PCR-RFLPs assay was used to investigate polymorphisms within the cloned introns 5 to 7. A MspI-RFLPs and a TaqI-RFLPs were detected in intron 6.The MspI-RFLPs is common in all the detected breeds, but the TaqI-RFLPs only was founded in Chinese breeds, and allele B(bearing T base) is rare in Chinese breeds. Associations between the founded MspI-RFLPs in intron 6 and carcass traits in the seven beef cattle breeds and two Chinese dairy cattle were tested , no relation between MspI-RFLPs and body fat in beef cattle were founded in present study, but the individuals bearing AA genotype had higher(P<0.05)dressing percentage. Individuals with genotype AA had higher milk fat percentage（P<0.01）and dry material percentage（P<0.05）in Sanhe .For the TaqI-RFLPs in intron 6, the present study only tested the carcass trait's difference between genotype AA and AB in Luxi cattle, but no associations were founded.

This report describes construction of specific labeling vector in CES cell. The putativeU3-R sequence regions of 1.2 kb of chicken Embryonic Normal Stem cell gene 2 was amplified by PCR and successfully sequenced. Based on sequence analysis and alignments of nucleotides sequence, found many factor or protein binding sites such as AP-1 ,GATA-1and an important elements: B region which exhibits a strong enhancer activity in CES and differentiated cells. The CMV promoter was cut off from pEGFP-N1, the putative U3-R fragment was inserted just upstream of GFP . Results of restriction digestion and DNA sequencing showed that the vector for expressing in CES cell was successfully constructed.

Nanyang and Luxi cattle were used as experimental population; the genetic variations of GH genes were researched by PCR-SSCP and PCR-RFLP method. The results were as follows: The polymorphic PCR-SSCP sites in the 3^rd, 4^th intron, the 5^th exon, and 5′flanking region and the polymorphic HaeIII site in the 3′flanking region of the bovine growth hormone (GH) were examined in two breeds. The polymorphisms of the 3rd intron were caused by C→T transition at 1 547 bp position. The polymorphisms of the 4^th intron were tested by T→G transition at 1 947 bp position in Nanyang, Luxi cattle. The polymorphisms of the 5^th exon were caused by C→G transition at 2 141 bp position, which resulted amino acid translation, leucine to valine. And the polymorphic HaeIII site in the 3′flanking region were caused by GCC→GGC transition at 2 639 bp position, and C→T transition at 303 bp resulted the polymorphisms of 5′flanking region.

A cDNA library was constructed with RNA extracted from the skin tissue of an adult goat, and 392 redundancy ESTs were registered in the GenBank, with the accession numbers of CD051766-CD052157. 319 function known gene were identified based on sequence similarity, among which there are 108 unidentified, 70 gene/protein expression, 43 metabolism, 45 cell structure, 30 cell signaling/cell communication, 15 cell defense /immunity and 8 cell division. Some important candidate genes that have possibly involved in cashmere growth are TGFβ binding protein, the EDRK enriched factor, growth factor receptor, G protein associated receptor, dermal papilla derived protein 2, homeobox gene otx2, kallikrein7, jagged1, thrombospondin1 and p53 inducible protein.

Microsatellite enrichment from AFLP fragments by magnetic beads can obviate the tedious work of library construction which save not only time but also expense. The genomic DNA was converted into pre-amplified AFLP fragments by using a few restriction enzymes combination and hybridized with biotinlabeled SSR probes. Then the hybrid mixture was used to incubated with magnetic beads coated with streptavidin. After washing to remove the non-SSR fragments, the eluted single-strand DNA which was cloned and sequenced was largely enriched for microsatellites. Primers can then be designed according to the sequence flanking the repeat motifs and used for polymorphism analysis. Thirty primers had been developed from six China indigenous geese breeds successfully. Eleven of them were highly polymorphic better specific and did not link each other. The mean Polymorplism Information Content(PIC) of six geese breeds was also all over 0.5. The highest was the Gushi goose (0.546), and the lowest was the Yan goose (0.513). The results showed that the 11 microsatellites had higher PIC . These 11 microsatellites could be used in the analysis of genetic diversity, construction of saturated maps, and in some cases, in marker-assited selection.

