE in comparison to the genotype EF,and this mutation resulted in an amino acid change of Met→Val. The difference of the LSM for milk yield between EE and EF was significant (P<0.05) in Saanen dairy goat,The milk yield of individuals with genotype EE was higher than that with genotype EF in third,fourth lactation milk yield and average milk yield (P<0.05).Based on these results,it was indicated that PRLR gene had significant positive effects on milk performance of dairy goats.Additionally, The exon 10 of PRLR gene can be regarded as effective molecular markers on MAS program for milk performance of dairy goat.

Association Analysis between DGAT1 Gene and Milk Production Traits in Chinese Holstein

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

2008, 39(12): 1661-1664. doi:

Abstract ( 838 )

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With PCR-RFLP technique, the polymorphism in the exon8 of DGAT1 gene were investigated in a population of 1 222 Chinese Holstein cows from 17 bull families. Three kinds of genotypes were found and the genotypic frequencies of KK, KA and AA were 0.143 2, 0.609 7 and 0.247 1, respectively-Using SAS (8.02) software with animal model, significant associations between the K232A mutation within exon8 of DGAT1 gene and three milk production traits were revealed. Duncan’s multiple-range test showed that the cows with AA genotype had higher 305day milk yield and protein yield than those with KA and KK genotypes (P<0.01), while had lower fat yield (P<0.01). The results suggested that DGAT1 gene has a great effect on milk production traits and could be a useful genetic marker in Chinese Holstein breeding.

The Affection of Different Activation Ways and Culture Medium to the Parthenogenesis and Development of Rabbit Oocytes

LI Shan-gang;CHEN Xue-jin;LI Hong-bin;WEI Cai-hong;DU Li-xin

2008, 39(12): 1665-1670. doi:

Abstract ( 822 )

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The development potentiality of the matured Newzeland rabbit oocytes that were activated by different ways and cultured in different media was compared. The result is that the electric pulse or chemical activation could not activate the oocytes perfectly when they were used separately, but the combination of electric pulse and chemical activation could activate the oocytes more perfectly. B2 medium is better than other media to support the development of the parthenogenetic rabbit embryos by comparison. The study offered an optimized system for the rabbit oocyte activation and cultures same as fertilization in vivo, which will supply good condition for rabbit nuclear transfer.

Effects of Dietary Energy Level and Source on Oocyte Quality and Gene Expression in Gilts

WANG Yan-zhong;WU De;XU Sheng-yu;ZHOU Dong-sheng;ZHOU Ping;TAN Xian-yi

2008, 39(12): 1671-1678. doi:

Abstract ( 929 )

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Fifty-four Landrace×Yorkshire crossbred gilts, with initial body weight of （59±4.2）kg, were allotted to 3×2 factorial experiment to study the effects of dietary energy level and source on follicle development, oocyte maturation in vitro and growth differentiation factor 9 (GDF-9), bone morphogenetic protein 15 (BMP-15), LH receptor (LHR), FSH receptor (FSHR) mRNA expression from cumulus-oocyte complexes (COCs) during the pre-ovulatory phase. Three energy levels were 87.5%(L), 100%(M) and 112.5%(H) accounting for NRC(1998) recommended energy requirement, respectively; two energy source were starch(S) and fat(F). Gilts were slaughtered on day 19 after second estrus. The results showed that the number of large follicles were higher for gilts fed H (P<0.05), energy sources had no significant effect on number of follicles in gilts(P>0.05); cumulus expansion and oocyte maturation were higher for gilts fed H (P< 0.05), energy sources had no significant effect on cumulus expansion(P>0.05); oocyte maturation were higher for gilts fed S than that fed F (P< 0.05); L energy level had higher GDF-9 and BMP-15 mRNA expression (P<0.05), energy sources had no effect on BMP-15mRNA expression, starch diets upregulated GDF-9 mRNA expression (P<0.05). GDF-9 and BMP-15 mRNA expression were higher in COCs from small follicle than that from large follicle in all treatments (P<0.01). LHR and FSHR mRNA expression was higher in H (P<0.05), energy sources had no significant effect on FSHR mRNA expression, however, starch diets increased LHR mRNA expression (P<0.05). The results of this study indicate that higher energy level could increase number of large follicle during the pre-ovulatory phase and oocyte maturation, although energy sources had no significant effect on number of follicles, starch diets increase oocytes maturation. The effects of energy level and source on number of follicle and oocyte maturation maybe modulated by GDF-9，BMP-15, LHR and FSHR mRNA.

