To investigate the role of ankyrin proteins encoding by Orf virus (ORFV) in the immune modulation to host infection, five ankyrin (ANK) genes of ORFV GDZC strain were amplified using specific primers by PCR amplification and cloning from the viral genome DNA which extracted from virus infection cells,and their sequences and coding protein structure bioinformatics analysis were performed. The results showed that ORFV008, ORFV123, ORFV126, ORFV128 and ORFV129 consisted of 516, 525, 497, 501 and 516 amino acids, respectively, which shared a nucleotide identity of 98.3%, 98.7%, 97.9%, 97.3% and 98.0% with those of ORFV OV-SA00 strain, respectively. All of five ANK proteins contained ANK and F-box structural domain that showed they were structure conservative proteins. The analysis of protein transmembrane showed that ORFV123 and ORFV128 had no potential transmembrane domains, while the ORFV129, ORFV008 and ORFV126 had one, two and three potential transmembrane domains, respectively. And these ANK proteins had no signal peptide. These results provided the basic data for further study of the ANK proteins in the pathogenic and immune evasion mechanisms of ORFV.

This study was aimed to build Wip1 over-expressed transgene mice, which could provide an animal model for clarifying the pathogenetic mechanism of atherosclerosis, and the transgene mice were screened at RNA level. Mice tissues cDNA were obtained by reverse transcription polymerase chain reaction (RT-PCR), which were used for the next PCR amplification template. The amplified fragments were used for gel recovery.Then, the amplified fragment and empty vector were double enzyme digested.Full-length gene of Wip1,which was obtained by gel recovery, was connected to pcDNA3.1(+) vector, and the Wip1-pcDNA3.1(+) over-expressed vector was constructed. Finally, Wip1 over-expressed transgene mice were obtained. The transgene mice were identified and screened by Southern blotting and qPCR. 30 Wip1 over-expressed transgene mice were got in this experiment, and the positive mice identified were 11 by Southern blotting, the positive rate was 36.67%. Moreover,the positive rate was 32.84% through screening by qPCR. Therefore, the Wip1-pcDNA3.1(+) over-expressed vector was constructed successfully, and the Wip1 over-expressed transgene mice were obtained through identifying and screening at DNA and RNA level of Wip1 in mice tissues.

This experiment was aimed to develop a method for simultaneous detection of H9 and H6 subtype avian influenza virus (AIV).Two pairs of specific primers were designed according to the conserved regions sequences of H6 and H9 AIV HA gene,a duplex RT-PCR simultaneous detection of H9 and H6 subtype AIV was developed by optimizing the PCR system such as the concentration of different primers and annealing temperature.It showed that all samples could be amplified specific bands from H9 subtype AIV single infection samples or H6 subtype AIV single infection samples,and the samples mix infection these two subtypes AIV.No specific bands of the same sizes were amplified from genomic materials of other avian pathogens.The detection limit of the duplex RT-PCR was 5×10⁴ copies/μL. It suggested that this duplex RT-PCR assay was a specific,sensitive,stable and repeatable method for detection of H9 and H6 subtype of AIV,and could provide technical support for the monitoring of H9 and H6 subtype AIV.

In this study,a quantitative Real-time PCR method using the specific primers according to CPS2J gene was established to detect Streptococcus suis serotype 2. The result showed that the equation of standard curve was y=-3.073x+36.87,r=0.995,which demonstrated that the assay had good linear relationship.The melting curve analysis showed that there was only specific peak.Sensitivity test showed that the method could detect the template at the lowest concentration of 1.0×10¹ copies/μL,which was 10 times higher than the ordinary PCR.The specific tests showed that this method could able to detect Streptococcus suis serotype 2 specially and had no cross-reaction with other serotypes or other bacteria from swine. The CV of repeatability test was 0.37% to 0.63%,lower than 2.5%. The clinical diagnosis showed this assay was more sensitive than ordinary PCR and bacteria isolation. All the results showed that the established method was sensitive,specific and reproducible,which could be used for the rapid diagnosis and quantitative detection of Streptococcus suis serotype 2.

In order to evaluate whether Nsp9 could enhance the replication of porcine reproductive and respiratory syndrome virus (PRRSV), pIRES2-EGFP-Nsp9 plasmid containing whole Nsp9 genome were transfected into Marc-145 cells,Real-time PCR and Western blotting were used to evaluate the expression of N protein after PRRSV was inoculated. The results showed that the level of N protein in the Nsp9 transfected cells was significantly higher than control group (P<0.05),which was 1.5 times higher. Meanwhile,the expression at protein level had the same trend as the mRNA level. When the dose of plasmid was increasing,the expression of N protein both at the mRNA and protein levels were increased. In conclusion,Nsp9 gene could enhance the replication of PRRSV in Marc-145 cells.

In order to construct the prokaryotic expressing vector of SLA-1 derived form Yorkshire pig and express the interest of protein, a pair of primers was designed to amplify the extracellular domain of SLA-1 gene from Yorkshire pig (named SLA-1-DYKe) by PCR. Then the PCR product was cloned into pMD^®19-T Simple Vector and transformed into Escherichia coli TOP10. After cleaved by Nde Ⅰ and Xho Ⅰ, the positive clones were selected to be sequenced. Analyzing by biological soft, the fragment from positive clone with correct sequence was inserted into pET-28a (+) and transformed into E.coli BL21(DE3). After induction and expression, the interest of protein was detected by SDS-PAGE. The results showed that the extracellular domain of SLA-1-DYKe was successfully amplified with the fragment length of 837 bp. The interest of SLA-1 gene was successfully cloned into pMD^®19-T Simple Vector and the positive recombinant plasmids with correct sequences were obtained. The SLA-1-DYKe from positive recombinant plasmids was further inserted into pET-28a(+). After transformed into E.coli BL21(DE3) and induction, the SLA-1-DYKe was successfully expressed. The molecular weight of the protein was about 34 ku. It was concluded that the prokaryotic expressing vector of SLA-1 was constructed successfully from Yorkshire pigs and then the expressed protein was obtained, which would lay a base for studying on the structure and function of SLA-1 from Yorkshire pig in the future.

