In this study,we cloned MAP3K5 gene cDNA sequence,compared the sequence of MAP3K5 with MAP3K families in pig and other species,and studied their conservative structure domain.Obtained the full-length of MAP3K5 gene (5 452 bp) by RACE-PCR,predicted the open reading frame (ORF) and amino acid (AA) sequence,and obtained the information of 31 exons.The low similarity were found in mRNA and amino acid sequences of MAP3K families in pig (<50%).And MAP3K5 had the comparatively high similarity with MAP3K6 in pig.The comparative analyzed the mRNA and protein sequence of pig MAP3K5 gene with other species MAP3K5 gene,founded that the ten species of MAP3K5 mRNA and protein sequence had the high similarity (>78%),and pig MAP3K5 had the comparatively high similarity with Bos taurus,Ovis aries,Canis lupus and Homo sapiens.Function domain analyzed the high similarity protein with MAP3K5 in pig,this research found that pig MAP3K6 protein had the similar conserved function domain with pig MAP3K5.However,the other pig MAP3K families didn't have the similar conserved function domain with pig MAP3K5.The ten species of MAP3K5 protein had the similar conserved function domain.The results obtained the sequence and molecular structure of MAP3K5 gene,and provided an important foundation for further function research of MAP3K5 gene in pig.

In this study,a multiplex RT-PCR assay was established to differentially detect porcine epidemic diarrhea virus (PEDV),porcine transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PRoV) after optimization of the reaction conditions.Three pairs of primers PEDV-N,TGEV-M and PRoV-VP6 were designed for specifically amplifying PEDV N gene,TGEV M gene and PRoV VP6 gene,respectively.The assay could specifically amplify PEDV,TGEV and PRoV,but not classical swine fever virus (CSFV),porcine foot and mouth disease virus (FMDV),pseudorabies virus (PRV),porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2).The detection limits of PEDV,TGEV and PRoV standard recombinant plasmids were 1.41×10³,1.41×10² and 1.41×10³ copies/μL,respectively.The repeated reaction under the same conditions obtained uniform results.The assay was used to detect a total number of 190 clinical samples,of which 42 (22.11%) samples were positive for PEDV,58 (30.53%) samples for TGEV and 34 (17.89%) samples for PRoV,and there were mixed infection among these viruses.The results indicated that this multiplex RT-PCR assay had the advantages of sensitivity,specificity and repeatability and provided a useful tool for differential detection and epidemiological investigation of PEDV,TGEV and PRoV.

To in-depth analyze the molecular structure characteristics of agouti gene,using bioinformatic methods for RNA-Seqs test to get the complete coding sequence of raccoon dog agouti gene,and its coding protein structure was forecasted and analyzed.The results showed that the length of raccoon dog agouti gene cDNA was 530 bp which contained 396 bp open reading frame (ORF),encoded 131 amino acids.Predicting the weight of the raccoon dog agouti protein molecular was 14.41 ku and isoelectric point (pI) was 9.68,as the unstable hydrophohbic protein;It contained 11 phosphorylation sites,1 glycosylation sites and a long reach 24 amino acid signal peptide;Random coil was the main secondary structure.Phylogenetic tree showed that the raccoon dog had a close genetics distance to vuipe and canis familiaris which was consistent with the traditional animal taxonomy.Through the analysis of the molecular structure characteristics of raccoon dog agouti gene,to provide the oretical basis for revealing the influence of the colour diversity molecular genetics mechanism.

The study was conducted to establish a quantitative Real-time PCR method for PRRSV Nsp9 gene rapid detection and study its expression in PRRSV infected cells.A pair of specific primers targeted to Nsp9 of PRRSV was designed and a Real-time PCR method based on SYBR Green Ⅰ fluorescent was developed for the quantization of PRRSV.The melting curve analysis using SYBR Green Ⅰ dye showed one specific peak,and no primer dimers peak was observed.No amplification was detected from HEV,SIV and PRV samples by this method.There was good reproducibility and low variation coefficient.The quantitative Real-time PCR method developed in this study would be useful for rapid laboratory diagnosis and epidemiology investigation of PRRSV.During the process of virus infecting cells,the expression level of Nsp9 increased gradually,and it got the highest at 36 h.This study laid the theoretical basis to explore the law of virus replication and clinical vaccine production.

To monitor TGEV antibody in pig,major antigenic fragments of TGE B/C zone was amplified by PCR,purified PCR product and pET-32a which were separately digested,and were connected and transformed into the BL21 competent cells.We picked out successfully transformed bacterial colonies to amplify by PCR,and put the remained bacterial colonies into LB solution to culture overnight,which was extracted to get recombinant plasmid and sequenced.We cultivated right sequenced bacteria to induce protein to express by IPTG,there was a right target band in 37 ku,which was purified by cutting polyacrylamide gel,then detected by Western blotting with homemade anti-rabbit serum,specific band (about 37 ku) could finally be seen in the right position,which proved that this recombinant protein was immunogenic.It would profitably create ELISA-kit to supervise TGEV antibody,and effectively provide some relevant value in immune situation of pig.

To develop a quantitative Real-time PCR method for detection of pigeon interleukin-8 (IL-8),a pair of specific primers was designed based on the conserved region of IL-8 gene sequence published on GenBank.A fragment of IL-8 gene was amplified from the pigeon lymphocytes template and cloned into pMD18-T vector.Plasmid DNA was extracted from the bacteria and was serially diluted to serve as a standard.A standard curve for the SYBR Green Ⅰ of quantitative Real-time PCR was established and the specificity,sensitivity and reproducibility of this assay were investigated.The results showed that the quantitative Real-time PCR melting curve only had one single melting peak.The assay was linear with R² values was 1.0;The reaction efficiency for the pigeon IL-8 gene was 103%.The detection limit of this assay was 16 copies per reaction.Other interleukin including IL-1β,IL-6 and IL-18 and double distilled water control were tested by this assay and the results were all negative.The CV values of intra- and inter-assay were less than 1.52%.The established quantitative Real-time PCR assay of this study was suitable for the detection of pigeon IL-8.It would provide basis for analyzing cytokine expression quantitatively after the host cell was infected by the virus.