Isolation, Culture and Identification of Goat Hair Follicle Stem Cells

SHI Ming-yan;GAO Xue;YANG Xue-yi;DOU Zhong-ying

2008, 39(12): 1679-1683. doi:

Abstract ( 807 )

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Hair follicles were isolated from goat ear skin by 2.4 U/mL Dispase digestion and the bulges were separated with special fine needle and digested with 0.5 mg/mL trypsin plus 0.2 mg/mL EDTA. The resultant cells were cultured in self-prepared serum-free conditional medium and studied through morphological observation, cell growth curve and colony-forming efficiency(CFE), and identified by immunohistochemical staining. The results showed that the cells were slide, small-sized, ball-shped, obviously threedimensional and undifferentiated, immunohistochemically positive to K19, integrin-β1 and p63 staining and so performed stem cells attributes and high CFE. The CFEs of the third, the fifth and the seventh passages were respectively 25.6%±2.6%, 31.8%±1.9 % and 27.0%±3.9%, significantly higher than that of control (P<001). And the data proved that the goat follicle stem cells could be subcultured for 19 times in vitro in the serum-free conditional medium self-prepared.

Study on the Determination of Endogenous Outputs and True Digestibility of Calcium and Phosphorus with Soybean Meal for Growing Pigs by Linear Regression Analysis Technique

ZHANG Tie-ying;ZHANG Yan-ling;YAN Su-mei;YIN Yu-long;FAN Ming-zhe;WANG Jing

2008, 39(12): 1684-1691. doi:

Abstract ( 1174 )

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This study was designed to determine endogenous fecal calcium and phosphorus losses and true fecal calcium and phosphorus digestibility with soybean meal for growing barrows by the Linear Regression Analysis Technique (REG). Six Yorkshire×Landrace barrows, with averaged initial body weight of （28.6±1.76） kg, were fed in the metabolic cage. According to a 6×6 Latin square design, six soybeancornstarch based diets, containing six levels of calcium (0.04%, 0.09%, 0.13%, 0.17%, 0.22% and 0.26%) and phosphorus (0.09%,0.18%,0.27%,0.35%,0.44% and 0.53%), were formulated in six experimental periods. Each experimental period comprised 8 d with 6 d adaptation and 2 d collection of fecal samples. When expressed as g/kg DM diet intake,increasing linear relationships（P=0.002<0.01）were found between fecal outputs and dietary inputs of Ca. The endogenous Ca output of barrow was calculated as 0.622 5 g/kg DMI and true Ca digestibility was 44.34% when the dietary inputs of Ca point to zero on the regression curve.When expressed as g/kg DMI, increasing linear relationships（P=0.025<0.05）were found between fecal outputs and dietary inputs of P. The endogenous P output was calculated as 1.077 1 g/kg DMI and true P digestibility was 48.78% when the dietary inputs of P point to zero on the regression curve. These results suggested that in the soybean diets of different Ca and P levels, apparent digestibility of Ca and P vary a lot,but true digestibility of Ca and P are relatively stable. Apparent digestibility of Ca and P were lower than true digestibility by 41.50% and 35.51%, respectively. Current diet formulation using apparent Ca and P digestibility leads to underestimation of Ca and P biological availability and excessive intake and excretion Ca and P in pigs. True Ca and P digestibility should be determined and used in diet formulation for pigs.

