Phytic acid exists in food and feed widely，which can be combined with phosphorus and other nutrients to form insoluble complexes and subsequently reduce the phosphorus and other nutrient utilization rate.Phytase can decompose phytic acid and its salts to release of phosphorus and other nutrients，thus，phytase can be added in feed to replace part of dicalcium phosphate.However，if we reduce the amount of calcium hydrogen phosphate and forget to add phytase in feed in the same time，which will decline the feed quality，and subsequently lead to poor growth performance and production quality of animals.The measurement of phytase activity in feed is very important to ensure the quality of feed products.At present，the commonly detection methods are cumbersome and unsuitable for on-site detection of phytase activity for feed and animal husbandry enterprises.In this paper，a conception of detecting phytase activity with immunological techniques is proposed.Furthermore，we prospect the application of immunological techniques in the detection of phytase activity.

The study aimed to amplify the coding region of DLDH gene，which could be used to further study the signal pathway of DLDH expression and the correlation between DLDH gene expression and sperm capacitation，acrosome reaction.RNA was extracted from testicle and cDNA was obtained by reverse transcription.Specific primers were designed to amplify the coding region of DLDH gene.The sequence of coding region and amino acid of DLDH were analyzed and predicted by Lasergene，BioEdit，ClustalX，Mega bio-softs，and the molecular phylogenetic trees of coding amino acids region of DLDH gene was constructed by NJ and ME，respectively.The sequence length of coding region of the BMI DLDH gene was 1 530 bp，encoding a protein of 509 amino acids.By biology software analysis，the nucleotide and amino acid sequences of DLDH among different species were conserved in evolution.The DLDH protein contained the abundant phosphorylated sites and was assembled with α-helixes and random coil.

In order to study the potential regulation mechanism of the USF1 on monosaccharide transporters in chicken intestinal epithelial cells，we built the eukaryote expression vector pcDNA3.1-USF1 on the basis of bioinformatics analysis and made the transcription factor USF1 over-expressed in chicken intestinal epithelial cells.Besides，we detected and analyzed the influence of USF1 on the mRNA expressions of SGLT1，GLUT2 and GLUT5 by Real-time PCR.The results indicated that the over-expression of USF1 gene significantly up-regulated the mRNA expressions of GLUT2 and SGLT1 in chicken intestinal epithelial cells(P＜0.01)，while the expression of GLUT5 were not changed significantly(P＞0.05).Combining the results with the bioinformatics analysis of the USF1 binding sites on the 5′up-stream regulation area of the 3 monosaccharide transporters，our results indicated that the transcription factor USF1 positively regulated the expression of SGLT1 and GLUT2 in chicken intestinal epithelial cells.

This experiment was conducted to study genetic differentiation between 2 sheep populations with distinct tail types by genome-wide detection of selection signatures and to search the candidate genes related to important traits of sheep.Based on the Illumina Ovine SNP 50K Breadchip genotyping data of Mongolian sheep and Tibetan sheep，population differentiation index FST was adopted to detect the selection signatures，and to found the genes located in selection signature regions.Based on the tail adipose tissue of Hulun Buir sheep(fat-tailed sheep，include large fat-tailed sheep and small fat-tailed sheep) and Tibetan sheep(thin-tailed sheep)，the relative expression study was adopted to explore the difference of gene relative expression level of PPARG and PDGFD，which were related to fat metabolism.This result showed that：(1) 465 selected SNPs were found，448 candidate genes were detected by gene annotation． And 50 genes related to lipid metabolism were selected from these candidate genes，the analysis of GO showed that these genes were related to lipid biosynthetic process，phospholipid metabolic process and lipid binding.In addition，4 genes were found，PPARG，RXRG，SLC27A2 and ACSL6，and enriched into the PPAR signaling pathway.(2)The gene relative expression levels of PPARG and PDGFD from Hulun Buir sheep was significantly higher than that of Tibetan sheep(P<0.01)， between fat-tailed and thin-tailed sheep，the gene relative expression level of PPARG from lager fat-tailed Hulun Buir sheep was significantly higher than that of small fat-tailed Hulun Buir sheep(P<0.05)，but the gene relative expression level of PDGFD had no significant difference between lager fat-tailed and small fat-tailed Hulun Buir sheep.F_ST could be used to detect the genes with selection signatures and some of the candidate genes were related to important traits in sheep.PPARG and PDGFD genes were proved to be closely related to tail fat deposition through the relative expression studies，these genes could be used as candidate genes for different tailed sheep breeding.This study can provide theoretical references for sheep breeding or breed improvement.

