CRISPR/Cas9 system is one of genome editing technologies developed in 2012.Compared with TALENs and ZFN technologies，CRISPR/Cas9 system has similar gene editing efficiency but is easily manipulated.The engineered dCas9 protein without endonuclease catalytic activity can be obtained by mutating D10A and H840A sites of Cas9 protein.The dCas9 guided by gRNA can be targeted to specific DNA site for the purposes of either inhibiting or activating gene transcription.This paper reviewed the application of CRISPR/Cas9 system in gene regulation as regarding the principle and affecting factors and so on，in order to provide a reference for related research.

Avian immunosuppressive diseases are a group of infectious diseases which can result in immunosuppression of infected poultry and they pose serious threat to the development of the poultry industry all over the world.Previous epidemiology studies of avian immunosuppressive viruses were mainly focused on poultry，and reports of infection in wild birds were quite limited.In recent years，an increasing number of reports showed that many avian immunosuppressive viruses were detected in wild birds.This article will review the avian immunosuppressive viruses spread by wild birds in the following ways: the epidemiology of avian immunosuppressive viruses in wild birds，the bird species of might hosts，the possible transmission mechanism and the surveillance and early warning on wild birds’ diseases.The review enriches the epidemiological data of avian immunosuppressive viruses and lays a foundation for the effective control of avian immunosuppressive diseases.

This experiment was conducted to analyze PCR detection ability of exogenous gene in appA-MxA transgenic pigs and to study the possibility of exogenous gene drifting to the environment through feces.To analyze PCR detection ability of exogenous gene，a series of pcDNA-appA-MxA plasmids 10-fold diluted were amplified by PCR which taking primers designed according to appA and MxA genes in appA-MxA co-transgenic pig.Appropriate fecal DNA in transgenic pigs also be amplified by PCR to detect gene drift between feces and environment.Results of PCR showed that，the minimal concentration of CMV-appA and CMV-MxA in 10 μL PCR system were 2.4×10⁴  and 2.4×10³ copy•μL^-1，respectively.Detection results of appropriate fecal DNA from transgenic pigs which could overcome PCR inhibition showed that appA and MxA were not existed in feces from appA-MxA co-transgenic pigs.These results indicated that exogenous genes in transgenic pig could hardly be drifted into the environment through feces，and to some extent，transgenic pigs are safe to the environment.

Transcription level and methylation in promoter region of Piwil1 gene in different types of spermatogenic cells were conducted to explore the mechanism of transcriptional regulation of Piwil1 gene in the process of spermatogenesis.By using fluorescence quantitative PCR technique to detect Piwil1 genes in different cells of mRNA expression level，and using Bisulfite sequencing PCR method for analysis of Piwil1 gene promoter region methylation，which included in modification，transformation，cloning and sequencing the promoter region.The results showed that Piwil1 gene expression of chicken was lowest in sperm，and the expression of spermatogonia was significantly higher than PGCs，SSCs and tetraploid(P＜0.01).And there was a CpG island founded in Piwil1 gene promoter（-233-+298 bp），which present 56 CpG sites.In the region of -233--129 bp(1-10 CpG sites)，the methylation sites were not founded in sperm cells，tetraploid cells，PGCs and SSCs，while the methylation ratio of spermatogonia was up to 0.600; however，in the region of -233--129 bp(39-55 CpG sites)，significant differences in methylation ratio between different cells were detected，in which the methylation ratio of spermatogonia was the lowest to 0.212，while PGCs and SSCs in DNA hypermethylation inhibited the expression of genes Piwil1 in a certain degree.It was concluded that methylation in +105-+252 bp promoter regions may have some influence on transcription regulation of Piwil1 gene，but there were other transcriptional regulatory mechanisms.