In order to investigate the effect of LAT1 on β-casein expression in mammary epithelial cells of dairy cows,the LAT1 gene of dairy cow was amplified by PCR and pIRES2-EGFP-LAT1 expression vector was constructed.Then pIRES2-EGFP-LAT1 expression vector was transfected into mammary epithelial cells.The effects of LAT1 overexpression on expression of LAT1,4F2hc and β-casein were detected by Western blotting after 48 h of transfection.The results showed that pIRES2-EGFP-LAT1 was transfected into mammary epithelial cells successfully.The expression of LAT1 was extremely significantly increased (P<0.01) and β-casein was significantly increased (P<0.05) when LAT1 was overexpressed,while the expression of 4F2hc was not significantly changed (P>0.05).These results suggested that LAT1 had an important role in promoting milk protein synthesis in mammary epithelial cells of dairy cow.

According to the sequence of canine interferon-α (CaIFN-α) gene published in GenBank,primers were designed,synthesized and introduced EcoRⅠand HindⅢ restriction sites.Using canine liver DNA as a template,CaIFN-α gene was amplified by PCR,then the gene was cloned into pBV220 and pET-32a(+) expression vector,and transformed into the BL21(DE3) expression system.Recombinant fusion protein was analysed by SDS-PAGE and Western blotting.Protein was purified and the antiviral activity was detected by MDCK/VSV cytopathic inhibition assay.The results showed that pBV220-α-IFN recombinant protein has a low activity,but the activity of pET32a-α-IFN recombinant protein was up to 2.56×10⁶ U/mL.This study laid a foundation for production of highly active IFN.

Gene mutation detection in single cell is a new method used for DNA or RNA analysis based on single cell lysis products,the establishment of this technology will provide an easy and quick way for gene expression and mutation detection in single cell level.In this paper,pig 4-cell-stage parthenogenetic embryos microinjected with Cas9 mRNA and 2 target gRNA from pig SS gene were used as experimental material,and two pairs of nested primers were designed for nested PCR.Using this detection method,long fragment deletion in SS gene was detected.This method can minimize the occurrence of false negative and false positive results,which may play an important role in the pig gene mutation detection.

This study was aimed to screen gRNA of efficient knockout activity targeting Macaca fascicularis NTCP gene by the CRISPR/Cas9 enzyme digestion method and PCR amplification methods in vitro.Comparing NTCP gene sequences between Macaca fascicularis and human,the sequences of NTCP gene coding amino acid 84 to 87 and 157 to 165 were chosen as gene knockout targets.3 to 4 candidate gRNA sequences were designed in two target sequence regions through gRNA software.By screening cleavage activity targeting NTCP gene in vitro,gRNA1.2 and gRNA2.1 were selected and inserted into pLV hUbC-Cas9-T2A-GFP plasmid,respectively.The genome DNA was extracted from primary hepatocytes after gRNA1.2 and gRNA2.1 being transferred,respectively.Then NTCP sequences were amplified by PCR and sequenced by being cloned into T vector.The results indicated that compared to gRNA1.2,gRNA2.1 had much higher activity to make a frame-shift mutation in NTCP gene.This study laid a theoretical foundation for further editing NTCP gene and its biological function in Macaca fascicularis.

6 Small-tail Han sheep ram,2 to 3 year's old,with body weight of 48.3 kg±5.6 kg,were fitted with permanent rumen fistula and fed with the diet containing 70% of corn stalk.The experiment was designed as 4×4 Latin square,the sheep were orally administered with formalin at 0 mL/kg diet (control,treatment 1),1.0 mL/kg diet (low dose,treatment 2),3.0 mL/kg diet (high dose,treatment 3) and completely fauna-free (treatment 4),to study the effects of various treatments on the rumen microbiocoenosis,activities of digestive enzymes,NH₃-N and VFAs in rumen fluid of sheep,and their correlation with voluntary intake.The results showed that,compared with control,the voluntary intakes of dry matter in treatments 1,2 and 3 were increased by 14.9% (P<0.01),-6.8% (P>0.05) and -16.5%(P<0.01),respectively;NH₃-N were decreased by 10.5% (P<0.05),13.0% (P<0.05) and 23.0%(P<0.01),respectively;The protozoa number in rumen fluid were decreased by 59.3%,93.2% and 100.0%(P<0.01),respectively;The total number of bacteria were increased by 41.1%,-8.2% and 84.5%(P<0.01),respectively;The fungi copies were increased by 16.5%(P>0.01),23.5% (P<0.05) and 38.1%(P<0.01),respectively;The activities of endocellulase in treatments 1,2 and 3 were increased by 22.6%,-14.6% and -27.5%(P<0.01),respectively;The activities of exocellulase were increased by 15.5%,-23.9% and -25.2%(P<0.01),respectively;The activities of cellobiose were increased by 20.0%,-25.7% and -26.2%(P<0.01),respectively;The activities of xylanase were decreased by 0.4%(P<0.05),0.5%(P<0.05) and 0.1% (P>0.05),respectively;The activities of pectinase were decreased by 12.0%,11.0% and 2.8%(P<0.01),respectively;The activities of amylase were increased by 12.2%,15.3% and 19.9%(P<0.01),respectively;The activities of protease were decreased by 22.1%,48.0% and 22.2%(P<0.01),respectively.The correlation coefficients (R²) of the activities of the endocellulase,exocellulase and cellobiose with voluntary intake of sheep were 0.994,0.897 and 0.901.It was concluded that when the low dose of formalin was orally administrated the voluntary intake of sheep was increased,whereas the high dose of formalin or fauna-free was decreased;The factors determined the voluntary intake of sheep were the activities of endocellulase,exocellulase and cellobiose,rather than the numbers of the protozoa,bacteria or fungi,or the activities of other digestive enzymes in rumen fluid.