In order to select the genes which regulate the growth of secondary hair follicle in cashmere goat.This study choose cashmere goat and dairy goats to be the material.Because these 2 goats get the similar genetic background，but significant differences traits of the secondary follicle.An Illumina Hiseq2000 platform was used to sequence the 32 skin samples from 4 cashmere goat and 4 dairy goat on March，June，September and December.The Sequencing obtained 38G data，check out 20 780 goats genes.We identified 2 409 differentially expressed genes between cashmere goat and dairy goat in the same period，1 175 cashmere goat genes were up regulated compared with dairy goat，1 234 were down regulated.And there were a total of 550 differentially expressed genes between different growth periods in cashmere goat，302 cashmere goat genes were up regulated，248 were down regulated.GO functional annotations showed that the main differentially expressed genes appear in the cells and cell parts，organs parts，binding，biological regulation，cellular process.KEGG pathway analysis found that 42 differentially expressed genes appear in WNT，SHH，TGF β，TNF，MAPK and Notch signaling pathways which intimately associated with hair follicle growth.This study provided valuable information to verify secondary follicle growth related gene function，provided an important basis to reveal the secondary follicle growth mechanism and provided the theory to increase production and quality of cashmere.

This study aimed to explore the association of single nucleotide polymorphism(SNP) of SIRT1 and SIRT2 genes with Qinchuan cattle meat quality traits，so as to search for molecular markers related with meat quality traits.DNA seuqencing and PCR-PFLP methods were applied to detect the genotype polymorphisms of SIRT1 and SIRT2 genes in Qinchuan cattle populations，and analyze the associations of single and combined genotypes with meat quality traits of Qinchuan cattle.Results showed that 2 SNPs(G25764A and A25846G) were detected in 3′-untranslated region of SIRT1 gene，and 2 mutants(C19501T and C19518T) in 3′-untranslated region of SIRT2 gene were identified，too.Association analysis indicated that，in Qinchuan cattle population with 505 individuals，at G25764A locus，individuals with AA genotype exhibited very significantly higher loin-eye area than those with AB genotype(P<0.01).At A25846G locus，the loin-eye area of animals with AA genotype was significantly higher than that of animals with AB genotype(P<0.05) and very significantly higher than that of animals with BB genotype(P<0.01).Moreover，the C19501T locus significantly affected loin-eye area and intramuscular fatty content(P＜0.05)，and AA was the preferable genotype.Besides，at C19518T locus，individuals with the BB genotype showed significantly higher backfat thinckness compared to those with the AA genotype(P<0.05).The optimal combined genotype of SIRT1-SIRT2 was AB-BB-AA-BB，which was more effective on meat quality traits than other combined genotypes(P＜0.01 or P＜0.05).These results suggested that the single nucleotide polymorphisms and combined genotypes of both SIRT1 and SIRT2 genes affected the meat quality traits of Qinchuan cattle，and they could be used as candidate genes for breeding Qinchuan cattle with better meat quality traits.

The study aimed to analyze the genetic characteristic and expression of bovine FABP3 gene in transgenic mice，which will play a key role in culturing the breeds that produced higher yield and improved quality by transgenic method.Four FABP3 transgenic mice were used，G1 generation mice were obtained by 4 mating schmes.The genetic characteristic and expression of bovine FABP3 in heart，liver，kidney and skeletal muscle of transgenic mice were investigated in this study by RT-PCR and fluorescent in situ hybridization.The results of RT-PCR showed that bovine FABP3 gene could be identified in G0 transgenic mice by RT-PCR and propagated to G1 offspring，too.The positive rate was 68.4% in G1 and FABP3 gene expression levels in heart，skeletal muscle of transgenic mice were much higher than that of negative controls by real-time PCR.Furthermore，the fluorescent in situ hybridization results showed that bovine FABP3 gene could be expressed in the heart，liver，kidney and skeletal muscle in G0，G1 mice，and the expression in G0 was higher than that in G1 mice.This study suggests that bovine FABP3 gene has been successfully integrated and expressed in the mouse DNA，and expressed in G1 mice.The result would help us guide beef cattle molecular genetic breeding in the future.

This study aimed to optimize bet gene of bacteriophage λ so that it can be efficiently expressed in cattle cells. The optimized bet gene was synthesized by genome-wide synthesis method and expressed in prokaryotic expressing vector，the polyclonal antibody was prepared and antibody titer evaluation was preformed.The sequence of bacteriophage λ bet gene was obtained from GenBank(GenBank accession No.:AP005153 ) and optimized with bovine preferred codons. Then synthesized the optimized gene.According to the optimized gene sequence,a pair of primers with the restriction enzyme sites were designed.The bet gene was amplified by PCR and cloned into the pET28a prokaryotic expressing vector. Fusion protein expressed in E.coli BL21 by IPTG induction,then we purified the protein products by affinity chromatography methods and identified by Western blot with His tag antibody. CD1 mice were immunized with the purified fusion protein. Serum with anti-Beta protein polyclonal antibody was obtained, and then the antibody titer was detected by ELISA assay. As a result,confirmed by restriction enzyme digestion and sequencing, pET-bet prokaryotic expressing vector was successfully constructed; The fusion protein was high efficiently expressed, which was confirmed by SDS-PAGE; CD1 mice were immunized with the purified protein, the polyclonal anti-Beta antibody with 1∶41 700 titer was prepared and it could bind His-Beta fusion protein specifically, which was confirmed by ELISA assay and Western blot assay. The successful preparation of anti-Beta protein polyclonal antibody will lay a solid foundation for further studies of the biological functions and applications in bovine and other mammals with bet gene.