The present study was designed to investigate the effects of different nuclear transfer methods and supplementation of culture media with vitamin C on the fusion rate and development of ovine cloned embryos.The abattoir-derived ovaries were collected for materials.After in vitro maturation of oocytes，micro-electrode fusion，zona-free and micro-injection cloning were performed to compare their efficiencies in construction of cloned embryos.Optimal concentration of vitamin C was explored by examining the effects of different concentrations of vitamin C on the parthenogenetic development of oocytes and subsequently applied to in vitro culture of cloned embryos.The results showed that fusion rate obtained from micro-electrode fusion with 25 volts was significantly increased compared with Chamber fusion (P<0.05).Twenty five volts resulted in less dead oocytes than 30 volts in micro-electrode fusion (P<0.05).Zona-free cloning significantly increased the morula rate of cloned embryos (P<0.05)，but failed to increase blastocyst rate significantly (P>0.05).Vitamin C supplementation with the concentration of 50 μmol•L^-1 significantly increased blastocyst rate of ovine parthenogenetic and cloned embryos (P<0.05).Altogether，fusion rate during somatic cell nuclear transfer could be increased by micro-electrode fusion.Blastocyst development of ovine cloned embryos could not be improved by zona-free cloning.Fifty μmol•L ^-1 vitamin C was beneficial for in vitro culture of ovine cloned embryos.

In order to determine the possible roles of Meg8 gene-internal CpG island in the regulation of the gene imprinting expression，the allele-specific DNA methylation status of 17 CpGs in intron3 of Meg8 in lung tissues was analyzed using bisulfite sequencing.The results demonstrated that the DNA methylation of both the parental strands were high （>80%），but T-strand showed significantly higher DNA methylation than that of C-strand（P<0.05） and the C-strand had only one kind of methylation model.Then the DNA methylation and imprinting status were analyzed in lung in somatic cell nuclear transfer(SCNT) bovine which died within postnatal 48 h.The results showed that the SCNT bovines exhibited significantly higher DNA methylation than that of naturally breed bovines（P<0.05）.All of 17 CpGs showed complete methylation pattern in SCNT bovine.Imprinting analysis showed that Meg8 was mono-allelic expression in tissues of SCNT bovines.The DNA methylation of Meg8 intron3 CpGs island may not play a role in regulating of Meg8 imprinting expression.

To study the in vitro maturation (IVM) of bovine oocyte pretreated with C-type natriuretic peptide (CNP).Compared with the conventional 24 h IVM culture，the effect of bovine oocytes pre-treated with CNP for 6 h following 28 h IVM culture (CNP pre-treated group) were studied.The results showed that the maturation of bovine oocyte in CNP pre-treated group were significantly improved，and the blastocyst rate and the cell number of blastocyst were significantly higher than those of the control group（(51.55%±2.50%)，（156.2±11.0）vs.（28.13%±3.80%）,(116.3±19.0)，P<0.05) after in vitro fertilization (IVF).The distribution of mitochondria ranged from peripheral distribution in unmatured oocyte to homogenous distribution throughout the cytoplasm in matured oocytes，while semi-peripheral distribution observed in the oocytes pretreated with CNP for 6 h，but then became homogenous distribution followed 28 h maturation culture.The mtDNA copy number of matured oocyte in CNP pre-treated group were (510 078.4±28 906.0)，which was significantly higher than that of control group((416 558.5±51 743.0)，P<0.05).The results indicated that the quality of bovine oocytes in vitro maturation pre-treated for 6 h with CNP could be improved effectively.

