In order to reveal the molecular marker that influenced Jinghai Yellow chicken's INF-γ level.This research was based on female Jinghai Yellow chicken in core group and had measured the INF-γ concentration in serum.Genome-wide association study was carried out using specific-locus amplified fragment sequencing technology to detect SNP associated with this trait.Finally,one SNP that potentially associated with INF-γ was detected and it reached the chromosome significant level.This SNP was located in MYOM1 gene on chromosome 2.The cellular component,molecular function and biological process were analyzed using GO database.Furthermore,the result showed that MYOM1 gene was an important candidate genes affecting INF-γ in Jinghai Yellow chicken.

The purpose of this study was to research expression pattern of clock,tim,per1 and cry1 genes in cashmere goats skin,and to analysis the role of Clock genes as well as relationship of tissue development in cashmere goat skin.Clock genes form a transcription-translation feedback loop,clock gene expression was higher through the growth cycle of skin in cashmere goat,activating per1, cry1 genes transcription-conversion process,which regulated mRNA and protein levels of the core Clock genes with rhythmic expression.The result showed that the expression levels of Clock genes in cashmere goat's skin was:clock >tim >per1 >cry1.The period of anagen in skin hair follicle,rhythmic expression of high-amplitude clock,per1,tim,cry1 appeared at 04:00,08:00,08:00,16:00;The periods of telogen in skin hair follicle,rhythmic expression of high-amplitude clock,per1,tim,cry1 appeared at 08:00,04:00,04:00,16:00.Furthermore,the cry1 and per1 genes expression were activated in the Clock genes positive feedback loop,the tim gene expression was activated in a negative feedback loop.In short,under the regulation of clock,tim,per1 and cry1 feedback loop,which operated cyclical phenomenon of pile.

In order to clone the phosphotyrosine interaction domain containing 1(PID1) gene of Luchuan pig,a pair of special primers was designed according to released sequence of Laiwu pig PID1 gene in GenBank.The PID1 gene was amplified by RT-PCR,its gene sequence characteristics and protein structure was systemically analyzed by bioinformatics techniques.The results showed that the cloned PID1 gene fragment included a 654 bp whole length CDS (coding 217 amino acids).The sequence multi-aligned results showed that Luchuan pig shared 99.08%,87.61%,93.88%,93.58%,90.06%,83.79%,66.94%,66.52% and 60.09% of similar nucleotide sequence with that of Laiwu pig,Bos,Macaca,Homos,Mus,Gallus,Rattus,Danio retio and Xenopus, respectively.The phylogenetic tree indicated that PID1 gene was highly conserved in the process of evolution of different species.The cloning and analysis of PID1 gene provided an important foundation for further study regulation mechanism of PID1 gene in Luchuan pig.

In order to study SLA-DRa gene (also named as SLA-DRa-HB) from one of special breed of domestic pig,Hebao,primers were designed to amplify the coding domain of SLA-DRa from three Hebao pigs.Then,the amplified fragments were cloned into pMD18-T vector followed by transforming them into Escherichia coli JM109.After cleaving by the restricted enzymes,the positive clones were selected to be sequenced.The differences between SLA-DRa-HB and other SLA-DRa alleles were analyzed by sequences comparing,and then the phylogenetic tree was constructed.The results showed that the interest of the fragments was successfully amplified by RT-PCR and the molecular weight was about 800 bp.By cloning and sequencing,the whole length of SLA-DRa-HB was 779 bp and the open reading fragments (ORF) of SLA-DRa-HB located at sites of 1 to 759 coding for 252 amino acids.Results of sequences comparision and analysis showed that the characterized amino acids of SLA-DRa-HB mainly focused on sites of 135,159 and 202,while the mutational sites in trans-membrane and cytoplasm were 206 and 248.Analyzing from the phylogenetic tree,it showed that SLA-DRa-HB was clustered into one branch and they were relatively closed to other SLA-DRa alleles.In this research,the SLA-DRa alleles were cloned successfully from Hebao pigs and it would lay a base for study the function of the SLA-DRa genes.

In order to predict the possibility of Toxoplasma gondii nuclear factor 3 (TgNF3) as anti-Toxoplasma gondii vaccine candidate,TgNF3 gene was amplified by one step RT-PCR using a pair of specific primers.Then the fragments were cloned into the pMD18-T vector.The sequence of TgNF3 gene was then translated into amino acids and analyzed by bioinformatics software.The physical and chemical characteristics,hydrophilic domain,transmembrane domain,epitopes,and advanced structure of TgNF3 were predicted by multiple bioinformatics approaches.The results revealed that TgNF3 gene was approximate 950 bp in length.TgNF3 protein was predicted to contain only one transmembrane domain,9 α-helix regions,4 β-fold regions,8 hydrophilic regions and 10 flexible regions,and predicted to contain 9 linear B cell epitopes.The results indicated that TgNF3 could be a vaccine candidate antigen against Toxoplasma gondii,which provided the basic data for development of novel Toxoplasma gondii vaccines.