In order to study genetic variation of porcine circovirus type 2 (PCV2) strains in Shanxi province, the genomic sequences of four PCV2 strains which were SXXZ1, SXXZ2, SXTY and SXJZ were isolated recently from some areas of Shanxi province, they were amplified, cloned and sequenced. The amplified PCV2 genomic sequences of these four strains were analyzed and compared with that of 34 published PCV2 stains by DNAStar and drawing phylogenetic tree. The results showed that the genomic sequences of SXJZ PCV2 strain was 1 768 bp, and the others were 1 767 bp, which accounted for 25% and 75%, respectively. The homologies of nucleotide sequences of the four strains were 95.9% to 100.0%, the homologies of nucleotide sequences of the four strains with the 34 isolates from different regions of the world PCV strain were 94.5% to 99.9%, and the homologies of nucleotide sequences of the four strains with the domestic vaccine strains were 95.6% to 99.8%. The phylogenetic tree analysis showed that SXXZ1, SXXZ2 and SXTY belonged to PCV-2b genotype, and SXJZ belonged to PCV-2e genotype. The evolutionary relationship among SXXZ1, SXXZ2 and the QY strain of Guangdong were closer, we also found a recent evolutionary relationship between SXTY and AUT5 of Austria, and a recent evolutionary relationship between SXJZ and FJ of Fujian. Besides the evolutionary relationship among SXXZ1, SXXZ2, SXTY and the DBN-SX07-2 of PCV2 vaccine of Shanxi province, a recent evolutionary relationship between SXJZ and LG of PCV2 vaccine were found. Thus PCV2 strains were popular based on PCV-2b in Shanxi province, also PCV-2e was isolated and existed. It laid a foundation for the molecular epidemiology, genetic variation, prevention and control of PCV2 in Shanxi province.

In this study,the immunogenicity protein MPB70 gene was amplified from Mycobacterium tuberculosis genome DNA which separated from deer, and about 590 bp fragment was obtained. Then the fragment was cloned and constructed prokaryotic expression vector of pET-30a-MPB70, and the recombinant plasmid was put into E. coli BL21(DE3).Purified after IPTG induction, and analyzed by SDS-PAGE, a specificity protein band was observed at 20 ku. Using the deer serum positive of tuberculosis in Western blotting, the fusion protein could be combined with deer serum positive of tuberculosis antibody and arise specific immune response. The protein could be used as a specific antigen to test the deer tuberculosis. The study laid a foundation for further studying the deer tuberculosis appraisal method.

Antibacterial synergist can enhance the antibacterial activity of the antibiotics, reduce bacterial resistance and improve treatment effect. Pharmacokinetic study is to develop dosage regimen and evaluate efficacy in clinical.Elimination of residual study can understand drug residual target tissue and residual marker to formulate appropriate withdraw time. This review briefly describes the aditoprim, trimethoprim,diaveridine of three kinds of antibacterial synergist pharmacokinetic and residue elimination rule, emphatically when contrast aditoprim to trimethoprim in the pharmacokinetic parameters of different animals. We can conclude aditoprim with longer elimination half-life and larger apparent volume of distribution, and trimethoprim elimination half-life is different in different animals, diaveridine is often used as intestinal antibacterial synergist. Recently researchers always use high performance liquid chromatography (HPLC) method or chromatographic combination method to study antibacterial synergist on pharmacokinetics and residues elimination research, it shows that the method has good specificity and high sensitivity. Currently due to the irrational use even abuse of antibiotics leading to drug failure and bacterial drug resistance problems, the presence of the antibacterial synergist for solving this problem provide a new direction, and three kinds of antibacterial synergist in different animals pharmacokinetic and residues elimination research is helpful to understand various kinds of drugs commonalities and differences in different animals, in order to find problems of this kind of drug in clinical using and continuously perfect applications of antibacterial synergist in clinical.

To prepare polyclonal antibodies against σB protein of new-type duck reovirus (NDRV) XX strain, the encoding sequence of σB gene of NDRV XX strain was amplified by RT-PCR and successfully inserted to expression plasmid pET-32a(+), and transformed in Escherichia coli BL21(DE3). The His-σB recombinant protein was achieved with IPTG induction. SDS-PAGE result showed that the molecular weight of the expression on fusion protein was about 55 ku, was major insoluble fractions. IPTG induced time and concentration were 3 h and 0.25 mmol/L, respectively. The soluble σB recombinant protein was highly purified which was purified using Ni²⁺ affinity chromatography and verified by Western blotting and protein mass spectrometry. Then the polyclonal antibodies could be obtained from the rabbits which had immunized by the purified σB recombinant protein with the reasonable procedure. The Western blotting result showed that they had the specific reaction. The results built a foundation of the further study of the NDRV σB protein function and the research of genetic engineering vaccine.