The aims of the present study were to investigate if high concentrate diets with high concentration blood glucose and insulin depress milk fat synthesis and the underlying mechanism.4 Laoshan dairy goats in early lactation fitted with abomasal and jugular artery catheters were used in a repeated 2×2 crossover experiment.Treatments were abomasal infusion of saline or 200 g•d^-1 glucose.The experiment was consisted of two periods.Each period lasted 8 d and there was a 14 d interval between the two periods.RT-PCR was used to detect the mRNA expression of ACC,FAS,LPL,H-FABP,GPAT and SCD.The results indicated that glucose infusion elevated blood glucose (P<0.05) and tended to elevated plasma insulin (P=0.06).Glucose infusion increased milk yield，decreased both milk fat yield and milk fat percentage (P<0.05).Analysis of fatty acid profile of milk fat reveled that glucose infusion decreased the yield of C18∶0 and c9C18∶1 (P<0.05) but had no significant effect on the yields of those de novo synthesized fatty acids (C4-C14) and those dual source fatty acids that may be preformed or de novo synthsized (C16).Glucose infusion had no significant effects on the mRNA experssion of acetyl coenzyme A carboxylase，fatty acid synthetase，lipoprotein lipase，fatty acid bonding protein，glycerol-3-phosphate acyltransferase，stearoyl CoA desaturase and fatty acid transfer protein.It was concluded that abomasal glucose infusion decreased milk fat synthesis mainly by decreasing the amount of pre-formed fatty acids used for milk fat synthesis，had no significant effect on the expression of genes related to milk fat synthesis.The regulation of abomasal glucose infusion on milk fat synthesis occurred after transcription.

 The effects of storage temperature，packing methods and storage time on the in vitro dry matter digestibility (DMD）and enzymatic hydrolysate gross energy (EHGE）of corn and soybean meal were investigated to provide the basis for the storage of feed samples of computer-controlled simulated digestion system (CCSDS).A 3×2×7 factorial arrangement in a Split plot design was used.The main plot was storage temperature at 4 or -20 ℃ and the sub-plots were packing methods with a zip-lock bag，vacuum package or vacuum & nitrogen filling package and storage time at 0，2，4，6，8，10 or 12 months.The DMD and EHGE of corn and soybean meal were determined by using a computer-controlled simulated digestion system.The results showed as follows：1) The DMD and EHGE of corn and soybean meal stored at -20 ℃ were significantly greater than that stored at 4 ℃ (P<0.05).Compared with the initial values，the variability of the DMD and EHGE of corn and soybean meal stored at -20 ℃ were less than that stored at 4 ℃.2) The DMD of corn and soybean meal stored in the vacuum package or in the vacuum & nitrogen filling package were significantly greater than that stored in the zip-lock bag(P<0.01).The EHGE of corn stored in the vacuum package or in the vacuum & nitrogen filling package were significantly greater than that stored in the zip-lock bag (P<0.01).Compared with the initial values，the variability of the DMD and EHGE of corn and soybean meal stored in the vacuum & nitrogen filling package were less than that stored in the other packing methods.3) The DMD and EHGE of corn and soybean meal decreased as the storage time increased from 0 to 12 months (P<0.01).In conclusion，the DMD and EHGE of corn and soybean meal were affected by the storage temperature，packing methods and storage time.The DMD and EHGE of corn and soybean meal stored in the vacuum & nitrogen filling package at -20 ℃ had low variability and good storage effect.

This experiment aimed to study the effects of substitution of different levels of urea for natural protein on haematological indices，serological parameters and the histopathology of digestive organs in mutton sheep and provide basis for the safe use of urea.Sixty four Dorper×thin-tailed Han crossbred male lambs with average body weight of (30.77±0.02)kg were randomly divided into 4 treatments as follows：control group and control group supplemented with 0.5%，1.5%，and 2.5% urea,respectively.The investigation lasted for 63 days including 21 days of adaptation.Each treatment including 8 sheep and blood was sampled on d 1 and d 42.Thirty two lambs were slaughtered at the end of the experiment，and the sample of duodenum，rumen，liver and kidney were taken for pathological examination.The detection results of haematological indices showed that the platelet count of 1.5% and 2.5% urea group were significant higher than that of the control group and 0.5% urea group (P<0.05)；the mean corpuscular hemoglobin concentration of 2.5% urea group was also remarkably higher than that of 0.5% urea group (P<0.05)；average red blood cell hemoglobin content of 2.5% urea group was higher than that of other 3 groups (P<0.05).Blood urea nitrogen of 1.5% and 2.5% urea groups were significant lower than that of control and 0.5% urea group (P<0.05).However，the effect of urea supplementation on triacylglycerol was opposite to the results of blood urea nitrogen (P<0.05)；the concentration of glutamate pyruvate transaminase，glutamic-oxaloacetic transaminase，creatinine total bilirubin and blood ammonia in 2.5% urea group were the highest among the 4 groups (P<0.05)，but the concentration of alkaline phosphatase of 2.5% group was lower than that of control group (P<0.05)；the rest indices of serological parameters were not remarkably different among different groups (P>0.05).The lesion of kidney of 2.5% urea group was more serious than that of other groups (P<0.05).These results indicated that，the haematological indices，serological parameters and the main digestive organs of sheep were not significantly affected when the level of urea in ration under 1.5%.However，the health of mutton sheep will be seriously harmed as the supplementation of urea increased to 2.5% in feed.