This research was conducted to investigate the effect of light controlling and ventilation design on cashmere growth and ammonia concentration of cashmere goat,which would provide theoretical basis for high efficient and ecological feeding of cashmere goat during the cashmere non-growing period.A total of 56 non-pregnant Shaanbei White cashmere goat with similar age,weight and parity were selected and randomly divided into 3 groups.Group I was received natural light,group Ⅱ was received controlling (illumination at 09:30 to 16:30,and light control at 16:30 to 09:30 of next day),and group Ⅲ was received same light condition except for additional 15% concentrate feed supplemented.Different ventilation schemes of goat house under the light-controlled condition were set and cashmere wool growth traits and barn NH₃ concentrations were determined.The results showed that illumination condition had no significant effects on body weight of Shaanbei White cashmere goat (P>0.05),but average daily gain of groups Ⅱ and Ⅲwere slightly higher than group Ⅰ.Compared with group Ⅰ,the fluff mixtures weight of groups Ⅱ and Ⅲ were extremely significantly increased (P<0.01),the fluff ratio were significantly increased (P<0.05),and the weight of cashmere wool,which were increased by 68.40% and 78.78%,respectively,were extremely significantly increased (P<0.01).Less illumination resulted in longer cashmere fiber stretched length,while it caused an increasing fiber fineness of cashmere by increasing illumination.No significant differences were observed between groups Ⅱ and Ⅲ (P>0.05).Under light-controlled conditions,using four mechanical ventilators during non-light controlled time (09:30 to 16:30) could significantly reduce the concentration of NH₃ (P<0.05).The results indicated that light controlling could increase cashmere production during the cashmere non-growing period of cashmere goat,however,less nutrients intake should be done to prevent the increasing of fiber diameter.Proper ventilation and clearance rate should be considered to keep air quality and goat health according to feeding density and house space.

This experiment was conducted to study the effects of eight microbial fermentation feed formulas and the different turning processes on the yeast viable bacterial number and three kinds of nutritional active substance contents to determine the optimal formula and its turning process.Nine kinds of local agricultural products,such as wheat bran,rice bran,cottonseed meal and corn flour,were used as fermentation materials and eight different formulas were designed by mixture design of DPS.Saccharomyces cerevisiae BC,XR4 and Bacillus subtilis A15 were mixed by 2:2:1 and inoculated in the eight formulas to conduct solid state fermentation.The yeast viable bacterial number and the content of mannan,β-glucan and polypeptide were measured to screen the optimal formula.Basing on the optimal formula,the turning process experiment was conducted.There were three groups:Control group,experimental group 1 and 2.The control group was without turning process,while the feed in the experimental group 1 was turned once and the feed in the experimental group 2 was turned twice.The material temperature,yeast viable bacterial number and three kinds of nutritional active substance contents were measured to determine the optimal turning process.The results showed that:① Formula 8 was the optimized formula which consisted of 8.62% wheat bran,5.85% vinegar residue,20.89% rice bran,12.55% cottonseed meal,11.35% corn flour,7.31% corn bran,1.10% corn residue,25.36% sugar residue,5.35% corn germ meal,1.02% (NH₄)₂SO₄,0.50% KH₂PO₄ and 0.10% MgSO₄.② The best turning process of formula 8 was turning twice during the solid state fermentation.When the material temperature reached 35℃,the first turning was conducted and then when the temperature reached 42℃,the material would be turned again.After that the material continued fermenting until the end.Its optimal indexes were viable bacterial number was 4×10⁵ CFU/g,and the content of mannan,β-glucan and polypeptides were 41.28 mg/100 mg,87.06 mg/100 mg and 10.32 μg/100 mg,respectively.Its improved by 100.00%,3.90%,4.89% and 1.67% separately than the control group and improved by 33.33%,3.07%,3.31% and 0.88% comparing with the experimental group 1.

β-hyroxy-β-methylbutyrate (HMB) is a metabolite of leucine.HMB has attracted considerable attention of clinical medicine and animal production field,because it is centrally involved in the regulation of multi-biological process and metabolism pathway.HMB has been reported to promote protein synthesis,inhibit protein degradation,improve livestock and poultry production performance and carcass traits,enhance immune function,reduce disease risk and mortality.The synthesis and metabolic pathways,possible mechanism and positive effect of HMB in livestock were reviewed in this paper aiming to supply a useful reference for the application of HMB in animal production.

Bacillus amyloliquefaciens is a kind of non-pathogenic bacteria and widely exists in the environment.During the metabolic process,Bacillus amyloliquefaciens can produce bioactive substances which highly inhibit the activation of multiple pathogens and fungus.In recent years,some bioactive substances are isolated,extracted and purified.With the deeper understanding of Bacillus amyloliquefaciens,it is gradually applied in animal husbandry and aquaculture.This paper summarizes the application and action mechanism of Bacillus amyloliquefaciens in animal breeding industry,it is expected to provide information for further exploration and application.

This research was aimed to study whether the lncRNA would have effects on the structure and constitution of the intestinal microbial colonies in mice.High throughout sequencing was used to sequence and analyze the intestinal microbial colonies in both transgenic and non-transgenic mice of three months old.We conducted comparison and t-test at the level of phylum and genus.The result showed that transferred into long non-coding RNA genes GTL2 (lncRNA-GTL2),the mouse intestinal microbial colony structure and composition had no significant differences in the overall,within the same gender,but there were individual differences.Therefore,this study didn't find that long non-coding RNA transfered had a significant impact on the intestinal microflorain the phylum and genus level.

This study was aimed to systematically analyze the difference of camel milk physicochemical index of different seasons,stage of lactation,production sites and milk station.Collected 18 372 testing data about raw camel milk quality which were purchased by a company in 2015.The range of each index was determined by the four point test method,SAS 8.1 least squares analysis of variance were performed on the processed data.The results showed that different area,milk stations,stage of lactation and season effect of camel milk had extremely significant influence (P<0.01) on the relative density,fat,non fat milk solid,milk protein,lactose,freezing point and ash.Therefore,the reproduction and childbirth of Bactrian camel had strong periodicity and seasonality,lead to a larger difference in camel milk yield and physicochemical index of different seasons and months within a year,at the same time,camel milk producers could learn about the quality of camel milk situation of different production sites,milk station,stage of lactation and season using such analysis methods,the quality of camel milk product was determines by the physical and chemical indicators and health conditions of the raw camel milk.