Effect of Dietary Protein Levels on Meat Quality in Wujin Pig

GE Chang-rong;ZHAO Su-mei;ZHANG Xi;LAI Hua;LI Chang-qiang;GAO Shi-zheng

2008, 39(12): 1692-1700. doi:

Abstract ( 848 )

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The aim of the study was to investigate the effect of dietary protein levels on meat quality so as to find the suitable dietary protein level according to the best meat quality by Fuzzy Comprehensive Judgment Model. Ninety pigs were randomly allotted into five groups fed diet containing different protein levels. There were eighteen pigs in each group with three replicates. Six pigs were used in per replicates. The experiment was designed by single factor random arrangement. The dietary protein levels were high high protein（HHP,18%, 16% and 14%）, high medium protein（HMP, 17%, 15% and 13%）,medium medium protein（MMP, 16%, 14% and 12%）, medium low protein（MLP, 15%, 13% and 11%）and low low protein（LLP, 14%, 12% and 10%）respectively during 15-30, 30-60 and 60-100 kg body weight. The dietary energy level, trace element and amino acids content in one group during different growth stages were constant. The meat quality traits were measured when the pigs were slaughtered at 30, 60 and 100 kg body weight. Fuzzy Comprehensive Judgment Model was analyzed to make sure the suitable dietary protein level to attain the best meat quality in Wujin pigs during different growth stages. The results showed that dietary protein level affected obviously the muscle pH value, water holding capacity, shear force, cooking loss and drip loss during the different growth stages, the differences were significant among high, medium and low groups (P<0.05 or P<0.01). The marbling score increased with the reducing of dietary protein level and which was significant between low and high dietary protein levels (P<0.05). The muscle crude protein contents increased with the dietary protein level increasing and the difference was significant at 100 kg (P<0.05). However, the contents of muscle crude fat, intramuscular fat and inosine monophosphate reduced with dietary protein level enhancing, and the differences were significant among high, medium and low groups during different growth stages (P<0.05 or P<0.01). Fuzzy Comprehensive Judgment Model was analyzed basing on the marbling score, shear force and drip loss which represented the meat quality. The suitable dietary protein levels were 15.88%, 14.13% and 11.42% during 15-30, 30-60 and 60-100 kg body weight from the fitting curve in order to attain the best meat quality in Wujin pigs. The present results could provide scientific materials for the reasonable feeding of Wujin pigs to improve meat quality.

Developmental Changes of Myofiber Types in Longissimus Dorsi Muscle of Rongchang and DLY Pigs under Different Nutrient Condition

YANG Fei-yun;CHEN Dai-wen;HUANG Jin-xiu;LIU Zuo-hua

2008, 39(12): 1701-1708. doi:

Abstract ( 1146 )

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This experiment was conducted to study the developmental changes of myofiber types in longissimus dorsi (LD) muscle of Rongchang (RC) and Duroc×Landrace×Yorkshire (DLY) pigs under different dietary nutrient condition, by determining the proportion of mRNA abundance of four isoforms of myosin heavy chain (MyHC I, 2b, 2b and 2x) with semiquantitative RTPCR. The results showed that: (1)From 10 to 20 kg BW, the composition of myofiber types in LD muscle of both breeds changed dramatically with significantly decreased MyHC I and 2x fibers but ramarkably increased MyHC 2b fiber. (2) From 20 to 120 kg BW, the developmental changes of myofibers in LD muscle varied in breed and myofiber type. Except MyHC 2b fiber, the developmental patterns of the other three myofiber types were different between the two breeds. (3)There was no difference in the proportion of four myofiber types in LD muscle during 10-50 kg BW between the two breeds, but at 80 kg BW, RC pigs showed significantly lower proportion of MyHC 2b fiber and higher proportion of MyHC 2a fiber compared with DLY pigs. (4) Dietary nutitional levels had no significant effect on the composition of myofiber types in LD muscle of both breeds. The results suggested that the developmental changes and composition of myofiber types in LD muscle varied in breed. The breed difference of the composition appeared at 80 kg, and RC pigs showed significantly lower proportion of MyHC 2b fiber, which might correspond to their better meat quality.