The objective of the present study was to optimizing the dietary forage NDF (FNDF) and rumen degradable starch (RDS) content and ratios by using the meta-analysis in dairy cows.We collected 39 peered review manuscripts，which were related to FNDF or RDS content on rumen health and milk performance in dairy cows.In this study，we adopted FNDF to RDS ratio (CBI_R) or their intake difference (CBI_D) as carbohydrate balance indexes (CBI)，and evaluated their regression relationship to the ruminal pH，fermentation characteristics，milk fat and feed efficiency.The results revealed that the CBI_R and CBI_D showed strong linear or quadratic correlation with the dependent variables.Dietary CBI_R and CBI_D above 1.28 or 1.09 kg•d^－1 could minimize the risk of subacute ruminal acidosis，respectively.To obtain appropriate feed efficiency (≥1.5)，milk fat (≥3.5%) and acetate to propionate ratio (2.2:1-3:1) in dairy cows，the CBI_D and CBI_R should be set in the range of －0.42-0.99 kg•d^-1 and 0.93-1.30，respectively.In this study，the appropriate CBI_D and CBI_R ranges were calculated based on meta-analysis，the CBI_D and CBI_R can reflect the change of rumen health and performance of dairy cow.

The congruent relationship between the degree of grassland degradation and plant community nutrient was studied from the point of view of nutrition，exploring how utilize grassland scientifically and reasonably.According to the method of grassland survey- Height，coverage，abundance，Frequency，the degree of utilization was divided into control(CK)，light degradation，moderate degradation and high degradation.Exploring the relation between plant community nutrient content and the degree of grassland degradation by the method of correspondence analysis could reflect the difference degree between grassland degradation gradient and correlation degree between plant community nutrient indexes.With the degree of degradation aggravated，total digestible nutrients of grassland plant community increase，whereas dry matter content reduce.The dry matter(DM) content was highest in the condition of CK.The content of ether extract (EE) of light degradation (LD) was higher.The content of nitrogen-free-extract (NFE) of moderate degradation (MD)was higher.The content of crude protein (CP) and total digestible nutrients (TDN) of high degradation (HD) was higher.The content of crude fiber (CF) was relatively stable，which had little to do with grassland degradation degree.The degree of grassland degradation had great influence on DM,EE,NFE,CP and TDN and had little influence on EE.

The current study was undertaken to study expression of E2 gene of bovine viral diarrhea virus (BVDV) in tobacco and to analyze reactionogenicity of the expressed products.A pair of primers was designed according to the major antigen region of the E2 gene derived from GenBank.The plant constitutive expression vector pBI121-E2 containing E2 gene was constructed，and was then transformed into tobacco plants via Agrobacterium tumefaciens.The transgenic anti-type tobacco plants were screened by screening with kanamycin.PCR amplification and real-time PCR analysis indicated that the E2 gene has been a low-copy in five tobacco plants.Western bolt analysis showed that the E2 protein could be specifically recognized by anti BVDV positive serum.Our study provides a basis for further analysis on plants as bioreactors development and the production of E2 oral vaccines.

