In order to elucidate the function and molecular mechanisms of buffalo signal transducer and activator of transcription 4 (STAT4) gene during folliculogenesis,embryogenesis and lactogenesis,buffalo STAT4 gene was studied with 3' -RACE,bio-informatics analysis,eukaryotic vector construction and cell transfection technologyin this study.The results showed that the coding region of buffalo STAT4 was 2 247 bp,3'-was 268 bp,and encoded 748 amino acids;BLAST analysis showed that the buffalo STAT4 gene shared 99%,99%,99%,95%,93%,93% and 92% of similar nucleotide sequence with that of Bos taurus, Capra hircus,Ovis aries,Sus scrofa,Equus caballus,Canis lupus and Homo sapiens,respectively.STAT4 protein was weakly acidic,without signal peptide,located in the cytoplasmic,and with the presence of STAT_int,STAT_alpha,STAT_bind and SH2_STAT4 domain.bta-miR-200a,bta-miR-2429 and bta-miR-2410 and so on were potential microRNAs of STAT4 3'-UTR.The buffalo STAT4 eukaryotic expression vector was successfully constructed,after transfected into HEK293T cell lines,STAT4-EGFP fusion protein was detectable.

To explore the mechanism of MHCⅠ molecule in immune response,chicken MHCⅠα and β₂m genes were cloned by PCR.Then the fragments were inserted into the eukaryotic expression vector with fluorescent protein,and the recombinant plasmids pEGFP-MHCⅠα and pmCherry-MHCⅠβ₂m were constructed.The recombinant plasmids were transfected into 293T cell with lipofectin reagent.The gene products of recombinant plasmids were mainly located to endomembrane system of the cells by fluorescence microscopy,and changed the intracellular localization of the fusion with the fluorescent protein.Moreover,the positive reactions were observed by the method of Western blotting,and the proteins had the molecular weight of 68.3 and 41.3 ku,respectively,in accord with the target proteins.The results showed that the recombinant plasmids were expressed in 293T cells with a good immunological activity,and the proteins had the binding reaction with specific antibodies.

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

In this study,through cloning buffalo MBD3 gene and analyzing the biological information of MBD3 gene sequence,and constructing the expression vector of buffalo MBD3,to provide a basis for the function research of buffalo MBD3 gene on embryo development and iPCSs.The total RNA was extracted from buffalo fresh ovary,and MBD3 gene was amplified and sequenced,the sequence was systemically analysed with bioinformatics techniques.And the MBD3 CDS was cloned into the pEGFP-C1 vector.Then the recombinant plasmid pEGFP-C1-MBD3 was transferred into the HEK293T cells and buffalo fetal fibroblasts (BFF),the expression was analyzed by RT-PCR,Western blotting and fluorescence microscope.The results showed that 898 bp of MBD3 gene fragment including whole 774 bp CDS was cloned and sequenced,and encoded 257 amino acids.The multiple sequence alignment and analysis of phylogeny tree showed that MBD3 gene was highly conserved in the process of evolution.Especially the MBD domain,the MBD domain of buffalo MBD3 gene shared 100% of similar nucleotide sequence with Bos taurus,and shared 97% of similar nucleotide sequence with Homo sapiens,Sus scrofa and Pan troglodytes.The recombinant plasmids pEGFP-C1-MBD3 were transferred into HEK293T cells and BFF,fluorescence observation,RT-PCR and Western blotting were used to analyze the expression of buffalo MBD3.The results suggested that the expression vector of buffalo MBD3 gene was successfully constructed.The study laid the foundation for the function research of MBD3 on embryo development and inducing of iPCSs.

In order to explore the biological characteristics of Oct-1 gene in mouse,CDS region of Oct-1 gene in mouse was cloned.And the sequence,physicochemical property,subcellular localization,target genes and conservative structure domain of Oct-1 gene were analyzed by bioinformatics method to predict whether the Oct-1 gene could regulate the production of melanin.The results showed that the length of Oct-1 gene in mouse was 2 313 bp.The formula of Oct-1 protein was C₃₄₂₅H₅₆₀₆N₉₇₆O₁₁₆₃S₁₅,which was an unstable and soluble protein.The secondary structure of Oct-1 mainly contained random coil and α-helices.Subcellular localization of Oct-1 was mainly in the nucleus,which Oct-1 might play a role of signal transducer or transcription regulation in the energy metabolism and cofactor biosynthesis.The Oct-1 gene regulated the expression of 13 genes associated with the melanin formation of mouse.The protein was encoded by Oct-1 gene including a homeo-domain and a POU domain.There were binding sites of Oct-1 on the promoters of MITF,TYR,TYRP1 and TYRP2. Oct-1 might interact with Brf2,Pou2af1 and Tbp to regulate the colour gene expression.We inferred that the transcription factor Oct-1 played an important role in melanogenesis and these results provided a theoretical basis for studying the coat colour formation mechanism.