To determine the content of total flavonoids from Rosa laevigata Michx. fruits, and optimize the process of cellulase-assisted extraction, the content of total flavonoids from Rosa laevigata Michx. fruits were determined with a double-wavelength spectrophotometry with detection wavelength were 500 nm and 580 nm as the reference, using rutin as a standard material, and the process of cellulase-assisted extraction designed by orthogonal test was optimized with the yield of flavonoids as the evaluation index, and the effects of extraction temperature, concentration of ethanol, cellulase dosage, ratio of liquid to material and other 4 factors on the content of total flavonoids were investigated. It showed a good linearity (r=0.9981) between absorbance difference(ΔA) and the rutin mass ranging of 19.90 to 398.00 μg, the standard curve equation was y=0.0008x-0.0047, and the yield of total flavonoids was 87.94 mg/g, the average recovery and RSD were 98.23% and 8.77%, respectively. And the optimumcellulase-assisted extraction conditions of total flavonoids from Rosa laevigata Michx. fruits by orthogonal array design were extraction temperature of 80℃, concentration of ethanol of 40%, cellulase dosage of 0.5%, ratio of liquid to material of 40:1, extraction time of 90 min, pH of 6.0, and extraction numbers of 2. Various factors affecting extracting yield were in a order of extraction temperature > cellulase dosage > ratio of liquid to material > concentration of ethanol. In this condition of extraction, yield of total flavonoids from Rosa laevigata Michx. fruits was (93.35±3.15) mg/g. Therefore, it was suggested that extraction of total flavonoids from Rosa laevigata Michx. fruits by cellulase-assisted extraction was feasible, and the yield of total flavonoids was higher and its conditions was mild.

This experiment used carbodiimide get egg albumin synthesis of artificial antigens coupling neomycin,through square test to determine the best antigen packaged concentration and antibody dilution ratio,mass concentration logarithm of neomycin as abscissa,inhibition rate of antibody as the ordinate drawing standard curve,established ELISA fast detection techniques of neomycin in edible animal for import and export.Methods of recovery and specific test had been done in further research.Results showed that the test dilution ratio of optimum synthetic antigen was 1:200,the best dilution multiple of antibodies was 1:2 000, half inhibitory concentration(IC₅₀) was 6.99 ng/mL;The minimum detection limit of the method was 40 ng/mL,and cross reaction rate of gentamicin, kanamycin, streptomycin,tobramycin,amikacin and dihydrostreptomycin were less than 0.1%,the average of recovery rate was 83.77%.The range of standarding curve in 1.5~40 ng/mL was linear,the correlation coefficient was 0.987,the equation of standarding curve was y=-37.66x+94.592;Compared with HPLC method,the operation was simple and fast.The test results showed that the enzyme-linked immunosorbent method of detecting neomycin was rapid,efficient and specific.

To improve the quality standard of Qingwenbaidu powder, TLC was used to qualitative identity of Coptis, Radix Rehmanniae,Gardenia, Forsythia, Rhizoma Anemarrhena and Lophatherum gracile Brongn in Qingwenbaidu powder.The content of gardenin was determined by HPLC. Stationary phase were performed on ODS C18 column (4.6 mm ID×250 mm,5 μm) and the mobile phase was acetonitrile and water (13:87),the detection wavelength was 238 nm. The results showed that in the relative position of the chromatography of the control medicinal material, the chromatogram of the test sample showed the same color spots, and the negative sample had no interference. There was a good linear relationship (R²=0.9999) when the garden in range of 0.062 to 0.370 μg. Its regression equation was y=1 789.1x-14.296 and R²=0.99984. The average recovery was 99.60% and RSD was 0.76%. This method was simple, accurate and reproducible, which could better control the quality of the Qingwenbaidu powder, and could be used as the quality standard of Qingwenbaidu powder.

This experiment was conducted to study the effect of supplementary feeding milk replacer on growth performance,nutrient digestibility,serum biochemistry indexes in piglets during lactation,and explore the feasibility of shorten the weaning age of piglets by feeding milk replacer. One hundred and twenty (twelve litters) 5 days old piglets with average body weight of (3.12±0.63)kg were assigned into two groups with sixty piglets per group (six litters)and one litter of piglets per replicate. The piglets in control group received supplementary creep feed and were weaned on 28 days old,whereas the piglets in experimental group received supplementary creep feed and milk replacer (creep feed:milk replacer=1:1) and were weaned on 21 days old. The piglets in experimental group were continued feeding with creep feed and milk replacer until 28 days old. All piglets were fed with same diets from 28 to 70 days old. The results showed as follows:① Comparing with control group,the body weight of piglets in experimental group were not significantly changed at 5,21,28 and 70 days old (P>0.05),ADFI and F/G were extremely significantly or significantly increased during 5 to 21 days old,22 to 28 days old and 5 to 28 days old (P < 0.01;P < 0.05),while ADG was extremely significantly decreased during 22 to 28 days old compared with control group (P<0.01).② The digestibility of GE,DM,OM and EE of experimental group were significant higher than control group (P<0.05).③ All serum biochemistry indexes between the two groups had no significant differences (P>0.05). In conclusion,supplementary milk replacer feeding could increase the feed intake and dietary digestibility of weaned piglets. The age of wean could be forward to 21 days old according to the final body weight of weaned piglets when fed milk replacer supplementation.

The study was conducted to determine the effects of different dietary cotton by-products levels on production performance,nutrient digestibility and blood biochemical indexes of sheep.30 healthy sheep were chosen and randomly divided into 5 groups with 6 sheep per group. The feed of each group was that 50% formula concentrate feed was mixed with 50% wheat straw (control group),25% wheat straw and 25% cotton stalk (group Ⅰ),50% cotton stalk (group Ⅱ),45% cotton stalk and 5% cotton seed (group Ⅲ),and 40% cotton stalk and 10% cotton seed(group Ⅳ), respectively. The experiment lasted for 60 d. The result showed that:① The total weight gain and ADG of sheep showed a decreased with the increase of cotton stalk added proportion in the diets, and the total weight gain and ADG of group Ⅱ were significantly lower than group Ⅲ (P<0.05).② The concentration of NH₃-N was increased or decreased at different levels after adding different proportions of cotton by-products,and the TVFA and propionic acid concentration were not significantly affected (P>0.05).③ There was no significant difference of intake nitrogen, fecal nutrogen and uninary nitrogen among groups (P>0.05),but apparent digestibility of NDF and ADF showed a downward trend with the increase of cotton by-products added proportion in the diets,which were extremely significantly lower than control group (P<0.01).④ There were no significant differences among groups of the blood white cell count,red cell count,hemoglobin concentration,total protein,albumin,globulin,serum iron,sodium,chlorine and potassium content (P>0.05),but serum magnesium was significantly increased with increases of gossypol content in the diets (P<0.05). In conclusion,when the cotton by-products addition level in diets reaches 50% caused the free gossypol concentration in the feed more than 300 mg/kg, it would has certain negative effects on the production performance and health of sheep,and the cotton stalks add ratio in the diet should be controlled in the range of lower than 25%.