Autophagy plays an important role in virus proliferation and release while there have no report about the relationship of classical swine fever virus (CSFV) and host cell autophagy.This study was undertaken to elucidate the impact of CSFV Shimen strain on host cell (ST) autophagy and the effect of autophagy to virus proliferation.The transmission electron microscopy (sem)，LC3 dots，biomarkers of autophagy，were adopt to get a preliminary determination.The protein expression of LC3 and P62，as well as conversion of LC3Ⅰto LC3 Ⅱwere semi-quantified by Western blot，the results suggested that the CSFV promotes cell autophagy.Autophagy promoter rapamycin，inhibitors 3-MA，chloroquine(CQ)，fusion blocker of autophagosome and lysosomes，were used to regulate the autophagy，as well as autophagy associated gene (ATG) Beclin 1 and LC3 protein were interfered to investigate the effect of autophagy on virus proliferation.The results showed that virus infection promotes cell autophagy and form complete autophagy flux，which facilitates virus proliferation.This research provides new information to the relationship of CSFV and host cell.

To evaluate immune responses of gE DNA vaccine of pseudorabies virus (PRV) and the enhancement potency of porcine interleukin-2 for its immunogenicity，the recombinant plasmid of pcDNA3.1(+)-gE1 and pcDNA3.1(+)-gE2-IL-2 were constructed and transfected into Vero cells.The expression of gE and IL-2 gene were identified by indirect immunofluorescent assay，RT-PCR and Western blotting.The results showed that recombinant plasmids were transiently expressed in Vero cells and the protein expressed in Vero cells had good reactogenicity.Furthermore，animals were immunized by recombinant plasmids，empty vector，inactivated virus and normal saline.Indirect ELISA and determination of T lymphocyte subsets showed that the recombinant plasmids were able to induce efficient immune responses in inoculated animals.The IL-2 gene showed the significant enhancement effects on the immunogenecity of PRV gE DNA vaccine.The animals immunized with the pcDNA3.1(+)-gE2-IL-2 recombinant plasmid and pcDNA3.1(+)-gE1+ pcDNA3.1(+)-IL-2 co-immunize provided over 80% protection in mice and over 60% protection in piglets against wild type virus challenge.These results confirm that the fusion protein and hybrid protein enhances the protection against PRV through both humoral and cell-mediated immunity.

Technology of cell culture，cyto-histochemistry staining and real-time fluorescence quantitative PCR （SYBR GreenⅠ） were used to research dynamic changes of the proliferation responses of T-lymphocytes to ConA，the quantity of T_ANAE⁺ cells and the transcription of IL-2 mRNA in immune organs of ducks infected with AIV at 21 days old，which were useful to reveal the mechanism and regulation of immunity.The experiment provided scientific evidence for the mechanism of morbidity and useful effective prevention.The results were as follows：the transcription of IL-2 mRNA，the proliferation responses of T-lymphocytes to ConA and the quantity of T_ANAE⁺ cells in immune organs were all decreased remarkably in initial period of AIV infection.And it indicated that molecular immune function of cytokines was inhibited，and the activation and proliferation of the immunocompetent cells，especially T-lymphocyte，were inhibited as well.The cellular immunity functions were much lower than that of controls，which could be the main reasons of the ducks death in initial period of AIV infection.

Avian reovirus (ARV) is a main viral pathogen of poultry，and its infection threatens broiler production due to severe arthritis and immune suppression，leading to huge economic losses.In this study，7-day-old specific pathogen free (SPF) chickens were infected with the ARV S1133 strain through footpad injection.Tarsal joints samples were collected on day 1，7，14，21，28，35 post-infection (PI) and tested for mRNA relative transcription levels of cytokines genes of IL-1β，IL-6，IL-17，IFN-γ and TNF-α by specific real-time PCR assays.Relative transcription levels of mRNA of IL-1β，IL-6，IL-17，IFN-γ and TNF-α in tarsal joint of SPF chickens were investigated.The results showed that ARV infection up-regulated the gene transcription of these cytokines in tarsal joints and the elevated transcription continued during the course of infections，the mRNA levels of IL-1β，IL-6，IL-17，IFN-γ and TNF-α genes were up-regulated (P<0.01) compared with control groups on day 21，28，35 PI.Moreover，cytokines genes transcription of IL-1β，IL-6，IL-17 and IFN-γ have the up-regulated trends.The results revealed that IL-1β，IL-6，IL-17，IFN-γ and TNF-α participate in the ARV-induced damaging processes in the tarsal joints.This study indicated that the changes of cytokines transcription in joints of chicken infected with ARV were helpful for further understanding pathogenesis of avian reovirus infections.