The Effect of Formic Acid or Previous Fermented Juice on Baled Afalfa Silage

XU Qing-fang;YU Zhu;LI Sheng-li;HAN Jian-guo;BAI Chun-sheng;XUE Yan-lin;BAI Jing-long;HUANG Yi

2008, 39(12): 1709-1714. doi:

Abstract ( 1428 )

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The effects of formic acid and previous fermented juice on fermentation and nutrient of baled alfalfa silage were studied. The wilted forage as CK, the amount of formic acid and previous fermented juice were 6 L/t and 2 L/t, respectively. Unchopped alfalfa baled and wrapped with 6 layers stretch film. After storage 300 d, the bale opened and sampled to analyze fermentation quality and nutrient value. Compared with formic acid and previous fermented juice treatment, which the pH value were 4.32 and 4.25, the wilted alfalfa silage pH increased significantly to 5.22 （P<0.05）. The lactic acid contents were 6.26% and 6.63% for formic acid and previous fermented juice treatments, which were higher than that of wilted alfalfa silage of 0.24%（P<0.05）. But the wilted alfalfa silage had the lowest ammonia nitrogen（P<0.05）. No difference of NDF, ADF in all treatment, adding formic acid improved significantly effective degradability of DM, OM, CP, NDF, ADF (P<0.05), wilted treatment decreased significantly effective degradability of DM, OM, CP, ADF (P<0.05) and with lower CP content.

Establishment and Application of Immunofluorescence Assay and Indirect ELISA for Detection of B.avium

YANG Chun-xiao;WANG Xiao-e;LIU Guan-hua;XIAO Hai-jun;CUI Jin-sheng;ZHU Rui-liang

2008, 39(12): 1715-1720. doi:

Abstract ( 792 )

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In this study, out membrane proteins of Bordetella avium strains P5 and P8 were prepared by improved Wooldridge method. The contents of Bordetella avium OMP were determined by Bradford method. SDS-PAGE electrophoresis was performed to identify Bordetella avium OMP. Prepared OMP immunogen was immunized to rabbits for preparation of high titer immuno serum. And then detections of indirect ELISA and indirect immunofluorescence were established by optimization and screening of conditions. The result showed that the contents of Bordetella avium OMP was 320 μg/mL. The protein molecular weight of OMP are 58，47，41，36，24 kDa in P5, and are 58,47,41,38,21,16 kDa in P8. The detection technique of I-ELISA and IFA were simple, rapid and specific. They can be used in study of epidemiology and serology of Bordetella avium. The two methods laid a foundation for fast diagnosis of Bordetella avium.

Effect of Experimental Infection with Two SS2 Strains on Several Immune Indices in Miniature Swine

SHEN Yan;HUA Xiu-guo;CUI Li; ZHU Jian-guo;YANG Zhi-biao

2008, 39(12): 1721-1730. doi:

Abstract ( 1495 )

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To investigate the effect of infection of different Streptococcus suis strains (HA9801, ZY05719) on the blood biochemical indices and immunological state in Bama miniature pigs，bacterial content and leucocyte of blood were determined after infection, some immunological indexes in the blood were analyzed. Results were as follows: Acute death cases, subacute infection and chronic infection were found after intramuscular injection of Streptococcus suis, the bacterial content of dead pigs reached 106 CFU/mL. Hypersecretion of IL-1, IL-8, TNF-α, IFN-γ were found at 12 hours post infection, the amount of TNF-α, in particular, increased significantly in all infected pigs. Increase of CD8+ and reduction of CD4+ were also found in all infected pigs, the ratio of CD4+ to CD8+ reversed in death cases even, suggesting inhibition of immunity in these pigs. The ability of lymphocyte transformation showed irregular in dead pigs, whereas that of the subacute infectious pigs recovered from a one week-descend after infections. In the first 2 days, the numbers of blood neutrophil reduced in the subacute infectious pigs, and then increasd and reached the peak at 5 days post infection, accompany with reduction of phagocytic ability of neutrophil. The number of blood neutrophil, neutrophil phagocytic ability and lymphocyte transformation ability varied with different prognosis. Our results suggested that the pathogenicity between ZY0571 and HA9801 was different, secretion of the cytokines and changes of the amount of T cell subset and other immunological examination in this research have some associations with the infection progression of SS2.