In order to clarify the genetic variation of three different endogenous avian influenza viruses subtype H9N2 A/Chicken/Jilin/22/13 (JL22),A/Chicken/Jilin/24/13 (JL24) and A/Duck/Jilin/37/13 (JL37),eight gene fragments of the three viruses were amplified by RT-PCR,and then cloned and sequenced.The results showed that the main pathogenic genes of the three H9N2 subtype AIV belonged to the classic Eurasian species.Compared with A/Chicken/Hong Kong/G9/97 and A/Duck/Hong Kong/Y280/97,the amino acid sequence of HA of JL22 had a more potential glycosylation site at the location of 551.The amino acid residue at position 226 of HA of JL22,JL24 and JL37 were all Leu,sialic acid receptors in mammals had the same characteristics and it showed that it enhanced human infection.Asn substituted Ser phenomenon was occurred on M gene at 31 site,indicating that these viruses were resistant to amantadine.Phylogenetic trees could be seen three viruses distantly related strains,each gene belonged to a branch not a unity,and some genes from chicken,duck and swine origin influenza virus strains derived three kinds of highly homologous,it was speculated that three different strains of different animals were naturally rearrangement products after a long period of evolution .

The test was aimed to study the molecular identification of self-excision of bacterial artificial chromosome in meq-null Marek's disease virus (MDV) clone genome.SC9-1 recombinant plasmid containing cre recombinase expression cassette was transfected into chickens embryo fibroblast (CEF) to rescue SC9-1 recombinant virus.SC9-1 recombinant virus was passaged on CEF cells continuously,using cre recombinase knocked out BAC sequence in process of virus passaged.Extracting 1 to 10 generations DNA of SC9-1 recombinant virus as template,PCR detected BAC sequence of viral genome by specific primers.Using specific primers for the BAC sequence of gpt gene,the MDV conserved gene pp38 as an internal standard,the content of BAC sequences in the viral genome of different passages were detected by fluorescence quantitative PCR.The residual sequence of both sides of loxp site were amplified by PCR,and future verifying the knockout of BAC sequence by sequencing.The results showed that,using the BAC sequence specific primers to detect BAC sequence of different generations in the viral genome, the first five generation of virus DNA could amplify 600 bp specific band meaning the viral genome contained the BAC sequence.The 6 to 10 generationes of virus DNA couldn't amplify 600 bp specific band meaning the viral genome didn't contain the BAC sequence.The result of fluorescence quantitative PCR showed that the content of BAC sequence in viral genome decreased gradually with the passage of the virus and couldn't detect BAC sequence completely until the sixth generation.In the process of the knockout of BAC sequence,PCR identifying the residual sequence of both sides of loxp site,the results showed only one loxp site left after the knockout of BAC sequence and the sequence homology was above 99.7%.The experiment confirmed that the cre/loxp system could delete the BAC sequence in MDV meq-null mutant SC9-1 completely by culturing virus on CEF cells for 6 generation of batches and the knockout of BAC sequence had high degree of consistency.

In order to demonstrate the nonstructural proteins 1 (NP1) molecular characterization,the target fragments was obtained from post-weaning multisystemic wasting syndrome piglets using PCR method with specific primers,targeting to the NP1 fragments.The results showed that the porcine bocavirus NP1 gene was 675 bp in length,coding an open reading frame (ORF) with 224 amino acids.The nucleotide sequence were compared with the porcine bocavirus NP1 downloaded from GenBank using MegAlign.The sequenced gene shared the highest homologies of nucleotides with the USA porcine bocavirus OH110 strain at 97.3%,and only shared 81.8% identity to IN109-1 strain; which shared the homogeneity with the United Kingdom porcine bocavirus F41 strain and 64-1 strain at 82.5% and 80.9%,respectively.However,the cloned sequence only shared the homologies of nucleotides with porcine bocavirus H18 strain at 43.8%.According to the phylogenetic tree,the porcine bocavirus could be divided into three genotypes,all of which the homologies of nucleotides were lower than 50.0%.The results suggested that the porcine bocavirus could be divided into three different genus in genus bocavirus.

To construct the prokaryotic expressing vector of SLA-3 derived from Hebao and Laiwu pigs (named as SLA-3-HB and SLA-3-LW) and to express the interest of protein,the extracellular domain of SLA-3 were amplified by PCR,and then they were cloned into the pMD19-T Simple vector followed by transforming into Escherichia coli Top10.By cleavage and sequencing,the positive clones were selected.After cleavage and purification of the recombinant plasmids,the interest of genes were further ligated to pET-21a(+) and transformed into the Escherichia coli BL21.The interest of genes were induced to express by IPTG and the interest of proteins were detected by SDS-PAGE.The results showed that the extracellular domain of SLA-3-HB and SLA-3-LW were successfully amplified with the molecular weight of 850 bp.It was also shown that the SLA-3-HB and SLA-3-LW were successfully cloned into pMD19-T Simple vector and the positive recombinant plasmids with correct sequences were selected.Further study proved that SLA-3-HB and SLA-3-LW were successfully ligated into pET-21a(+) with the 831 bp inserted fragments.After induction,SLA-3-HB and SLA-3-LW were all expressed successfully and the molecular weight of the interest of proteins were about 31 ku,and the contents of the expression of the proteins reached 40 percent.In this study,the prokaryotic expression vector of SLA-3 gene derived from Hebao and Laiwu pigs,which would lay a base for furter study the structure and function of the interest of proteins.

