This study aims to establish a reporting system for stem cells tracer, EGFP cDNA together with LoxP-flanked PGK-neo cassettes were inseet into the Oct-4 gene of Wuzhishan Miniature pig at the stop codon via TALENs mediated homologous recombination, expression of EGFP gene was under control of Oct-4 5'-regulatory region. TALENs directed against sequences flanking the stop codon of Oct-4 were cotrasfected with targeting vector, 514 drug-resistant cell clones were screened, and 36 correctly targeted clones were confirmed by PCR from them, the targeting efficiency were 5.6% and 13.0%. Oct-4-EGFP transgenic cell line was establinshed in this study, and it was also proved that TALENs could significantly improve the efficiency of homologous recombination.

Using goat UniGene to identify miRNA and RT-PCR was used to validate the predicted miRNA, and then Real-time PCR was performed to detect the miRNA expression during one year in goat skin and muscle. Results revealed that 5 new miRNA were identified and all of these miRNA were expressed in goat skin and muscle, in which a miRNA cluster was discovered which was composed of chi-miR-421*,chi-miR-421 and chi-miR-374b. miRNA expression results indicated that chi-miR-1839,chi-miR-374b and chi-miR-2284n in skin gave a highest expression level on February, chi-miR-421 and chi-miR-421*expression increased remarkably on October. The identification of 5 new miRNA in goat(chi-miR-2284n, chi-miR-421*, chi-miR-421, chi-miR-1839 and chi-miR-374b,accession number:JQ002550-JQ002554) was a useful complement for goat miRNA database.

A pair of primers was designed according to the porcine follistatin(FS) gene mature peptide sequence(GenBank:M36512.1), the mature peptide sequence of FS gene was achieved by PCR. The PCR product of the expected length was cloned into the pMD18-T vector and transformed into E.coli DH5α. The positive clones were identified by PCR, enzyme digestion and sequencing. Then insert the FS gene mature peptide between the BamHⅠ and HindⅢ sites of the pRSET-A vector generating the recombinant plasmid pR-FS and transformed it into E.coli BL21(DE3). The positive clones were also identified by PCR, enzyme digestion and sequencing. Bacteria transformed by pR-FS were expressed in E.coli BL21(DE3) after induction with IPTG and produced a product showing expected molecular mass of 26.1 ku in SDS-PAGE, which achieved the highest level induced by 0.2 mmol/L IPTG for 3 h. The product can be purified by 50% Ni-NTA agarose. These results demonstrated that the porcine FS gene mature peptide sequence was successfully cloned and expressed in E.coli BL21(DE3).

The study was designed to establish a method for the efficient gene transfection in vitro and rapid screening of amniotic fluid-derived stem cells (AFSC), while maintaining their pluripotency. pCMV-DsRed was used as the reporter plasmid to transfect amniotic fluid-derived stem cells by liposome. 36 h later, transient transfection efficiency was 69.17% by flow cytometry. The purified transgenic sheep AFSC monoclones were obtained through the selection of G418 at the concentration of 6 mg/mL after the treatment of 5 h. The transgenic sheep AFSC was positive for Oct-4, SSEA-1 by RT-PCR. AFSC could also formed embryoid bodies by in vitro suspension culture. The results implied a possibility that the transgenic sheep AFSC could be used to track down in vivo differentiation path and the definitive host.

In order to develop an indirect ELISA method for detecting antibody against Brucella, the bp26 gene of Brucella was inserted into pET-32a(+) vector and the recombinant plasmid was transformed into E.coli BL21(DE3) and expressed. Using the purified recombinant protein bp26 as coating antigen to establish indirect ELISA method, and the reaction conditions were optimized and kit was assembled. The recombinant protein was expressed in high level in BL21(DE3) as confirmed by SDS-PAGE, and showed strong immunological reaction with Brucella positive serum detected by Western blotting. The test confirmed that the best coating concentration of the recombinant antigen was 3.6 μg/mL, and the critical value of positive serum samples was 0.370. The specificity test showed that there were no cross-reactivity between the recombinant protein and TB, HPS, SC and other pathogens positive serum. The sensitivity tests indicated that the diluted serum ratio was 1:12800. The coefficient of variation of intra-assay and inter-assay were less than 10%. There was 97.1% coincidence rate between the indirect ELISA kit and SAT based on detection of 241 clinical bovine serum samples. In conclusion,the prokaryotic expression of recombinant protein bp26 had good immunogenicity, and the established indirect ELISA method had good sensitivity, specificity and reproducibility, which could provide a serological method for distinguishing between bp26 gene-detected marked vaccine immunization and natural infection.

In this study, some PMWS-suspected samples from a swine farm in Liaoning province were detected by PCR method,then we amplified the complete genome of PCV2 and made some sequence analysis. The results showed that the complete genome of PCV2 Liaoning strain was 1768 bp in length. The nucleotide homology between the PCV2 Liaoning strain and other strains ranged from 95.9% to 99.5%. Among these strains, the PCV2 Liaoning strain showed the highest homology with Denmark (EF565365), Australia (AY424405) and Jiangsu (FJ158606), all about 99.5%, the lowest homology was with Guangdong (JX912915) about 95.9%.