Construction of an Infectious Clone within the V3 Region of gp90 Surface Protein of EIAV by N-glycosylation Reversmutations

HAN Xiu-e;ZHANG Ping;WANG Xue-feng;KONG Xian-gang;XIANG Wen-hua;ZHOU Jian-hua

2008, 39(12): 1731-1736. doi:

Abstract ( 1392 )

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The Chinese equine infectious anemia virus (EIAV) donkey-leukocyte attenuated vaccine (DLV) provides a unique natural model system, by which the mechanism of virulent attenuation and the immunological control of lentivirus replication can be studied. In this study, we compared gp90 sequences of the env gene from both virulent and attenuated EIAV strains and found that all theses sequences lost the potential N-linked glycosylation sites in the principal neutralization domain of the V3 region of the attenuated envelope gp90. To determine whether the loss of this potential N-linked glycosylation site also changes the neutralization sensitivity of the vaccine strain, this glycosylation site was restored in the infectious clone pLGFDg5. The derived virus, pLGFDg5V, demonstrated a decreased replication rate in vitro when compared with the parental vaccine strain. More importantly, the reverse mutation of the glycosylation site in the V3 region significantly enhanced the resistance of pLGFDg5V to serum neutralizing antibodies, which can recognize parental viruses. This study improves the understanding of the mechanism that the Chinese EIAV vaccine provides solid immunity to the infection of virulent strains.

The Analysis of the Immune Enhancing Effects of CpG-ODN on the Recombinant Plasmid which Contain the H Protein of Canine Distemper Virus

XU Xiang-ming;ZHU Shan-yuan;ZHANG Quan;XUE Zheng-feng;LI Hou-da

2008, 39(12): 1737-1742. doi:

Abstract ( 1324 )

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To enhance the immunocompetence of recombinant plasmid against canine distemper virus, four CpG-oligodeoxynueleotides (CpG-ODN) were designed and used in this research. Following lymphocytes proliferation test determined by 3H-TdR incorporation, one CpG-ODN was selected with optimistic immunostimulatory activity which sequence is 5′TCGTCGTTTTGTCGTTTTGTCGTT-3′. 30 healthy Beagle dogs were assigned into two groups and each group was further divided into 5 subgroups. All the subgroups were tested with different dose of CpG-ODN and recombinant plasmid pcDNA-H (which contain the haemagglutinin gene of CDV) respectively. On the 2nd, 4th and 6th week after immunization, the neutralization antibody specific to CDV and the level of gama-interferon (IFN-γ) were determined, and the data showed that the CpG-ODN could elicit a better antibody response to CDV and increase the level of IFN-γ. Furthermore, the effect of different levels of CpG-ODN were dissimilar on improving immune response, while the optimistic CpG-ODN level is 30 μg with 50 μg pcDNA-H for each animal. Five Beagle dogs were inoculated with optimistic level of CpG-ODN and pcDNAH, while another 10 dogs were only injected with pcDNA-H or normal saline water as control, all the animals were challenged with CDV 4 weeks post immunization, and the data showed that there were no Canine Distemper clinical symptom and no CDV infection in the first group, while 2(40%) and 5(100%) animals were infected with CDV in the pcDNA-H and normal saline water control group respectively.

