Residue Detection of Tetracyclines in Eggs by High Performance Liquid Chromatography

The study was aimed to establish the method of simultaneous detection of four kinds of tetracyclines,including oxytraoycline,tetracycline,chlortetracycline and doxyeycline in eggs by high performance liquid chromatography (HPLC).The eggs samples were detected with HPLC after through the procedure of extraction of 0.1 mol/L Na₂EDTA-Mcllvaine buffered solution,purification of Oasis MAX solid-phase extraction column,gradient elution of taking acetonitrile-0.01 mol/mL trifluoroacetic acid as mobile phase,separation with reverse liquid chromatography.The results showed that the calibration curves of four kinds of tetracyclines were linear correlated in the concentration range of 30 to 1 000 ng/g which the limits of detection was 10 ng/g and the limit of quantification was 50 ng/g.The average recoveries were 67.3% to 81.4%,relative standard deviations of intraday was 2.7% to 14.3%,relative standard deviations of inter-day was 0.9% to 5.0%.This mothod established in this study could simultaneously detect oxytetracycline,tetracycline,chlortetracycline and doxycycline drug residues in egg and it had high sensitivity,accuracy and precision.The method was rapid and simple to operate.     

Effect of Weaning Stress on Blood Physiology and Biochemistry Indexes and Immune Function in Dairy Buffalo Calves

To study the effect of weaning stress on dairy buffalo calves,5 healthy calves were chosen and weaned,and the blood samples were collected at 1 day before weaning,and 1,14 and 21 days after weaning to analysis blood physiology and biochemistry indexes and immune function.The results showed that the count of white blood cell,lymphocyte,erythrocyte,monocytes and hematocrit,aspartate aminotransferase activity,blood glucose,cortisol and immunoglobulin G contents of dairy buffalo calves were no significant difference between before and after weaning treatments (P > 0.05).The count of neutrophils at 21 d after weaning were significantly lower than that of 1 d before weaning of dairy buffalo calves (P < 0.05).However,lactate dehydrogenase and alanine aminotransferase activity in weaned treatments were significantly higher than those of animals in the treatment before weaning (P < 0.05).The serum urea nitrogen of dairy buffalo calves at 21 d after weaning was significantly higher than that of 1 d before weaning (P < 0.05) and hemoglobin content at 21 d after weaning was significantly lower than that at 1 day before weaning (P < 0.05).It indicated that the physiological and immunity function of dairy buffalo calves were challenged under weaning stress.     

Study on Clinical Efficacy of Oligosaccharide Sulfate and Dextran Sulfate Against Bovine Mastitis

In order to detect the clinical efficacy of oligosaccharide sulfate(DEAE) and dextran sulfate (DS-4000) against bovine mastitis,56 cows with bovine mastitis were divided into 5 groups (DEAE group,DS-4000 group,DEAE＋penicillin group,DS-4000＋penicillin group and penicillin group),and their breasts were perfusion with different drugs twice one day for 3 days.The effective rates of drugs were observed by clinic symptoms.Milk samples were collected before and after treatment of drugs,and the bacterial negative conversion rate of each group were detected by microbial examination.The results showed that total effective rates of 5 groups were 10.00%,20.00%,58.33%,75.00% and 50.00%,respectively,and the bacterial negative conversion rates were 0,12.50%,50.00%,54.55% and 44.44%,respectively.In conclusion,the curative efficiencies of the polysaccharides against bovine mastitis were weak,but it could be improved by combination with penicillin.     

Production and Identification of Clone Sheep Using Bone Marrow Mesenchymal Stem Cells as Donor Cell

In order to obtain the cloned sheep using bone marrow mesenchymal stem cells（BMSCs）as the donor cells,the BMSCs of sheep were chosen and reconstructed embryos were built to transfer.10 published microsatellite markers were chosen,and the DNA samples from clone sheep,donor cells and surrogate ewes were amplified,and the relationship of father-son（RCP）was analyzed using the Quantity One for genotyping.The results showed that the reconstructed embryos were successfully built for electric fusion using sheep BMSCs as nuclear donor,and making nuclear transplantation into enucleated mature oocytes of which the fusion rate was 80.62%.20 surrogate ewe were chosen to be implanted with the reconstructed embryos at morula stage by implant surgery,and 5 lambs were born and only 3 were survived.The genotype of cloned sheep was in line with the dornor cell and the RCP were more than 99.999%.In conclusion,the first clone sheep were obtained successfully by using BMSCs as a nuclear donor in this experiment.     

The Fluorescent Characterization and Analysis of Two Brucella Attenuated Vaccine Infecting Mouse Macrophagocyte

The experiment was conducted to discuss the difference of binding time of green fluorescent protein B.melitensis M5 (GFP-M5) and B.abortus S19 (GFP-S19) infecting the mouse macrophagocyte (RAW264.7),lysosome,endoplasmic reticulum and golgi body in the initial stage and compare the binding rate of GFP-M5,GFP-S19 with organelle in different timeline,respectively,by confocal laser scanning microscope (CLSM) and flow cytometry.The result showed that GFP-M5 and GFP-S19 were successfully constructed.The intracellular survival ability of Brucella M5,Brucella S19,GFP-M5 and GFP-S19 were not obvisouly affected after infecting RAW264.7.GFP-M5 and GFP-S19 could enter the macrophagocyte in 30 mins,and in 2 h the Brucella could reach lysosome,endoplasmic reticulum and golgi body.In addition,the binding time for two attenuated vaccine did not show differences in 1,2,3 and 4 h.The content of GFP⁺ cell produced by RAW264.7 infected by GFP-M5 and GFP-S19 did not show significant differences (P>0.05).Therefore,the two strains did not have significant differences in the invasion ability in the initial stage of infecting host cell.     