400 AA broilers of 1 day were allocated into 4 treatments, 5 replicates per treatment, 20 broilers per replicate. The basal diet was wheatcasein diet. Biotin was added in the basal diet at 0.3 mg/kg, 0.9 mg/kg and 2.7 mg/kg. There were 4 diets including the control of basal diet. The results showed as follows: (1) Addition of biotin enhanced their development and elevated their weight index. The antibody titer to Newcastle and IgG content were elevated by addition of biotin. The antibody titer to Newcastle of 0.9 mg/kg and 2.7 mg/kg treatments were higher significantly than that of the control and 0.3 mg/kg treatment (P<0.05). The IgG level of 2.7 mg/kg treatment was higher markedly than that of the control (P<0.05). (2) Addition of biotin enhanced significantly B-lymphocyte transformation ratio in blood (P<0.05), and Tlymphocyte and Blymphocyte (P<0.05) transformation ratio in spleen. In spleen, the cell content of expressing CD3⁺ , CD4⁺ and CD8⁺ were reduced significantly by addition of biotin (P<0.05). The cell content of expressing CD3+, CD4⁺ and CD8⁺ of 2.7 mg/kg treatment was lower markedly than that of the control and 0.3 mg/kg treatment (P<0.05). In blood, addition of biotin reduced significantly the cell content of expressing CD3⁺ (P<0.05). The cell content of expressing CD3⁺ of 2.7 mg/kg treatment was lower markedly than that of the control and 0.3 mg/kg treatment (P<0.05). (3) Biotin addition elevated serum T3, T4, growth hormone and cortisol (P>0.05), and what’s more, no noted difference existed among the biotin addition treatments (P>0.05).

Effects of wheat NSP and xylanase on intestinal microflora and bacterial community in broilers were determined by traditional method and denaturing gradient gel electrophoresis (DGGE) analysis of 16S ribosomal RNA. 90 AA broilers were allocated randomly into 3 groups, fed 3 diets separately. The diets were corn based, wheat-based or wheat-based supplemented with xylanase. The results showed that the wheat NSP significantly increased (P<0.05) the microbial population of total anaerobic bacteria in cecum or ileum and increased (P<0.05) the streptococcus faecalis in cecum of broilers. However, the wheat NSP significantly decreased (P<0.05) the bacterial community in intestine of broilers. The xylanase-supplemented diet had no markedly effect (P>0.05) on microbial population and bacterial community in cecum and ileum of broilers.

144 Duroc×Large white×Landrace crossbred piglets weaned at 18 d, weighted 5.6 kg, sorted by weight and gender, were allotted to 6 treatments, with 3 replicates per treatment and 8 piglet per replicate．The basal diet was corn-soybean, without antibiotics or probitotics, which was fed to the 1st group as negative control(CTL)．The 2nd group was fed with basal diets adding chlortetracycline hydrochloride (CTC) in 150 mg/kg as the positive control．The 3rd-5th group were fed with basal diets adding 1 000 mg/kg Bacillus Natto sawamura (Natto) and 1 000 mg/kg or 2 000 mg/kg mannan oligosaccharides(MOS) respectively, the 6th group was fed with basal diets adding 1 000 mg/kg MOS and 500 mg/kg Natto．Intestinal pH, colonic gut flora population were examined, and the intestinal morphology were detected．The results were as follows: Dietary Natto and MOS supplementation have a trend of reducing pH value of jejunum, ileum, caecum and colon cotent, the intestinal pH were reduced significantly when Natto used combining with MOS. Dietary Natto increased lactobacillus and bifidobacterium population in colonic content, and lactobacillus population in mucous membrane increased, dietary MOS increased population of lactobacillus both colonic content and mucous membrane, group of 2 g/kg MOS reduced population of E.coli both colonic content and mucous membrane. In colonic content, cooperation of Natto and MOS had a suppressoion to E.coli compared with CTL and an enrichment of lactobacillus compared with CTC group(P<0.05), while in colonic mucous membrane, population of lactobacillus increased and E.coli decreased, with a remarkable difference to CTL and CTC group(P<0.05). Natto increased villus height of mucous membrane, as same as group of cooperation of Natto and MOS , There is no difference between each group for crypt depth of mucous membrane(P>0.05), the ratio of villus height and crypt depth of group Natto, 1 g/kg MOS, and the group of cooperation of Natto and MOS increasing remarkable compared to CTL group.

