Mutation in myostatin (MSTN) gene resulted in double muscle effect,generating more mutton.To knock out MSTN gene in sheep fetal fibroblast by CRISPR/Cas9 system and obtain MSTN gene knockout cell lines,four plasmids were designed and constructed to target MSTN gene,and confirmed correctly by sequencing.The correct plasmids were delivered into the fetal fibroblast cells.The targeting efficiency was detected using SURVEYOR assay Kit.The stable transfected cell colonies were obtained via limiting dilution procedure.The sequence results demonstrated that the pX330-target 1 and pX330-target 4 plasmids could successfully knockout MSTN gene,and the targeting efficiency were 24.20% and 10.18%,respectively.Twelve MSTN gene knockout cell colonies were obtained via limiting dilution,and one of them was homozygous mutation.Several indel mutations were discovered at specific site,and some of them were frame-shift mutation.Therefore,we concluded that the CRISPR/Cas9 system could apply to the gene editing of sheep efficiently,and the gene knockout cell clones had potential application in generating MSTN gene knockout sheep.

The assay was aimed to study the effect of 18β-glycyrrhetinic acid derivatives containing pyridine carboxamide 6c,10c and 11c on the proliferation of HeLa,SMMC-7721 and SGC-7901 cells.Detected the cellular inhibition rate of 18β-glycyrrhetinic acid derivatives containing pyridine carboxamide 6c,10c and 11c to HeLa,SMMC-7721 and SGC-7901 cells by MTT method;Detected the conditions of apoptosis by Hoechst-33258 Staining Kit;Detected the apoptosis rate by Annexin V-FITC/PI method;The related protein expression levels were detected by Western blotting;To further clarify the situation of apoptosis induced in treatment group by detecting the activity of Caspases-3.18β-glycyrrhetinic acid derivatives containing pyridine carboxamide 6c,10c and 11c had the inhibition and promoting apoptosis effects on the proliferation of HeLa,SMMC-7721 and SGC-7901 cells,and had the concentration dependence.The results all showed statistical significance in t test (P<0.05).18β-glycyrrhetinic acid derivatives containing pyridine carboxamide 6c,10c and 11c could induce the apoptosis of HeLa,SMMC-7721 and SGC-7901 cells.

In this study,the 5'flanking sequence of FSHR gene was cloned,and bioinformatics analysis and transcriptional activity detection were studied.One pair of primers was designed among the homologous of 5'flanking sequence of FSHR gene in different species,5'flanking sequence of FSHR gene was cloned by PCR and constructed into the EGFP reporter vector,the transcriptional activity of recombinant expression vector pFSHR-EGFP-1 was analyzed by transfecting into HEK-293T and CHO cell lines.The results showed that 2979 bp of 5'flanking sequence of FSHR gene and partial CDS region of Guangxi local swamp buffalo was successfully cloned and sequenced.The homology comparison result showed that buffalo FSHR gene shared 100%,99%,93%,92%,91% and 75% homologies of nucleotide sequence with that of the river type buffalo,cattle,sheep,goat,pig and human,respectively.Promoter and transcription factor binding site predictions showed that there were a TATA box in the -147 bp area of the upstream of translation initiation site,and trans acting element binding sites for GATAs,FOXO1,FOXO3,Nobox,STAT1,STAT3,STAT4,STAT5a,STAT5b,STAT6 and YY1.There were multiple binding sites for GATAs family genes in the FSHR gene promoter region,and in the same site of FSHR promoter,it displayed multiple GATAs bound.The FSHR gene promoter could initiate EGFP expression in HEK-293T cell lines,however very weakly,the expression of EGFP promoted by FSHR promoter was similar with by CMV promoter in CHO cells lines,and suggested that buffalo FSHR was a strong promoter.In conclusion,this study had successfully cloned FSHR gene promoter,analyzed its promoter sequence characteristics and successfully verified the tissue specific transcriptional activity.It laid the foundation for clarifying the transcriptional regulatory mechanism of buffalo FSHR gene and produced transgenic buffalo base on ovary specific expression of exogenous gene.

In order to analyze the immunogenicity of 120 ku hemagglutinin-related outer membrane protein from F.necrophorum,specific primers were designed to amplify the 3 truncated overlapping gene fragments covering the whole open reading frame (ORF) based on the result of antigenic epitope analysis.The gene fragments were ligated into the prokaryotic expression vector to construct pET-28a-p1,pET-32a-p2 and pET-32a-p3,respectively.Then recombinant plasmid was induced expression in E.coli.The SDS-PAGE results showed that the expression of recombinant proteins P1,P2 and P3,molecular mass of the expressed proteins were about 48,59 and 62 ku,respectively.Western blotting indicated that the recombinant proteins could be recognized by antiserum against F.necrophorum from mouse.Humoral immunity response against recombinant proteins was detected in the immunized rabbits.Altogether,these findings suggested that 120 ku hemagglutinin-related outer membrane protein from F.necrophorum had good immunogenicity,and this study provided a reference for further research on genetic engineering subunit vaccine of F.necrophorum.

The 128 bp specific and consensus sequence of Eastern equine encephalitis virus (EEEV) was amplified by RT-PCR and cloned into pMD20-T vector,and conducted in vitro transcription to prepare standard cRNA.10 fold serial diluted cRNA were used as standard templates for RT-PCR to quantify the genomic copy number of EEEV.We developed a TaqMan MGB Real-time RT-PCR to detect EEEV.Sensitivity assay result showed that the established TaqMan MGB Real-time RT-PCR could detect 10 copies/μL cRNA.The specificity assay exhibited that negative control and the other equine pathogens (EHV-1,EAV,EIV H3N8,WNV,WEEV and JEV) could not be detected.A good linear correlation was demonstrated in the standard curve for TaqMan MGB Real-time RT-PCR within the range of 1.0×10¹ to 1.0×10⁶ copies/μL with a correlation coefficient of 0.998 and a standard curve of y=40-3.35logx.The results demonstrated that TaqMan MGB Real-time RT-PCR was 100 fold more sensitive than conventional RT-PCR.The results suggested that the convenient,specific,high performance and sensitive method of TaqMan MGB Real-time RT-PCR for the detection of EEEV was successfully established in this study.