The test was conducted to study the effect of different proportions of sun-dried cassava stem and leave in diet on growth performance,serum biochemical indexes and nutrient apparent digestibility of Hainan Black goat. 20 weanling Hainan Black goats with similar day old and body weight were selected and divided into four groups (groups Ⅰ,Ⅱ,Ⅲ,Ⅳ) which the concentrate to cassava stem and leaf ratio were 3:7,4:6,5:5 and 6:4,respectively. The test period was 60 d. The results showed that:The ADFI and ADG of group Ⅲ were higher than the other three groups. And the ADG of group Ⅲ was significantly higher than that of group Ⅰ (P<0.05);ALT of group Ⅰ was significantly higher than group Ⅳ (P<0.05),while there was no significant differences in the other indexes (TP,ALB,GLB,AST,BUN,TC,TG and GLU) between the experimental groups (P>0.05).The nutrient apparent digestibility in group Ⅲ were the highest,and all gradually increased from groups Ⅰ to Ⅲ, and then decreased in group Ⅳ. Therefore,in the actual production,the recommend ratio of concentrate to cassava stem and leaf was 5:5 when feed fattening Hainan Black goat. On this basis,it could also be appropriate to reduce concentrated content and increase cassava stem and leave content to reduce the feeding cost,but the proportion of cassava stem and leave should not be more than 60%.

The objective of this experiment was to study the effects of dietary supplement of fermented corn straw (fermented by bacillus subtilis, lactic acid bacteria and saccharomycetes) on production performance, nutrients digestibility, and methane emission for mutton sheep. 30 ram lambs (Dorper × Small tailed han sheep, F1) were randomly allotted to two dietary treatments supplemented with 0 (control group) and 27.5% fermented corn straw(experimental group), forage-to-concentrate ratio was 45:55.The results indicated that feeding fermented corn straw could significantly increase the ADG and nutrients digestibility of sheep; the methane emission of experimental group decreased 21.5% compared with control group by using respiration calorimetry unit. In conclusion, microbial fermentation could improve straw quality, and feeding this fermented corn straw could enhance the production performance and nutrients digestibility of mutton sheep, and significantly decrease the methane emission and methane energy.

The present study was aimed to study the effects of plant additives on growth performance, immune function and morphology of internal organs and tissues in Yellow-feathered broilers. 400 Yellow-feathered broilers at 10-day-old were divided into 4 groups. The broilers in control group were fed with basal diets, while in experimental group A,B and C were fed with basal diets added 3,5 and 8 g/kg plant additives, respectively. Each group had 10 replicates (10 birds per replicates), and the experiment lasted for 50 d. The results showed that group C got the best experimental results of all groups:ADG increased by 5.53% (P<0.05), F/G was decreased by 6.11% (P<0.05), the survival rate was increased by 2.16%(P<0.05), and immune organ index, duodenal villous length and serum antibody titer were significantly increased (P<0.05) compared with the control group. All the experimental groups supplemented with the plant additives could enhance development of the duodenum, liver and kidney, and NDV antibody titer. In conclusion, adding the plant additives in the diet could improve the growth performance, immune function and development of internal organs in Yellow-feathered broilers, and the recommended amount was 5 g/kg.

The experiment was conducted to study the effects of composite deodorant with different supplemental levels on growth performance,immune organ index,intestinal tract pH value,contents of volatile fatty acid and microflora in cecum of broilers. A total of 900 14-day-old healthy AA broilers were randomly assigned to 5 groups (groups Ⅰ,Ⅱ,Ⅲ,Ⅳ and Ⅴ) with 6 replicates per group and 30 broilers per replicate. The experimental treatments were divided as follows:The broilers in group Ⅰ(control group) were only fed basal diet.0.5‰,1.0‰,2.0‰,3.0‰ composite deodorant were added in groups Ⅱ,Ⅲ,Ⅳ and Ⅴ,respectively. The experiment lasted for 28 days. The results showed as follows:The index of immune organ in group Ⅴ were the highest. The index of spleen and bursa increased by 37.74%,18.40% than that of control group on 28 days,respectively. The change of intestinal tract pH in group Ⅱ were the largest. pH value of ileum and cecum were significantly lower by 1.04,0.79 than that of control group (P<0.05),respectively. Compared with the control group,the contents of acetic acid,butyric acid and propionic acid in group Ⅲ were significantly reduced by 41.74%,35.96%,30.56% on 28 days old (P<0.05),respectively. The contents of acetic acid and butyric acid in group Ⅴ were significantly reduced by 38.04% and 44.52% on 42 days old (P<0.05),respectively. The numbers of Lactobacillus from cecum in treatment groups were more than that of control group,while the numbers of E.coli were less than that of control group. In conclusion,treatment groups could reduce the contents of volatile fatty acids obviously,improve the environment of digestive tract,enhance the immune organ index and there was no significant negative effect in improving growth performance. Considering all of the results,it was calculated that the broilers diet with 1‰ composite deodorant was the most appropriate.