This study focus on the affection of BMP1 gene on proliferation and apoptosis of in vitro cultured buffalo granulosa cells.Expression pattern of BMP1 in different in vitro cultured time of buffalo granulosa cells including 0，24，48，72 and 96 h were determined by qRT-PCR separately.Different concentrations of BMP1 recombinant protein or BMP1 antibody were added to the culture medium，and the effect to proliferation of granulose cells were detected，further effect on expression pattern of cell proliferation and apoptosis associated genes were also analyzed by qRT-PCR and Western blotting.BMP1 gene was significantly increased in the in vitro cultured granulosa cells with the extended recording time(P<0.05)，and reached a peak at 96 h.Adding BMP1 recombinant protein significantly promoted the proliferation of granulosa cells(P<0.05)，as well as up-regulated the expression of Cyclin D1，Cyclin D2 and PCNA genes in mRNA level.In addition，mRNA expression level of pro-apoptosis factors(Fas，FasL，TNFR1， TNF-α，Cytochrome C，Apaf1，Chop，Caspase-3，Caspase-8 and Caspase-9) were down regulated.Furthermore，Western blotting results showed that the protein expression level of Fas，Bax and Caspase-9 was also down-regulated.The contrast results were observed by adding BMP1 antibody investigation.BMP1 promotes proliferation and inhibits apoptosis of in vitro cultured buffalo granulosa cells by regulation the expression pattern of associated genes，which indicated that it took an important role in the follicular development of buffalo，and laid a theoretical foundation to clarify the livestock follicular genesis.

For developing efficient cryopreservation technology of chicken blastodermal cells (BCs) and storing female poultry genetic resources，the BCs was frozen using dimethyl sulfoxide (DMSO) and straw，and the different DMSO concentration，freezing and thawing rate were compared by calculating the cell viability (CV) using fluorescein diacetate (FDA) and cell adherent rate cultivated for 24 h (CAR).Results showed that：(1) For the concentration of DMSO，the highest CV was in 20% group (0.58) and had no significant difference with group 15% (P>0.05)，but had extremely significant difference with group 5%，10% and 25% (P<0.01).The highest CAR was 30.5% in group 20%，and had no significant difference with group 15% (P>0.05)，but had significant difference with group 25% (P<0.05)，and had extremely significant with group 5%,10% (P<0.01).(2)The 3 steps to freeze BCs was used，and the first step was cooling at a rate of -1 ℃•min-1 to -7 ℃ and kept 10 min，the second step was to continue cooling the temperature at -15,-35,-55 or -75 ℃ respectively，the third step was to plunge the straws into liquid nitrogen.The highest CV was 0.53 of group -75 ℃，and had no significant difference (P>0.05) to group -35 or -55 ℃.The CAR had extremely significant difference within each groups (P<0.01)，and the highest was 36.6% in group -35 ℃.(3) The thawing rate at 37 ℃ water bath for 10 s，1 min，2 min and 3 min，the CV of each groups had no significant difference (P>0.05)，the highest one was 0.60 in 10 s group.The CAR was 43.9% in group 1 min，and it had no significant difference (P>0.05) with group 2 min，had significant difference (P<0.05) with the group 10 s and had extremely significant difference (P<0.01) with group 3 min.The thawing rate of 50 ℃ water bath for 5 s，20 s，40 s or 1 min，the highest CAR was 0.7 in group 5 s，and it had significant difference (P<0.05) with group 20 s，had extremely significant difference (P<0.01) with group 40 s and 1 min.The CAR thawed at 50 ℃ had extremely significant difference within the 4 groups (P<0.01).The highest CAR was 36.9% in group 5 s，and the lowest was 5.6% in group 1 min.In conclusion，the BCs frozen using 20% DMSO and straw，cooling at -1 ℃•min-1 to -7 ℃ for 10 min，continuing to cool at -35 ℃ at the same ratio and plunging into liquid nitrogen，then thawed by 37 ℃ water bath for 1-2 min，could get not only good CV，but also CAR.