Construction and Identification of the Expression Recombinants for PEDV COE Gene in Lactococcus Lactis

DONG Li-na;GAO Feng-shan;XU Chong-bo;HU Gui-xue;JIANG Hai-long

2008, 39(12): 1743-1747. doi:

Abstract ( 830 )

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The partial S glycoprotein gene of porcine epidemic diarrhea virus (PEDV) was amplified by RT-PCR and sequenced. The gene consisted of 531 bp and shared 99.4% nucleotide homology with PEDV CV777 strain (AF353511) published on GenBank. The COE gene (about 501 bp) in S glycoprotein gene was subcloned into the Lactococcus lactis vectors pNZ8149 and transformed into Lactococcus lactis NZ3900 by electroporation. The recombinant protein was detected by Western blot and IFA experiments after the bacteria were induced by 1 ng/mL nisin. The results indicated that the molecular weight of the expressed recombinant protein was about 20 kDa; The protein had the reactionogenicity with antibody against PEDV as expected；The protein was secreted and located on the surface of the bacteria.

Binding Research Between the Tissues Membrane of Chick Embryo and pMGA

PENG Xiu-li;GAO Jun-wei;HU Si-shun;HU Fu-li;BI Ding-ren

2008, 39(12): 1748-1752. doi:

Abstract ( 1440 )

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The GST-pMGA was expressed in E. coli BL21 with the pGEX fusion expression system. GST-pMGA expressed in supernatant was purified using GST Bind Resin and a plenty of soluble protein was obtained, the final preparation was 96% after purification by SDS-PAGE analysis. GST-pMGA was incubated with thrombin protease overnight, the pMGA was obtained and showed well immunological activity by Western-blot analysis. The tissues membrane protein from trachea, heart, lung, liver, kidney, thymus, bursa of Fabricius, spleen, proventriculus and the duodenum of SPF chick embryo were obtained through centrifugation, Dot-ELISA was applied to detect the specific binding relation between the membrane protein and pMGA. The results showed that pMGA and the tissues membrane protein of chick embryo had a specific binding except the spleen, this binding indicated receptor of pMGA on the tissues membrane protein of chick embryo. In membrane protein extracted from these tissues except spleen, there are the same product with Molecular weight approximately 30 kD by SDSPAGE analysis, so it was concluded it’s the possible protein interacting with pMGA. All this makes it possible to do further studies of receptor of pMGA.

Preliminary Studies on Prokaryotic Expression,Refolding of Duck IFN-α Mature Peptide and Its Activity against Virus

GONG Yong-qiang;CHENG An-chun;WANG Ming-shu;YANG Mei;ZHANG Yao;CHEN Bin;ZHONG Xiao-rong

2008, 39(12): 1753-1758. doi:

Abstract ( 804 )

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In order to obtain DuIFN-α and investigate its biological function, duck interferon-α(DuIFN-α) mature protein gene was cloned in to pMD18-T vector and inserted into the expressing plasmid/ pET32a⁺. Then,this recombinant plasmid was transformed into engineering bacteria/ BL2l(DE3) and a 37 kD protein was expressed in the form of inclusion bodies after 5 hours induction by 0.4 mmol/L IPTG. The expressed product which was purified and refolded by metal immobilization affinity chromatography(MIAC) have an ideal activity against virus. Its activity of anti-VSV in DEF was up to 12 800 U/mg and DuIFN-α at 15 U/mL can reduce duck plague virus replication by 80% as measured by FQ-PCR.