The objective of the present experiment was to determine the effect of different types of N-carbamoylglutamate on growth performance,serum biochemical indexes and fat distribution of Tan sheep. Sixty male Tan lambs with average weight of (20.14±0.31)kg and similar age were randomly assigned three groups with five replications per group and 4 lambs per replication. The lambs in control group were fed basal diet, while lambs in test groups were fed diets containing 0.1% NCG and 0.1% RP-NCG,respectively. The results showed that:① Comparing with control group,the ADG was significantly improved (P<0.05) and F/G was significantly decreased (P<0.05) when 0.1% NCG or 0.1% RP-NCG were supplemented,while there was no significant difference between two experimental groups (P>0.05).② Dietary supplemented with 0.1% NCG or 0.1% RP-NCG significantly decreased the serum urea nitrogen (P<0.05),but there were no significant differences in serum total protein,creatinine content and creatine kinase activity (P>0.05).③ The ration of meat weight to carcass weight of two experimental groups was significantly improved (P<0.05).As 0.1% NCG or 0.1% RP-NCG were supplemented in the diets, the content of subcutaneous fat and GR value were significantly decreased (P<0.05),and the content of intramuscular fat (IMF) was significantly increased (P<0.05).In conclusion,dietary supplemented with 0.1% NCG or 0.1% RP-NCG could improve the growth performance and slaughter performance while there was no significant difference between NCG and RP-NCG.

Copper is one of the essential trace elements, which plays an important part in growth and development of animals. To solve the shortcomings of high inorganic copper doses in practical production,development and effective utilization of new organic copper source has become the focus of the current industry. The author introduced the nutrition function, distribution, absorption, transshipment and excretion of copper in the body,summarized the research situation and application prospects of different copper sources in livestock production, hoping to provide the reference for popularization and application of organic copper in actual production.

In order to investigate the differentiation of sheep umbilical cord mesenchymal stem cells (UCMSCs) into muscle cells induced by mouse MyoD gene. This study based on the previous work constructed the eukaryotic expression vector of MyoD-pcDNA3.1 in mice, and the vector was transfected into sheep UCMSCs. The morphological changes of cells were observed by fluorescent microsco, the expression of MyoD, Desmin and MyoG genes were detected by immunofluorescence, the percentage of cells expressing the cell specific factor (MyoD, Desmin and MyoG) was analyzed by flow cytometry and Real-time quantitative PCR to detect the relative expression of mRNA relative to muscle cell specific factor. Compared with the control group (no transfection), the vector was transfected into sheep UCMSCs, it was found that the cells were transformed into a long, slender, muscular cell state, and the cell spiral gradually disappeared at 21th day. It was found that MyoD and Desmin showed positive expression by immunofluorescence assay at 8th day, the expression of MyoG was also found after 16 d of induction, and the expression of MyoD decreased, the amount of Desmin expression was no change;By flow cytometry, the percentages of the expression of MyoD, MyoG and Desmin were 93.5%, 97.4% and 99.5%,respectively;Real-time quantitative PCR results showed that the relative expression of MyoD, MyoG and Desmin were increased and compared with the control group (non transfected cells), the cells were increased by 2.046, 2.389 and 5.489 times, respectively. The results showed that mouce MyoD gene could induce the differentiation of sheep UCMSCs into muscle cells.

To explore the cytotoxicity of palmatine in vitro,goat endometrial epithelial cells (EECs) were stimulated by different concentrations of palmatine,then the growth curve of cell was drew by MTT assay which could detect the cell proliferation. The cell growth state was observed by inverted microscope,and Wright-Giemsa staining was used to observe morphological changes of the cells while LDH test was used to check the membrane permeability of EECs. The results showed that when the concentration was 10 to 227 μg/mL,palmatine had a role in promoting proliferation of the EECs,while when the concentration was more than 227 μg/mL,cell proliferation was inhibited,and 50% inhibitory rate of proliferation (IC₅₀) of this drug on EECs was 360 μg/mL.Wright-Giemsa staining results showed that themorphology of the nucleus and cytolymph were not significantly changed when 100 and 200 μg/mL palmatine effected EECs. LDH results showed that when the palmatine concentration was more than 250 μg/mL,LDH content was extremely significantly higher than that of normal cell group(P<0.01).In conclusion,palmatine presented certain toxic effect on EECs with a significant dose-response effect,and the safe dose range of palmatine was less than 250 μg/mL.

The assay was aimed to study the cytotoxicity and mechanisms of mitoxantrone (MTN) in rat hepatama cell line RH35. MTT assay was performed to assess the cytotoxicity of MTN, and microscope was used to observe cellular morphologic changes. Apoptotic ratio and intracellular ROS generation were measured by flow cytometric analysis. The protein expression was examined by Western blotting analysis. The results showed that MTN inhibited RH35 cell growth in a time-and concentration-dependent manner. The morphologic changes were observed in the cells, including cell shrinkage, membrane blebbing and apoptotic bodies when treated with 15 μmol/L MTN for 24 and 48 h. And MTN also induced the increase of apoptotic ratio and generation of ROS in a time dependent manner. In addition, the intracellular ROS generation ratio and the apoptotic ratio of MTN treated group were extremely decreased by the ROS scavenger NAC (P<0.01). The pretreatment of RH35 cells with 15 mmol/L NAC thoroughly reversed the MTN-induced enhancement of caspase-3, Bax and CytC level and attenuation of Bcl-2 level. In conclusion, MTN induced apoptosis in RH35 cells through increasing the generation of ROS.