To explore the antigen harvest time of Mycoplasma bovis (M.bovis) and the antigen quantitation alternative method,M.bovis 08M strain was inoculated in the Thiaucourt's medium.Four growth curve plottings were made by measuring color change units (CCU),colony forming units (CFU),protein concentration and nucleic acid levels.Both the results of CCU and CFU counts showed that the growth of M.bovis was divided into four phases,the logarithmic phase appeared after being cultrured 10 h,the stationary phase appeared at 30 h with the highest number of viable cells up to 1.0×10⁸ CCU/mL and 7.7×10⁷ CFU/mL,and the decline phase started at 75 h.The protein concentration of M.bovis increased rapidly from 15 to 35 h,reached 72.06 μg/mL at 35 h,then maintained at 58.38 to 70.65 μg/mL.The nucleic acid levels of M.bovis increased rapidly from 15 h,and the cycle threshold (Ct) values were maintained between 15.32 to 17.84 after 25 h.These results indicated that there was a good correlation between the protein concentration and viable count of M.bovis at the early stationary phase,which was the best time period to harvest antigen.The protein concentration determination could be an alternative method to quantify antigen contents of M.bovis when preparing inactivated M.bovis vaccine.

Single immunoglobulin IL-1 related receptor (SIGIRR) is a member of TIR superfamily.Recent evidences suggested that it could negatively modulate ILRs/TLRs-mediated innate immune response,and in particular its important role focused from infectious and intestinal inflammation to autoimmunity or cancer-related inflammation had been illuminated.Here,we summarized our current understanding of the expression patterns of SIGIRR gene and protein in different specises including human,mouse and bovine,its downregulate function of the IL-1/TLRs singal pathway and specific negative regulation mechanism ascribed to its structure specificity,and also its elaborate regulating process related to the occurrence and development of different pathogen condition.And then it can not only provide theoretical references for clinical inflammatory related disease,but also broaden the application scope of SIGIRR gene in the treatment of animal diseases.

The experiment was aimed to explore the influence of water extract from fresh betelnut's different parts on physiological indexes in mice.Four-week-old and seven-week-old SPF KM mice at the weight of (18±2)g and (35±2)g were used in the experiment,respectively.48 four-week-old mice with half male and half female were randomly divided into four groups.These mice were intragastricly administered with distilled water (control group,CK),water extract from betelnut,water extract from fresh betelnut's peels and seeds once a day and were observed for 14 days to determine the survival rate,feed intake,water intake and body temperature.36 seven-week-old male mice were randomly divided into 4 groups to determine deformity rate of sperm in mice,and the intragastric administration was the same as above.The results showed that the survival rate of betelnut's seeds were 0 at the sixth day,and other treatments had no death;Compared with CK,the experimental treatments' feed intake and water intake were reduced,and they were the lowest in the betelnut's seeds treatment and had significant difference with other treatments (P<0.05),the betelnut treatment took the second place.All the treatments' body temperature were lower than CK,and the peels treatment's body temperature was the most stable one and was similar to CK(P>0.05),the body temperature in the betelnut's seeds treatment was the lowest and had significant difference with other treatments (P<0.05).Compared to CK,the other 3 treatments all increased sperm deformity rate of mice,the highest sperm deformity rate went to betelnut's seeds treatment (P<0.01),and betelnut treatment took the second place (P<0.01),then was the peels treatment (P<0.05),moreover,there were extremely significant difference among these 3 treatments (P<0.01).The results showed that the water extract from fresh betelnut's seeds had the greatest influence on mice,then was the water extract from betelnut,while the impact of water extract from fresh betelnut's peels was less.

The purpose of this study was to understand the change trend of growth hormone (GH) and growth hormone receptor (GHR) in serum of Mashen and Large White pigs during the period from 0 to 6 months of age,and to analyze the influence of GH and GHR on growth rate.The method of ELISA was used to detect the concentration of GH and GHR in serum of Mashen and Large White pigs from 0 to 6 months of age.The results showed that the variation trends of GH in Mashen pig was roughly the same as in Large White pig,the GH concentration was increased with age increasing after birth and reached the peaks at 4 and 5 months of age for Mashen and Large White pigs,respectively,and then decreased gradually.The serum GH concentration in Mashen pig was a little greater than that in Large White pig at 3 and 4 months of age,on the contrary,the serum GH concentration in Large White pig was greater than that in Mashen pig at other months.During the period from 0 to 6 months of age,the difference of GHR concentration in serum was not significant in Mashen pig (P>0.05).In Large White pig,the serum GHR concentration at 1 month of age was lowest,and was significantly lower than that at 4 and 6 months of age (P<0.01;P<0.05).During the period of 0 to 2 months of age,the GHR concentration in Mashen pig was greater than that in Large White pig,but the difference was extremely significant only at 1 month of age (P<0.01).Conversely,the serum GHR concentration in Large White pig was greater than those in Mashen pig during the period from 3 to 6 month of age,there was significant difference at 4 and 6 months of age (P<0.05),and there was extremely significant at 5 months of age(P<0.01).The concentration of GH and GHR in serum was related to the developmental stages and genetic background of pig,and its change trend was in accordance with the trend of growth rate.

Buffalo mammary epithelial cell,cumulus cell and fibroblasts were transfected by adenovirus vectors and compared their transfection efficiency.293 cells were transfected with pBHGloxdelE13cre and pDC316-eGFP by liposome,the virus was collected and titer was detected.Buffalo mammary epithelial cell,cumulus cell and fibroblasts were exposed to different multiplicity of infection (MOI) of adenovirus vectors.After 72 h,the cells were observed with inverted fluorescence microscope,and transfection efficiency was calculated.When the MOI was 25,50,100,200 and 400,the transfection efficiency of fibroblasts were 0.7%,7.0%,9.0%,12.5% and 34.0%,the transfection efficiency of cumulus cells were 42.5%,55.3%,57.4%,76.0% and 80.0%,the transfection efficiency of mammary epithelial cells were 88.7%,100%,100%,100% and 100%.The results showed that the transfection efficiency of mammary epithelial cell was the best,followed by cumulus cell,and fibroblast was poor.