The goal of this study was to assess how different microinjection conditions influence chimeras production after ES/EG cells injection into embryos in mouse and pig.Mouse ES cells were micrioinjected into embryos at morula or blastocyst stages.Hatching rate and blastocyst rate after cell injections were measured in vitro.Non-injected embryos were used as controls.In mouse，embryos showed significant higher developmental rate when ES cells were injected into morulae compared to those injected into blastocysts(P<0.01).In contrast，cells injected into blastocysts had higher hatchability than those injected into morulae(P<0.01).Compared to non-injected controls，ES cell injected embryos had higher hatchability but lower developmental rate after in vitro culture(P<0.01).We further assessed porcine embryonic development after EG cells microinjection into morulae，early blastocysts，blastocysts，or hatched blastocysts，and we also investigated the optimal injecting/ holding pipette sizes for pig EG cell microinjection.Our data showed that pregnancy rates of EG cells microinjected into morulae，early blastocysts，or blastocysts，were significant higher than those injected into the hatched blastocysts(P<0.01).The hatched rate was significantly lower in microinjected embryos compared to non-injected controls(P<0.01).Two chimeric piglets were obtained after embryo transfer.In addition，we found that the optimal inner and outside diameters for pigs EG microinjection were 25 and 30 μm for the injecting pipette，and 40 μm and 130-150 μm for the holding pipette，respectively.Upon comparison of different conditions，we determined optimal injecting/holding pipette sizes for pig EG cell injection.Furthermore，ES/EG cells microinjection into morulae could achieve higher contribution rate in chimera.Microinjection had lower impact on mouse embryonic development compared to pig，suggesting that pig embryonic microinjection condition has to be further optimized.

This experiment was conducted to investigate the effect of dietary zinc(Zn) on laying performance，egg quality，hatching performance of eggs and plasma antioxidant capacity of broiler breeder hens under different temperatures，and discussed the anti-heat stress effect of dietary Zn on broiler breeder hens preliminarily.One hundred and forty-four twenty-three-week-old female broiler breeders were randomly allotted to 6 treatments with 6 replicates of 4 birds per replicate based on body weight(BW).A completely randomized factorial design involved in 2 environmental temperatures：(21±1) ℃ normal temperature and(32±1) ℃ high temperature and 3 dietary Zn treatments：a no Zn addition corn-corn starch-soy isolate protein meal basal diet(9.98 mg•kg^-1 Zn)，and the basal diet supplemented with 110 mg•kg^-1 Zn of diet as either ZnSO₄•7H₂O or Zn proteinate with the moderate chelation strength(Q_f=30.73).The adaptation period was 7 weeks，the Zn depletion period was 3 weeks and the experimental period was 3 weeks.The result showed as follows：1) Feed intake had not significant difference(P>0.10) between normal and high temperature.On that condition，heat stress significantly decreased laying rate，average egg weight，eggshell thickness，egg yolk color，eggshell weight(P<0.01)，eggshell strength(P<0.10)，and plasma CuZn superoxide dismutase(CuZnSOD) activities(P<0.01) of broiler breeder hens，and significantly increased the broken egg rate，feed-egg ratio(P<0.01)，and plasma malondialdehyde(MDA) content(P<0.10).2) Compared with the no Zn addition group，broiler breeder hens fed the diets added Zn regardless of sources(P>0.10) had higher birth rate and healthy young chicken percentage(P<0.05).Broiler breeder hens fed the diet added organic Zn had higher yolk color score，plasma Zn，and plasma CuZnSOD activities(P<0.10) than those fed the diets added inorganic Zn and no Zn addition group，which had not significant difference(P>0.10).3) No interactions(P>0.10) between temperature and dietary Zn were observed in all of above indices.The results indicate that the high temperature impaired laying performance，eggshell quality，and yolk color score，and plasma antioxidant capacity of broiler breeder hens.Diet added either organic or inorganic Zn improves hatching performance of eggs of broiler breeder hens，and especially the effect of the organic Zn on increasing the yolk color score，plasma Zn，and plasma CuZnSOD activities is better than that of the inorganic Zn.

The aim of this experiment was to investigate the dynamics of nutrient composition and rumen degradation characteristics of Kinggrass at different plant heights in Hainan Black goat.Kinggrass was cut and sampled when the plants height were 90，120，150，180，210，240 and 270 cm，respectively，and their nutrient compositions were determined.Rumen degradability and effective digestibility (ED) of dry matter (DM)，crude protein (CP) and neutral detergent fiber (NDF) were determined by the rumen nylon-bag technique with 3 Hainan Black goats with permanent rumen cannulas.The results showed as follows：The nutrient composition of Kinggrass changed with increasing plant height，CP content decreased but acid detergent fiber (ADF) and NDF content increased，and Kinggrass had the lower mixture quality standard when the plant was higher than 180 cm.Kinggrass rumen degradability at different plant heights increased faster before 48 h，from 48 to 72 h gradually stabilized，and the rate of degradation of slowly degradable fraction from low to high and then decreased.Rumen degradability of DM，CP and NDF at different periods decreased with increasing plant height.The slowly degradable fraction of DM，CP and NDF were highest at 180 cm and the rate of degradation were highest at 150 or 180 cm.The rapidly degradable fraction，potentially degradable fraction and ED of Kinggrass decreased with increasing plant height.In conclusion，considering nutrient composition，mixture quality standard and rumen degradation characteristics of Kinggrass at different plant heights，it have the higher feeding value when the plant is lower than 180 cm.