To investigate the antioxidant effects of the Cordyceps militaris polysaccharides(CMP),90 male BALB/c mice were randomly divided into six groups, three experimental groups (CMP groups), model control group (CY group), blank control group (BC group) and positive control group (PC group). The mice in CMP and CY groups were given cyclophosphamide at 80 mg/kg via intraperitoneal injection for 3 d. Then the CMP groups were administrated 17.5, 35.0 and 70.0 mg/(kg·BW) CMP via gavage, respectively, BC and CY groups were given physiological saline, and PC group was orally administered 70 mg/(kg·BW) CMP, lasting 18 d. At 24 h after the last administration, heart, liver and kidney were collected to measure the levels of MAD, CAT, SOD, GSH-Px and T-AOC in the homogenate.SOD and TAOC activity of three CMP groups increased significantly (P<0.01), and the level of MDA decreased significantly (P<0.01) compared with CY group. As for CAT activity of heart, the value in low-dose CMP group had no significant change (P>0.05), while in middle and high-dose groups had significant (P<0.05) and extremely significant(P<0.01) increase respectively. Except that the hepatic GSH-Px activity increased significantly (P<0.05) in low-dose CMP group, the rest values of GSH-Px activities in all CMP groups increased extremely significantly (P<0.01). The results indicated that CMP could effectively improve the antioxidant function of mice and had the potential to be a good resource of new antioxidant drugs.

The blood physiological and biological indexes were determined in both polled and horned yaks, to investigate the differences of body condition and metabolism between the two breeds, and provide a comprehensive understanding for new yak breeds. Thirty physiological and biochemical indexes of the polled and horned yaks were measured, and all these data were analyzed by independent-samples t test to compare the same index between the two groups. The results showed that there were extremely significant differences (P<0.01) in 11 indexes,5 indexes reached significant differences (P<0.05) and no significant differences (P>0.05) in 14 indexes between the polled and horned yaks. The results suggested that the polled yak was easier to adapt to the plateau hypoxia and there were many differences in adaptability of plateau hypoxia, kidney metabolism, liver metabolism and the growth and development of the bone between the polled and horned yak. This study could provide scientific basis for better protection and utilization of yak and the selection of the new yak breeds.

Apoptosis is programmed cell death process, which plays an important role in the process of development of germ cells. In the process of growth and development of mammalian oocytes, in addition to the mature ovulation fertilization of the oocytes, the others are apoptotic at different stages of follicular oocytes. This paper summarizes the research progress of development of the main apoptosis mechanism and apoptosis pathway of mammalian oocytes, and lays a foundation for further studying growth, development and ovulation mechanism of oocytes.

The objective of this study was to analyze two SNPs of HMGA1 gene,and investigate its association with feed efficiency related traits in Duroc populations.HMGA1 gene SNPs(g.-543 T > C and g.1356 C > T) in Large White pig and Min pig were detected by re-sequencing technology,SNP genotyping of g.-543 T > C and g.1356 C > T sites in Duroc populations were performed using Sequenom mass spectrometry,and the association of HMGA1 gene SNPs with growth and feed efficiency related traits in Duroc populations were analyzed.The results indicated that the individuals of CT genotype in g.-543 T>C increased 2.15 kg on 90 d body weight (P<0.05),decreased 3.21 d at 30 kg weight age (P<0.05),weaning weight was increased and 100 kg age were decreased,residual feed intake,30 to 100 kg average daily gain (ADG),40 to 90 kg average daily feed intake (ADFI) and 40 to 90 kg ADG were decreased,comparing with CC individuals.Moreover the individuals of CC genotype in g.1356 C>T had significantly increased 90 d body weight (0.37 kg,P<0.05),ADFI (less 0.13 kg,P<0.05),back fat (BF, less 0.43 mm,P<0.05),RFI (less 70.40 g,P<0.05),30 kg age (less 0.56 d,P<0.05) and 40 to 90 kg ADFI (less 0.17 kg,P<0.05) were significantly decreased;Weaning weight and 100 kg age were increased;30 to 100 kg ADG,40 to 90 kg ADG were decreased than TT genotype individuals.And the CT genotype individual in g.1356 C>T had the similar results with CC genotype individuals that compared with TT genotype individuals.All in all,the two SNPs of HMGA1 gene would be helpful in feed efficiency related traits in Duroc population.

Fiber and wool quality not only affects the economic benefits of cashmere goat breeding, but also affects the quality of wool textiles. In recent years, fluff quality has declined with cashmere yield improvement. In order to improve production efficiency, improving fluff quality is imperative in the production process. At the same time, fiber and wool quality is important parameters of selection in breeding of goats. The main traits of controlling the fluff quality are quantitative traits. It can be to find the number of main effect genes by quantitative genetics and molecular genetics and to study its growth mechanism and gene expression. In this review, we summarize the recent achievements of the quality of cashmere, including the use of phenotypic select to improve the quality of the fluff and finding the genetics of controlling the quality of cashmere, for example, HOX,BMP,KAP genes and pigments, and beginning to find the corresponding control target trait QTL and different sequences of SNPs by GWAS analysis to improve the quality by genotype selection, in order to improve the quality of cashmere and provide reference to study the cashmere quality of genetic factors for later research.

This study was aimed to understand the characteristics of length polymorphism with repeat sequence of keratin associated protein 1 (KAP1) family genes in yak. KAP1 family genes of yak and cattle were sequenced, and compared with sheep KAP1 family gene sequences. The results showed that cattle KAP1 family genes were located in chromosome 19, according to location of sheep KAP1 family genes in the chromosome and similarity with cattle KAP1 family genes, renaming the cattle KAP1 family (according to the gene location of chromosome) B2D, B2A, KAP1-1 and B2C genes into KAP1-4, KAP1-1, KAP1-2 and KAP1-3 gene, respectively. KAP1 family genes in the 3'and 5' flank were highly conserved, the difference between family genes mainly in the the repeat sequence region, which yak KAP1 to KAP4 genes were found 30 bp length polymorphism. There were B(CCQTS)A₁(CCQPT) repeat sequence and a new repeat sequence C(SIQTS). The results indicated that the repeat sequence was the key of the polymorphism of KAP1 family genes, which might be relate to combination with keratin protein.