This study aims to develop a differential assay for popularizing the use of E2-based marker vaccines against classical swine fever (CSF). Based on the recombinant E^rns produced by E. coli, an indirect ELISA (E^rns-ELISA) was established and optimized using the E^rns fusion protein as coating antigens. The optimal amount of coating antigen was optimized to be 0.15 μg, and the serum dilution was optimized to be 1∶100. The established E^rns-ELISA was shown to be sensitive, specific and reproducible, and has a good agreement with E2-ELISA based on the recombinant fusion E2 protein (96.7%) and Dot-ELISA based on whole viral antigens (77.5%). The E^rns-ELISA combined with E2-ELISA can be used as differential diagnostic assays accompanying marker or DIVA vaccines against CSF to differentiate animals infected with wild-type viruses from those vaccinated with E2-based marker vaccines.

By cotransfection of PRV Fa SA215 DNA and PP63LacZE2 DNA in Vero cells,12 recombinants named SA215(A)1,SA215(A)2…，and SA215(A)12 were obtained with calcium phosphate transfection system. The recombinant strains were determined preliminarily by biotin-labeled probe of HCV E2， then SA215(A)1 strain were identified by BamHI digestion and Southern-blot acid hybridization. The results indicated that the recombinant virus of PRV and HCV named SA215(A) was successful. IFT，SDS-PAGE，electrophoresis and Western-blot all confirmed that HCV E2 gene was expressed in recombinant virus, about 51 ku fragment of envelope glycoprotein E2 were acquired. Research on culture characteristics of SA215(A) strain indicated recombinant virus could be replicated very well on Vero cells, BHK21 cells and so on, but showing variability in culture on different cell lines. 21-day-old SPF pigs were inoculated with SA215(A) strain. After 28 days, the pigs were affected with 10⁶PFU high-dose virulent PRV Fa strain; and 14 days later affected with 10⁵ MLD high-dose virulent HCV SM strain. All of the inoculated pigs were protected. The results indicated SA215(A) was high efficacy.

Some characteristics of multiplication and expression of classical swine fever virus Cstrain,Shimen strain and constructed viral RNA were studied by RT-PCR, direct fluorescent antibody method, sandwich ELISA. The results indicated that Shimen strain can easily adapted SK6 cell;but C strain and constructed viral RNA were sensitive to PK15 cell.Meantime RT-PCR, direct fluorescent antibody method, sandwich ELISA is the useful methods for identifying the transfected cell.

A pseudorabies virus (PRV) transfer vector pLTK-HA containing a LacZ gene expression cassette and the HA gene of swine influenza virus (SIV) H3N2 subtype under the control of human cytomegalovirus (CMV) promoter was constructed and used to cotransfect with genomic DNA of strain Bartha-K61 into Vero cells using lipofectin transfection procedure. A recombinant PRV haboring SIV HA gene, designated as rPRV-HA, was generated after ten cycles of blue plague purification and PCR identification. The expression of the HA by rPRV-HA infected cells were characterized by Western blotting and indirect immunefluorescence assay (IFA). Compared to its parental virus Bartha-K61 strain, rPRV-HA showed no obvious difference in virus multiplication and cytopathogenic effects in Vero, PK-15, IBRS-2, chicken embryo fibroblast cells. The results of analyzing 30th passage rPRV-HA indicated that HA gene can be stably expressed by rPRV-HA.