In order to optimize the preparating conditions on pig intracytoplasmic sperm injection (ICSI) embryos,the Piezo operating system was used in this experiment and the embryonic survival rate,cleavage rate and cell numbers of blastocysts developed in various stages were measured as traditional and Piezo operating system,the concentration of CB,the operating temperature with Piezo operating system and different sperm processing method in preparating pig ICSI embryos.The results showed that Piezo operating system could significantly improve the survival rate and cleavage rate of injected oocytes (P<0.05),but there was no significant effect on blastocyst rate.There was a slight damage to oocyte added CB in the operating fluid,and adding 7.5 μg/mL CB was most concentration,30℃ was most appropriate operating temperature,but there was no significant effect of early development on pig ICSI embryos with breaking sperm tail with ultrasonic wave.The results suggested that Piezo operating system could significantly improve the survival rate.The results suggested that breaking sperm tail with ultrasonic wave,adding 7.5 μg/mL CB in operating fluid and 30℃ of operating temperature and using Piezo operating system was more suitable for preparating pig ICSI embryos.

To compare the histological changes of the adult yak's epididymis in different breeding seasons,six adult yak testis in breeding season and nine adult yak testis in breeding interval were collected for structure investigation by HE staining,Masson's and Gomori's histochemistry methods,and IPP (Image-Pro Plus) statistics method was used to quantitative statistics.The results showed that comparing with the adult yak in the breeding interval,the epididymis ducts of adult yak in the breeding season were covered with the columnar ciliated epithelium.The collagen and reticular fiber in cauda epididymis were obviously more abundant than caput and corpus epididymitis.And the thickness of columnar epithelium cells in caput and corpus epididymitis,the length of the cilia in caput,and also the internal and external lumen diameter of caput and corpus epididymitis were all significantly increased in the breeding season (P<0.05),but the external lumen diameter of the cauda epididymis had no significant differences (P>0.05).In conclusion,the research showed that the distribution of collagen and reticular fiber in adult yak's epididymis interstitial were similar,and they were more rich in cauda epididymis,which might relate to the capacity and the sperm transport;The changes of the epithelial thickness,the length of cilia,the internal and external lumen diameter were close related to the different breeding seasons,and it might be a common phenomenon in plateau mammals that the enlargement and reduction of the epididymal duct in different breeding seasons.

In order to investigate the genetic diversity and the origin of evolutionary relationship of Zhongdian yak,we analyzed the complete sequence of 15 individuals Cytb gene,its sequence polymorphism was analyzed,and the phylogenetic tree was constructed.The results showed that the length of the nucleotide sequence were 1 140 bp,with nucleotide frequencies of 26.3%,31.8%,13.1% and 28.8% for T,A,G and C,respectively.Three haplotypes were identified of 15 individuals,with 3 polymorphic sites,including two conversions,one transversion,haplotype diversity was 0.2571 and nucleotide diversity was 0.00035.Phylogenetic analysis suggested that Zhongdian yak and Bos mutusc clustered firstly,then gathered with Bison bison,which indicated that there were high genetic similarity and closer genetic relationship,genetic similarity with other cattle genus was relatively low.Combining with the proof of molecular biology and paleontology,the result supported the point that Bos grunniens and Bos mutus were classified as an alone genus in Bovinae.

The latest research progress on domestic and international rabbit genetics and breeding in 2015 were reviewed in the present paper based on three aspects of traditional breeding,molecular breeding and reproductive technology,so as to provide references for rabbit breeders and researchers.At abroad,the researchers focused on the analysis of carcass traits in molecular breeding,genetic diversity in rabbits were also studied.While the key point was still efficient reproductive technique in foreign breeding studies,including semen cryoprptectants development and semen motility.In china,traditional breeding mainly contained evaluation and comparison of Chinese local breeds and cultivating breeds.In domestic rabbit genetic and breeding,molecular bases were still the point of importance for traits of growth,carcass and fur.The researchers focused on breeding performance and semen motility.Overall,the genetic and breeding studies focus on partial different aspects at home and abroad.In China,researchers on molecular bases of reproductive traits should be given further emphasis.

Molecular breeding technology is direct selection at the DNA level by randomly distributed molecular markers throughout the genome polymorphism comparison,it is helpful to carry out more comprehensive study of polymorphism on investigated subjects,meanwhile,more information can be got,and the accuracy of selection will be higher,thereby,the milk production efficiency and economic benefits can be improved.From the perspective of the dairy cattle molecular breeding technology,the author expounds the research challenges,problems and research progress on several aspects,such as localization of QTL,MAS,transgenic breeding and GWAS,points out that molecular breeding techniques have a very important role in dairy cattle breeding and improvement process.Further research and application of molecular breeding technology not only can accelerate the development of dairy cows in the core group of comprehensive construction,but also is very necessary to cultivate a bull.

Epigenetics means that there are heritable changes in gene expression while without a change in DNA sequence.In the recent years,the researches of epigenetic regulation on livestock field have developed rapidly.The main studied domestic animals include poultry,swine,bovine,sheep and goat,thus a new subject——livestock epigenetics is developing.Livestock epigenetics mainly uses various kinds of epigenetic modifications involved in DNA methylation,histone modification and non-coding RNAs regulation to study livestock growth and development,disease resistance,reproduction and economic traits,and so on.Here,we reviewed the mainly research fields and progress on livestock epigenetics,the trends of livestock epigenetics studies were also discussed to comprehensively understand the molecular basis of the complex traits formation of livestock and poultry,which greatly broadened the research and strategy to improve the economic traits of livestock and poultry.