To understand the pathogenicity of Escherichia coli O157:H7,the secondary structures,hydrophilicity plot,antigenic index,flexibility plot and surface probability plot of the flagellin FliC of Escherichia coli O157:H7 were analyzed by bioinformatics.The α-helix,β-sheet,turn and coil regions of flagellin FliC were analyzed by the methods of Garnier-Robson and Chou-Fasman of DNAStar software,hydrophilicity plot was used the method of Kyte-Doolittle,the flexibility plot was based on the Karplus-Schulz method,surface probability plot and antigenic index were analyzed by the methods of Emini and Jameson-Wolf,respectively.The results showed that 63 to 74,236 to 247,338 to 349,460 to 471 and 542 to 553 amino acid sites of flagellin FliC might be B cell antigen epitopes.338 to 349 and 460 to 471 peptides were synthesized by chemical synthesis,and then subcutaneously immunized BALB/c mice three times.Antibody response was evaluated by ELISA.The result showed that 338 to 349 and 460 to 471 peptides could induce BALB/c mice to produce high titer antibody.

In order to express the F protein of duck Newcastle disease virus (NDV) in insect cells,one pair of specific primers was designed according to the published genome sequences of duck NDV to amplify F gene by PCR,the amplified fragment was cloned into baculovirus expression vector pFastBac1.Then the recombinant vector pFastBac-F was transformed into E.coli DH10Bac competent cells,and the positive recombinant bacmid rBacmid-F was screened according to the resistance and blue-white plague screening.rBacmid-F was transfected into Sf9 insect cells by liposome,once the cytopathic effect was found,the rBac-F could be aquired.The results of Western blotting and indirect immunofluorescence test showed that the recombinant proteins could be recognized by positive serum,indicating that the protein had good reactiongenicity.These results suggested that F protein of duck NDV had been successfully expressed in insect cells,which laid the foundation for the prevention and control of duck Newcastle disease.

Adipose tissue had two different types:White adipose tissue (WAT) that stored energy and brown adipose tissue (BAT) which could consume energy by specific expression of uncoupling protein 1(UCP1).In contrast with BAT,WAT had strong plasticity and possessed capacity of conversing phenotype,which was called thermogenesis (browning) under cold exposure.According to the results of RNA transcriptome sequencing of interscapular brown adipose tissue (iBAT) and subcutaneous white adipose tissue (sWAT) in the cold exposure group and control group,we preliminary screened one differentially expressed lncRNA (lncRNA-6030408B16Rik).The lncRNA-6030408B16Rik expression pattern was detected by qRT-PCR in spleen,muscle,kidney,duodenum,brain,liver,sWAT and iBAT from the wild type mice.Twenty-four 8-week-old C57BL/6 wild type mice were randomly divided into two groups respectively under cold exposure 6℃ and 28℃ as control,followed by qRT-PCR detection of lncRNA-6030408B16Rik expression in sWAT and iBAT.Separating preadipocytes from 1-day-old wild type mice,then inducing differentiation,and collecting cells on 0,1,3 and 5 days,expression of UCP1 and lncRNA-6030408B16Rik were detected by qRT-PCR.The results showed that the expression of lncRNA-6030408B16Rik were greatly enriched both in sWAT and iBAT compared with other six tissues.The expression of lncRNA-6030408B16Rik were significantly or extremely significantly up-regulated both in sWAT and iBAT of mice under cold exposure compared to control group (P<0.05;P<0.01).In contrast with UCP1,lncRNA-6030408B16Rik expression level presented an up-down trend at 0,1,3 and 5 days of brown preadipocytes differentiation.lncRNA-6030408B16Rik showed tissue-specific expression both in sWAT and iBAT,these results suggested that it might play important roles in WAT "browning" and brown preadipocytes differentiation.

The experience was conducted to screen the genes associated with hair follicle development from the expressed sequence tags (ESTs) derived from the skin tissues of Xinji fine wool sheep and explore the association between their expression patterns and the fineness of wool.Using Trinity and Blat,we screened out the candidate genes by assembly and homologous annotation of ESTs,and further studied their expression patterns through detecting the correlation value 2^-△△Ct by Real-time PCR method.As a result,four genes (KRT27,KAP3.2,KAP11.1 and KAP8.1) were carried out to be responsible for hair follicle development,and 4 keratin genes were extremely significant more highly expressed in skin tissues of super fine wool sheep than in fine wool sheep (P<0.01);The expression level of 4 keratin were as follow:KRT27 >KAP3.2 >KAP11.1 >KAP8.1.The study provided certain data for the research on transcript level of fine wool sheep keratin gene.

The present study was designed to construct recombinant plasmids,which could express porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 gene.RNA was extracted from spleen and lung samples of the suspected pigs which were infected with PRRSV.According to PRRSV ORF5 gene,a pair of primers was designed for RT-PCR amplification.The ORF5 target gene was cloned into pMD19-T vector and then the recombinant pMD19-ORF5 was achieved.According to the sequencing results and the characteristics of expression vectors,a pair of primers with NcoⅠand XbaⅠenzyme cleavage sites was designed.Target fragment dORF5 was amplified and then connected to pProEXHTb and pNZ8149 vectors,respectively.And recombinant HTb-dORF5/DE3 and pNZ8149-dORF5/NZ3900 was induced by IPTG and Nisin,respectively,and analyzed by SDS-PAGE and Western blotting.Recombinant HTb-dORF5/DE3 induced by 1.5 mmol/L IPTG was expressed in the highest quantity.There were specific band at about 22 ku with reactionogenicity when it was tested by SDS-PAGE and Western blotting.Recombinant pNZ8149-dORF5/NZ3900 induced by 20 ng/mL Nisin was expressed in the highest quantity.There were specific band at about 19 ku with reactionogenicity when it was tested by SDS-PAGE and Western blotting.The IFA result showed specific green fluorescence.This study successfully constructed recombinant plasmids HTb-dORF5 and pNZ8149-dORF5 and expressed,the result laid a solid foundation for further development of PRRS vaccines.

To prepare the mono-specific serum to diagnose H9N2 avian influenza virus (AIV),this test extracted H9N2 subtype AIV RNA and then amplified the upper HA1 gene,the middle HA2 gene and the lower HA3 gene by RT-PCR,respectively.Then they were inserted into expression vector pET-32a(+) and transformed into BL21(Rosetta) expression strain.The expressed proteins were used to immune Kunming White mice to prepare antiserum.Recombinant fusion proteins of HA1 HA2 and HA3 were obtained successfully and they showed good immunogenicity.Indirect immunofluorescence assay (IFA) showed that the two serums obtained by the upper HA1 and the middle HA2 could react with the H9N2 subtype AIV,while that of the lower HA3 could not.Recombinant Marek's disease virus (MDV) MZC12 HA/NA also proved that the serums prepared by HA1 and HA2 could recognize the expression of HA gene.The mono-specific serum of H9N2 subtype AIV was prepared successfully,which could lay the foundation for the diagnosis and research of H9N2 subtype AIV.