In order to provide a scientific basis for the diagnosis and prevention of porcine circovirus disease,ORF1 gene of porcine circovirus type 2 (PCV2) GZ strain was obtained by PCR with specific primers designed according to PCV2 sequence in GenBank. Amplification product was connected with pMD18-T vector. After the identification, subclone it into pET-30a(+) prokaryotic expression vector. It was sequenced after double enzyme identification.The results showed that the purpose fragment of ORF1 gene of PCV2 was in the right position of pET-30a(+) vector,and we successfully constructed pET30a-PCV2-ORF1 prokaryotic expression vector.The recombintant plasmid was transformed into Rosetta, expressed by IPTG induction, and detected by SDS-PAGE.It showed that PCV2-ORF1 had efficient fusion expression in the pET-30a(+) and the expressed protein was 42 ku.The expression products, which existed mainly as inclusion bodies,were purified by Ni²⁺-affinity chromatographic column.Western blotting analysis showed that the purified products could react with anti-His MAb.

To investigate the major steps in the replicative cycle and ultrastructural changes during pseudorabies virus (PRV) infection, the swine testis (ST) cell line was used as the target cell to be infected with PRV Ra strain. The results demonstrated that PRV could cause obviously cytopathic effect, with a close relation with the PRV infected time. According to the observation by electron microscopy, PRV virions attached to the cell surface were found in the initial stage of infection, virus entered into cells and cell nuclear area by membrane fusion and endocytose, then began to replicate in the nucleuses, virus envelop was obtained from the membrane structure in the cytoplasm, especially in the Golgi body;the mature PRV particles were released into the extracellular to infect other cells during the early stages of infection;and the virus particles were released by cytolysis in the later stages. Ultrastructural changes of infected cells showed mitochondria swelling,decreased,cristae vague, intranuclear inclusions, cell fusion, serious cavitation and cytolysis.

In this assay,cloning, expression and identification of outer membrane protein H (OmpH) of Pasteurella multocida were conducted to provide the foundation for study of immune effects of OmpH. OmpH gene was amplified from the genomic DNA of P.multocida CVCC448 by using PCR. After being cloned into pMD18-T, OmpH-pMD18-T was sequenced to confirm its identity, and then cloned into expression vector pET-28a. The constructed recombinant plasmid OmpH-pET28a was transformed into E.coli BL21 and induced with IPTG, and the expressed product was identified by SDS-PAGE and Western blotting. Sequence analysis showed that the full coding length of OmpH was 978 bp, which could encode 326 amino acids, expressed recombinant protein was 37 ku. Western blotting result showed that recombinant OmpH could react with sera of rats immunized with P.multocida, which showed that the recombinant protein had good immunogenicities. OmpH-pET28a was constructed successfully, and fusion protein was expressed in E.coli BL21,which laid the foundation for the study of immune effects of OmpH.

The single nucleotide polymorphisms(SNPs) in exon 2 and part of intron 2 of H-FABP gene in 96 individuals from Black cattle were detected by PCR-SSCP methods and sequencing analysis technique. The results showed that there were two SNPs in intron 2 at primer 2 and primer 4, no variance at primer 1 and primer 3. Three genotypes (AA, AB, BB) determined by SNP at 323 (G→A) were detected using primer 2.Their genotype frequencies were 0.448, 0.146, 0.406, which were no in Hardy-Weinberg equilibrium with chi-aquare analysis. Statistical analysis revealed an intermediate polymorphism information content (PIC) in Black cattle. Three genotypes (HH, Hh, hh) determined by SNP at 872 (C→G) were detected using primer 4.Their genotype frequencies were 0.104, 0.417, 0.479, which were in Hardy-Weinberg equilibrium with chi-aquare analysis. Statistical analysis revealed an intermediate polymorphism information content (PIC) in Black cattle.

Chicken epithelial cells (IECs) from eighteen day-old chicken embryos were separated by the digestion of thermolysin. The chicken epithelial cells were identified by morphological observation and immunocytochemical method. TLR mRNA expression was detected with reversing transcription polymerase chain reaction (RT-PCR). TLR receptor expression in the intestinal epithelial cells of different varieties of chick were compared. The results showed that the epithelial cells of chicken embryo grew well. 90% for the purification of the IEC was obtained by removing single cells by low-speed centrifuge and differential velocity adherent. The result of identification by detecting the cyto-keratin was positive. IECs all expressed of 10 kinds of TLR mRNA with different intensity. There was no significant difference in TLR receptor expression in the intestinal epithelial cells of different varieties of chick.

The BQ-C isolate of porcine parvovirus (PPV) was propagated on PK15 cell, and identified by polymerase chain reaction (PCR), haemagglutination test (HA) and electron microscope. To express the VP2 structural protein of PPV BQ-C strain, the VP2 gene was cloned into pET-30a(+) and expressed in E.coli BL21(DE3). SDS-PAGE analysis showed that the recombinant protein was about 71.5 ku. Western blotting assay confirmed that the protein was able to specifically react with PPV positive serum. In this study, the BQ-C isolate of PPV was successfully obtained, and the recombinant VP2 protein could be used for the diagnosis of PPV and vaccine development.

The assay was aimed to establish a method for detecting the residual DNA in recombinant interleukin-1 receptor antagonist (IL-1ra) using digoxigenin labeled probe. By optimizing experimental conditions, with ultrasonic means,genomic DNA of engineered bacteria containing IL-1ra was labeled by digoxigenin,as a probe for slot blotting assay to dectect residual DNA in bulk products. The residual DNA in 24 batches of bulk products was less than 10 ng per dosage for per person. This method was sensitive, specific and convenient,and could be well repeated, which could be used for detecting residual DNA of recombinant IL-1ra protein bulk products and semi-finished products.