This paper was aimed to study the impact of hatching egg weights and egg shape indexs on hatching of Zi goose,and then sum up the best scheme for hatching eggs screening,so as to provide scientific reference for the selection and retention of Zi goose individuals and family hatching eggs,as well as the breeding and incubation production.2469 hatching eggs of Zi geese of the second year were chosen with the weight range of 94.8 to 154.4 g and an average weight of (123.3±9.5) g;the egg shape index range was 1.24~1.63 and the average index was 1.45±0.05.Using 2 factors 4 levels test design,the weight and the shape index of eggs were divided into four levels and all the eggs were separated into 16 hatching groups.The weight and the shape index were measured and labeled on eggshells.Two hatching experiments were conducted in the same instrument and the hatching conditions were same.The results showed that:① Egg fertility rate was the highest in egg weight <118.0 g group,followed by >131.9 g group;Hatchability of fertilized eggs was the highest in >131.9 g group,followed by 125.0~131.9 g group,and the hatchability of fertilized eggs increased along with the increase of egg weight;The highest healthy chick rate of breeding egg hatching lay in 125.0~131.9 g group,followed by >131.9 g group;The highest breeding egg hatching gosling birth weight lay in >131.9 group.② When egg shape index was between 1.47 to 1.51,the egg fertility rate and healthy chick rate were the highest,followed by >1.51 group;In the group 1.47 to 1.51,hatchability of fertilized eggs was the highest,followed by 1.42 to 1.46 group.In conclusion,hatching egg weight of Zi goose had significant impact on egg fertility rate,hatchability of fertilized eggs,healthy chick rate and gosling birth weight (P<0.05),and the egg weight of Zi goose between 125.0~154.4 g was advisable;on the other hand,egg shape index also had significant impact on egg fertility rate,hatchability of fertilized eggs and healthy chick rate (P<0.05),but had no impact on gosling birth weight (P>0.05),and the egg shape index between 1.47 to 1.51 was appropriate.Egg fertility rate,hatchability of fertilized eggs,healthy chick rate and gosling birth weight were affected by interaction of egg weight and shape index.Compared with the weight of eggs,the egg shape index had greater influence on healthy geese rate.The egg shape index had no direct impact on birth weight of young geese,but it could be influenced by the interaction between the egg shape index and the weight.Thus it was not very scientific to take either the weight or the index as the only basis for choosing qualified hatching eggs.

To study the immune enhancement activity of CpG motifs on the recombinant Eg95 antigen of Echinococcus granulosus,Escherichia coli BL21(DE3) containing pET-32a-3Eg95 was grown in LB medium,and IPTG was then added for induction.The recombinant Eg95 protein was expressed and later harvested by affinity chromatography.The obtained antigen was mixed either with the conventional adjuvant Quli-A or with different types of CpG motifs (pUC18-CpG or CpG ODN),to prepare vaccine candidates for mice immunization.The average levels of serum IgG antibody were tested by indirect ELISA and the quantification of cytokine expression were determined by Real-time quantitative PCR after in vitro stimulation.The results identified that the size of the recombinant protein was 56 ku as predicted,and the recombinant protein could be recognized by serum from Echinococcus granulosus-infected sheep.The average levels of serum IgG antibody (D_{450 nm}) of the pUC18-CpG and CpG ODN groups on 14,28 and 42 d were 3.10,3.03,3.22 and 2.98,3.12, 3.27,respectively.Antibody levels from both CpG groups were significantly higher compared to the conventional adjuvant Quli-A group (P<0.05) The average level of serum IgG antibody in CpG ODN group was a little higher than that in pUC18-CpG group(P>0.05).After stimulation by recombinant protein 3Eg95,the relative expression levels of IFN-β,IFN-γ and TNF-α of pUC18-CpG and CpG ODN groups were 6.88,2.35,6.28 and 5.03,2.85,7.07,respectively,and both were higher than that of the Quli-A group (P<0.05).The results indicated that pUC18-CpG and CpG ODN had significant immune enhancement property on Eg95 antigen in both humoral and cellular immune response,and the immune enhancement activity of CpG ODN was slightly better than that of the pUC18-CpG.Hence,pUC18-CpG and CpG ODN could be used as efficient immune adjuvants enhancer in future vaccines against Echinococcus granulosus infection.

The purpose of this study was to evaluate drug safety of Dichroa febrifuga oral liquid (DFOL),and the acute toxicity test of DFOL was established in mice based on modified Karber's method and dose increasing method,and histopathological examination had been applied to explore the main target organs of its toxic injury.The dosage was determined by preliminary experiment,a total of 60 Kunming mice were selected and divided randomly into one control group and five experimental groups,DFOL was administered by intragastric gavage at five different doses (1.00,1.40,1.96,2.74,3.84 g/(kg·BW)),and observed for 7 days continuously,the LD₅₀ value and 95% confidence interval of LD₅₀ were determined by the modified Karbers method.It showed that LD₅₀ was 2.0961 g/(kg·BW) and its 95% confidence interval was 1.7414 to 2.5429 g/(kg·BW).The liver and kidney of some mice in 1.96,2.74 and 3.84 g/(kg·BW) drug groups appeared severe edema,congestion and white necrotic foci by necropsy.Acute liver injury was found in mice by histopathological examination and the liver cell showed degenerative change,kidney damage showed renal congestion,interstitial edema,renal tubule epithelial cloudy swelling and degeneration.The liver was damaged seriously in the main organ and the main toxic organ of DFOL was liver.The test showed that the toxicity of DFOL was lower and it had high security in a conventional doses for clinical anti-coccidiosis application,however,high dose,long course of medication had toxicity,and the toxicity of DFOL should be analyzed and evaluated combining with long-term toxicity tests and many other inspection results.

In order to figure out the antiviral effects of Radix Isatidis,Astragalus herb,Artemisia annua and their polysaccharides on porcine reproductive and respiratory syndrome virus (PRRSV) in vitro,the antiviral activity of the three Chinese herbal medicine and their polysaccharides,Isatis root polysaccharide (IRPS),Astragalus polysaccharides (APS) and artemisinin maximum were evaluated by adding to Marc-145 cell cultured with PRRSV respectively.The results showed that the safety concentrations of IRPS,APS and artemisinin to Marc-145 cells were 0.03,0.03 and 0.06 mg/mL;The safety concentrations of Radix Isatidis,Astragalus herb,Artemisia annua to Marc-145 cells were 6.25,6.25 and 3.13 mg/mL.Almost all the cells (about 100%) were protected from PRRSV when Astragalus herb were added early to PRRSV,while IRPS had the same effect on PRRSV among three extracts with 48.03% cells were protect and artemisinin could not protect cells when the concentration was lower than 0.16 mg/mL.IRPS had no effect on PRRSV while APS and artemisinin got a protection rate for cells at about 20% when simultaneously added with PRRSV,under the concentration of 0.32 mg/mL.In terms of simultaneously added with PRRSV,Chinese herb Astragalus could exterminate the virus directly with its protect cells coming to 100% and more.Artemisia annua could promote the growth of cells but its stability was weak.Its protection rate decreased as its concentration reducing.In the experiment of exploring the antivirus effect of infected cells,polysaccharides of these three herbs had nearly the same effect.When in a concentration of 0.01 mg/mL,IRPS,APS and artemisinin respectively protected 24.57%,24.30% and 26.20% cells.However,the protection rate to cells of the three herbs themselves was respectively up to 100% or more.In conclusion,in the extracts of the three herbs,APS had the best antiviral effect,followed by artemisinin and IRPS,and in the single herbal medicines,Astragalus was the best and Artemisia annua was worst.Therefore,Astragalus and APS had the best antiviral effect on PRRSV.It needed more research on Astragalus and APS,and try to use them as antiviral drug in clinical application.