For preparation of specific IgY against the ETEC strains from the pig farms,types of fimbriae gene of an ETEC strain isolated from these pig farms were identified by PCR.The pilin was purified and used to immunize hens to obtain the IgY.The titer,specificity and in vitro inhibitory effect of IgY were detected.The results showed that this ETEC strain had two types of fimbriae gene of K88 and 987p,and the immunogenic of the strain fimbriae was high.The titers of the isolates fimbriae IgY against the K88 and 987p fimbriae were both 1:64 000.The IgY could bind with K88 and 987p but could not either with K99 or F41. 5 mg/mL fimbriae IgY could completely inhibit the growth of K88 and 987p in vitro.

The F81 cells were divided into three groups,control group,feline panleukopenia virus (FPV) infected group and ribavirin treatment group,we used Real-time fluorescent quanlitative PCR to detect the expression of Bcl-2,Bcl-xl,Bax and Caspase 3.Real-time fluorescent quanlitative PCR results showed that the number of apoptotic cells increased in FPV infected group compared with the control group,and mRNA levels of Bcl-2 was inhibited,which was an anti-apoptotic gene,while the expressions of Bax and Caspase 3 were both increased.In ribavirin treatment group,expression levels of Bcl-2 and Bcl-xl were significantly higher than that of FPV infected group (P<0.05),and had a peak at 48 h in all time points.However,mRNA levels of Bax and Caspase 3 in ribavirin treatment group were significantly lower than that of FPV infected group (P<0.05),and the lowest levels were at 72 h in all time points.In a word,ribavirin could inhibit the apoptosis of F81 cells infected by FPV at the genetic level.

Viral protein 4 (VP4) is an important non-structural protein with proteolytic enzyme activity encoded by infectious bursal disease virus (IBDV) genome that catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3 to form viral proteins pVP2,VP4,VP3,which plays an important role in the process of viral proteins maturity.To study the interaction between VP4 and the host cell,VP4 full-length cDNA sequence was amplified and inserted into pGBKT7 vector to construct a bait vector (pGBGtVP4).The result of autoactivation assay showed that pGBGtVP4 vector had no autoactivation and virulence in Y2H gold yeast cell.After screening,sequencing and confirming interaction,22 kinds of potential interacted proteins were obtained from the expression library of chicken embryo fibroblasts (CEF) using the matchmaker gold yeast two-hybrid system.This study laid the foundation for further researches on the effect and mechanism of the interaction of IBDV VP4 and its host cell.

β1 toxin gene was amplified from Clostridum perfringens type B C58-1 strain by polymerase chain reaction (PCR),PCR products were connected to pMD18-T vector screening positive clones,and then cleaved with restriction endonucleases BamHⅠand SalⅠ,the 927 bp gene fragment was recovered and inserted into the same site of pET-32a vector.The recombinant plasmid pETβ927 was studied in detail by restriction endonuclease analysis and nucleotide sequencing.The recombinant plasmid could produce β1 toxin protein by SDS-PAGE.Expressed products were purified by pre-installed column of His-Trap FF,the size and distribution of the target protein were detected by SDS-PAGE,and its immunorectivity was confirmed by Western blotting.The results showed that the β1 toxin gene was 1 011 bp and the homologies with B and C type Clostridum perfringens protein sequences of GenBank were greater than 99.4%;In SDS-PAGE analysis,the fusion protein was 54 ku as expected and distributed in ultrasonic lysis supernatant as well as in inclusion bodies,but mainly existed in inclusion bodies.Western blotting analysis showed that the β1 toxin protein had a good immunorectivity with specific serum antibody.

In order to establish a loop-mediated isothermal amplification assay (LAMP),a set of LAMP primers targeting to NP gene of porcine epidemic diarrhea virus (PEDV) were designed and synthesized.Calcein and manganese chloride were added as the indicator instead of SYBR Green Ⅰ after reaction.The LAMP could amplify PEDV specifically at 65 ℃ in 1 h with detection limit of 3.73 pg/μL and the result could be judged by naked eye.The LAMP didn't react with CSFV,TGEV,PoRV and E.coli.93 clinical samples of PED suspected case collected from Veterinary Institute of Longyan University were submit to detect PEDV by LAMP,the result showed that the positive rate of the samples was 43.01%,which was higher than that (38.7%) of conventional PCR.The established visual LAMP could be used for diagnosing PED.

Capsid (Cap) protein was the main structural protein of porcine circovirus type 2 (PCV2),and used as a main basis for PCV2 serological diagnosis in the clinical.In this study,the complete gene encoding Cap protein of PCV2 TJ strain was amplified by PCR using the specific primers designed according to the strain sequence (GenBank accession No.:KC751546),and then the amplified products were cloned into pET-32a(+) vector to construct recombinant plasmid pET32a-Cap.The recombinant fusion protein with about 48 ku was expressed by IPTG induction.The result of Western blotting analysis showed that the fusion protein had the positive reaction with anti-6×His tag monoclonal antibody and porcine serum against PCV2.We had successfully cloned Cap gene,constructed the prokaryotic expression vector,and then expressed Cap fusion protein,it provided the support for the establishment of PCV2 detection and development of subunit vaccine,and also made a foundation for the study of the function of PCV2.

microRNAs (miRNAs) are evolutionary conserved small noncoding RNAs of 18 to 25 nt that were found in all metazoans,and it regulates gene expression by base pairing with their target mRNA,leading to mRNA cleavage or translational repression.let-7 is a small RNA molecule originally found in worms,and it is highly conserved and plays a vital role in posttranscriptional regulation.The study found that let-7 takes part in the development of organs and tissues.This review summarized the recent progresses about the function of let-7 miRNA in the development of brain,nervous system,lung and cardiovascular system,and muscle development,and elucidated the possible mechanism of regulating the developmental processes of animal tissues and organs.It will provide the basis for further exploring the function of let-7 miRNA in animals.