It was aimed to lay a foundation for epidemiological survey of porcine circovirus type 2(PCV2) in Liaoning province and selection of highly homologous strain inactivated vaccine.Some pigs suspected of having postweaning multisystemic wasting syndrome (PMWS) from Shenyang,Chaoyang,Tieling,Dalian and other places in Liaoning province,were detected by PCR method,then the whole genome of 10 positive samples were cloned and sequenced.The result showed that all of them were virulent strains,9 strains were 1767 bp and 1 strain was 1768 bp,and there was a T base additon at 1039 bp.The nucleotide homology was 95.1% to 98.5% with HM038034 and JQ692110.In the phylogenetic tree,JHZ2 was PCV2a type,the other 9 strains were PCV2b type,DLS38,LYD32 and SYX7 were in the same branch and same onset time.The amino acid homology of ORF2 was 89.6% to 100.0%,there were 35 mutation points,large degree of variation,the only glycosylation site (NYS) was conserved.10 strains did not change significantly,mutants were not due to PCVD,PCV2b was the predominant type,and PCV2 was related with seasonality.

In this study,the dung samples of a sick cattle from an animal hospital were detected by bacteria cultivation.The bacteria were cultured by different differential media,and analyzed by biochemical test,16S rDNA gene was amplified by PCR,then sequenced and constructed phylogenetic tree,drug sensitivity test and growth curve were carried out.The results showed that the bacteria could grow smooth bulge,rounded edges,different size,different colors of the bacterial colonies in blood agar,chocolate agar,LB agar and so on,but could not grow in MRS agar and high salt mannitol agar.The results of lactose,maltose,mannitol and so on were negative,the results of glucose,simmon's citrate and urea were positive,this bacteria were identified as Streptococcus bovis by these preliminary results,and confirmed by phylogenetic tree construction.This isolate exhibited 99% nucleotide sequence identity with Streptococcus bovis RD09.The drug sensitivity test result of Streptococcus bovis showed that it was extremely sensitive to doxycycline and kanamycin,highly sensitive to erythromycin,sensitive to streptomycin,novobiocin,cotrimoxazole,ceftriaxone and gentamycin,resistant to chloramphenicol,cephalosporins cefradine and amoxicillin.The growth curve of Streptococcus bovis indicated that a rapid growth phase of the bacteria was from 2 to 23 h,the stationary phase was from 23 to 28 h,the decline phase was after 28 h.These experimental data provided the prerequisite for further study of Streptococcus bovis.

Transcriptome sequencing technology (RNA-seq) is to inform all RNA sequences on a specific developmental stage or functional state in a particular tissue or cell through the high throughput sequencing method.The technology is widely used in biology,medicine,agriculture and other basic research,mainly because of accurate and high-throughput,as a powerful research tool.During the different stages of parasites,including development,infection and propagation,a series of complex biological changes occur.The differential expression genes in different stages of parasite development,function about the genes and the interactions in host-parasite are the key focus of current research.On the basis of the above,the summary of the application of RNA-seq technology in parasite research is summarized.

This study was aimed to detect FUT1 gene,analyze its structure,and reveal the distribution of SNP in Wuzhishan pig.FUT1 gene was amplified by PCR.Then the gene mutation was identified by sequencing.PCR-RFLP method was used to detect the FUT1 gene of 120 Wuzhishan pigs.Some characters of FUT1 gene were analyzed by bioinformatics tools.Two mutations were detected in the FUT1 genomic fragment,one in the coding region (A+307G) and the other in the 3'regulatory region (A+1856C).At the A+307G locus of coding region in FUT1 gene,100% of Wuzhishan pigs were GG genotype.In addition,bioinformatics analysis revealed that FUT1 gene promoter and the second exon region contained CpG islands.In this study,we obtained the sequences of FUT1 gene which laid a foundation for further studying its functions in Wuzhishan pig.

microRNAs is a class of small non-coding RNA,whose size is 20 to 22 nucleotides and widely exist in eukaryotic cells.It makes the target gene degradation or translation blocked by completely or incompletely complementary with the 3'UTR of target mRNA.microRNA involve in the expression of many genes,refer to cell differentiation,development,apoptosis,viral infection,immunity,metabolism,tumor and so on many processes of life.The relationship between microRNA and virus is complicated,microRNA encoded by host cells can control the gene itself or virus so as to affect the virus infection,microRNA encoded by virus also can control the gene itself or host cells so as to affect the virus infection,the virus can also influence the formation and fuction of host microRNA.To clarify the relationship between microRNA and virus will help researchers to explore new ideas,to better carry out the related research work,to provide theoretical support for scientific researches.Therefore,the author reviewed microRNA discovery and naming,formation,function mechanism and the current new view of the interrelation between microRNA and virus.

In the cell,proteomics is a discipline that explores the interactions and change rules between proteins.It is widely used in various fields of bioscience in recent years.Proteomics provides a set of tool on the whole and dynamic level.The establishment of large-scale proteomics technology greatly promote the research progress of proteomics.These techniques are not only used for protein extraction and separation,but also can undertake the identification and analysis of target protein.This paper briefly described the results about the research of proteomics technology in disease,skin,hair follicle growth cycle,etc,in order to provide some references for further research of wool-sheep and cashmere-goat.