The study was aimed to construct prokaryotic expression vector of NDV HN gene using attenuated Salmonella typhimurium SL1344ΔcrpΔasd as a carrier. The HN gene was cloned from the plasmid pMD18-T-HN by polymerase chain reaction (PCR) and inserted into the vector pYA3493. Electricity transformation method was used in this recombinant plasmid pYA3493-HN transformating to recepted state Salmonella typhimurium SL1344ΔcrpΔasd. The recombinant plasmid pYA3493-HN was identified by digestion of endonuclease, PCR and sequencing. The results confirmed that recombinant attenuated Salmonella typhimurium vaccine SL1344ΔcrpΔasd(pYA3493-HN) was constructed successfully, which provided a foundation for research and development of NDV HN orally genetic engineering live vaccine.

According to the gene sequences of duck Newcastle disease virus (NDV) L gene and Muscovy duck parvovirus (MDPV) Vp3 gene in GenBank, two sets of specific primers were designed by Primer Premier 5.0. The duplex PCR assay was developed through optimization of reaction conditions and validation of specificity, sensitivity and repetitiveness of the method. The sensitivity of the assay was 30 fg for NDV and 16 fg for MDPV. No specific bands of the same sizes were amplified from other duck pathogens, such as duck plague virus, duck hepatitis virus, duck circovirus, H9 subtype avian influenza virus, duck flavivirus, Salmonella, E.coli, avian Pasteurella multocida. This duplex PCR assay was a quick, sensitive and specific test for detection of duck NDV and MDPV, and would be useful for the control of these viruses in ducks.

An experiment was conducted to investigate the effects of supplemental phosphorus (P) level on carcass traits and meat quality of broilers from 22 to 42 days of age. A total of 500 day-old Arbor Acres male broilers were fed the same corn-soybean meal complete diet containing 0.39% P for 21 days posthatching. On 22 days of age,360 birds were selected according to body weight and randomly divided into 10 treatment groups with 6 chicks per cage of 6 replicate cages for each treatment in a completely randomized design. Birds were fed a P-unsupplemented corn-soybean meal basal diet containing 0.09% P and the basal diets supplemented with 0,0.05%,0.10%,0.15%,0.20%,0.25%,0.30%,0.35%,0.40% and 0.45% P as CaHPO₄ (feed grade) for 21 days,respectively. The results showed that dietary proper P levels improved dressing percentage (P<0.1) and whole net carcass percentage (P<0.1) of broilers on 42 day of age. However,there were no significant differences in meat quality and other carcass traits among treatments(P>0.1).

Three rumen fistulated Xinjiang Kazakhstan rams were applied as experiment animals to compare ruminal degradation kinetics of lavender hay before and after essential oil extraction using a nylon bag technique. The hays placed in nylon bags were incubated with duplicate for 6, 12, 24, 36, 48 and 72 h in the rumen of each ram. No obvious differences between essential oil unextracted and extracted lavender hay were observed for crude protein, neutral detergent fibre and acid detergent fibre, the rumen disapperance rates of DM and CP decreased significantly after the essential oil extracted (P<0.05). Although the rapidly degradable portion a value and slowly degradable portion b value did not differ between unextracted and extracted lavender hays (P>0.05), the degradation rate c and effective degradability values for DM and CP were significantly lower in the extracted hay than the unextracted hay (P<0.05), and such results might be due to greater contents of vanillic (26.5%), protocatechuic (33.3%) and ferulic acids (24.8%) occurred in the oil extracted hay. In summary, essential oil unextracted lavender hay exhibited high nutritive value in term of rumen degradation rate.

A method based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) had been developed for the determination of vancomycin in feeds. The analyte was extracted with phosphate buffer -acetonitrile(85:15,V/V),and cleaned by strong cation exchange solid phase extraction cartridge. Quantification of the analyte was achieved by UPLC-MS/MS with multiple reaction monitoring (MRM) using external standard method. The calibration curves of vancomycin were linear over the concentration ranges of 25 to 1000 ng/mL in different matrix. Detection and quantification limits of the method for the analyte were 0.05 and 0.1 mg/kg in feeds. The average recoveries ranged from 86.7% to 117.1% at the spiked level of 0.1 to 100 mg/kg in feeds. The inter-run and between-run relative standard deviation was 1.0% to 17.4% and 2.9% to 10.1%,respectively. The method was reliable,sensitive and reproducibility,its performance could meet the requirements of the domestic and international legislation.The method adapted to the determination of vancomycin in feeds.

Humic acids (HA),a class of compounds resulting from decomposition of organic matter are natural constituents of soil,lignite,drinking water and plants,etc. There is no doubt that HAs have many beneficial effect like antibacterial,antiviral and anti-inflammatory in animals,can improve immune system,promote growth,increase feed conversions and reduce loss of nitrogen. This paper summarized recent advances about the health value of animal and application in animal production from the aspects of an alternative to antibiotics.

Unconventional feed plays an important role in sheep cultivation with increasingly tight supply of feed. This review focuses on the application and additional precautions of several kinds of fruit marc utilized normally in sheep industry and provides a theoretical basis for reasonable application of unconventional feed in sheep cultivation.