Porcine Hokovirus(PHoV) is a new parvovirus of pigs which was recently discovered in Hong Kong，China in 2008.In present study，a total of 147 blood samples were collected from pig farms and slaughter houses in Lanzhou，Zhangye and Tianshui city，Gansu province.Two pairs of primers were designed based on VP1 regions of PHoV genome(GenBank accession No.EU200677).One hundred and forty seven samples were detected by nested PCR(nPCR) assay for PHoV.The nPCR showed an overall prevalence of PHoV as high as 38.10%(56/147).Of which，>12-week-old pigs showed a significantly higher positive rate(55.56% ) than <6-week-old piglets(25.00%) （P<0.05），while there was no significant differences among three cities （P>0.05）.Out of the 56 positives，12 were selected in terms of geographical distribution for sequencing(GenBank accession No.：JQ177084-JQ177095).Phylogenetic analysis together with 11 reference VP1 sequences of PHoV from GenBank revealed a homology of 95.2%-99.0% among the present 12 VP1 sequences.Of which，JQ177093，JQ177085 and JQ177089 formed a separate branch in the phylogenetic tree，and other 9 together with 12 reference sequences from GenBank formed the second branch.Of the later 9，JQ177095，JQ177087 and JQ177091 formed a sub-branch closely related to JF738366 from wild boar in Romania，whereas JQ177086，JQ177090 and JQ177094 formed another sub-branch just next to HK3 from Hong-kong pigs，and the third sub-branch of JQ177088，JQ177092 and JQ177084 closely related to HK5.The present 12 PHoV-VP1 sequences shared a homogeneity of 95.2%-99.0% with Romania wild boar isolate (JF738366)，and 96.7%-99.7% with Hong Kong-originated HK1-HK6.The present findings confirmed a high prevalence of PHoVs both evolved from Romania wild boar isolate and Hong Kong pig isolates in Gansu province.

This study aimed to investigate the epidemiology of bee viruses in Apis cerana and Apis mellifera and analyze the influence of different locations and beekeeping environment on virus infection.During 2012-2013，50 colonies of Apis cerana and 108 colonies of Apis mellifera were collected from 8 provinces and municipalities.RT-PCR was used to detect seven viruses，deformed wing virus(DWV)，black queen cell virus(BQCV)，sac brood virus(SBV)，Kashmir bee virus(KBV)，chronic bee paralysis virus(CBPV)，acute bee paralysis virus(ABPV) and Israeli acute paralysis virus(IAPV).The results showed that no KBV was detected in any samples.BQCV was most prevalent among the six detected viruses，and DWV was the second in both honey bee species.Infection rate of SBV in Apis mellifera is significantly higher than in Apis cerana(P<0.01).CBPV，ABPV and IAPV were present at low levels in both species.Among the four provinces for Apis cerana samples，IAPV was found in Liaoning and Hainan，CBPV was present in Fujian and Liaoning while ABPV was only detected in Guangdong.DWV，BQCV and SBV distributed in all seven provinces where Apis mellifera were collected.But SBV infected at lower level in Fujian than the other locations(P<0.01).Five viruses were present in Xinjiang，Beijing and Liaoning，and four viruses were detected in Shanxi，Shandong，Hainan and Fujian.For the three types of beekeeping areas，six viruses were found in Apis mellifera isolated areas，while five viruses in mixed Apis mellifera and Apis cerana areas and only three viruses in Apis cerana isolated areas.Simultaneous multiple viruses infection was common in both species，with most samples were infected with DWV and BQCV or DWV，BQCV and SBV.These results indicated that BQCV and DWV were the most prevalent viruses in both species.The distribution and infection rate of the six detected viruses varied in different locations and beekeeping areas，but multiple-viruses infection was common in two honeybee species.

The study was conducted to develop the specific method for detecting antibody against Actinobacillus pleuropneumoniae(App) serotype 7(S7)，the most prevalent serotype all over the world，and apply to the inspection and quarantine for antibodies of imported and exported pigs.Hybridoma cell line secreting monoclonal antibody(McAb) against App S7 was screened，and the McAb was purified from mouse ascitic fluid and labeled with HRP.The competitive ELISA（cELISA）was developed and its specificity and stability were tested.The sera collected from pigs immunized with reference strain of App S7 or field samples were assayed by this cELISA and other commercial ELISA kits parallelly.The McAb，characterized as IgM，can react specifically with reference strain of App S7，and no cross-reaction with other serotype strains and 27 other strains.The developed cELISA was also specific for detecting antibody against App S7.The antibody against App S7 from the immunized pig can be detected by this method at 14 days post injection，the antibody titer reached and kept at the peak from 35 to 143 days post immunization.The data detected from the samples of 260 clinical sera and the immunized swines，showed that the cELISA have 97.2% coincidence with IDvet test kit and 97.9%-98.2% consistent with Biovet test kit.This cELISA can be used to detect antibody against Actinobacillus pleuropneumoniae serotype 7 with high specificity.