This experiment was conducted to study the effect of dietary supplementation of maggot dry on meat performance,nutritional quality and flavor compounds of meat in Qingjiao Ma chickens.A total of 1200 Qingjiao Ma cocks at 35 days of age were randomly allocated to 2 groups with 4 replicates per group and 150 chickens per replicate.The chickens in control group were only fed the basic diet (without maggot dry),and those in the experiment group were fed basal diet supplemented with 3% maggot dry.The chickens in two groups were raised in the same conditions.At 70 days of age,eight cocks were randomly selected and slaughtered in each group.The results showed as follows:①The meat performance in control group and experiment group were all good,and had no significant difference (P>0.05).② Compared with control group,the crude protein contents of chest and leg muscle in experiment group were increased by 8.8% and 4.4% (P<0.05),respectively,and the amino acids (especially essential amino acid) contents were increased by 11.0% and 2.3% (P<0.05),respectively;The relative contents of nutmeg acid and pentadecane acid in chest and leg muscle of experiment group were significantly higher than those of control group (P<0.05).③ Compared with control group,the delicious amino acid content in chest and leg muscle were increased by 11.9% and 3.5%,respectively;the inosine acid content in chest muscle of experiment group was increased by 21.1%,The four kinds of special volatile flavor substances (dipentene,methoxyacetone,3-nonanone and isobutyl lactate) of meat in experiment group were obtained,and the flavour components of aldehyde and mushroom alcohol were higher than those of control group.Therefore,dietary supplementation of 3% maggot dry could increase the contents of crude protein,amino acids,delicious substance and volatile flavour compounds,and helped to produce chickens with flavor and nutritious meats.

In order to explore different concentrations of mixed acids in improving quality of whole-plant corn silage and their effects on growth performance of Dorper×Han F1 sheep.4 levels of 45% formic acid,45% propionic acid and 10% sodium propionate were mixed,and 0(control),0.3%,0.6% and 1% were added to 4 groups of ripening period whole-plant corn.The results showed that compared with control group,the experimental groups could significantly decrease the proportion of AA/PA in silage (P<0.05);0.3% and 0.6% groups had the better effects.Based on the result of CNCPS,0.6% group significantly improved the PB₃ component (P<0.05),reduced the CC component (P>0.05),and improved the quality of silage.Compared with control group,the experimental groups effectively inhibited the mold and yeast reproduction,improved the aerobic stability of silage.The feeding trial results showed that ADG and F/G of sheep in experimental groups were better than control group,and F/G of both 0.6% and 1% groups were significantly better than that in control group (P<0.05).In conclusion,treatment of mixed acids could improve the quality of whole-plant corn silage,and silage treated with 0.6% levels of mixed acids could significantly improve the growth performance of Dorper×Han F1 sheep.

In this research,12 heads of 28 to 30 months (low months age group) and 8 heads of 32 to 34 months (high months age group) Xinjiang Brown cattle steers which fed in the same mode of nutrition were chose for the slaughter test,in which the carcass traits,nutrient composition and some edible quality index were measured and comparative analyzed,a research which through comparative analysis the carcass traits and meat quality of nutritional components of different months of Xinjiang Brown cattle steers was carried out.The results indicated that the marbling,carcass grade,fat,meat color of high months age group were better than that of low months age group,the traits of the low months age group on fat color,moisture,protein,cooking loss and shear force value were better than that of high months age group,and there were significant difference on the traits of fat and meat color L^* between the two groups (P<0.05),while the differences of other traits between them were not significant (P>0.05);As high-grade beef,in addition to the traits of meat color and pH value of the chunk roll,tenderloin and striploin,each site of the meat traits on the fat,protein,moisture,cooking loss,shear force value indicators were all better than other parts,and showed the best of nutritional value and edible quality.28 to 34 months of Xinjiang Brown cattle steers could produce high-grade beef,the meat could be used as the raw materials of steak and boiled beef.

Six adult Small-tail Han rams at the age of 1 to 1.5 years,with the average body weight of 55.3 kg±3.8 kg,fitted with the permanent duodenum and ileum T-type canula,were fed with concentrate-type diet(cornstalk as 30%),collecting the chyme of the duodenum and ileum to study the effect of supplement of aerosol OT(0.8 g/kg of diet (as DM basis))on the digestion and absorption in the former stomach and small intestine by self-control experimental design.The results showed that by supplementing of aerosol OT the voluntary intake of dry matter was increased by 23.6%(P<0.01),the digested DM,OM,CP and cellulose in former stomach were increased by 39.2%(P<0.01),37.3%(P<0.01),21.8%(P<0.01) and 13.4%(P<0.01) respectively;DM,OM,CP,microbial protein and by-pass protein arrived at small intestine were increased by 8.9%(P<0.01),8.8%(P<0.01),21.3%(P<0.01),29.3%(P<0.01) and 13.1%(P<0.01),respectively;The total amino acids,lysine,methionine,threonine,valine and phenylalanine arrived at small intestine were increased by 45.6%(P<0.01),62.7%(P<0.01),40.8%(P<0.01),32.8%(P<0.01),32.0%(P<0.01) and 32.3%(P<0.01),respectively.DM,OM,CP,calcium and phosphorous absorbed in small intestine were increased by 28.7%(P<0.01),29.8%(P<0.01),28.7%(P<0.01),26.6%(P<0.01) and 13.1%(P<0.01),respectively;The total amino acids,lysine,methionine,isoleucine,arginine and histidine absorbed in small intestine were increased by 37.6%(P<0.01),56.9%(P<0.01),28.6%(P<0.01),40.9%(P<0.01),41.5%(P<0.01) and 51.7%(P<0.01),respectively.It was concluded that the supplement of aerosol OT could increase the voluntary intake and the digestion and absorption in forestomach and small intestine,therefore significantly modify the nutrient supply for sheep.

This study was performed to evaluate the effects of alfalfa flavonoids on rumen fermentation function and cellulolytic enzymatic activity of crossbreed sheep.A single factor random block design was used,and twelve crossbreed sheep (F1) Suffolk×Small-tail Han sheep with permanent rumen fistulas and the body weight of 27.02 kg±3.03 kg were selected and randomly divided into 4 groups with 3 replicates per group and 1 single sheep per replicate.The control group was fed a basal diet without alfalfa flavonoids,and the experimental groups were fed the basal diets supplemented with 0.1%,0.2% and 0.4% alfalfa flavonoids,respectively.The pre-test period lasted for 7 days and the trial period lasted for 41 days.The results showed as follows:①Compared with control group,the pH of 0.2% group was significantly increased at 12 h after adding alfalfa flavonoids (P<0.05),and no significant differences were observed in other groups (P>0.05);There were no significant differences in NH₃-H concentration among all groups (P>0.05),and the concentration was increasing with the dose rising (P>0.05).②Compared with control group,the average concentrations of acetate,propionate,butyrate,valerate and TVFA in 0.1% and 0.4% groups were increased,whereas the concentrations in 0.2% group showed contrary pattern.The isovalerate concentration in 0.1% and 0.2% groups were significantly higher than that in 0.4% group at 2 h after adding alfalfa flavonoids (P<0.05).③Compared with control group,the activities of xylanase,endoglucanase,cellobiase and cellulase in 0.1% and 0.2% groups were decreased,but those in 0.4% group were increased and the difference was not significant (P>0.05).In conclusion,dietary supplementation of alfalfa flavonoids could keep the stability of rumen fermentation function of crossbreed sheep and this compound in different dose had different effect on cellulolytic enzymatic activity.The recommended supplemental level of alfalfa flavonoids was 0.4% in diet for crossbreed sheep under this test condition.