Piglet diarrhea is one of the most challenging problems what swine industry has faced at home and abroad,which causes a huge economic loss annually.Enterotoxigenic Escherichia coli (ETEC) is the main pathogenic bacteria of piglet diarrhea,whose pathogenicity depends on the co-effect of adhesion and enterotoxin.As the research methods innovate,the researchers at home and abroad have got important breakthroughs in the development of ETEC vaccines.This article introduced the pathogenic characters of ETEC,and summarized the progress on the development of all kinds of vaccines against ETEC.

To confirm a suspected Klebsiella pneumonia (K.pneumoniae) infection,and study the pathogenicity,drug sensitivity and homologies of the 16-23S rRNA ITS genes of the pathogen,one Gram-negative,rod-shaped bacteria named as KP14013 was isolated from livers and gut of the piglets with diarrhea,high-mobility and high-mortality.The isolate was preliminarily identified as K.pneumoniae by biochemical test and further identified as K.pneumoniae by 16S rRNA sequence analysis which shared 99% homology with 23 representative isolates from GenBank.Lethality tests indicated that the 50% lethal doses (LD₅₀) in mice were 3×10^1.8 colony-forming units (CFU) for the isolate and that 100% mortality in piglets by intraperitoneal inoculation with 3×10⁸ CFU.The 16-23S rRNA ITS sequence analysis indicated that KP14013 belonged to one subgroup with 15 represent isolates from GenBank and shared homologies of 98.4% to 99.2%.This study confirmed the K.pneumoniae infection with diarrhea and high mobility and high motality in piglets.One highly virulent K.pneumoniae strain was isolated from liver and gut of piglets.The KP14013 isolate was high virulent to mice and piglets, and showed multiple-drug resistance among common antibiotics.The 16-23S rRNA ITS of KP14013 proved to be a feasible and rapid way for identification of different K.pneumoniae species for its moderate conservatism and variability.

To identify the epitope of porcine transmissible gastroenteritis virus (TGEV),the NSP8 gene was cloned into the vector pGEX-6P-1 for expression in E.coli.BALB/c mice of 6 week-old were immunized with the recombinant NSP8 protein.Then the monoclonal antibody against NSP8 protein was prepared by lymphocyte hybridoma technique.The monoclonal antibody belonged to IgG1 subtype with κ chain.The titers in cell culture medium of the hybidomas was 1:3 200.Western blotting assay showed that the monoclonal antibody was able to recognize the recombinant NSP8 protein.Epitope on NSP8 protein was identified by truncated expression and the linear epitope of ³¹SPQILKQLTKAFNIAKSDFEREASV⁵⁵ was identified by Western blotting with the monoclonal antibody.This monoclonal antibody and its epitope mapping provided a basis for further study of the function of the TGEV NSP8.

To diagnose a suspected sheep pseudorabies case,the tissue samples of sheep with clinical symptom of sheep pseudorabies from a farm in Shanghai suburb were collected for pathological observation,isolation and identification of the virus.The histopathological observation of sheep showed extensive neuronal degeneration and necrosis occurred in the brain tissue accompanied with neurotropic phenomenon and gliosis.BHK-21 cells inoculated by tissue samples showed lesions.Pseudorabies virus (PRV) in BHK-21 cells was confirmed by specific fluorescence of indirect immunofluorescence assay and fluorescence PCR.Epidemiological investigations showed sheep pseudorabies was possibly caused by swine pseudorabies virus.Sheep pseudorabies was controlled in time by inoculating swine pseudorabies attenuated vaccine.

The research aimed to study the expression of Kiss-1 in different developmental stages of Wuzhishan pig testis.The expression of Kiss-1 mRNA in 30,60,80 and 120 day-old Wuzhishan pig testis were studied using Real-time PCR.Cellular localization of Kiss-1 in testis was examined by immunohistochemistry.The results showed that the expression of Kiss-1 mRNA was increased with the age before puberty (P<0.05),and reached the highest level in 80-day-old testis (P<0.05),then decreased at 120 days of age (P<0.05).There was a positive expression of Kiss-1 in interstitial tissue,spermiogonium,primary spermatocyte,second spermatocyte,and round spermatid of adult Wuzhishan pig testis.However,the Kiss-1 immunopositive production was not found in sperm.The results confirmed that Kiss-1 gene played important role in spermatogenesis of Wuzhishan pig.

The objective of this experiment was to study the effect of synchronizing the rate of dietary energy and nitrogen release in diets on microbial protein synthesis and rumen fermentation in dairy cows using in vitro gas production method.Four healthy Holstein cows in mid-lactation were used to offer rumen juice.A single factor experimental design and three isoenergetic and isonitrogenous diets varying in synchronization index (SI) were used in this study (diet A,SI=0.74;diet B,SI=0.83;diet C,SI=0.91).The results showed that microbial protein production was significantly increased (P<0.05),and NH₃-N concentrtatin was extremely significantly decreased in 8 and 12 h as synchrony level of diets increased (P<0.01).In 8 h,group A compared with group B,gas production,pH eached significant or extremely significant difference (P<0.05;P<0.01),in 12 h,group A compared with group B,the volatile fatty acid content was extremely significant difference (P<0.01).The results indicated that synchronization of dietary energy and nitrogen release had significant influence on microbial protein synthesis and NH₃-N concentration for dairy cows.