Effects of Donor Cells Treated with Egg Extracts on the Development Competence of Cloned Embryos

Japanese Balck cattle fetal fibroblasts (JBCFF) were induced with Xenopus leavis egg extracts and somatic cell nuclear transfer (SCNT) was carried out with the reprogrammed JBCFF as donor cells in order to investigate their effects on SCNT efficiency.Three samples of egg extracts were acquired from different Xenopus laevis.The protein contents and kinds in extracts were evaluated with BCA Protein Quantification Kit and SDS-PAGE.Concentration of Digitonin to permeabilize JBCFF was optimized and assessed with PI staining.Reprogrammed cells treated with egg extract were used as donor in SCNT.Additionally the reconstructed embryos were activated with ionomycin+6-DMAP and A23187+6-DMAP to compare their effects on the development competence.The protein contents of extracts samples were 56.2255,64.6570 and 71.2158 μg/mL,respectively,the each extract had the same composition about 40-55 and 70-100 ku.The optimal concentration of Digitonin was 7 μg/mL and the permeabilization rate was 55.44%.After extracts treatment and continuous culture for 6-7 d,JBCFF formed well-defined colony structures.No significant composition difference was found in rates of fusion (92.83% vs 96.04%),cleavage (89.64% vs 89.78%) and blastocyst formation (24.06% vs 23.12%) of cloned embryos when the colony cells and JBCFF without extracts treatment were used as donor cells (P>0.05).Similarly,the two activation methods had no significant effect on the developmental competence of cloned embryos (cleavage rate 92.16% vs 92.28%,blastocyst rate 23.21% vs 24.18%).Conclusively,Xenopus leavis,egg extracts could induce JBCFF reprogramming to a low differentiated state.However donor cells with reprogramming partially could not improve the development of cloned embryos and its mechanism requires further research.     

Isolation and Identification of Cellulase-producing Bacillus in Yak Rumen

To investigate the species of cellulase-producing Bacillus in yak rumen,10 samples of rumen content were aseptically collected from 10 adult Maiwa yaks to isolate the heat-resistant Bacillus by water bath at 80 ℃ for 20 min.The cellulase-producing strains were screened using the CMC-Na medium and Congo red staining.The 16S rRNA gene sequence of those cellulose-producing strains were amplified and sequenced.The results showed that 64 strains were isolated from the 10 samples.Total 23 strains were identified as cellulase-producing bacillus,including 16 strains of Bacillus cereus,7 strains of Bacillus thuringiensis.Furthermore,phylogenetic analysis showed that the 16 Bacillus cereus strains were clustered into two branches:One isolate was clustered into a branch alone,the other 15 isolates were clustered into a branch which clustered into 5 small branches,showing that there was certain genetic diversity in the isolates of Bacillus cereus.And all 7 Bacillus thuringiensis strains were clustered into a branch.Hence,the results layed the foundation of investigating the species of cellulase-producing Bacillus in yak rumen and developing probiotics special for yak.     

Effects of Supplemental Complex Water Soluble Vitamin on Production Performance,Blood Biochemical Parameters and Oxidation Resistance of Dairy Cows

The objective of the study was to investigate the effects of supplemental complex water soluble vitamins on production performance, blood biochemical parameters and oxidative resistance of dairy cows in spring and summer.The experiment was designed by 2×2 factorial randomized blocks design.Eighty healthy dairy cows with similar parity and body weight were randomly divided to four groups, with 7 days of adaptation and 70 d experimental phase.The dairy cows in group Ⅲ and Ⅳ were fed a basal diet supplemented with complex water soluble vitamins in spring.The dairy cows in group Ⅱ and Ⅳ were fed a basal diet supplemented with complex water soluble vitamins in summer.The results showed that the ADFI and daily milk yield of dairy cows receiving water soluble vitamin in spring were extremely significantly higher (P<0.01);The ADFI of dairy cows receiving water soluble vitamin was significantly higher in summer (P<0.05) and daily milk yield were extremely significantly higher (P<0.01);The butterfat rate was extremely significantly increased and SCC was extremely significantly decreased in spring and summer (P<0.01);Supplementation of water soluble vitamin was also significantly increased LYM (P<0.05).In summer, T-AOC, T-SOD were extremely significantly increased (P<0.01), and TP, CK, LDH, TP, HDL and ALB were extremely significantly decreased (P<0.01) by water soluble vitamin supplementation than that of control group.In conclusion, complex water soluble vitamins could increase the production performance and non-specific immune function, improve the oxidative resistance and relieved the heat stress of the dairy cows.     

Isolation and Identification of BPIV3 Genotype C

To identify the infection agents from Ningxia Hui Autonomous region, where feedlot cattle indicated bovine respiratory disease complex (BRDC), the M gene of the bovine parainfluenza virus type 3 was amplified by RT-PCR.The PCR product was ligated to pMD18-T vector and cloned to E.coli DH5α.The positive clones were sequenced and compared with the reference strains in GenBank by the molecular biology software.Sequence alignment results showed that a BPIV3 strain was isolated from the samples and named NX49, the M gene of NX49 included 1 056 nucleotides.Evolutionary analysis showed that the NX49 belonged to BPIV3 C genotype and shared 99.4% nucleotide identity with that of the SD0835 isolated in Shandong province.The characterization of the NX49 demonstrated that it was sensitive to temperature, acid and organic matter.The presence of Mg²⁺ showed no protection against the treatment at high temperature.The HA test suggested that the NX49 enables to agglutinate the guinea pig RBC at 4 ℃ and the titer was 1∶4.The study isolated a BPIV3 genotype C strain successfully, which facilitate the study of molecular evolution and epidemiology of BPIV3 in China.