This study was conducted to investigate the effect of partial replacement of corn by Dioscorea alata L.on microflora and short-chain fatty acid content in cecum of Wenchang chicken.A total of 288 healthy Wenchang chickens at 50 days of age with the similar body weight were divided into 4 groups with 4 replicates per group and 18 chickens per replicate.Chickens in the control group were fed a basal diet (corn-soybean diet),and the others in the experimental groups (group Ⅰ,Ⅱ and Ⅲ) were fed three isonitrogenous and isoenergetic experimental diets which using Dioscorea alata L.replaced 10%,15%,20% corn,respectively.The trial lasted for 28 days.The results showed that after 14 days of feeding trials,replacement of different proportion of corn by Dioscorea alata L.could not significantly affect the microflora in cecum (P>0.05),but could significantly affect the short-chain fatty acid content (P<0.05),the contents of propanoic acid and butyric acid in group Ⅲ were significantly lower than that in control group (P<0.05).After 28 days of feeding trials,replacement of different proportion of corn by Dioscorea alata L. could significantly affect the microflora and the short-chain fatty acid content in cecum (P<0.05),the number of Escherichia coli in group Ⅱ was significantly lower than that in control group (P<0.05),and the number of Streptococcus faecalis and total anaerobic bacteria in group Ⅲ were significantly and extremely significantly higher than that in control group (P<0.05;P<0.01),respectively;The content of butyric acid and propanoic acid in group Ⅰ were significantly and extremely significantly lower than that in control group (P<0.05;P<0.01),respectively.In conclusion,partial replacement of corn by Dioscorea alata L.could promote the number of beneficial bacteria in cecum,but suppress the short-chain fatty acid content in cecum of Wenchang chicken in a relatively short time,however,no significant effect showed in the short-chain fatty acid content after 28 days of feeding.The suitable replacement proportion was 15% under this experiment conditions.

In order to determine the appropriate level of the crush process of alfalfa hay in field,this study used alfalfa as research material with the process of crushing stalk in varying degrees,and analyzed the rule of the moisture loss,and the characteristic in drying process and nutrients content in the modulation process,and used the in vitro digestion experiments to validate the result.The results showed that the content of moisture demonstrated the tendency which was decreased fast at first and then slow down,the speed of the process of crushing was significantly faster than unflattened treatment during alfalfa drying process (P<0.05),the degree of crushing was greater,the speed of drying was faster.The treatment of crushed made the stem and leaf went synchronous during the process of drying,and the rate of leaf reservation was higher.Moderate flattening alfalfa was drying faster,and the better preservation of nutrients,and it had higher DM,CP in vitro digestion rate,it was concluded that the moderate crushed was the appropriate degree of flattening alfalfa hay in feild.

This paper was conducted to investigate the effects of a diet supplemented various Bacillus complex preparation on the hematological indice,blood biochemical indice and immunologic function in weaned piglets.The control group was fed the basal diet.The experiment groups Ⅰ,Ⅱ,Ⅲ and Ⅳ were supplemented Bacillus coagulans+Bacillus megaterium,Clostridium butyricun+Bacillus megaterium,Bacillus coagulans+Clostridium butyricun,Bacillus coagulans+Clostridium butyricun+Bacillus megaterium complex preparation,respectively.The supplemented content was 0.2%,and the experiment last for 43 days.All of the groups had no significant differences on the red blood cell count,white blood cell count,blood platelet count and hemoglobin concentration (P>0.05).All of the groups had no significant differences on the serum levels of alanine aminotransferase,total protein,albumin,urea nitrogen,creatinine and glucose (P>0.05).The experiment groups Ⅰ,Ⅱ and control group had no significant differences on the serum levels of aspartate aminotransferase (P>0.05),which was significantly increased in experiment groups Ⅲ and Ⅳ (P<0.05).The experiment groups Ⅰ,Ⅱ,Ⅲ and control group had no significant differences on the serum levels of total cholesterol and triglyceride (P>0.05),which were significantly increased in experiment group Ⅳ (P<0.05).The experiment group Ⅰ and control group had no significant differences on the serum levels of IgG and IgM (P>0.05),which were significantly increased in experiment groups Ⅱ,Ⅲ and Ⅳ (P<0.05).The investigate results in this paper provided some theory evidence and reference value for the application of Bacillus in the aquaculture field.

The objective of this study was to investigate the effect of cowherb seed extract on serum biochemical indexes,lactation related hormones and immune function of mid-lactating dairy cows.32 Chinese Holstein cows in parity 2 to 4 with similar weight and milk yield were selected and randomly divided into four groups and eight cows per group.Dairy cows were fed the diets supplemented with 0(control group),5.7 g/(head·d) (low dosage group),19 g/(head·d)(medium dosage group) and 38 g/(head·d) (high dosage group) cowherb seed extract.The pre-test lasted for 7 days,and the trial period lasted for 35 days.The results showed that compared with control group,the serum TP and ALB contents of each dosage group had no obvious change (P>0.05).The serum GLU content of each dosage group had a rising trend on the 30th day,while the serum TG and BUN contents of each dosage group had a decreasing trend,but the differences were not significant (P>0.05).Compared with control group,the serum PRL and GH contents of each dosage group increased at a certain degree,the medium and high dosage groups were significantly increased on the 30th day (P>0.05).Compared with control group,the serum IgA and IgG contents of each dosage group had a rising trend,while the serum IgM content of each dosage group had a decreasing trend,but the differences were not significant (P>0.05).In conclusion,dietary supplementation of cowherb seed extract could improve the energy metabolism and protein utilization,promote the lipid metabolism and increase the milk production of dairy cows.And the optimum supplemental level of cowherb seed extract in the diet of mid-lactating dairy cows was 19 to 38 g/(head·d).