This experiment was conducted to study the effects of dietary tannins levels on growth development,pedicle initiation and primary velvet antler growth of young male sika deer (Cervus nippon).Twenty four 7-month-old male sika deer with similar body condition were randomly separated into four groups (6/group).The control group was fed with basal diets without tannins,groups Ⅰ,Ⅱ and Ⅲ were fed with basal diets complementary with 1%,2% and 4% tannins,respectively.The trial lasted for 4 days,and 140 days for experiment.The results showed that the daily growth of body weight and height in group Ⅱ were significantly higher than that in control group (P<0.05). There were no significant differences in the daily growth of body length and circumference of canon among all groups (P>0.05).The pedicle initiation time of sika deer from the tannins-treated groups was earlier than that of control group.The length of both branch from groupⅠwas longer than the other groups,and the right branch of groupⅠwas significantly longer than that of group Ⅲ (P<0.05).The production of primary velvet antler in groups Ⅰ,Ⅱ and Ⅲ was significantly higher than that in control group (P<0.05).In conclusion,supplementation of tannins promotes time of pedicle initiation,production of primary velvet antler and growth development of young sika deer.The optimum supplemental level of tannins in the diet of young sika deer was 2%.

This study was aimed to research the effect of intestinal membrane protein and fermented soybean meal replacing fish meal on the immunity and digestive tract development of weaned piglets.A total of 180 weaned piglets,half male and half female,Duroc×Landrace×Yorkshire at 28 days old with body weight (8.0 ± 0.5)kg,were randomly divided into 3 treatments with 6 replicates of 10 piglets per pen.The diet of control group contained 17.0% soybean meal + 6.0% fish meal,the other two diets contained 16.6% soybean meal + 4.0% fermented soybean meal + 4.0% imported or domestic intestinal membrane protein.The pre-test period lasted for 3 days and the trial period lasted for 14 days.The results showed that compared with the control group,the indexes of spleen,mandibular lymph and the serum content of ALB in imported intestinal membrane group were significantly increased by 43.53%,49.18% and 16.20%,respectively (P<0.05),and in domestic membrane group,the index of spleen was significantly increased by 41.77%,and the serum content of complement C3 was significantly decreased by 16.22% (P<0.05);there were no significant differences in the indexes of thymus,inguinal lymph,iliac lymph,popliteal lymph,superficial cervical lymph,and the levels of serum TP,BUN,IgA,IgG and NO among all groups (P>0.05).Compared with the control group,there was no significant difference in the weight of stomach and duodenum in imported intestinal membrane group (P>0.05),but the weight of stomach and duodenum in domestic intestinal membrane group were significantly decreased by 19.68% and 48.55%,respectively (P<0.05).The length of duodenum in imported intestinal membrane group was significantly increased (P<0.05),while in domestic intestinal membrane group was significantly decreased (P<0.05).The length of jejunum and the weight of ileum in imported intestinal membrane group and domestic intestinal membrane group were significantly decreased (P<0.05),the villus height of ileum in domestic intestinal membrane group was significantly decreased (P<0.05).There were no significant differences in the weight of liver and jejunum, the length of ileum and the villus height and crypt depth of duodenum and jejunum among all groups (P>0.05).It was concluded that imported or domestic intestinal membrane protein and fermented soybean meal replacing fish meal could not affect major immunity organ index,serum items and intestine morphology of weaned piglets,but there were significantly decreased in the weights of stomach and duodenum in domestic membrane protein group.

As one of the important nutrition,meat quality gets increasing attention of consumers.Magnesium as organisms important cations in the body,plays an important biological function in the body's immune,stress,antioxidant,also has important influence to the meat.This paper discusses the biological functions and effects on meat quality of magnesium,in order to provide basis for exploring the feasible measures of improving meat quality.

The aim of the study was to prepare ivermectin nanoemulsion for transdermal drug delivery.The physicochemical property,stability,in vitro drug release and transdermal property were evaluated.The optimal drug loading with good stability and larger nanoemulsion region was investigated by ternary phase diagram.The response surface methodology was used for selecting the optimal prescription.The average diameter,zeta potential,morphology,pH and viscosity were also investigated.In vitro drug release and transdermal property of ivermectin nanoemulsion and market ivermectin skin varnish were compared by dialysis bags and Franz diffusion cells.The nanoemulsion region was largest and most stable when the drug loading was 2.00%.The optimal prescription was Cremophor EL-35 :Transcutol HP :ethyl oleate :ivermectin :H₂O=26: 12: 7: 2: 53 and its average diameter was 18 nm.The ivermection nanoemulsion was stable when it was stored in 4 ℃ refrigerator and room temperature for 1 year.The cumulative permeation and retention of ivermectin nanoemulsion in 24 h were 3.24 and 2.05 times over the commercially available varnish,respectively.These results indicated that the ivermectin nanoemulsion had the advantages of simple preparation process,excellent stability and transdermal property,and had good application prospects.