The development of modern biotechnology provided a new way for the use of exogenous animals,plants,microorganisms genetically bred the excellent genetically modified (GM) rice varieties.Along with people's special concern to food safety,the food security of GM rice was increasingly becoming the focus of attention.A brief overview of the latest progress in the research status and feed security evaluation of GM rice domestic and abroad had been reviewed in this paper.

This experiment was conducted to study the effects of Chinese herbal additive level on production performance and diarrhea of weaning Rex rabbits and compare with the western medicine.A total of 80 30-day-old weaning rabbits with the similar weight were randomly divided into 4 groups (Ⅰ,Ⅱ,Ⅲ,Ⅳ).Rabbits in the 4 groups were fed basic diets containing 0.1% olaquindox premix and 1.0%, 1.5%, 2.0% of Chinese herbal medicine,respectively.The results showed that compared with the group Ⅰ,the diarrhea frequency of weaning Rex rabbits in groups Ⅰ,Ⅲ and Ⅳ was reduced,average daily gain in Ⅲ and Ⅳ groups was extremlely significantly different (P<0.01), dietary crude protein digestibility in Ⅲ and Ⅳ groups was significantly increased (P<0.05),crude fat digestibility in Ⅲ group was extremely significantly different compared with that in group Ⅰ(P<0.01),there were no significant differences in all groups about slaughter performance,meat quality and fur quality of weaning Rex rabbits (P>0.05),the cooked meat rates,skin area and hair density were increased,but the differences were not significant (P>0.05).Therefore,adding 1.5% Chinese herb additive for preventing diarrhea and improving production performance of weaning Rex rabbits is the best.

The study was conducted to investigate the effects of time-limited grazing on the digestive tract development of Tan sheep. A total of 50 male sheep,1.5 months after weaning with similar initial body weight,were randomly divided into 5 groups,respectively,A group was grazing for 12 h,without supplementary feeding;B group was grazing for 8 h with supplementary feeding;C group was grazing for 4 h with supplementary feeding;D group was grazing for 2 h with supplementary feeding;E group was feeding with no grazing. All sheep were slaughtered after 4 months to collect digestive tract for analysis. The results showed that with increasing time of grazing,the weight of sheep stomachs and jejunum increased, and they were significantly higher in group A than those in group E (P<0.05). In addition,as a result of time-limited grazing,the height of rumen papillae increased,but the width of papillae decreased, the height of papillae in the grazing for groups C and D were significantly higher than those in group E (P<0.05),while the width of papillae in groups B and C were significantly lower than that in group E (P<0.05). The height of intestinal villus of jejunum also increased due to the grazing,they were significantly higher in groups A and B than those in group E (P<0.05). The results indicated that the gastrointestinal tract had a fine development in groups C and D under the condition of appropriate grazing and supplementary feeding.

Casein is the main component of milk protein which has high nutritional value and plays an important regulatory role in the organism.The factors such as genetic,lactation stage,breeding and diet affect the content of casein.Regulation of diet is a common method to improve the content of milk casein,which also has a significant effect to optimize diet utilization and the quality of dairy products.The paper summarizes the influence of different diet nutrition levels on the content of milk casein and provides a theoretical foundation for improving the content of milk casein in dairy cows.

The objective of this study was to investigate the regulation and variation of milk amino acids composition from different dairy animals. Milk samples from cows,goats,yaks and buffalos(n=20 for each species) were obtained from the local farm of Beijing,Shanxi,Yunnan and Qinghai province,respectively.Amino acids were extracted by hydrolyzed with 7.8 mol/L HCl for 24 h,and concentrations of amino acids were determined by high speed amino acid analyzer.Data was performed statistical analysis with the SAS 9.0 and principal component analysis with The Unscrambler 9.8 software. The results showed that concentrations of all tested amino acids in yak milk were significantly higher than those of buffalo,goat,high-protein and low-protein cow milk(P<0.05). Except for Thr,Ser,Val and Tyr,other amino acids of buffalo milk were significantly higher than those of goat,high-protein and low-protein cow milk(P<0.05).Thr,Ser,Val,Met,Phe and Tyr of goat milk were significantly higher than those of cow milk(P<0.05).Except for Met,amino acids in high-protein cow milk were significantly higher than low-protein cow milk(P<0.05). Amino acids of the cow,yak,buffalo and goat milk were significantly different(P<0.05).According to the PCA scores and loading plots,goat milk with Ser,Thr,Val and Tyr contents were different from the cow,buffalo and yak milk, which were grouped together. It was concluded that the different specials showed distinct amino acids profiles and had a certain species.

Mammary stem cells (MaSCs) possess the ability to generate all differentiated cell types in the mammary gland.During the process of mammary development,MaSCs are critical for the growth and reconstruction of mammary tissue in puberty,pregnancy stage,lactation and lactation recession stage.The MaSCs under in vitro culture are ideal experimental model for studying the proliferation,differentiation,survival and apoptosis of mammary cells,and potential approach for producing mammary gland bioreactor.Therefore,the researches about MaSCs are of great importance to the understanding of mammary development,therapy of mammary cancer and agricultural production.This paper will give a systematic review for the localization,purification and in vitro culture of MaSCs,and show prospects of the researches and applications about MaSCs.