Effect of Monochromatic Light on the Muscle Growth and Muscle Fiber Development and Testosterone Secretion in Broilers

LIU Wen-jie;CHEN Yao-xing;WANG Zi-xu;DONG Yu-lan;CAO Jing;XIE Dian;JIA Liu-jun

2008, 39(12): 1759-1764. doi:

Abstract ( 1509 )

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The effect of monochromatic light on muscle growth and muscle fiber development in broilers were studied, and the internal mechanism was discussed in this paper. A total of 260 newly hatched broilers were divided randomly into four light treatments, and were reared under blue (480 nm, BL), green (560 nm, GL), red (660 nm, RL) and white (400—760 nm, WL) light by using LED for 49 days, respectively. The developmental characters of muscle fiber and the concentration of testosterone were investigated by histological chemistry and radioimmunity. The results showed that: ① At 21 days of age, weight of breast muscle and thigh muscle were significantly higher in GL group than that in other groups (6.46%—13.57% more in breast muscle and 6.37%—16.34% more in thigh muscle, P<0.05). Until 49 days of age, weight of breast muscle and thigh muscle were significantly higher in BL and GL group than that in other groups (21.42% more in breast muscle and 16.67%—25.44% more in thigh muscle, P<0.05). ② At 21 days of age, the area of major pectoral muscle fiber in GL group and gastrocnemius muscle fiber in GL and RL group were higher than that in other groups (Increased by 23.19%—54.01% and 33.46%—56.67% resepectively, P<0.05). Until 49 days of age, the area of major pectoral muscle and the gastrocnemius muscle fiber were higher in BL group than that in other groups (Increased by 9.92%—21.69% and 21.11%—57.44% resepectively, P<0.05). ③ At 21 days of age, the density of major pectoral muscle fiber in RL group and gastrocnemius muscle fiber in BL group were greater than that in others (Increased by 19.17%—42.94% and 10.13%—49.33% resepectively, P<0.05). Until 49 days of age, the density of major pectoral muscle fiber in WL and RL group and gastrocnemius muscle fiber in RL group were greater than that in others (Increased by 27.36% and 57.02%—112.03% resepectively,P<0.05). ④ At 21 days of age, the testosterone concentration was 35.37%—37.13% greater in BL and GL group than that in other groups (P<0.05). By 49 days of age, the testosterone concentration was 14.36%—28.77% greater in BL group than that in other groups (P<0.05). These date indicated that green or blue monochromatic light can be used effectively to increase broiler’s muscle growth and to elevate testosterone secretion.

The Study on the Distribution of PRL and Its Receptor in Skin of Young Male Alpaca(Lama Pacos)

HE Xiao-yan;ZHU Zhi-wei;YOU Rong-li;HAO Huan-qing;FAN Rui-wen;GAO Li;CAO Jing;DONG Yan-jun;DONG Chang-sheng

2008, 39(12): 1765-1771. doi:

Abstract ( 840 )

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Alpaca is one of the most important hair-producing animals. However, the genetic regulation of the growth of alpaca hair is largely unknown. Here, we report a surprising finding that prolactin (PRL), PRL receptor (PRL-R) and other PRL related genes were expressed in the male alpaca skin. The expression levels of PRL in the skin were relatively lower than that in pituitary gland. The PRL and its receptor protein were detected in root sheath of hair follicles, epidermal and sebaceous gland of alpaca. The expression of PRL and PRL-R in the male alpaca suggested that PRL may regulate the growth of alpaca hair through an unknown mechanism in addition to its established role in milk production.

Simultaneous Determination of Nitroimidazole Residues in Swine Meat by Gas Chromatography-Ion Trap Tandem Mass Spectrometry

WANG Ji-cang;MA Su-ying;WANG Da-ju;HAN He-you;LIU Deng-cai

2008, 39(12): 1772-1778. doi:

Abstract ( 902 )