This study was aimed to investigate the effect of the Chinese herbal medicine on immune function and serum biochemical indexes of Rex rabbits.12030-day-old healthy Rex rabbits (half male and half female) with similar body weight were randomly divided into 6 groups with 4 replicates in each group and 5 rabbits in each replicate.The diets of these groups were basal control diet (control group),positive control diet containing 4 mg/kg diclazuril (group Ⅰ),and experimental diets containing 0.5% (group Ⅱ),1.0% (group Ⅲ),1.5% (group Ⅳ) and 2.0% (group Ⅴ) Chinese herbal medicine,respectively.The pre-test lasted for 7 days,and the trial period lasted for 50 days.The results showed as follows:①Compared with control group,dietary supplementation of Chinese herbal medicine increased the thymus index,spleen index and serum IgG content of Rex rabbits,but had no significant effect on spleen index (P>0.05).②Compared with control group,dietary supplementation of Chinese herbal medicine increased the serum TP,ALB,GH,IGF-Ⅰ and T4 contents of Rex rabbit,and that in 1.5% group were significantly increased by 4.40%,7.46%,12.76%,23.53% and 6.24% (P<0.05),respectively,but had no significant effect on serum GLB and T3 contents (P>0.05).In conclusion,Chinese herbal medicine had obvious regulation effect on serum immune to Rex rabbits,improving the development of immune organs and immune function of animal body.And the recommended supplemental level of Chinese herbal medicine was 1.5% in diet for Rex rabbits.

The experiment was aimed to establish a method for determining the content of praziquantel in praziquantel injection and investigate its stability.HPLC method was used to determine the content of praziquantel.HPLC analysis was carried out using a Diamonsil C18 column (150 mm×4.6 mm,5μm) with ultraviolet detector at a wavelength of 210 nm.The column temperature was 25℃.The mobile phase,a mixture of acetonitrile and water (60:40) with the flow rate of 1.0 mL/min.The injection volume was 20μL.Stability of praziquantel injection was studied by stress test,accelerated stability test and long-term stability test.Excellent line relationship was obtained in the range of 6.037 to 90.555μg/mL (R²=0.9993).The average recovery was 99.24% and RSD was 0.77%.The average content of praziquantel injection (percent of the labeled amount) was 100.3%.Praziquantel injection was stable to temperature (60℃) and unstable to light (4500 lx±500 lx) during 10 days in stress test.No obvious changes of the appearance and content of praziquantel injection were observed during 6 months in accelerated test and 24 months in long-term stability test.The results suggested that the established method was simple,sensitive and accurate with good repeatability,which could be used for quality control of praziquantel injection.It could be forecasted that praziquantel injection was stable and its period of validity was two years stored under room temperature (25℃) and away from light.

In the present study,SFRS18 gene CDS region of Tibetan goat was cloned and analyzed by bioinformatics method,further its expression regulation in organs or tissues of goat were investigated and the correlation between SFRS18 mRNA expression and intramuscular fat (IMF) content in muscles was analyzed.Thereby providing data for further investigation on the gene functions in goat IMF deposition.The RT-PCR technique was employed to amplify the SFRS18 gene of Tibetan goat.Physicochemical property,structure and homology of SFRS18 encoded protein were analyzed by bioinformatic methods,and the tissue expression specificity as well as the development stage expression specificity of this gene were analyzed using the Real-time fluorescence quantitative PCR.The results showed that 1299 bp for SFRS18 cDNA,1272 bp for ORF and 423 amino acids for protein encoded.The formula of SFRS18 protein was C₂₀₀₃H₃₄₂₄N₇₆₀O₆₉₆S₄,and the molecular weight of SFRS18 protein was 49.42 ku,and its theoretical isoelectric point was 11.20.SFRS18 protein was unstable and hydrophilic,without signal peptide.There were 103 phosphorylation sites,2 N-glycosylation sites and 39 O-glycosylation sites within the SFRS18 protein.This protein respectively located in nuclear (82.6%),cytoplasmic (8.7%),cytoskeletal (4.3%) and plasma membrane (4.3%) through the sub-cellular level prediction.It was an untransmembrance protein.Moreover,the secondary structure of the SPRS18 protein was composed of 99.29% random coil and 0.71% α-helix,indicating an unconventional protein.The nucleotide sequence and the deduced amino acids sequence of Tibetan goat SFRS18 shared 99% homology with the SFRS18 mRNA of Bos taurus,Ovis aries and Bubalus bubalis.Accordingly,the close genetic relationship between Tibetan goat and Bos taurus was indicated by the phylogenetic tree analysis.The results of Real-time PCR exhibited that the SFRS18 gene was expressed in various tissues at different levels.The mRNA expression level of the SFRS18 gene was the highest in spleen and the lowest in longissimus dorsi.The results of correlation analysis showed that the mRNA expression level of the SFRS18 gene in longissimus doris and in crureus was correlated positively with IMF content (r=0.081,P<0.05;r=0.373,P<0.05).The SFRS18 gene was involved in the regulation of IMF deposition in goat.

In order to disclose the genetic variation of PRRSV,65 materials from some areas were identified during the period from 2013 to 2014.Viruses were isolated from the suspected PRRS samples.Four isolates were isolated and identified by RT-PCR and IFA.Partial Nsp2 amino acid sequences of the isolates were obtained by sequencing,and comparison analysis showed that all of the isolates were variant strains.Then we found a discontinuous deletion of 30 amino acids in Nsp2.It shared the same feature with JXA1 and TJ.Besides,we sequenced and analyzed the GP5 amino acids of 23 currently epidemic strains with some typical strains published online earlier,the results showed that all strains were American strain,21 of which had high homology with highly pathogenic variant strains (JXA1,TJ and HUN4).Moreover,further analysis of the GP5 protein suggested that these strains exhibited variations in the primary neutralizing epitope and N-linked glycosylation sites.The other two strains had the same genetic features with RespPRRSV MLV.The results showed that the highly pathogenic PRRSV was still the dominant strain and GP5 amino acid mutations were frequently,there were some genetic differences between PRRSV in different regions.