This study was conducted to investigate the effects of liquorice extract on subcutaneous fat deposition and the activities of metabolism related enzymes in sheep. In this single-factor experiment,50 healthy male lamb of Ningxia Tan sheep on 20 to 30 d after weaning with similar initial body weight were randomly divided into 5 groups and experiment Ⅰ to Ⅳ groups were fed with diets supplemented with 0,1000,2000,3000,4000 mg/kg basic ration liquorice extract (LE),respectively. After a trial period of 120 days,the sheep were slaughtered and the subcutaneous fat weight,serum lipid metabolism indices and subcutaneous fat metabolism related enzyme activity were tested. Results showed that compared with control group,the subcutaneous fat weight of trialⅠ to Ⅳ groups reduced by 20.16%,14.40%,18.11% and 20.16%,respectively. The effect of 3000 mg/kg LE was the best to decrease the serum total cholesterol(TC). But LE had no significant effect on total triglycerides (TG) and high density lipoprotein cholesterol (HDL-C) levels (P>0.05). The glucose 6-phosphate dehydrogenase (G6PDH) activity in subcutaneous adipose tissue was significantly suppressed by LE (P<0.05),and 3000 mg/kg LE was the best. But the influence of LE on the activities of fatty acid synthase (FAS),malic dehydrogenase(MDH),isocitrate dehydrogenase(ICDH) and 6-phosphate dehydrogenase (6PGDH) had no significant effect (P>0.05).Thus,LE tended to reduce the subcutaneous fat weight and serum total cholesterol, and 3000 mg/kg LE adding level had the best lipid-lowering effect,and LE reduced fat deposition mainly by down regulating the key enzymes in fat synthesis approach,however,the specific regulation mechanism of LE on subcutaneous fat metabolism key enzymes in sheep still needed to further study.

Intestinal stem cells (ISCs), a kind of cells which located in the gastrointestinal tract have ability of continuous proliferation and differentiation. Wnt signaling pathway is an embryonic development and cancer-related protein networks which first discovered in Drosophila, the pathway maintains the intestines in a multi-level regulation of the steady state. This article outlines research progress in the field of intestinal stem cells, the function of Wnt signaling pathway, understand intestinal stem cells involved in the pathological inflammatory process, then reveal dysplasia and the molecular mechanisms of chronic inflammation which leads to cancer.

The growth of hair is associated intimately with hair follicle morphogenesis and cyclical variation, hair follicle morphogenesis and cyclical variation depend on intensive interactions between epithelial keratinocytes and dermis mesenchymal cells. The paper reviewed the structure, morphogenesis and cyclical variation of hair follicle, discussed the effect of signal factors on the progress of hair follicle morphogenesis and cyclical variation. The paper aims to not only provide foundation for elaborating regulatory mechanisms of hair follicle morphogenesis and cyclical variation at the molecular level, but also provide reference for the increase of cashmere's production and quality.

The polymorphisms of estrogen receptor (ESR) and follicle-stimulating hormone β (FSHβ) genes in 84 Landrace pigs were detected with PCR-RFLP method and the relationship between reproductive performance and different genotypes (include single gene and combined gene) in this study. The results showed that ESR and FSHβ genes could be divided into AA, AB and BB genotypes, and the pattern types of ESR and FSHβ genes were fit with Hardy-Weinberg equilibrium. Single gene analysis showed that reproductive performances with AB genotype were significantly higher than AA genotype in ESR gene, and AB genotype was the beneficial genotype in ESR gene. In first farrowing sow, the reproductive performances with BB, AA and AB genotypes increased gradually, and in multiparous sow, reproductive performances with AA genotype were higher than others, but the difference was not significant. Combined gene analysis showed that the effect of combined genotype was not simple sum of single genotype. In the third parity which reproductive performances had already stable, reproductive performances with AA-AB combined genotype were lower than other combined genotypes. So this combined genotype was detrimental combined genotype and should be culled in marker-assisted selection.

The polymorphism of toll-like receptor 4(TLR4) gene and its correlation with somatic cell score (SCS) in Chinese Holstein cattle were analyzed for the purpose of providing molecular markers in the marker-assisted selections (MAS) to accelerate the disease-resistant breeding progress in Chinese Holstein cattle. The polymorphism of TLR4 gene was detected by PCR-RFLP and PCR-SSCP technique in 610 Chinese Holstein cattle cattle from 30 bull families. The associations between polymorphic sites of TLR4 gene and SCS were analyzed by the least square mean analysis. Two SNPs were found, SNP -226 G>C was found in the 5' flanking region of TLR4 gene, three genotypes (GG, GC and CC) were found in 610 Chinese Holstein cattle by PCR-RFLP, and the frequencies of GG, GC and CC genotypes were 0.208, 0.482 and 0.310, respectively. SNP 1760 C>T was found in the exon 3 of TLR4 gene, three genotypes (GG, GC and CC) were found in 610 Chinese Holstein cattle by PCR-SSCP, and the frequencies of CC, TC and TT genotypes was 0.738, 0.225 and 0.007, respectively. Chi-square tested showed Chinese Holstein cattle was not in Hardy-Weinberg equilibrium at the two sites. There were no significant differences in SCS among genotypes for SNP -226 G>C, least square mean of SCS for CC genotype was very significantly lower than that for TT and TC genotypes(P<0.01). CC genotype for SNP 1760 C>T site had a great genetic effect on SCS in Chinese Holstein cattle, which could be used as a candidate marker for mastitis resistance selection in dairy cattle.