The aim of the present study was to clone the cathepsin B (Etcat B) gene involved in E.tenella using homologous method，and lay the the foundation for further gene expression and gene function study.Through analysis of E.tenella sequence published，the cDNA sequence of Etcat B was cloned by RT-PCR from unsporulated E.tenella Houghton strain.The Etcat B was recombined in trans-E1 prokaryotic expression vector.After sequence identification，the recombinant plasmid was transfected into E.coli rosetta to induce expression.The purified protein was used to immunize rabbits to produce polyclonal antibody，which was verified by ELISA，and was also analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gelatin zymography.With the method of real-time PCR and Western blot，we detected the expression of Etcat B and Etcat L at different stage.Results showed that an 1 539 bp gene was obtained，which encoded 512 amino acid residues.The Etcat B cDNA fragment was sub-cloned into the prokaryotic expression vector trans-E1.After inducing in 1 mmol•L^-1 IPTG at 37 ℃ for 4 h，SDS-PAGE electrophoresis showed it was expressed mainly in the form of inclusion bodies；while at 4 ℃ overnight the protein was inducted into soluble expression.With the presence of reducing agent at low pH，the recombinant protein can degradate the gelatin，which proved that the recombinant enzyme having a certain activity.Etcathespin B was higher in the phase of gametophyte and merozoite，and gradually increased during sporulation process.Cloning and expression the cathepsin B in E.tenella，provides theoretical basis for further exploration of the role of the gene in chicken coccidia invasion and other biological functions.

DF-1 cells with over-expression of chicken PrP^C (DF-1-PrP)，DF-1 cells with pcDNA3.1(DF-1-NC) and DF-1 cells were used as cell models in order to study the effect of PI3K/Akt pathway on DF-1-PrP cells proliferation，adhesion，invasion and apoptosis and its relationship with ChPrP^C expression.After cells were treated with 0，10，20，50，100 and 200 nmol•L^-1 Wortmannin，adhesion assay，transwell assay，MTT assay，flow cytometric assay and RT-PCR analyses were used to detect cell adhesion，invasion，proliferation，apoptosis and expression of PRNP mRNA，respectively.The data showed that the expression of PRNP mRNA of DF-1-PrP，DF-1-NC and DF-1 cells were all decreased with the increasing of wortmannin concentration，and adhesion，invasion and proliferation ability were reduced，but apoptosis rate were increased.Furthermore，under the same concentration of less than 100 nmol•L^-1，adhesion，invasion and proliferation ability of DF-1-PrP cells was higher than DF-1-NC and DF-1 cells，apoptosis rate was lower.All these results indicated that over-expression of ChPrP^C promoted DF-1-PrP cells adhesion，invasion and proliferation and inhibited apoptosis，in which processes PI3K/Akt pathway may play an important role.PI3K/Akt pathway can be effectively blocked by wortmannin，but it may not be the only pathway that ChPrP^C regulated cell apoptosis.

CD8α is an important surface marker of cytotoxic T lymphocyte.This molecule is critical for cell-mediated immune response and defense mechanisms，widely involved in recognition of specific antigens in association with MHCⅠ molecules and transmembrane signal transduction.In this study，the yak(Bos grunniens) CD8α was cloned using RT-PCR，and the expression of CD8α mRNA and protein in the major immune organs(thymus，spleen，mesenteric lymph nodes and haemal nodes) of adult male yaks were detected by means of quantitative real-time fluorescence quantitative PCR，Western blots and immunohistochemical SP methods.The RT-PCR results showed the coding sequence of yak CD8α contained 729 bp，encoded for 243 amino acids.The cDNA sequences of yak CD8α revealed significant homology with Bos taurus.The quantitative real-time fluorescence quantitative PCR and Western blot results showed that CD8α mRNA and protein were widely expressed in immune organs.The expression levels in the thymus was the highest than other immune organs(P<0.01)；additionally the levels of haemal nodes was similar to that of spleen，and both of them were higher than those for mesenteric lymph nodes(P<0.01).Moreover，the immunohistochemical results showed the CD8α ⁺ cells were distributed in the cortex and medulla of yak thymus.In the spleen，many CD8α ⁺ cells were located in the periarteriolar lymphoid sheaths and red pulp.Moreover CD8α⁺ cells in mesenteric lymph nodes and haemal nodes were mainly presented in the mantle zone and medulla.The results indicated the thymic production might influence the CD8α⁺ T lymphocytes extent of secondary immune organs.Then haemal nodes might have similar functions in cellular immunity as the mesenteric lymph nodes and spleen.These datas would provide new insights into timely immunization，the pathogenesis and disease prevention.