The ORF2 of porcine circovirus type 2 and BVP22 of bovine herpesvirus were amplified by PCR. The cloned ORF2 gene was inserted into eukaryotic expression vector pCDNA3.1(+)for constructing nucleotide vaccine vector pCORF2. Then nucleotide vaccine vectors which express BVP22-ORF2 fusion protein were constructed by inserting BVP22 gene,which has the function of protein transduction into pCORF2 at the upstream or downstream of the ORF2 gene respectively. All the above vaccines were intramuscular injected to BALB/c mice for twice at two weeks interval. 2 and 6 weeks after the first immune injection, serums were collected and the humoral antibodies were tested by ELISA. The result of ELISA showed that all the experimental groups produced anti-ORF2 antibodies, and high antibody level showed four weeks after the second immune injection. Results also showed that BVP22 could enhance the efficiency of ORF2 nucleotide vaccine. Our research provided a candidated vaccine for controlling PCV2 infection.

In order to provide the effective vaccine for the immunoprophylaxis of swine pseudorabies, a genedeleted vaccine against pseudorabies was prepared using a previously constructed mutant, PrV HB-98 (TK^- /gG^- /LacZ⁺ mutant) , that were grown with primary embryo fibroblast cells. The optimal doses for the oral and intramuscular administration of the vaccine were determined, and the safety, efficacy, immunoprotective duration and shelf life of the gene-negative vaccine were measured. In addition, the performances of four batches of the gene-negative vaccine were assessed through field trials, in which the vaccine was clinically applied to sows, piglets and growing pigs in 23 pig farms. The optimal doses of vaccine for oral and intramuscular administration for pigs at different stages were determined to be 10^5.0TCID₅₀. The vaccine was demonstrated to be safe to one-, fifteen-day old piglets and pregnant sows when it was used at dose of 10^6.0TCID₅₀, ten fold of the normal immune dose. The immunized pigs could be protected from challenge with the virulent PrV strain. The vaccine could be stored for six and twelve months under 4℃ and-20℃ respectively without fluctuation of performance. The immunoprotective duration could last for six months in PrV antibody-free commercial and breeding pigs . The highly safety and good efficacy of the vaccine were further confirmed by the results obtained from the field trials, and the vaccine could be used in PrV-suffered piglets to quickly cease the prevalence of Aujeszky's disease. In conclusion, the research provided the useful hints for vaccine production and field applications of the vaccine.

The PPV VP2 gene obtained by PCR was inserted into pPI-2.EGFP vector, and the expression vector pPI-2.EGFP.VP2 was constructed. The results of Dot-blot hybridization and restriction enzymes digestion suggested that the construction of pPI-2.EGFP.VP2 was successful. PRV SA215 DNA and recombinant plasmid pPI-2.EGFP.VP2 DNA were co-transfected into Vero cells with calcium phosphate, and several recombinant viruses were screened out with fluorescence microscopy assay and Dot-blot hybridization. The recombinant virus named SA215-B was analyzed with BamHⅠrestriction enzyme digestion, Southern blotting, SDS-PAGE and Western blotting, the result confirmed that PPV VP2 gene had been inserted into the PRV SA215 DNA and expressed about 93 ku EGFP fusion proteins in recombinant virus. The successful construction of PRV-PPV recombinant virus would lay a foundation on the development of an genetically engineered PRV bivalent marker vaccine.

In the first part of the study, the concentration of enrofloxacin and its main metabolite in chicken feces were examined at the dosage of 2.5, 5 and 7.5 mg/kg body weight respectively by high-performance liquid chromatography (HPLC) with fluorescence detection. In the second part of the study, the degradability of enrofloxacin in chicken feces was tested under different form of illumination. The results were as follows: After administration in healthy chicken, enrofloxacin was excreted in feces as the parent compound (main form) and its metabolite as ciprofloxacin. When the oral dosage was 2.5 mg/kg and 5.0 mg/kg body weight, the concentration of enrofloxacin and ciprofloxacin in chicken feces reached the peak at the 6th hour and at the 4th hour after administration respectively. When the oral dosage was 7.5 mg/kg body weight, the concentration of enrofloxacin and ciprofloxacin reached the peak at the 9th hour and at the 6th hour after administration respectively. At 10th day after administration, enrofloxacin and ciprofloxacin couldn’t be detected in chicken feces. The degradation of enrofloxacin in chicken feces was affected by illumination. Enrofloxacin in chicken feces was very stable in photophobic condition. But under daylight illumination, the degradation rate of enrofloxacin was very fast in chicken feces. The degradation process of enrofloxacin in chicken feces could be evaluated on the basis of the first order kinetic equation as C_t=C₀e^-kt. Calculated by this equation, the half-life of enrofloxacin in chicken feces was(2.23±0.25)d under daylight illumination.