In this research, we studied the genetic variation of porcine bone morphogenetic protein 6(BMP-6) gene and the associations between the genetic variation and the litter size. Genetic variation of BMP-6 gene was detected by PCR-SSCP in 268 individuals from five swine breeds (Wild boar, Min pig, Landrace, Beijing Black pig and French Yorkshire), and association analysis were carried out to evaluate the effects of genotypes on litter size in French Yorkshire. The G1104A transition mutation, a synonymous mutation, was found within CDS of BMP-6 gene, and there were three genotypes of AA, AB and BB in all breeds. The A allele frequency was greater in Wild boar and Min pig, whereas the B allele was superior in Landrace, Beijing Black pig and French Yorkshire. The results showed that there were significant differences among sows with different BMP-6 genotypes in TNB and NBA, which implied that the G1104A mutation within BMP-6 gene had the significant influence on litter size in swine.

Fearful behavior remained to be an important cause of loss in poultry production,it would be hardly picked out by traditional methods. With the advances of genome-wide association study, comparative genomics and QTL mapping, great progress had been made in identifying genes related to fearful behavior in poultry. Expression in fearful behavior were significantly differences between breeds and sex. Chicken, quail and pheasant had been selectively bred to show differences in fearful behavior through bidirectional selection. It had been reported that polymorphisms of candidate genes, such as serotonin genes, dopamine receptor, were associated with fearful behavior. However, many of these results had needed further confirmation. Based on the results from genome-wide linkage and association analyses on different subject populations, 37 loci located on 9 chromosomes were reported to harbor fearful genes in chicken. It's expected that the dissections in genetic architecture of avian fearful behavior would provide a basis on selection and genetic improvement on local breed.

In this study, we analyzed the expressional pattern of Shp2 gene on the transcriptional level in mice testicles in different developmental stages. General RT-PCR and Real-time PCR were uesd to investigate the Shp2 expression in 16 groups of postnatal mice according to the differently developmental stages. The result showed that the temporal expression of Shp2 gene in mice testicles presented successively regardless of the daily growth according to the general RT-PCR assay. The result of Real-time PCR showed that, during the period from twentieth day to thirty-first day, the expression quantity of Shp2 gene fluctuated from the extremly significantly increase (P<0.01) to extremly significantly decrease (P<0.01) to extremly significantly increase again (P<0.01), and finally, the amount was back to normal gradually after the thirty-first day (P>0.05).The results indicated that Shp2 involved in the development of mouse testis, but the functions in details needed to be explored and investigated in depth.

In this study, myogenin (MyoG) gene as a candidate gene of slaughter traits, the genetic relationship between the single nucleotide polymorphism sites (SNPs) of MyoG gene and slaughter traits of 13 weeks old Royal chickens were analyzed. MyoG gene was amplified by PCR, the information of DNA sequences were obtained by direct sequenced of the PCR products. The results showed that four SNPs were found in the exons of MyoG gene. There were four haplotypes ((T-T-A-G (H₁), T-T-A-A (H₂), C-C-A-G (H₃) and C-C-G-A (H₄)) and five diplotypes(H₁H₁ ,H₁H₂,H₁H₃,H₁H₄,H₂H₃). By statistical analysis, it showed that there were no significant associations between diplotypes and slaughter traits of Royal chickens.

The generation of RNAi transgenic mice made it possible to knock down gene expression at the whole organism level in mammalian species. At present, for larger livestock animals, the RNAi transgenic pigs, hit a shot in the arm for the application of RNAi technology to breed new disease-resistant transgenic pigs. This review described the special phenomenon of RNAi in mammalian cells as well as one of the most promising approaches to prevent viral escape, and gave the details of the research progress of RNAi technology in the disease-resistant transgenic pigs.

To study the molecular genetic evolution characteristics of Haemophilus parasuis(Hps), PCR was used to determine the 16S rRNA gene of the strain XY0501 isolated from local pigs in Henan province, and genetic evolution analysis was conducted. The results showed that the amplication of 16S rRNA of the isolate XY0501 was 822 bp, the nucleotide sequence similarity among the reference strains was 97.1% to 99.3%, and 99.3% with the strain serotype 5. The phylogenetic tree based on 16S rRNA revealed that the Hps isolate XY0501 from local pigs belonged to the same branch with reference strain serotype 5. The results identified that the Hps infection could cause severe clinical symptoms in pigs, but infection source need to be further investigation,in addition, 16S rRNA of Hps of different serotypes was conserved and no species difference.

In this study, meat quality and muscle fiber were tested, at the same time, relationship between fiber and meat quality were analyzed of Princess,Huaixiang and Beijing You cocks raised under the same conditions for 120 days. The results showed that the muscle color was significant difference (P<0.05), Beijing You cocks was higher than Princess cocks by 1.66, while Princess cocks was higher than Huaixiang cocks by 2.54, there was no significant difference in pH value of three different broilers (P>0.05), in the drop water loss, Beijing You cocks was significant lower than Princess cocks by 2.16%, and was significant lower than Huaixiang cocks by 2.59% (P<0.05). In the muscle fiber characteristic, Beijing You cocks was better than Princess cocks, while the Princess cocks was better than Huaixiang cocks, the leg muscle diameter were lower by 0.68 and 0.59 μm, respectively, however, the leg muscle density were lower by 373.67 and 142.07 root/mm², respectively (P<0.05). There were negative correlation between fiber diameter and density of Princess, Huaixiang and Beijing You chicken, however, the muscle fiber characteristic of Princess, Huaixiang and Beijing You chicken was positive correlation among meat color, pH value and drop water loss, it was no significant difference (P>0.05).