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A gas chromatography-ion trap tandem mass spectrometry method was developed for the determination of DMZ, RNZ, SNZ, MNZ and ONZ residues in swine muscle. The nitroimidazole residues were extracted with dichloromethane, followed by reextracted with hydrochloric acid. The acid extract was made basified with K2HPO4, followed by extracted with dichloromethane. The final solution was carefully evaporated just to dryness with a vacuum evaporator (water bath at 40 ℃). The remaining residues was dissolved with 3 mL ethyl acetate. The solution was transferred into a derivation tube, evaporated to dryness under a weak nitrogen stream and derivatised with a solution of 50 μL of BSA-50 μL of i-octane for 60 min at 50 ℃ to produce trimethylsilylderivatives (TMSderivatives). The resulting mixture was directly injected into the GC-MS/MS system. It was shown that the calibration curves were linear with a correlation coefficients (r) over 0.998 in range of 0.005-1.6 μg/mL. The limits of detection were 0.2 μg/kg for RNZ, 0.5 μg/kg for SNZ, 1.0 μg/kg for DMZ, MNZ and ONZ. Recoveries of five nitroimidazoles in tissues spiked at 0.2-4.0 μg/kg were 74％-82％, the interday relative standard deviation was less than 13%. These results suggested that the developed method was a sensitive and accurate method for the determination of nitroimidazole residues in swine muscle.

Variation of Complete Coding Sequence of GH Gene in Chinese Bovinae Species

GENG Rong-qing;CHANG Hong;LI Yong-hong;JI De-jun;CHANG Chun-fang;WANG Lan-ping;CHANG Guo-bin

2008, 39(12): 1779-1784. doi:

Abstract ( 815 )

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CDS of GH gene of 5 bovine species Bos taurus, Bos indicus, Bos grunniens, Bos frontalis and Bubalus bubalis was connected from PCR fragments by standard double-strand DNA sequencing method to analyze the variation and molecular evolution of complete coding sequence (CDS) of growth hormone (GH) gene in Chinese bovinae species. The result showed that CDS of GH gene was 654 bp in length and mutation rate was between 0.1% and 1.84% of different bovine species. Total 10 haplotypes were defined according to sequences. Bos indicus possessed the highest haplotype diversity and no haplotype diversity was observed in Bos frontalis and Bubalus bubalis. There was codon usage bias in CDS and 25 preferenced codon were discovered. The predominant substitution model of nucleotide was transition and transition was higher than transversion with the ratio of 3.0. Nonsynonymous sites were far less than synonymous sites. The ratio of synonymous and nonsynonymous substitution were all less or equal to one manifesting that it was not effected by Darwin positive selection. Molecular tree based on haplotype of GH sequence showed that differentiation was very apparent between Bubalus bubalis and Bos taurus, Bos indicus, Bos grunniens, Bos frontalis. There was no apparent differentiation among Bos taurus, Bos indicus, Bos grunniens, Bos frontalis and they shared an alike ancestral sequence. CDS of GH gene in Chinese bovinae species was fairly conservative under functional constraint during the course of evolution together with few variation and low evolution velocity.

Improved and Selective Breeding in Low Nitrogen Excretion Quails

LI Yong-zhu;KATSUNORI Sato

2008, 39(12): 1785-1793. doi:

Abstract ( 1488 )

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An experiment was conducted to breed low-excretion type Japanese quails for decreasing the environmental pollution source. Japanese quails from Normal ，Brown breeds and crossing group were used in this eight generations (6-20 week-old) selection experiment. Quails were allocated into selected and nonselected groups with 18% CP diets and control group with 24% CP diet, with feed intake as main select traits and laying rate and nitrogen excretion as subsidiary select traits. The results showed that adaptive capacity of selected and non-selected groups were both increasing with the increasing generation (P<0.05) and the genetic improving capacity of N strain was 0.26. Laying rate between generations had an increasing trend, the average cumulative laying rate of selected group was higher than that of non-selected group (P<0.05), and the genetic improvement of selected group was significant (P<0.01). There were no difference on feed intake between generations(P>0.05), while the feed intake of selected groups was higher than that of non-selected group but lower than that of control group (P<0.05) in the same generation. The nitrogen excretion and serum uric acid content of selected group was lower than that of non-selected and control group after improved two generations (P<0.05), and the nitrogen excretion was degreed by 15%-17%. To sum up, laying performance was improved and nitrogen excretion was reduced of Japanese quails in the N and B breeds after selective breeding, especially in BN group.

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