This study aimed to investigate the correlation between ESRβ gene exon polymorphism and egg production of Black plumage egg quail, and provide the theoretical foundation for molecular selection. The healthy Black plumage egg quail were selected as research object, and the egg production were analyzed using SPSS 13.0 software. Estrogen receptor β(ESRβ) subunit gene was selected as candidates gene to screen the genomic variation by PCR-SSCP, and the association of the ESRβ gene polymorphisms with egg production were analyzed. The results of polymorphisms detection showed that exon 1, exon 3, exon 4, exon 5,exon 6,exon 7 and exon 8 of ESRβ gene all had nucleotide variation. The association analysis results of ESRβ gene exons and egg production showed that the egg weight of first laying of exon 3 CD genotype were significantly higher than that of CC and DD genotypes (P<0.05). The age at first laying of exon 5 was also significant difference between DD and CC, CD genotypes (P<0.05). Moreover, the average egg weight of exon 7 CD genotype was significant higher than that of CC and DD genotypes (P<0.05). These data suggested that ESRβ gene could significantly affect egg production of Black plumage egg.

The aim of this study was to evaluate the polymorphism and evolution in royal chicken using the tandem repeat microsatellite LEI0258 located within the chicken major histocompatibility complex B region (MHC-B). Genomic DNA was extracted from forty blood samples of royal chicken and used to be genotyped and sequenced. Combining the published sequences,the analysis of allele and nucleotide polymorphism and median-joining network were carried out. A total of nine alleles ranging from 193 to 297 bp in 40 samples were found,five of which was novel for royal chicken. The observed heterozygosity,expected heterozygosity and polymorphic information content were 0.625,0.716 and 0.666,respectively. Three alleles were corresponding to six available serotypes. Alleles were classified into five clusters,based on the SNPs and indels found within the sequences flanking the repeats. The alleles of royal chicken existed in clades A and B,both of which were also found in Red junglefowl. The results suggested that royal chicken had a middle or high level of genetic diversity in microsatellite LEI0258,and that might originate from Red junglefowl.

Rotavirus infections are a major cause of viral diarrheas in young animals and children. Isolation and identification of rotavirus make a contribution to epidemiological survey and molecular biology study.A strain of porcine rotavirus was isolated in MA104 cell cultures from the intestinal contents of piglets with diarrhea in Beijing.The virus was identified to be porcine rotavirus by immunochromatography strip test, Real-time RT-PCR, immunofluorescence test and sequencing analysis.According to the sequence analysis, the virus was classified as group A porcine rotavirus, the genotype of VP4, VP6 and VP7 genes belonged to P[13], I5 and G11, respectively.The virus was designated Rotavirus A pig/China/BJ/2015/G11P[13].

The objective of this study was to isolate and identify Streptococcus bovis from yak, and detect their hemolytic, pathogenicity and antimicrobial susceptibility. 45 fecal samples collected from the diarrheal yaks in Aba state in Northwest Sichuan province were used to isolate Streptococcus bovis with sheep blood culture media under 37℃ and 5% CO₂ condition cultured for 24 h. The isolated bacteria were identified by 16S rRNA amplification and sequencing. Total 14 Streptococcus bovis were identified, among which, 8 Streptococcus lutetiensis strains and 6 Streptococcus gallolyticus strains. 9 Streptococcus bovis strains showed α hemolytic, and 5 Streptococcus bovis showed β hemolytic. Both Streptococcus lutetiensis and Streptococcus gallolyticus caused the experimental mice mild diarrhea. The isolated 14 Streptococcus bovis were sensitive to penicillin, cefotaxime, vacomycin, acetylspiramycin, ciprofloxacin, rifampicin, teicoplanin, ampicillin and gentamicin, and highly tolerant to streptomycin, kanamycin, lincomycin, erythrocin, tetracycline and clindamycin.This study illustrated the biological characteristics of the isolated Streptococcus bovis from yak, which made the basis for the prevention and control of diseases caused by Streptococcus bovis.

The purpose of this study was to investigate the molecular types and antimicrobial resistance of Salmonella isolated from pigeon in Shanghai from 2011 to 2014. A total of 92 fecal samples were collected from markets. XLD plate and Salmonella chromogenic medium were used to isolate suspected Salmonella colonies, and then determined the number of Salmonella through gram staining and biochemical tests. Among them, 24(26.1%) were positive for Salmonella. Kauffmann-White method and the K-B method were used respectively for serotype identification and susceptibility testing. In result, serological identification showed that 24 isolates from pigeon could be divided into 3 serotypes including S.typhimurium (66.7%), S.agona (25.0%) and S.corvallis (8.3%). Drug susceptibility test showed that 75.0% of the isolates were resistant to one antibiotic at least. The highest level resistance were found for tetracycline as well as sulfisoxazole (62.5%), followed by streptomycin (58.3%), nalidixic acid (50.0%) and ampicillin (20.8%). Multi-drug resistant strains accounted for 62.5% (15), in which the largest number of strains (7, 16.7%) were resistant to four drugs. In addition, isolates were 100.0% susceptible to cefepime, cefotaxime, ceftazidime, imipenem and ofloxacin, but 12.5% were moderately sensitive to the amoxicillin/clavulanic acid and ciprofloxacin. The study showed that Salmonella had a high separation rate of pigeon in Shanghai farmers market and performed serious multidrug resistance, which would bring great challenges and risks to the prevention and control of Salmonella in food, so that the prevalence and drug resistance of Salmonella in pigeon deserved close attention.

To confirm the pathogen of a suspected mycoplasmal pneumonia of goat in a farm of Fujian province,the pathogen in the lung tissue was isolated and purified using medium for Mycoplasma. It was identified by biochemical test and PCR method,and 16S rRNA gene of the isolate was sequenced. The results showed that the isolate colonies were like fried eggs with brown protrusion in the center,and it could ferment glucose,not hydrolyze arginine and decompose urea,meanwhile,four azole nitrogen chloride reduction reaction,cholesterol test,hemadsorption test,blood adsorption experiment and hemolysis test of isolate were all negative,however,Meilan reduction reaction was positive.The productions of PCR amplification was about 505 bp which was Acholeplasma laidlawii specific band.The result of sequence analysis indicated that there was 99.8% homology between the nucleotide sequence of 16S rRNA gene of the isolate and that of Acholeplasma laidlawii strain PG8.The identification results showed that the isolated from the lung tissue of goat was Acholeplasma laidlawii called FJ-NP strain,while futher study would be needed for the relationship between the pathogentic isolates and goat disease.