Taenia solium (Ts) is an sort of important zoonotic parasite，and thioredoxin peroxidase (TPx) is a protein with important function both in Taenia solium and its larvae Cysticercus cellulosae.The aim of this study was to clarify the biological roles of TsTPx and screen an efficient immunologic target against Cysticercosis.The gene tstpx was obtained by RT-PCR with the total RNA of Taenia solium as template and was confirmed by sequencing.Then the recombinant plasmid pET-30a(+)-tstpx was constructed，subsequently transformed into E.coli BL21，and induced for expression with IPTG.After purification of TsTPx by Ni²⁺-NTA affinity chromatography，the biological properties of TsTPx including antioxidative activity and immunogenicity were analyzed.Protein TsTPx prokaryotically expressed in this study predominantly located in the supernatant as a soluble protein confirmed by SDS-PAGE.Its antioxidative property was proved by，positive correlation of TsTPx concentration to protection percentage of plasmid’s super-coiled DNA structure in metal catalytic oxidation system，where 20 μg•mL^－1 TsTPx achieved 100% protection confirmed by disappearance of nicking slower band in agarose gelelectrophoresis；and also positive correlation of TsTPx concentration to H₂O₂ clearance rate in H₂O₂ reduction system，where 100 μg•mL^－1 TsTPx achieved 69.9% and 54.5% of H₂O₂ clearance with and without dithiothreitol，respectively.Its antigenicity was demonstrated by special recognition between Cysticercus positive serum from pig in Western blotting and antibody titers up to 1：320 000 detected with indirect ELISA after TsTPx vaccine was prepared and injected into rabbits.The results showed that the prokaryotically expressed TsTPx in present study was biotically active and showing satisfied antigenicity，which laid the foundation for a comprehensive study of its biological function and for the development of a vaccine against cysticercosis.

In the viral genomes of Marek’s disease virus (MDV)，dozens of miRNAs have been identified and some of them are suggested to potentially play important roles in MDV pathogenesis and oncogenesis.Previous studies have shown that the MDV-1-encoded miR-M7-5p is expressed at a high level during different phases of the developing disease.To investigate the potential self-regulation of miR-M7-5p on MDV during virus infection，we have presently scanned the MDV protein-coding genes utilizing bioinformatics method and obtained 10 potential target candidates for miR-M7-5p，including the most important viral oncogene meq.Dual luciferase reporter assay (DLRA) showed that miR-M7-5p can interact with the 3′-UTR of meq gene.Using real-time quantitative RT-PCR，we further confirmed that the transcription level of meq gene in MDV-infected CEF was down regulated by miR-M7-5p.Our data suggests that the oncogene meq is self-regulated by the viral miRNA miR-M7-5p in the infection processes，which may contribute to MDV oncogenesis.

 The purpose of this study was to investigate the possible mechanism of neuron cell damage in Prion diseases.And we studied the respiratory chain function of N2a cells by testing the NADH activity after treatment with ferric ions and PrP106-126，as well as the depolarization of the mitochondrial membrane potential and the distribution of cytochrome C.It is proved that the activity of NADH of N2a cells，which were treated with ferric ions and PrP106-126，was significantly decreased (P <0.01) to about 65%.And the mitochondrial membrane potential of N2a cells had been depolarization significantly.It is showed that the cytochrome C were released into cytoplasm and activated the pro-caspase-9.