To select the most suitable cell line for research on the pathogenic mechanism and vaccine of pseudorabies virus (PRV) Ra strain, different kinds of cells (ST, PK-15, Vero, F81, DF1, MDCK and CEF) were chosen to be inoculated with PRV. Cytopathy effect (CPE) characteristics, time and titers of the cell cultures were compared in this study. According to the morphology observation of infected cells, we found that the PRV Ra strain could proliferate in these seven kinds of cells and cause significant CPE, and the CPE obviously appeared firstly with in 24 h on the PK-15 and ST cells. The TCID₅₀ of PRV in the seven kinds of cell cultures performed differently, and the TCID₅₀ of infected ST cell was the highest. Therefore, it suggested that ST cell could be used in the further study of PRV Ra strain in vitro.

Porcine reproductive and respiratory syndrome (PRRS) was one of infectious disease, its main manifestations were reproductive disorders in sows and respiratory symptoms in piglets. Due to constantly emerging of porcine reproductive and respiratory syndrome virus (PRRSV) variant, and enable virus to evade immune surveillance, which had brought huge challenges to the swine industry. The inoculation of gastrointestinal way with live or inactivated PRRS vaccine was ineffective in inducing complete protection to the swine herds. In order to reduce the economic losses in the swine industry, new control methods and design of new vaccine approaches should be figured out soon. The research progress on the effective prevention and treatment of PRRSV with mucosal immune method was discussed briefly in this review. The mucosal immunity including mucosal sites, respiratory protective mucosal immune response was introduced, selection of mucosal immune route and adjuvant as well as the immunosuppression were elaborated. This information mightbe a help in understanding the mucosal immunity against PRRSV variants and designing innovative mucosal vaccines.

After MDCK cells were seeded in 6-well culture plate at a density of 8×10⁴/cm²,avian influenza virus subtype H9 were inoculated with those cells at different doses (0,10^-3×EID₅₀,10^-4×EID₅₀ and 10^-5×EID₅₀).Experimental results showed that compared with the control group, hydrogen peroxide (H₂O₂) and hydroxyl radical (·OH) in MDCK cells inoculated high-dose of avian influenza virus subtype H9 enhanced significantly (P>0.05),and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity significantly decreased (P>0.05) in those cells. In addition,intracellular malondialdehyde (MDA) had a significant increasing (P>0.05) in MDCK cells treated with high-dose of virus. In conclusion,avian influenza virus subtype H9 could significantly enhance the content of reactive oxygen species (ROS) in MDCK cells, reduce the activities of antioxidant enzymes,and hinder ROS scavenging mechanisms within the cell,cause the cell lipid peroxidation,which damaged cells.

Total of 34 chicken rectal swab samples were collected to isolate and identify Escherichia coli. And all isolates were tested for susceptibility to 8 antimicrobial agents by using the broth micro-dilution method. As a result, 34 intestinal Escherichia coli isolates recovered from chicken were identified and 4 of them were hydrogen sulfide producing stains. Their MIC₅₀ to kanamycin, chloramphenicol, ampicillin, cyprofloxacin, ceftriaxone sodium, gentamicin, sulfadiazine and doxycycline were more than 256 μg/mL, equal to 256 μg/mL, more than 256 μg/mL, equal to 16 μg/mL, less than 0.125 μg/mL, equal to 256 μg/mL, more than 512 μg/mL and equal to 16 μg/mL,respectively. And their resistant rates to these 8 antimicrobial agents were ordered as 100.0%, 100.0%, 100.0%, 70.6%, 11.8%, 94.1%, 100.0% and 82.3%, respectively. All isolates were multi-drug resistant strains. The minimal and maximum kinds of antimicrobial agents to 34 Escherichia coli strains were 5 and 8, respectively. The percentages of resistant isolates against 5, 6, 7 and 8 antimicrobial agents were ordered as 5.9%, 32.4%, 58.8% and 2.9%.It was suggested that wide resistant spectrum and high resistant ratio were presented in 34 intestinal Escherichia coli isolates. Medicinal proposals in clinical should be adjusted according to the antimicrobial susceptibility test.

A new virus named XZ strain,was isolated from layer cherry duck flocks with typical eggs drop symptoms and death in Xuzhou district of Jiangsu province.The virus was observed by scanning electron microscope, hemagglutination test, ELD₅₀ measurement, amplification specific DNA by RT-PCR, sequence analysis and animal regression experiment. The results showed that the isolated virus caused lethal hemorrhage in SPF chicken embryo and duck embryo.The appearance of virus particles showed spherical under election-microscope, and the virus had no hemagglutinating activity. The samples from infected ducks and the allantoic fluid of infected duck embryos were PCR positive for duck flavivirus.Sequence analysis to the gene fragment of the isolated virus revealed 98.7% nucleotides identity with Tembumu virus Fengxian strain, and had high homologywith other Tembumu virus strains from 86% to 98%. Experimental infections with the isolated virus resulted in reproduction of the disease in healthy egg laying duck. The results demonstrated that the isolated virus was Tembumu virus of duck flavivirus.

Bonamiosis, caused by Bonamia ostreae, was a highly mortal disease of oysters and could result in enormous economic losses to shellfishery. Up till now, the study on the pathogen and the cause of the disease was much limited. To lay a foundation for the further pathogenic and diagnostic research of the disease, B.ostreae culture in vitro was preliminarily performed in this study. Based on PCR result, the B.ostreae positive oyster was selected and the tissue was separated for B.ostreae culture in vitro. Chosen in situ hybridization as a confirmation method, the results showed that the parasite could be detected after culture for one month. And the infection results determined by PCR assay and in situ hybridization were consistant. In this study,we established a simple and feasible culture method in vitro for B.ostreae.