In order to further verify the influence of early application of astragalan and thiamphenicol on the ALV-J congenital infection，5-day-old-SPF chickens were inoculated with 1000 TCID₅₀ ALV-J through the yolk sac and count the mortality of the SPF embryos during incubation.Then，105 SPF chickens at 1 day of age were randomly assigned into three groups of 35 each.The experimental group was respectively given with astragalan(100 mg•L^-1) and thiamphenicol(100 mg•L^-1) lasting for five days，three times with 5 days of the internal，and the last group was the virus infected control.A comprehensive evaluation about astragalan and thiamphenicol on the influence of ALV-J congenital infection was carried out by cloaca detoxification and viremia of ALV-J，vaccine immunization level of ND，the immune organ index and body weight，tumor incidence and histopathological examination.The results indicated that the two drugs had no significant influence on viremia elimination，occurrence of ALV-J antibody and the increase of ND antibody.Similarly，the weight difference between astragalus group and virus infected group was not significant at the whole experimental process；Although early weight of thiamphenicol group was obviously higher than that of other two groups，its weight was lower than that of other two groups after 60 d.The significant deduction of peripheral blood leucocyte/lymphocyte at 6 d and immune organ index of the spleen and bursa of Fabricius at 42 d in thiamphenicol group was observed lower than those of other two groups.Furthermore，tumors incidence at thiamphenicol group was the highest，28.57%(10/35)，followed by infection control group’s 20%(7/35) and astragalus group’s 11.43%(4/35)，and tumor types also appeared diversification including the hemangioma，myelocytomas，lymphatic sarcoma，fibrosarcoma and adenocarcinoma.Therefore，the early application of thiamphenicol can lead to immunosuppression，promote the tumorigenic effect and diversify the tumor types of ALV-J.Although the astragalus can reduce the pathogenicity of ALV-J，it can’t contribute to the elimination of viremia.

The experiment was designed to preliminary study on the effect of Jinqiancao compound potion to the formation of calcium oxalate stones in the urine of canine.Twelve healthy male Canis Lupus familiaris were randomly divided into 3 groups，which were the control group，the model group and the treatment group.The dogs in control group were fed with basal diet.The dogs in model group and treatment group were fed with basal diet plus glycol，ammonium chloride，calcium carbonate and vitamin D.Once 80% of the microscope view appeared calcium oxalate crystals in the urine of the experimental dogs，the Jinqiancao compound potion was orally administrated to the dogs of treatment group.During the experiment，the urine index(including the content of microalbumin，the enzymatic activity of N-acetyl-β-D-Glucosaminidase and γ-glutamyltransferase) and the serum index(including the content of creatinine，blood urea nitrogen，uric acid and malondialdehyde，the enzymatic activity of lactate dehydrogenase and superoxide dismutase) were detected.Results were as follows：compared with model group，by treating Jinqiancao compound potion，the treatment group appeared lower activity of N-acetyl-β-D-Glucosaminidase，lower activity of γ-glutamyltransferase，lower concent of serum uric acid，lower malondialdehyde，lower activity of lactate dehydrogenase and higher activity of serum superoxide dismutase than that of modle group.Our study demonstrates that Jinqiancao compound can effectively reduce the damage to renal during the formation of calcium oxalate calculus，improve the enzyme activity of blood antioxidant.In other words，kidney cells were effectively repaired to ensure the function of kidney by the Jinqiancao compound，which to prevent the formation of calcium oxalate calculus in the urine of canine.

The objective of this study was to investigate whether or not dandelion aqueous extract combated bacterial growth or affected biofilm formation by S.suis.In this study，the chlorogenic acid content of the dandelion aqueous extract was detected by high efficiency liquid chromatography (HPLC).Using a microdilution broth method，the MIC or MBC of dandelion aqueous extract or chlorogenic acid against S.suis was determined.The S.suis biofilm was determined by crystal violet staining.The S.suis biofilm was also examined by scanning electron microscopy.The results revealed that the chlorogenic acid content of the dandelion aqueous extract was 5.833 mg•mL^-1.The MIC，MBC of dandelion aqueous extract to S.suis was 62.5，250 mg•mL^-1，respectively.The MIC，MBC of chlorogenic acid to S.suis was 2.5，10 mg•mL^-1，respectively.In the negative control，scanning electron microscopy analysis revealed that bacteria were embedded within the biofilms，and the thick slimes revolved around the bacteria.However，the biofilms were significantly affected following growth in the presence of dandelion aqueous extract or chlorogenic acid.Moreover，biofilm formation by S.suis was dose-dependently decreased by sub-MICs of dandelion aqueous extract or chlorogenic acid.Sub-MICs of dandelion aqueous extract or chlorogenic acid also diminished bacterial population by S.suis.There were no slimes and extracellular matrix components in sub-MICs of dandelion aqueous extract or chlorogenic acid treated s.suis biofilm.In summary，our study showed that sub-MICs of taraxaci herba aqueous extract significantly decreased biofilm formation by S.suis，and one of the active ingredients in dandelion aqueous extract was chlorogenic acid.