This research aimed to identify single nucleotide polymorphism (SNP) of Myf5 and Pax7 genes in Xiangxi cattle，investigate the population genetic characters of SNPs and search the molecular markers for growth.SNPs of intron 2 in Myf5 gene and exon 3 in Pax7 gene were detected by PCR and sequencing methods.Population genetic polymorphisms for 217 Xiangxi cattle were detected by PCR-RFLP，and the association analysis between genotypes and body measurement traits was performed by least-squares linear model.The results showed that the total of 9 SNPs were detected on intron 2 in Myf5 gene，in which，7 SNPs were novel.While，only one SNP was detected on exon 3 in Pax7 gene.The results of population genetic polymorphism indicated，for g.1948A>G locus of Myf5 gene in Xiangxi cattle，the genotype frequencies of AA，AG，GG were 0.046 1，0.235 0，0.718 9.Allele frequencies of A and G were 0.163 6 and 0.836 4，respectively.For g.31G>A locus of Pax7 gene，only two genotypes of AG and GG were detected in Xiangxi cattle，and the genotype frequencies were 0.073 7 and 0.926 3，the allele frequencies of A and G were 0.036 9 and 0.963 1，respectively.Both loci were low polymorphism，and fitted with Hardy-Weinberg equilibrium (P>0.05).The results of association analysis for g.1948A>G locus in Myf5 gene revealed that the animals with AA and AG genotypes had higher body weight than those with GG genotype in Xiangxi cattle (P<0.05)，while there were not significant differences for withers height，body length and heart girth among the individuals with different genotypes (P>0.05).Allele A positively affected on body weight in Xiangxi cattle.These results indicated that there were 9 SNPs in Myf5 gene，in which，g.1948A>G locus might be molecular marker for body weight.

 To explore the relationship between the changes of DNA methylation level and the differentiation of BMSCs，the effect on cell viability of DNA methylation inhibitors (5-aza-dC) in different concentrations at different times was detected by MTT method.The cells were treated with 5-aza-dC at the concentration of 1.0 μmol•L^-1 and differentiate into islet cells combining with DMSO and nicotinamide，which was compared with the induction group that was not treated with 5-aza-dC with Dithizone staining method and the immunofluorescent staining method in the key regulatory genes of pancreatic islet (PDX-1).The results showed that the proper concentration of 5-aza-dC for the treatment of rabbit BMSCs ranges from 0.2 to 1.0 μmol•L^-1.The combine use of 5-aza-dC at the concentration of 1.0 μmol•L^-1 and DMSO，nicotinamide could promote the expression of the key regulatory genes of pancreatic islet (PDX-1) and established an efficient system for the differentiation of BMSCs into islet cells.It reveals that the degree of methylation could influence the differentiation of BMSCs into islet cells and the low degree of methylation could promote the expression of specific gene of pancreatic islet (PDX-1).

This study was performed to explore the effect of the monochromatic lights supplement on pigeons’ performance and provide the scientific basis for the illuminating system of the pigeon breeding industry.Monochromatic lights supplement experiment was conducted in the four same lofts with close number of pigeons (510-522 pairs•loft^-1) with blue light (480 nm)，green light (540 nm)，red light (660 nm) and white light (400-760 nm)， respectively，the white light group used as control group.Each group was divided into three subgroups according to the light intensity.The production data of adult pigeons and the weights of 0，7，14，21 days of squabs were recorded.The results showed that：1) Under the red light，pigeons’ had the highest laying rate and fertility rate，lowest broken egg rate.2) Under the light intensity of 1-10 lx，the birth weights of squabs had no significant difference(P>0.05)，but at the age of 21 days，the weights of blue light group were significantly higher than red and white light groups(P<0.05)；Under the light intensity of 10-20 lx，the birth weight and the weight of 21 days of four groups had no significant difference (P>0.05)；Under the light intensity above 20 lx，the birth weights of squabs had no significant difference (P>0.05),but at the age of 21 days，the weights of red，blue and green light groups were significant higher than white light group(P<0.05).These results indicated that using natural light during the day and taking monochromatic light supplement measures in the morning and evening could improve production efficiency effectively；Compared with the white light group，the pigeons of the red light per hundred could lay 28.68 more eggs and hatch 11.76 more squabs；In terms of specialized production，under the 1-10 lx illumination intensity，the blue light could promote squabs’ weight gain.