This study established a primary culture model of sheep thyroid cells,and paved the way for in vitro study on thyroid functions in sheep.In this experiment,thyroid gland was dissected,digested with trypsin and collagenase Ⅰ,purified by centrifugation and seed on 6 well plates.The cells were characterised bymorphologic observation,mRNA detection of genes specifically expressed in thyroid cells,including thyroglobulin (TG),thyriod peroxidase (TPO) and thyroid-stimulating hormone receptor (TSHR) using RT-PCR (reverse transcription-polymerase chain reaction) and specific antigen protein (TG) expression with immunofluorescence technique.Meanwhile,secretion of thyroid hormones T3 & T4 was analysed using ELISA (enzyme linked immunosorbent assay) at different culture time points.The results showed that the cells could adhereceto plate and grew well.The cells also presented all the characteristics of the epithelioid cells,expressed thyroid specific genes and maintained the functions of secreting T3 & T4 thyroid hormones,though the secretion amount of the hormones tended to decline along with the culture time.It indicated that thyroid epithelioid cells were obtained in vitro.

This research was aimed to further improve the ability of protease produced by Bacillus subtilis JNB001.By series single factor and orthogonal analysis to optimize the enzyme-producing conditions from initial pH,fermentation temperature,inoculation volume,bottling volume and cell age,etc.The results showed that the optimum conditions were:Initial pH 7.0,inoculation volume of 7%,bottling volume of 35 mL in the 250 mL flask,cell age was 18 h,and cultured for 36 h at 35 ℃.Cultivated at these conditions,the protease activity of the strain JNB001 could reach to 371.66 U/mL.These consequences laid the foundation for the subsequent applications such as the design of harmless biological treatment tank,etc.

In order to provide guidance for selection of replacement gilts,this study investigated the effect of partial non-genetic factors (parity,birth month and pure/cross-breeding) on the farrowing performance (total number born,number born alive and birth weight) of Landrace,Yorkshire and Duroc sows.The results showed that the birth weight (BW) from 2nd to 5th parities was heavier,and the BW of 2nd and 3rd parities was extremely significantly higher than the 1st and after 6th parities' (P<0.01).The total number born (TNB),number born alive (NBA) and BW in March were extremely significantly higher than other months (P<0.01),and the BW in November was significantly lighter than other months (P<0.05).The NBA of pure-breeding Landrace was extremely significantly higher than pure-breeding Yorkshire and Landrace×Yorkshire (P<0.01).The BW of Yorkshire×Landrace was extremely significantly higher than pure-breeding Yorkshire and Landrace×Yorkshire (P<0.01).There was extremely significant positive correlated with 2nd and 3rd to 4th parities in TNB,and so was 3rd and 4th parities (P<0.01).In conclusion,the farrowing performance of 3rd to 6th parities and January to April were the best;Pure-breeding Landrace and Landrace×Yorkshire were the advantageous ways of hybridization;The farrowing performance of 2nd parity could be regarded as an important basis for replacement gilts selection.

To investigate the genetic structure of inbred line of Guangxi Bama miniature pig from the 10^th to the 15^th generations,33 individuals of the inbred line and 19 microsatellite loci were used to estimate their number alleles (N),polymorphic information content (PIC) and inbreeding coefficients (F).The results showed that there were a total of 31 alleles at the 19 loci,and the number of allele on F10,F11,F12,F13,F14 and F15 groups were 27,29,28,27,26 and 25,respectively;Average loci alleles were 1.42,1.53,1.47,1.42,1.37 and 1.32,respectively;The average inbreeding coefficients of animals from the 10^th to the 15^th generations were 0.8070,0.8263,0.8491,0.8710,0.8904 and 0.9118,respectively.In addition,the average PIC was 0.1044.The results indicated that the inbred line of Guangxi Bama miniature pig had low gene polymorphism and high inbreeding coefficients and it was concluded that this inbreeding line had already become a genetically stable animal herd.

In the present study,Cherry Valley ducks were subjected to diets with 4% linoleic acid and eicosapentaenoic acid (EPA) from week 2 to the end of week 10,respectively,and detected the mRNA expression of SCD-1 in 4,6,8 and 10 weeks,and analyzed its association with meat quality and serum biochemical indexes in 10 weeks.The Real-time fluorescence quantitative PCR results showed that the linoleic acid and EPA could significantly reduce the SCD-1 mRNA levels in liver,fat and breast muscles (P<0.05),meanwhile,the SCD-1 gene mRNA expression level had no significant difference between linoleic acid and EPA in every period (P>0.05).Correlation analysis showed that the mRNA expression levels of SCD-1 had significant positive correlation with serum TG,TCHO,HDL and LDL (P<0.05),and no significant correlation with all meat quality traits in 10 weeks (P>0.05).The research suggested that the transcription of SCD-1 gene was inhibited by linoleic acid and EPA,and played important role in the regulation of duck lipid metabolism.