In order to know the law of seasonal reproduction of yak (Bos grunniens) at protein level,the composition changes of follicular fluid and plasma protein were analyzed by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS).The study employed Qinghai plateau yak follicular fluid and plasma as the research objects.The maps were obtained by 2-DE.After silver staining,the differential proteome was analyzed using the Image Master 2D Platinum software and MALDI-TOF-MS (matrix-assisted laser desorption ionization time of flight mass spectrometry).First we described the purification of follicular fluid and plasma samples using ProteoExtract Albumin/IgG Removal Kit,and then described the maps of protein from these purified follicular fluid and plasma using the 2-DE system.24 different expression protein spots were found with Image master 2D platinum soteware,2 of these proteins were up-regulated,and 22 of these proteins were down-regulated in yak follicular fluid.Through MALDI-TOF-MS identification,8 protein spots were successfully identified and 5 protein spots were unknown in NCBI_Bos tarus.Establishment of 2-DE maps,isolation and identification of differential protein were successfully to provide experimental basis for revealing the law of follicular development and developmental microenviroment of oocytes at protein level in yak.

The objective of this study was to investigate the effect of climatic traits on body size traits and reproductive performance in Chinese native Black-bone chicken breeds using canonical correlation analysis. In this study, canonical correlation analysis (CCA) was applied to estimate the relationship between local climatic traits and body size traits (9 traits), climatic traits (4 traits) and reproductive performance (5 traits) 17 Chinese native Black-bone chicken breeds. The result of climatic traits and body size traits was that the first canonical correlation coefficient was 0.992(P<0.01), which accumulative pct represented 83.368% for the total correlation information. The second canonical correlation coefficient was 0.953(P<0.01), the value of pct was 12.566%, It showed that the correlation between the two sets of traits mainly by annual sunshine hours, frost-free and chest width closely related to the cause, the longer annual sunshine hours, the longer frost-free season, the greater chest width of Black-bone chicken. The result of climatic traits and reproductive performance was that the first canonical correlation coefficient was 0.867(P<0.01), the value of pct was 60.797%. The second canonical correlation coefficient was 0.754(P<0.01), the value of pct was 26.496%, The results from CCA indicated that the longer annual sunshine duration, the longer frost-free season, the wider of chest breadth, the later average of age at first egg. The research provides reference basis for genetic breeding and reproduction on Black-bone chicken.

The generation of functional sperm in vitro has been a goal for almost a century.Fortunately,researchers reported the successful in vitro production of functional sperm,thereby potentially opening up a new era of reproductive biology.In vitro cultural methods for spermatogenesis include tissue cultural method and cells cultural method.Take together transplanting isolated cells into testicular tissue for tissue culture.Here,we summarize the history of research directed toward reproducing steps of spermatogenesis in vitro,suggest ways that can be applied toward clinical applications and address fundamental questions about the underlying mechanism of spermatogenesis.

The present study was conducted to evaluate the effect of areca nut extract on blood indexes and anticoccidial efficacy of chickens infected with Eimeria tenella.270 Wenchang chickens at 1 day-old were randomly divided into six groups,which were non-infected control group,negative control group,positive control group and areca nut groups 1,2 and 3.Areca nut groups 1,2 and 3 were fed the basal diets supplemented with 200,400 and 600 mg/kg areca nut extract powder throughout the experiment,respectively,while three control groups only fed the basal diets.At 15 days of age,30 chickens from each group that had similar weight except non-infected control group were inoculated with an oral dose of a normal saline mixture of 1×10⁵ sporulated oocysts (1 mL/head).The results showed as follows:①On post-inoculation days 6 and 9,in contrast with non-infected control group and positive control group,blood RBC,Hb and PCV in areca nut groups 1,2 and 3 and negative control group were decreased.②Compared with non-infected control group,the contents of serum TP,ALB and GLB in positive control group and areca nut group 1 were increased on post-inoculation days 3 and 9;On day 9 post-inoculation,the ALT content in serum of areca nut groups 2 and 3 were significantly decreased than that of non-infected control group and positive control group (P<0.05),the serum UA content of areca nut group 1 was significantly decreased than that of negative control group,positive control group and areca nut group 2 and 3 on day 9 post-inoculation (P<0.05);On day 6 post-inoculation,the serum Na⁺ and Cl^- concentrations of negative control group were significantly decreased than that of non-infected control group and positive control group (P<0.05),there were no significant differences in the Na⁺ and Cl^- concentrations among areca nut group 1 and three control groups (P>0.05).③The ACI of areca nut groups 1,2 and 3 were 128.48,126.53,141.11,respectively.In conclusion,200,400 and 600 mg/kg areca nut extract powder which were added to basal diet had middle anticoccidial effect.In addition,200 and 400 mg/kg areca nut powder could maintain the normal Na⁺ and Cl^- concentrations in serum and reduce the liver injury of chickens infected with Eimeria tenella.

The assay was aimed to clarify the reasons why pneumonia and arthritis had happened in a dairy farm,and provide effective control and treatment.A newborn calf was autopsied and the samples were collected.Mycoplasma bovis (M.bovis) and other pathogens were isolated and cultured,and PCR identification and drug sensitivity test were performed.Bovine viral diarrhea virus (BVDV),foot-and-mouth disease virus (FMDV) and infectious bovine rhinotracheitis virus (IBRV) were detected by PCR.Pathological slices from calf lung tissue were made to evaluate pathological changes.M.bovis and bovine capsular serotype A Pasteurella multocida (Pm A) were isolated,respectively.There were negative results for BVDV,FMDV and IBRV detection.Pathological slices of lung tissue showed damaged visible alveolar structure,bleeding and a large number of inflammatory cells infiltration.Drug sensitivity test results showed that M.bovis and Pm A were sensitive to tylosin and cefazolin,respectively,but resistance to penicillin,gentamicin,clindamycin and ampicillin.It was confirmed that M.bovis pneumonia with Pm A infection had happened.The disease in the farm was controlled by using tylosin and cefazolin at the same time.Furthermore,symptomatic treatment and standardized management should be carried.

In order to obtain a producing amylase Clostridium butyricum,the chicken small intestinal mucous was collected to isolate and screen Clostridium butyricum.First of all,the samples were heated at 80℃ for 10 min for removing most non-spore bacteria,then the samples were inoculated on TSN medium.The suspected Clostridium butyricum strains were screened by colony morphology and microscopic morphology observation.The producing acid ability and amylase activity of the isolates were successively tested,so the C.B1 strain,both producing acid and producing high-yielding amylase,was selected to be identified by physiological and biochemical test and 16S rDNA sequence analysis.The results showed that C.B1 strain had the characteristics of gram-positive anaerobe,rod-shaped and forming a circular or oval spore,16S rDNA sequence was 1450 bp.Homology comparison analysis indicated that it was most closest to Clostridium butyricum with 99% homology.So C.B1 strain was identified as Clostridium butyricum,this study would lay the foundation for developing new microecological preparations.