The objective of this study was to evaluate the effect of nutritional level of ration on the growth performance of Chinese Holstein heifer with body weight of 300-350 kg，to reveal metabolic rule of energy requirement.Twenty-one 10-month-old heifers ((307.67±2.99)kg of BW) were divided into 3 groups(A，B and C) in a randomized experiment design，each group was fed different diet consisted of concentrate，corn silage and Leymus chinensis.The DE and CP of group A,B and C were 10.98 MJ•kg^-1 and 10.06%，11.43 MJ•kg^-1 and 11.04%，11.90 MJ•kg^-1 and 12.04%(DM basis)，respectively.The ADG of heifers in 3 groups reached 620.09，820.30 and 887.30 g•d^-1，respectively.The apparent and metabolic digestibility of gross energy(GE) were 67.86% and 57.94%,respectively.Average digestibility for DE was 85.35%.The study showed models of DER and MER requirement of heifers were：DER（MJ•d^-1）=0.730 14W^0.75+44.38△W，MER（MJ•d^-1）=0.616 90 W^0.75+39.50△W，W is BW of heifers(kg)，△W is ADG(kg•d^-1).

 RT-PCR-RFLP was established to detect and differentiate Japanese encephalitis virus genotype Ⅰ (GⅠ) and genotype Ⅲ (GⅢ).A pair of primers was designed according to the sequences of Japanese encephalitis virus to amplify the C-PrM-E gene.One restriction enzyme (SpeⅠ) was selected to generate the polymorphisms in different restriction fragments length for genotype discrimination.Seventy-eight clinical samples were detected by RT-PCR-RFLP for detecting and genotyping.The results showed that a 819 bp target fragment was amplified by RT-PCR from genomic RNA of JEV GⅠ strains which could be digested with SpeⅠ into 2 fragments of 568 and 251 bp when analyzed with RFLP，the GⅢ strains could be digested into 3 fragments of 446，251 and 122 bp，while the mixture of GⅠ and GⅢ strains could be digested into 4 fragments of 568，446，251 and 122 bp，respectively，but not amplified from swine fever virus，porcine parvovirus，porcine reproductive and respiratory syndrome virus，porcine circovirus and pseudorabies virus.The lowest concentration of RNA could be detected was 1.5 pg•μL^-1.Seventy-eight samples were tested by RT-PCR-RFLP，and the RT-PCR-RFLP results showed that 6 of these were GⅠ and 8 of these were GⅢ，and those results by RT-PCR-RFLP correlate well to those determined by sequencing.The RT-PCR-RFLP technology is a useful tool for detecting and rapid genotyping of JEV GⅠ and GⅢ.

To express the porcine epidemic diarrhea virus (PEDV) N protein by prokaryotic expression system and to identify its antigenicity，truncated N protein (tN) genes were amplified by RT-PCR and recombinant prokaryotic expression plasmid (pET32a-PEDV-tN) with tN protein gene was constructed.After the recombinant plasmids had been transferred into competent cells of E.coli BL21 and the bacteria had been inducted by IPTG，the expression and immunoreactivity of tN protein were detected respectively by SDS-PAGE and Western blotting.Then，BLAB/c mice were subcutaneously immunized with purified tN protein and the protein immunogenicity was detected by ELSIA and Immunoperoxidase monolayer assay.The tN protein genes of 705 bp were amplified and the recombinant plasmids carrying this gene could solubly express recombinant tN protein under induction of IPTG in E.coli BL21.The protein displayed specific binding to swine anti-PEDV antibody and induced mice to produce anti-PEDV antibody.These results showed that the soluble expression of PEDV tN protein with favorable complete antigenic activities was implemented by prokaryotic expression system.