Two sets of specific primers were designed according to the sequences of Newcastle diseases virus (NDV) and duck plague virus (DPV) in GenBank. It was shown that all samples containg NDV and DPV could be amplified into two specific bands, 419 bp for NDV and 602 bp for DPV by this duplex PCR, but no specific band was amplified from other avian pathogenic virus and bacteria. As little as 0.4 pg of NDV RNA and 126 pg of DPV DNA could be detected by this duplex PCR.

In order to further understand the structure,function and genetic variation of ALV-J gp85 gene,the gene was cloned by PCR in Anhui province,which named as ALV-J-WH. We compared ALV-J-WH with fifteen amino acid sequence of ALV-J gp85 genes in GenBank database. The results showed that the homology between ALV-J-WH and the other sequences ranged from 85.9% to 96.2%, the highest homology with ALV-J-WH were JN378888 sequences isolated from China. Evolution analysis further revealed that they had a close genetic relationship. Moreover,the deletion of a 10 amino acid was observed in the ALV-J-WH between position 223 and 224 of amino acid sequence. Alignment of the full-length sequences revealed 71 variable amino acid sites,10 sites had high mutation frequency,expecially sites in hr1 and hr2 domains. The results confirmed that ALV-J-WH and partial genes of strains isolated from China evolved from common strains,but the sequence was unique. ALV-J gp85 gene was highly variable,and some amino acids with high degree of mutation frequency might be action sites of ALV-J antibody under immune selective pressure.

A virulent strain of Pasterrella multocida was isolated and identified from ducks in Jiangxi province. The isolate was highly sensitive to cephalexin, cefatriaxone, cefazolin, sulfamethoxydiazine, SMZ-TMP and chloromycetin,medium sensitive to spectinomycin and kanamycin,lowly sensitive to cefotaxime, tetracycline,lincomycin and norfloxacin while insensitive to amoxicillin, penicillin, ampicillin and cefepime. The mortalities rates of chickens, ducks, mice and rabbits infected with the isolate within 24 h were 100%. The result indicated that the isolate was a virulent Pasteurella multocida.

The QseBC two-component regulatory system is widespread in the gram-negative bacillus, which plays an important role in the regulation of bacterial virulence. In this article, the composition of the QseBC two-component regulatory system and its role in bacterial pathogenicity, especially in E.coli, S.typhimurium, Actinobacillus, E.tarda and A.hydrophila were summarized. The further study on QseBC may aid to drug design based on the bacterial virulence as the new targets, and provide new ideas for resolving the problem of antimicrobial resistance.

Livestock and poultry genetic resource,which is the crucial part of biodiversity,is facing a severe test.The conservation work is no mere an extremely urgent task,but rather a necessarily persevering mission.Several main preservation methods and technologies,including in vivo preservation as well as technologies of cryopreservation,gene library,somatic cell cloning,molecular genetic markers.In particular,the induced pluripotent stem cells(iPSCs)technology developed in recent years are summarized in this paper to lay the foundation of further conservation.

The experiment was carried out to compare the real impression between two different text methods in rumen pH.Four multiparous Chinese Holstein cows fitted with rumen cannulas were used to compare two different test methods in rumen pH of dairy cows,the two methods were simple pH meter (MANpH) and continuous recording wireless equipment (LRCpH).The results showed that there were no significant differences between two different test methods in rumen pH of dairy cows(P>0.05).Meanwhile,there were also no significant differences in 24 h observed value between MANpH and LRCpH(P>0.05).This experiment indicated that LRCpH wireless equipment not only could test rumen pH,but also could Real-time and continuous record the rumen pH after feeding.

Microecological preparation had many advantages,such as green,environmental protection,pollution free and so on. It had been used in food,medical care,animal disease prevention, healthy breeding and other aspects. Probiotics would also have a broad prospect with the increased attention on the safety concern of animal products by the people,as well as self health care and the urgent need to improve their own living environment. The author reviewed the application of Bacillus subtilis as probiotics in animal ecological breeding,animal disease prevention and control,ecological environment improvement and some other fields in this paper.

In response to recent claims that synthetic antioxidants have the potential to cause toxicological effects and consumers' increased interest in purchasing natural products,the meat and poultry industry has been seeking sources of natural antioxidants. Due to their high phenolic compound content,fruits and other plant materials provide a good alternative to conventional antioxidants.A lot of researches used the tea polyphenol,grape seed extract,pomegranate,pine bark extract,rosemary and other nitrogen-containing compounds as antioxidants in meat products.This paper reviews the application of several kinds of natural antioxidant in meat products.

Penaeidin belonged to a kind of the antibacterial peptide in innate immunity system,and it was also an immunity effector molecular what aquatic animals used to defend bacterial invasion. In this paper, Litopenaeus vannamei penaeidin-2 (PEN-2) was the research object, and the antibacterial functions were explored by studying the bacteria binding activity, protease activity inhibitory function, and antibacterial activity that were related to the antibacterial function. The results showed Litopenaeus vannamei PEN-2 possessed strong bacteria binding activity in vitro, and could inhibit the avtivity of secretory protease, but the antibacterial activity was very weak.