The assay was aimed to explore the combination of aqueous extract of Radix Paeoniae Rubra and antibiotics against drug-resistant E.coli, we used the broth micro-dilution method and micro-dilution checkerboard technique to determine the minimum inhibiotory concentration (MIC) and fractional inhibitory concentration index (FICI).The results showed that the FICI of aqueous extract of Radix Paeoniae Rubra combined with ceftriaxone sodium, amoxicillin, ciprofloxacin, norfloxacin, levofloxacin, fosfomycin, mequindox, clinafloxacin, sulfamonomethoxine, gatifloxacin, amikacin, ceftazidime, lincomycin, ceftiofur sodium, florfenicol, azithromycin, cefotaxime and rifampicin were 0.27, 2.50, 1.02, 1.01, 3.00, 0.28, 1.02, 1.02, 1.02, 2.02, 1.02, 0.75, 0.56, 0.75, 0.50, 3.00, 0.75 and 0.38, respectively.The results indicated that the aqueous extract of Radix Raeoniae Rubra combined with ceftriaxone sodium, fosfomycin, florfenicol and rifampin had synergy effects;Combined with lincomycin, ceftiofur sodium, cefotaxime and ceftazidime had additive effect;Combined with amoxicillin, levofloxacin, gatifloxacin and azithromycin had antagonism effect;Combined with ciprofloxacin, norfloxacin, mequindox, clinafloxacin, sulfamonomethoxine and amikacin showed independent action.

In order to study the colonization pattern,localization and quantity of Bordetella avium (B.avium) in the tissues and organs of chicks infected with B.avium,a method of indirect fluorescent immunohistochemistry suitable for B.avium detection was established and applied to detect tissues of artificially infected chicks in this study.Firstly the fixation agent for fixing tissues and organs of chicks was screened.Then the fixed tissues were sliced to sections and good pasting agent was detected.Rabbit anti-B.avium outer membrane protein IgG was used as the first antibody.After repeatedly optimizing the experiment conditions,10% neutral formalin was used as a fixation agent to fix tissues for 24 h and 0.1% poly-L-lysine as a section pasting agent.Optimal working conditions were as follows:2% skimmed milk powder closing section at 37℃ for 30 min;1:100 dilution of rabbit anti-B.avium outer membrane protein IgG incubating at 4℃ overnight;Fluorescein marked goat anti-rabbit secondary antibody was diluted 1:20,incubating at 37℃ for 30 min.This method had good reproducibility and specificity in B.avium detection.Detection results of chick tissues infected with B.avium showed that this method could specifically localize B.avium in the tissues and organs of chicks.Specific fluorescence signals could be found in tracheas lung,liver,heart,spleen and kidney.It had coincidence rate of 99.27% with bacterial isolation and culture.The results showed that the established indirect fluorescent immunohistochemistry could be used as an effective method in research of B.avium pathogenic mechanism.

The assay was aimed to optimize the best preparation conditions of lactic acid bacteria fermented traditional Chinese medicine preparation against chicken coccidiosis,and study the prevention and contol effect.First,explored the best lactic acid bacteria,inoculational amount,time,temperature and pH of lactic acid bacteria fermented traditional Chinese medicine preparation,and then carried out the effect experiment of 150 AA+ broilers.The broilers were randomly divided into control group,infection group,lactic acid bacteria fermented group,western medicine group and traditional Chinese medicine group,after 21 d,calculated the growth performance and cytokine levels of IgG,IL-12,IFN-γ,IL-10.The results showed that lactic acid bacteria grew well under pH 6.0,37℃ cultivated 12 h,3% P.pentosaceus PP cultivated in the concentration of 1.0 g/mL traditional Chinese medicine medium;Moreover the average daily gain of lactic acid bacteria fermented group was significantly higher than infection group (P<0.05),not sigificantly higher than traditional Chinese medicine group and western medicine group (P>0.05),equivalent to control group (P>0.05);F/G of lactic acid bacteria fermented group was significantly lower than infection group (P<0.05),not sigificantly lower than traditional Chinese medicine group and western medicine group (P>0.05);The levels of IL-12 and IFN-γ of lactic acid bacteria fermented group were significantly higher than infection group (P<0.05),not sigificantly higher than traditional Chinese medicine group and western medicine group (P>0.05);The levels of IL-10 and IgG of lactic acid bacteria fermented group were significantly higher than infection group (P<0.05),slightly higher than traditional Chinese medicine group and western medicine group (P>0.05).The results indicated that the optimized lactic acid bacteria fermented traditional Chinese medicine preparation against chicken coccidiosis could improve the growth performance and enhance the immune level of chicken.

Type 1 diabetes mellitus (T1DM) is a chronic disease,which has an inherited tendency.T1DM is an insulin-dependent diabetes.Insulin-secreting β-cells of patients with T1DM are selectively and irreversibly destroyed by autoimmune,so that these cells lose their function to secrete insulin.Traditional methods to cure T1DM include insulin injections and pancreas islet transplantation.Recently with the development of stem cells technique,we can get insulin-secreting β-cells from different types of stem cells.These new techniques have provided a new chance to heal T1DM.The review summarizes thepathophysiology of T1DM and we also show some new advances in stem-cell-based approaches that could be applied to treat diabetes in the future.

With the continuous improvement of people's living standards,the number and scale of Chinese dairy cow farming continues to expand.A large number of gases (CO₂ and CH₄) which were produced by cows normal physiological activities caused increasingly serious air and water pollution.More and more nations put attention to the greenhouse effect caused by increased atmospheric concentrations of the trace gases CO₂ and CH₄.The CH₄ fermented by dairy cow gastrointestinal tract is one of the main sources of greenhouse gas emissions.The control of methane emissions by dairy cow is a effective method in mitigation of greenhouse gas emissions.Therefore,it is necessary to reduce the methane produced by ruminants in the production of animal husbandry.The methane emission in dairy cows production is related to feed nutrition,regulation and control of rumen fermentation,genetic selection and farm management,etc.This paper reviewed literatures on reduction of methane emission in dairy cow industry.