Bactericidal/permeability-increasing protein (BPI) has a strong effect on sterilization (mainly for G^- bacteria),neutralizing the activity of lipopolysaccharide (LPS) and enhancing the phagocytosis of mononuclear cells and neutrophils to pathogenic bacteria.The biological functions of BPI have been researched widely in recent years,which is known as "super antibiotic" and has been explored by many scholars as a candidate gene for resistance.This author summarized the research progress and application prospect of the BPI gene in the pig resistant breeding by introducing the structure,biological function of BPI gene and its relationship with the resistance,which was aimed to provide theoretical references and basis for the function research of pig BPI gene and its practical application in resistance breeding in future.

Cephalonium is a second generation cephalosporin.It is effective to prevent and treat mastitis during dry period.It has a broad antibacterial spectrum,strong bactericidal activity,less allergic reactions and low toxicity,and so on,especially showing a good antibacterial activity to Staphylococcus aureus and Streptococus.In this paper,the physical and chemical properties,pharmacokinetic,pharmacology and toxicology,residue and withdrawal periods,application in the prevention and treatment of mastitis in dairy cows,and the prospect of the development of cephalonium were investigated and reviewed.

A detection method of dexamethasone sodium phosphate in houttuynia injection,bupleurum injection,vitamin C injection,lincomycin hydrochloride injection,and asragulus polysacharin injection was established by UPLC-PDA.The Waters ACQUITY UPLC^® BEH C18 column(2.1 mm×50 mm,1.7 μm) was used,the mobile phase consisted of 0.75% triethylamine phosphatic liquor/solution (pH 3.0)-methyl alcohol-acetonitrile(50:45:5),the flow rate was 0.3 mL/min,the column temperature had remained 25℃,photodiode array detector wavelength range was 190 to 400 nm,the recorded wavelength was 242 nm,the injection volume was 10 μL.The linear range of dexamethasone sodium phosphatel was 10.0 to 200.0 mg/L (R²=0.9999),the mean recovery and RSD for dexamethasone sodium phosphate in houttuynia injection,bupleurum injection,vitamin C injection,lincomycin hydrochloride injection and asragulus polysacharin injection were 97.54% and 1.14%,101.59% and 0.19%,100.92% and 0.92%,99.82% and 0.73%,100.08% and 0.62%,respectively.This method was accurate,easy and fast,it was suitable for detection of dexamethasone sodium phosphatein houttuynia injection and other four kinds of injections.

To investigate the reason of the diarrhea in Guizhou pony,we used the feces of pony as experimental material to isolate and detect pathogenic bacteria.S6 strain was isolated from SS and XLD medium,and identified using Gram staining,biochemical tests and molecular phylogeny methods.The results showed that S6 strain could growth on SS and XLD medium,and the Gram staining was negative.Biochemical test suggested that its phenotype features were accordance with Salmonella.The 16S rRNA gene sequence of S6 strain was determined in a nucleotide sequence identity of 99% with Salmonella typhimurium ATCC 13311 (NR_119108).Based on the morphological and molecular phylogenetic results,the strain was identified as Salmonella typhimurium S6 strain.It was virulent to mice with the median lethal dose (LD₅₀) of 4.71×10² CFU.Then,we amplified the invasion protein A (invA) gene by PCR method.The invA gene isolated from S6 strain contained 6 to 8 bp different from the known gene,which resulted in only one amino acid substitution.The mutant sites of invA gene might attribute to the pathogenicity of S6 strain.The detection rate of Salmonella was 57% in Guizhou pony population.It was inferred that the diarrhea in Guizhou pony might be caused by virulent Salmonella typhimurium.

Hemagglutinin protein plays an important role in disease prevention and treatment of the swine influenza virus (SIV).In order to clone and express subtype H3N2 SIV A/swine/Henan/1/2010 (H3N2) HA1 and HA2 genes with chicken embryo allantoic fluid containing the H3N2 SIV after extraction of RNA.The HA1 gene and HA2 gene were amplified from the total RNA using RT-PCR and they were inserted into prokaryotic expression vector pET-28a (+) to construct recombinant expression vector.Then the vector was transformed and expressed in E.coli BL21 (DE3) pLyS.Then the bacteria were induced by IPTG and their lysates were analyzed by SDS-PAGE and Western blotting,respectively.The monoclonal antibody 1C10,which was specific to HA protein,was used as primary antibody in Western blotting analysis.It was found that the expressed recombinant HA1 protein was 34.8 ku and recombinant HA2 protein was 23.2 ku analyzed by SDS-PAGE.As demonstrated by Western blotting,this HA1 expressed products showed the capacity of reacting with monoclonal antibody 1C10.All the results suggested that the expressed HA1 and HA2 proteins of H3N2 influenza virus would be very helpful in the development of rapid diagnosis method and vaccine development,which would facilitate further study on the function of HA at the same time.

To design chimeric DNA vaccine targeted to antigen-presenting cells with enhanced efficacy to induce immunization.The plasmid containing the gene encoding the extracelluar domain of canine cytotoxic T-lymphocyte antigen 4(CTLA-4₁₂₅),was directly fused to the gene fragment for major antigenic epitopes of VP2(VP2₂₂₈) by molecular engineering technology.The plasmid containing VP2₂₂₈ alone was also constructed to serve as control and transfection of COS-7 cells with the two resultant plasimds was performed respectively,followed by assay of Western blotting.The mice were immunized with the constructed two plasimds,respectively.After immunization,the antibodies anginst CPV in the immunized mice at different times were measured by HI.The spleen lymphocyte proliferation response was determined by lymphocyte proliferation assay,and the interferon-γ (IFN-γ) expression level of the mouse lymphocytes were measured by ELISA.The eukaryotic expression plasimds of CTLA-4₁₂₅-VP2₂₂₈ fusion protein or VP2₂₂₈ alone were cloned.Western blotting showed that the two recombinant proteins could be expressed.Immunization results showed that the antibody levels in serum of CTLA-4₁₂₅-VP2₂₂₈-immunized mice were significantly higher than that of VP2₂₂₈-immunized mice (P<0.05).The lymphocyte stimulation indexes and secreted IFN-γ levels of the CTLA-4₁₂₅-VP2₂₂₈-immunized mice were significantly higher than that of VP2₂₂₈-immunized mice (P<0.05 and P<0.01),respectively.CTLA-4₁₂₅-VP2₂₂₈ chimeric DNA vaccine stimulates strong immune response in mice,making it possible for further exploration into chimeric DNA that target the antigen to APCs.