In order to establish AMA1 recombinant adenovirus of Neospora caninum (N.caninum) and Toxoplasma gondii (T.gondii), and analyze the immunogenicity of it, cross universal primers were designed according to the open reading frame of N. caninum and T. gondii AMA1 gene sequences. Based on pMD18T-NcAMA1 and pMD18T-TgAMA1 cloning plasmid, recombinant adenovirus shuttle plasmid ADV4-Nc/TgAMA1 was constructed. Then, ADV4-Nc/TgAMA1 and pacAd5 backbone plasmid were linearized and co-transfected 293T cells. After packaging recombinant adenovirus and measuring the virus titer, collected virus was inoculated into BALB/c mice, confirmed the IgG antibody levels by indirect ELISA method. The results showed that Nc/TgAMA1 was expressed in Ad5-Nc/TgAMA1 recombinant adenovirus, Ad5-Nc/TgAMA1 recombinant adenovirus titer was 10⁹ PFU/mL. IgG antibody levels in the Ad5-Nc/TgAMA1 vaccinated group were significantly higher than pVAX1-Nc/TgAMA1 plasmid group and PBS control group. This result indicated that the constructed Ad5-Nc/TgAMA1 recombinant adenovirus could induce specific humoral immune response in mice, this research laid a solid foundation for the development of a recombinant adenovirus vaccine against N. caninum and T. gondii.

One hundred and ninety four avian pathogenic Escherichia.coli (APEC) isolated from different pathotypes of chickens in Hebei province,Shandong province,Henan province,Jiangsu province,Shanxi province and Liaoning province in China between May 2005 and March 2016 were investigated by determination of biochemical characters,O serogroup identification and drug sensitivity test.Out of 194 isolates, 124 strains(63.9%) could be grouped to serogroups O1(3),O2(26),O57(15),O65(47) and O78(33).The drug sensitivity test used eight antibiotics (ampicillin, neomycin, doxycycline, sulfamonomethoxine,tilmicosin,ciprofloxacin ceftiofur and florfenicol) by one strain of O1,O2,O57,O65,O78 serogroup,respectively.The results showed that all strains were sensitive to ciprofloxacin and neomycin, and resistance to other drugs at varying degrees.This study demonstrated that the high prevalence of O65,O78,O2 and O57 isolates existed and provided the basis for the development of multivalent bacterin in future.

Rabies is a lethal zoonotic infectious disease caused by rabies virus (RABV), which infected the central nervous system. The RABV glycoprotein exists on the surface of the virus particles, it is a major protective antigen which stimulate the body to produce the humoral immunity and cellular immunity;It is the identification of structure of the cell receptors;It plays an important role in the viral pathogenicity and tropism in the nerve, directly related to the virulence of virus.In this review,we summarized research progress of structure and function of RABV glycoprotein,and also reviewed the detection and evaluation of the neutralizing antibody. All these fundamental studies are important for the control and elimination of the RABV,to provide research basis for further revealing the biological function of RABV glycoprotein.

The study was aimed to learn the infection of dairy cows with fungi in Ningxia and take effective measures to prevent and control the fungi infection. Milk samples were collected from dairy cows that did not respond to antibiotics in Ningxia, fungi in milk samples were separated and identified, and the biological characteristics of isolated bacteria were analyzed. Milk samples infected by fungus were screened out using fungal selective medium. The yeast pathogen was shown to be Candida albicans by biochemistry and molecular biology methods. Determination of enzyme activity and biofilm-forming tests confirmed that it had different levels of virulence with strong pathogenicity to experimental animals. Drug sensitive test was carried out by K-B programe, the results showed that the isolates were resistant to clinical commonly used antibiotics, and were sensitive to clinical common antifungal drugs. The fungal mastitis was caused by Candida albicans in Ningxia, the ratio of fungal infection was 43%. Candida albicans had strong virulence and were resistant to common antibiotic and sensitivity to antifungal drugs and traditional Chinese medicine.

Antimicrobial agents play a crucial role in the prevention and treatment of bacterial diseases. However, the widely clinical use of antibiotics makes the bacteria antibiotic-resistance problem much more severe, which could increase the difficulty of clinical treatment. Recently, the method of using traditional Chinese herbs to control bacterial infection by suppressing the bacterial virulence factors has got researchers'attention around the world. Hemolysin is one of the crucial virulence factors in many kinds of bacteria, therefore hemolysin is an ideal target to combat the infection caused by bacteria. The suppressing effect of Chinese herbs to the hemolysin is synthesized and reviewed here to lay theoretical foundation for the clinical treatment of bacterial diseases.

Antibiotic resistance genes have become a recognized environmental pollutant, threatening the ecological safety and human health. The application of antibiotics in the clinical and animals breeding, making the environmental microorganisms living with the impact of the residue of antibiotics and elements of resistance genetic and leading to antibiotic resistant bacteria to gain a competitive advantage and destroyed the stability of ecosystem. In this paper, we expounded the concept of resistance gene transmission by the view of macro environment. Through analyzing the mechanism of resistance development,environmental pollution caused by drug resistance and the impact of environmental microorganism for drug resistance, we clarified the key role of the environment in the development of the characteristics of bacterial resistance and analysis environmental resistance.Such as the ecological diversity of flora, the types and the spread of resistant bacteria, the residues of antibiotics and the transmission of the resistance genes.