The concentrations of tilmicosin residues in swine tissues were determined by HPLC. Swine tissues were extracted with acetonitrile and potassium dihydrogen phosphate buffer. The extracts were eluted by the C18 solid-phase extraction (SPE) cartridge. The residues were eluted from SPE cartridge with methanolacetonitrileammonium acetate solution, and the eluate was determined by HPLC at 290 nm. The limits of quantitation (LOQ) were 0.02 μg/g and 0.05 μg/g for tilmicosin in muscle and liver, respectively. The limits of detection (LOD) were 0.01 μg/g and 0.025 μg/g for tilmicosin in muscle and liver, respectively. Recoveries of tilmicosin in muscle fortified at 0.02~2.0 μg/g and in liver fortified at 0.05~5.0 μg/g were in the range of 84.2%~96.6% and 82.0%~92.8%, respectively. Coefficients of variation were in the range of 5.0%~8.2% and 5.8%~11.3%, respectively. The method can meet the requirement for tilmicosin residue analysis.

In order to find the morphological evidence that leptin plays a role in control of reproductive endocrine in the level of the hypothalamus, coexistence of OB-Rb mRNA and LHRH-like immunoreactive (LHRH-IR) in neurons of the pig forebrain was studied by combination of in situ hybridization and immunohistochemistry methods. The results showed that both OB-Rb mRNA and LHRH-IR containing neurons(dual labelled neurons) were seen in the hypothalamus, hippocampal formation, amygdala and cerebral cortex of the pig, and that some neurons expressing OB-Rb mRNA also contained LHRH-IR. It meant coexistence of OB-Rb mRNA and LHRH-IR in these neurons of the pig forebrain. In the hypothalamus, dual labeled neurons were seen in the paraventricular nucleus, periventricular nucleus, ventromedial nucleus, lateral area and arcuate nucleus. In the hippocampal formation, dual labeled neurons were mainly found in the polymorphic layer and the granular layer of the dentate gyrus and in the pyramidal layer of the hippocampus. In the cerebral cortex , dual labeled neurons were mainly observed in the Ⅲ~Ⅴlayers. The results suggested that leptin probably regulate endocrine and reproduction of animal through directly imposing on the GnRH neurons expressing OB-Rb in the hypothalamus.

Metabolism and deposition of exogenous DNA from transgenic glyphosate-tolerant soybean meal in grower pigs were studied in the experiment. The conventional and transgenic soybean meal diets were fed to barrows fitted with a simple T-cannula at distal ileum for collection of ileal chyme and feces. In addition, feeding experiment with 30% of conventional or transgenic soybean meal was performed in grower for 42 d. Then the tissue samples were obtained after slaughter for detection of exogenous DNA. The results showed that the exogenous gene cp4 epsps derived from glyphosate-tolerant soybean meal was detected in chyme and feces both only at 6.7%, which indicated that integrity of exogenous DNA was greatly or utterly devastated in distal intestine. Based on 5 pg of limit by PCR, there were no deposition of cp4 epsps gene in liver, muscle, spleen and thymus tissue of grower, and this implied that exogenous DNA has already been metabolized and degraded.