In order to produce high titers of infectious bursal disease virus(IBDV) antigen,the proliferation technology of chicken IBDV BJQ902 strain in DF-1 cell line was studied by the selection and optimization of the following five culture conditions,including the amount of inoculated virus,harvest time,inoculation time,culture temperature and serum concentration in maintenance media.The results showed that the optimal proliferation conditions of IBDV BJQ902 strain in DF-1 cell line were obtained as follow:The culture temperature was 37℃,the inoculum concentration was between 0.01% and 0.1% (V/V),the inoculation time was between 48 and 72 h after cell passage,the serum concentration in maintenance media was between 1% and 2%,the harvest time was between 60 and 72 h after inoculation.Under the optimal conditions,the virus titers were between 10^8.3 and 10^8.7 TCID₅₀/0.1 mL.

The objective of this study was to investigate the infection and pathogenicity of E.coli O157:H7 from 564 fecal samples in three parts of Xinjiang,such as Akesu,Yili and Tacheng.Suspected strains were detected by biochemical and PCR after enrichment by EC broth,being chosen by SMAC plate and screened by MUG test.On the basis of this,the isolated strains were used in mice attack drug test.The results showed that there were two strains of E.coli O157:H7 (named Y166 and Y226,respectively) were isolated from the collected samples of Yili,and the detection rate was 0.88%.In the mice attack drug test,the mice of Y166 and Y226 groups were all died within 48 h.In conclusion,two strains of E.coli O157:H7 were isolated and identified with pathogenic from samples in Yili area,and no E.coli O157:H7 were identified in the samples from Akesu and Tacheng.

The purpose of this experiment was to study the immunization rule of the egg yolk antibody affected by different vaccines,immunization dose and injection ways and further to discuss the optimal immunization procedures of the laying hens for the preparation of egg yolk antibody against swine Japanese encephalitis virus.180 brown laying hens without any vaccines were selected and divided into 18 groups randomly,each group of 10 hens.Groups 1,2 were the control groups,injected with the sterile saline;Groups 3 to 10 were injected with subcutaneous or intramuscular injection,and the vaccine was injected with 0.2,0.5,1.0 and 1.5 mL successively.Groups 11 to 18 were also adopted two kinds of injection,followed by the same dose of vaccine immunization.Six eggs of each experimental group were gathered before immune day and after 3,7,10,14,18,21 and 28 days,the egg yolk antibody was extracted and the titer was determined.As a result,the egg yolk antibody titers of groups 1 to 6,11 and 12 were all 0,and no significant immune response produced;The hens from 7 to 10 groups were injected with the inactivated vaccine.After 7 days,the average antibody titer reached the peak,and the duration of the antibody was 14 days.The hens from 13 to 18 groups were injected with the attenuated virus vaccine.After 14 days,the average antibody titer reached the highest value,and the duration of the antibody was 21 days.The egg yolk antibody titers were not significantly different in the two compared experiment groups with the same injection dose but with different injection ways (P>0.05).With the same injection way of each experiment group,and the difference was significant (P>0.05).Compared with some groups with the same injection and vaccine,the titer of yolk antibody was gradually increased with the increase of the immune dose,and the difference was significant (P<0.05).The results showed that,no matter intramuscular or subcutaneous injection,in order to produce a significant immune response to hens,the immune antigen dose was 1.0 mL inactivated vaccine or 0.5 mL attenuated vaccine at least.Compared with the attenuated and inactivated vaccine,inactivated vaccine stimulated the body to produce the antibody faster,but the maintenance time was shorter;The lower dose of attenuated vaccine could stimulate the body to produce antibodies,but the speed was slower,the maintenance time was longer.

The assay was aimed to investigate a fast,simple and efficient method with microwave heating for extracting DNA of Bacillus and Lactobacillus.Treated in a microwave oven at rated power (800 W) for a certain time (40 to 150 s),then centrifuged for supernatant.The supernatant was transferred to a sterile tube for PCR testing and agarose gel electrophoresis.Sequencing was carried out after determining the purity and yield.The results showed that heating for 40 to 150 s at 800 W microwave power conditions could effectively extract DNA of Bacillus and Lactobacillus.The obtained DNA quality was good (D_{260 nm}/D_{280 nm} was 1.8 to 2.1).The DNA concentration in line with the PCR testing requirements;Sequencing results was satisfied with the routine molecular biological research.The microwave extraction method had simple,fast and efficient features,and a wide range of practical,which provided a simple means for rapid molecular assay.

Measuring subsection body measurements of Thoroughbred×Yili horses,Orlov trotter×Yili horses,Russia trotter×Yili horses and Hanoverian horse,and calculated the proportion and index,analysing differences in each body measurement,index and proportion.The results showed that the body height,body length,chest circumference,cannon bone circumference,neck length,humerus length,back length,lumbar spine,rump length,rump high,thighbone length and tibiae length of Thoroughbred×Yili horses,Orlov trotter×Yili horses and Russia trotter×Yili horses were extremely significantly lower than Hanoverian horse (P<0.01),the trunk index of Thoroughbred×Yili horse was significantly higher than Orlov trotter×Yili horses (P<0.05),the humerus length/back length,thighbone length/back length and tibiae length/back length of Thoroughbred×Yili horses,Orlov trotter×Yili horses and Russia trotter×Yili horses were extremely significantly lower than Hanoverian horse (P<0.01),all body measurements of Orlov trotter×Yili horses had no significant difference with Russia trotter×Yili horses (P>0.05).The experiment results showed that the difference in body structure of horses for different uses could provide data reference for breeding these horses.