The aim of this study was to analyze polymorphism of angiopoietin-like protein 3 (ANGPTL3) gene and its association with meat quality traits in order to find markers in Chinese Simmental cattle. 98 Chinese Simmental cattles under identical feeding condition were selected randomly,the polymorphism of exon 4 of ANGPTL3 gene was analyzed by PCR-SSCP and direct sequencing methods.The association between different genotypes of exon 4 of ANGPTL3 gene and meat traits was analyzed in SPSS 19.0 program.One polymorphic site G7358C which had 2 genotypes of CC and CD was found in ANGPTL3 gene.Association analysis of polymorphisms of ANGPTL3 gene showed that the genotypes of different mutation in ANGPTL3 gene were significantly related to fat coverage of carcass,loin muscle area,marbling and IMF (P<0.05).The fat coverage of carcass,loin muscle area,marbling and IMF of individuals with CC genotype was significantly different from that of individuals with CD genotype (P<0.05).The results indicated that CC genotype was favorable genotype at this SNP locus and associated with fat coverage of carcass,loin muscle area,marbling and IMF,which suggested that ANGPTL3 gene could be studied as a candidate gene for molecular marker assisted selection.

This study analyzed one 116 mitochondrial DNA sequences from Chun'an spotted pigs and other five domestic pig breeds in Zhejiang province.The referenced pig breeds were Landrace and Wannan spotted pigs.The results showed that the 581 bp mtDNA control region sequence of Zhejiang domestic pig breeds included 14 variable sites.The nucleotide diversity was 0.00403±0.00036 and the haplotype diversity was 0.835±0.018.There were 15 haplotypes which from Hap_5 to Hap_19 in domestic pigs,and Landrace haplotypes were from Hap_1 to Hap_4.The highest haplotype frequency in Chun'an spotted pigs and Wannan spotted pigs was Hap_8 (0.680 and 0.833).Hap_5 frequency was highest in Jiaxing Black pigs (0.625), and in Shengxian spotted pigs and Bihu pigs that was Hap_6 (0.500 and 0.375).Hap_5 and Hap_8 frequencys were high mainly in Chalu Black pigs (0.429 and 0.429).Jinhua pigs had eleven haplotypes and the highest frequency was Hap_5 (0.241).A Neighbor-Joining phylogeny tree revealed 5 clades which signed from A to E among 6 Zhejiang domestic pig breeds.Chun'an spotted pigs belonged to E clade mainly,and the samples percent in Chun'an spotted pigs was 68.0% (17/25).C clade were all Jinhua pigs,and the samples percent in Jinhua pigs was 29.3% (17/58).Meanwhile,the percent of Jinhua pigs in D clade was 24.1% (14/58).It was concluded that the mtDNA control region polymorphism in Zhejiang domestic pig breeds were rich relatively and its mutations of base were different obviously from Landrace.There was close relationship between haplotype composition and frequency of domestic pigs and their geographic distribution and breed characteristics.

To investigate the antibiotic resistance and resistance genes in Escherichia coli (E.coli) isolated from beef,susceptibilities to 11 antibiotics were conducted on 117 isolates,antibiotic resistance determinants were detected by using conventional and/or multiple PCR.The results showed that resistance rates of 117 isolates to tetracycline,ampicillin,streptomycin,sulfisoxazole were 89%,42%,38%,22%,respectively.As revealed by detecting resistance determinants,tet(A),bla_TEM1,strA-strB,sul2 were the most prevalent genes among tetracycline-resistant isolates,ampicillin-resistant isolates,streptomycin-resistant isolates and sulfisoxazole-resistant isolates,respectively.The detection rates of tet(A),bla_TEM1,strA-strB and sul2 were 55%,73%,38% and 77%,respectively.The conclusion was that E.coli isolated from beef was seriously resistant to antibiotics,and antibiotics administration in beef cattle industry promoted the emergence and the propagation of antibiotic resistance in E.coli.Data reported here clearly emphasized the need for a stricter application of antibiotics restriction policies in feedlot setting.

The assay was aimed to investigate prevalent Salmonella strains from animal,their antibiotic susceptibility,and distribution of florfenicol resistant genes in Salmonella isolates with resistance to florfenicol.Pathogens were isolated from clinical samples with suspected salmonellosis,and identified by mPCR.Antibiotic resistance to 23 antibiotics was determined by antibiotic susceptibility test according to the K-B method.The isolates with resistance to florfenicol were chosen to PCR amplification for floR,fexA,fexB,cfr and pexA genes.The results showed 61 Salmonella strains were isolated,and the numbers of S.Enteritidis,S.Pullorum and S.Typhimurium were 10,12 and 39,respectively.All strains were resistant to penicillin,erythromycin and vancomycin,and 90.16% of the isolates were resistant to six or more than six actibiotics.The floR genes were detected in 8 out of 12 florfenicol-resisitant S.Typhimurium strains,and no other genes were detected.These data indicated that S.Typhimurium was the dominant serotype of Salmonella isolated from geese.The floR gene played a major role in its florfenicol resistance.However,it might exist other antibiotic resistance mechanism.

One strain of virus was isolated by passage culture in bovine testis cell,lamb testis cell,MDBK cell and BHK-21 cell of lip crust tissue collected from suspected Orf sheep in Chifeng,Inner Mongolia.The isolated virus was identified through negative staining by TEM observation and PCR detection method.One pair of specific primers was designed and synthesized referring to nucleotide sequence of Orf virus (ORFV) ORF059 (F1L) gene in GenBank.F1L gene of isolated virus was successfully cloned and sequenced,the homology was analyzed with many reference strains.The result showed that typical brick-shaped virion was found by TEM observation,and the isolated strains had high homologies with reference strains,the homologies were all more than 96%,illustrating that the isolated strains was ORFV,named as OV/nm-hd.