MTDFREML software was used to estimate the genetic parameters of main economic traits of high quality merino sheep in the natural ecological conditions of Xinjiang. The estimated values of heritability were 0.56±0.07，0.16±0.06，0.33±0.08，0.20±0.08，and 0.31±0.08 for staple length, fineness, total fleece grade, greasy weight and live weight after shearing respectively. Large positive genetic correlation estimates were obtained between greasy weight and live weight after shearing(0.77±0.11) , greasy weight and staple length(0.80±0.12); there was a negative genetic correlation (-0.45±0.15) between staple length and fineness. This suggests the attention should been paid to greasy weight and body weight when selection for fineness.

Lung of the somatic cell cloning goat “yuan-yuan” was observed with eyes, LM and TEM, the results are as follows: The left lung have two lobes and the right lung have four lobes. The fissures are incomplete which are connected with lobes by serosa. Many epithelial cells are full of osmotin. The respiratory bronchioles were lined with cuboid cells. The alveoli epithelium differentiated into flattened(type I cell) and cuboid (type II cell)epithelial cells. The capillary endothelium were pressed close to the alveolar epithelium. The blood-air barrier was composed of type I cell, basement membrane and endothelium. The osmiophilic lamellar bodies and ribosomes are seen abundantly in type II alveolar cells, rough endoplasmic reticulum pools expand, Mitochondria denatured, microvilli of the epithelial cells appear on the luminal surface.

A strain of virus was isolated from faeces of diarrhea piglet using the Vero cells， and was identified as PEDV by RT-nested PCR, then was named as LJB/03.The 854 bp M gene of LJB/03 was amplified by RT-PCR，then was directly inserted into pMD18-T, and sequenced. The 854bp PM gene was subcloned into a prokaryotic expression vector pGEX-6P-1. A positive colony pGEX-6P-PM was translated into BL21（DE3）plysS.High-level expression of recombinant fusion protein was obtained after induced by IPTG. 43 ku fusion protein was tested by SDS-PAGE and can be recognized by the positive serum of PEDV by Westernblot. Through gel thin layer scanning analysis, the amount of target protein is over 33.7%. So the recombinant M protein can be a valuable diagnosis or therapeutic agent for the diseases.

The glycoprotein E2 contains major antigenic determinants and involved in inducing neutralization antibodies. A truncated form of the CSFV glycoprotein E2 was expressed in baculovirus. The signal sequences of E2 gene were replaced with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway in insect cell. In addition, the hydrophobic transmembrane anchors at the carboxyl termini of E2 proteins were removed to enable secretion rather than maintenance in the cellular membranes. Recombinant E2 protein was purified from the supernatant of infected cells by employing Ni²⁺ affinity chromatography. Protein purified was recognized by at least three antiE2 pig sera and WH211 monoclonal antibody. WH211 monoclonal antibody couldn′t recognize the protein expressed in E.coli, indicating that it retain native structure. Thus, E2 expressed in insect cells can be used as a tool for diagnostic tests as well as obtaining material that could be suitable for X-ray crystallography studies.

Two fragments of 985 bp and 934 bp were amplified by PCR technique from the DNA genome of PRV Guangxi B and W wild strains according to the published gE gene of PRV Rice strain. These PCR products were purified by agrose gel, then were cloned into pMD18-T vector and transformed into E. coli DH5α. Four positive recombinants (pMD18-T GXB1, W1 and pMD18-T GXB2, W2) were obtained. The recombinants were determined by PCR and digestion of HindⅢ and EcoRⅠ. Then the inserts fragments were sequenced. The results revealed that the gE genes were both composed of 1 743 base pairs and coded for 580 amino acids. The homologe of nucleotide sequence and deduced amino acid sepuence between GXB and GXW stvains were 99.4%,99.1% respectively.GXB shared 98.0% and 95.7% homology of nucleotide sequence and deduced amino acid sequence with Rice strain,GXW shared 97.8% and 95.2% homologe respectively. The homology of nucleotide and deduced amino acid sequence between the two strains from Guangxi and other domestic PRV strains were 98.4%~99.5% and 97.4%~99.1% respectively. Phylogenetic tree analysis showed there was a high relationship between GXB, W strains and Ea strain.