The aim of this study was to analyze the immunological response of the recombinant bactericidal/permeability-increasing protein(rBPI) in Argali Hybrid Sheep(AHS) infected with Mycoplasma ovipneumoniae (MO). Six Bashibai Sheep(BS) was taken as group A，while 12 AHS were randomly divided into group B and C.All the sheep were artificially infected with MO.From the 4th day after infection，group C was treated with rBPI protein，150 mg•sheep-¹•day^-1，and group A and B with the same dose of placebo（Normal saline）.The concentrations of IL-8，IL-10，TNF-α and IFN-γ from serum at different times were detected by ELISA.At the end of the test，all the sheep was slaughtered，and the therapeutic effect was evaluated with pathological section of their lung tissue.Results were as follows：In the early phase of infection，the levels of IL-8，TNF-α，IFN-γ were elevated，while IL-10 revealed a lower trend.On the 7th day post-infection(DPI)，the concentration of IL-8 in group C was lower than group A(P<0.05).From the 7th to 21st DPI，IL-8 in group B was significantly higher than group A(P<0.01).On the 5th DPI，IL-10 in group C was higher than group A(P<0.05)，but lower than group B(P<0.01).On the 14th to 21st DPI，IL-10 in group C was lower than group A(P<0.05)，but higher than group B(P<0.05).On the 14th to 21st DPI，IFN-γ in group C was higher than group A(P<0.05).On the 14 th to 21st DPI，TNF-α in group C was higher than group A(P<0.05，P<0.01)，and lower than group B(P<0.05).Histopathological scores of the A，B，C group were 9.4，19.9 and 12.8，respectively.The score of group B had significant difference with group A and C(P<0.05，P<0.05).The results showed that rBPI has some treatment effect on AHS infected with MO and a regulatory role on serum cytokines.

The study aimed to study the effect of phytase supplementation in nursery pigs fed diets with different levels of inorganic phosphorus(P) on the growth performance，serum biochemical parameters and apparent digestibility of nutrients.A total of 224(40±2)-day-old nursery pigs were divided into 7 treatment(TRT) with 4 replicates per group and 8 piglets in each replicate.Pigs in the TRT Ⅰ(control group) were fed a basal diet prepared according to the NRC(1998)，adding dicalcium phosphate to meet phosphorus requirement.TRT Ⅱ，Ⅲ，Ⅳ，Ⅴ，Ⅵ，750 FTU•kg^-1 of phytase added，were supplemented with 100%，75%，50%，25%，0% of dicalcium phosphate in control group，respectively.No dicalcium phosphate and phytase were added in TRT Ⅶ(negative control group).Periods of pre-trial and trial were 7 and 35 d，respectively.The results showed that the average daily feed intake(ADFI)，average daily gain(ADG) and the feed conversion ratio(F/G) of the groups of 100%，75%，50% dicalcium phosphate added phytase were not significantly different with the control group(P>0.05).The group without dicalcium phosphate added phytase could significantly improve the F/G(P<0.05).With the reduction of dietary inorganic P levels，ADFI，ADG and the serum P concentration of the phytase groups decreased with linear and quadratic effects(P<0.01)，meanwhile the serum alkaline phosphatase(ALP) activities，apparent digestibility of calcium(Ca)，P，crude protein(CP) and dry matter(DM) of the groups added phytase increased with linear and quadratic effects (P<0.01).The apparent digestibility of Ca and P of the groups without dicalcium phosphate added phytase were significantly higher than that of negative control group(P<0.05).In summary，under the experimental conditions，the phytase addition in the diet reducing the amount of inorganic Pimproved the digestibility of nutrients and production performance of nursery pigs，and effect of phytase was more significant in diet with lower inorganic P level.It is suggested that adding phytase at 750 FTU•kg^-1 might reduce 25%-50% of dicalcium phosphate supplementation in the diets of nursery pigs.

Host fators are widely involved in transcription，duplication and translation of influenza virus genome，and host factors are reruired for the infection of influenza virus.PA-N182 is a recently identified trunctated form of influenza PA gene，and little is known about its host interaction factors.COPD(coatomer protein complex，subunit delta)，with the function of translocation of newly translated peptides from endoplasmic reticulum to Golgi apparatus，has an essential role in duplication of influenza virus and its knock down results in decrease of virus titer.We co-transfected PA-N182 and COPD expression vectors into chicken DF1 cells and found that PA-N182 interacted with COPD by the method of immunoprecipitation and Western blot.Our results reveal that COPD might affect the infection of influenza virus through ineracting with PA-N182 and translating it from endoplasmic reticulum to Golgi apparatus.