To investigate the prevalence of Salmonella in clinical healthy goats in Sichuan province,a total of 196 fecal samples collected from 5 goat farms in Sichuan province were tested in this study.Each sample was pre-enriched by buffered peptone water (BPW),and then selectively enriched by tetrathionate broth base (TTB).The enrichments were detected by PCR assay targeting the invA gene to determine the presence of Salmonella.The positive samples detected by PCR were isolated and identified; 25 Salmonella isolates were randomly selected to test the antibiotic resistance against 15 antibiotics.The results showed that the positive rate of Salmonella in fecal samples was 54.59%.Antibiotic sensitivity analysis showed that all of the tested strains were sensitive to amikacin,while the strains were resistant to the other 14 antibiotics in certain degrees.The rate of multi-drug resistance was 88%,and among the 25 isolates,52% were resistant to 2 to 7 antibiotic and 36% were resistant to 9 to 13 antibiotics.The results indicated that Salmonella were prevalent among clinical healthy goats in Sichuan province,and the multi-drug resistance of the Salmonella isolates from goats were quite common,which had significant implication for public health.

This study was conducted to investigate the effect of dietary Bacillus coagulans on growth performance,intestinal lesions and immune organs index of broilers infected with Clostridium perfringens.A total of 312 Kebao 500 male broilers were randomly divided into 4 groups with 6 replicates per group, and 13 birds per replicate.The 4 treatment groups were an uninfected Clostridium perfringens group,an infected Clostridium perfringens group and the level supplemented with Bacillus coagulans at 0 and 400 mg/kg of feed using a 2×2 factorial completely randomized design.The whole experiment period was 35 days.The results showed that Clostridium perfringens infection decreased the ADG at days 15 to 35 (P<0.05),severed the necrotic enteritis-related lesions at day 28 (P<0.01),reduced the bursa index at day 35 (P<0.01).Bacillus coagulans supplementation increased the ADG and decreased the F/G at days 15 to 35(P<0.05),improved the lesion score (P<0.05),increased the thymus index at day 28 and the bursa index at day 35 (P<0.01).In the case of necrotic enteritis-related lesion,there was significantly interacted between Clostridium perfrigens and Bacillus coagulans.In conclusion,dietary supplementation of Bacillus coagulans could promote growth performance,improve immune index and enhance immune function against necrotic enteritis of broilers infected with Clostridium perfrigens.

Rabies is a zoonosis caused by rabies virus,which has been characterized by infection of central nervous system.Rabies virus,which can cause fatal infections in humans and other mammals,has the characteristic of infecting neurotropic,and the mortality rate caused by rabies is almost 100%.Routine diagnosis of rabies include clinical symptoms,while a final diagnosis depends on laboratory diagnostic methods,such as etiological diagnosis,serological diagnosis and molecular biological diagnosis.As a zoonotic infectious disease,there is a global distribution situation of the rabies,and the number of deaths caused by rabies virus infection in China is the second in the world.Therefore,strengthening the awareness of disease etiology and epidemiology is very important to comprehensive prevention measures of rabies.The author mainly introduced the research progress on pathogen,epidemiology and diagnosis technology of rabies virus.In addition,the advantages and disadvantages of various methods were compared respectively,in order to provide scientific basis for the further study and rapid diagnosis of rabies.

It plays an important role for the endocrine and immune function of progesterone in pregnant establishment and maintenance during early pregnancy in ruminants.At present,there is a high rate of embryonic loss and early abortion in ruminant, which plagues the farmers,while the mechanism of progesterone-induced immune tolerance is not well understood.In this review,the effect of progesterone on immune tolerance is introduced during early pregnancy in ruminants,including the source of the progesterone,mode of progesterone action,effect of progesterone on organism immunity,progesterone-mediated immune tolerance.This will provide a theory reference for the study of immune tolerance induced by progesterone during pregnancy in ruminants.

In this study,we extracted total DNA from fresh fecal collected from Mongolian horses through phenol-chloroform technique and two bacterial genomic DNA extraction Kits.To find more suitable methods,we had compared the concentration and purity of DNA and the landscape of amplifying the 16S rDNA V3 region.The results showed that all three methods can be used for DNA extraction from gut bacterial in Mongolian horse.Meanwhile,the quality and purity of DNA extracted by B Kit was the most effective which could be more suitable for the extraction of genomic DNA from gut bacterial and the subsequent analysis of the diversity of bacterial population and biology experiments in Mongolian horse.

The Muslims in Yining of Xinjiang Uygur Autonomous region comprise more than half of the population,as a result,the local people need a larger amount of beef compared to the other places.To understand the status of beef consumptions of the local people in Yining city,and to promote the development of livestock husbandry in the minority frontier,we had conducted a consumer random sampling survey (more than one thousand respondents) and analysis on beef consumption in five urban areas with a dense population in Yining city.The results showed that the main local family's meat consumptions in the city were beef and mutton,which hold 43% and 29% of the total meat consumption,respectively.The most concerned factors were meat quality,safety and price when the consumers buying beef.The accepted highest purchasing price for beef was related to the consumers'age and family income.The investigation and analysis suggested that Yining city had a huge beef consumption market and a great development prospect on beef cattle,which could be a useful reference for the development of beef market on similar regions.