In this study,IBV HH06 complete E gene was firstly cloned and sequenced.According to the hydrophilicity and antigenic index analysis,its partial gene (193 to 327 bp) was subcloned into prokaryotic expression vector pET-32a(+) and eukaryotic expression vector PVAX1.The recombinant plasmid pET-32a-E1 was transformed into E.coli Rosetta (DE3) and induced with IPTG.The recombinant IBV truncated E1 protein with molecular weight of 23 ku was observed as expected.It could be recognized by positive IBV antisera in Western blotting with high reactivity.Then the purified recombinant protein was used as antigen for immunization of rabbit to prepare polyclonal antibody.Indirect ELISA showed that the titer of polyclonal antibody was 2²⁰,and it had high reactivity and specialty with recombinant protein.Furthermore,IFA test demonstrated that this polyclonal antibody could react with Hela cells transfected with PVAX-E1 plasmid and IBV-infected CEK cells.The IBV E polyclonal antibody obtained in this study laid a foundation for further functional research of E protein in IBV pathogenesis.

In this study,the CDS sequence of buffalo Keap1 gene was cloned and analyzed,then its expression pattern in different tissues was also investigated.A pair of primers of buffalo Keap1 gene was designed based on the nucleotide sequence of Bos taurus Keap1 gene from GenBank,and then the buffalo Keap1 gene was amplified.Using the bioinformation techniques,the gene sequence and the protein structure were analyzed.The expression level of Keap1 gene in different tissues were detected with Real-time quantitative PCR.The results showed that the length of buffalo Keap1 gene coding sequence was 1 875 bp and encoded 624 amino acids.The multiple sequence alignment results showed that buffalo Keap1 gene shared 99%,96%,92% and 90% of similar nucleotide sequence with that of Bos taurus,Ovis aries,Sus scrofa and Homo sapiens,respectively.And the phyogenetic tree also showed the conservatism between several different species.The second structure of buffalo Keap1 protein was predicted as 24 alpha regions,40 beta regions,38 turn regions and 27 coil regions.In addition,the results of Real-time quantitative PCR showed that Keap1 mRNA exists in all seven tissues,but the most abundant expression was in heart and the minimal expression was in liver and spleen.The results provided an foundation for further study of Keap1-Nrf2-ARE signal pathway,for enhancing the ability of antioxidant of buffalo embryo in vitro culture.

The study was aimed to use colloidal gold immune chromatography technology to establish a rapid method for detection of canine serum canine parvovirus (CPV) hemagglutination inhibition (HI) titer and CPV vaccine immunization effect assessment.Double antibody sandwich method and monoclonal antibodies of anti-CPV hemagglutination antigen were used to prepare CPV antigen test strip.Canine serum with different proportion respectively was mixed with quantitative CPV antigen for full reaction,then dropped the mixture into the CPV colloidal gold test strip,so according to the highest serum dilution ratios when the test strip line T (line T) vanishes,it was to judge CPV antibodies in serum of the HI titer.This method had been used to detect 86 canine serum samples,at the same time,analyzing and comparing it with traditional hemagglutination inhibition test method.The results showed that the CPV antigen detection test strip was successfully prepared,and the reaction conditions and results of the test strip for detecting the titer of CPV-HI in canine serum were determined.The results indicated that when detecting CPV antigen after the dilution of different ratios of canine serum,the highest serum dilution ratios when the strip line T vanished and the HI titer had positive correlation.The highest dilution ratios of canine serum multiplied by 4 was the HI titer.The results of two methods had 90.7% consistency.This experiment established the colloidal gold immune chromatography test strip for the detection of CPV-HI titers method initially.This CPV-HI detection provided a simple and fast test method for the effect evaluation of CPV vaccine immune.

This experiment was conducted to investigate the effect of mulberry leaf meal and fermented mulberry leaf meal on slaughter performance,meat quality and cecum microflora of Huxu chicken at the late growth stage.Three hundred and ninety-two Lingnan Ⅲ Huxu chickens aged ninety-five days were assigned to seven dietary treatments randomly,they were control group,5%,10%,20% mulberry leaf meal groups and 5%,10%,20% fermented mulberry leaf meal groups.Every treatment had 4 replicates,14 chickens per replicates (7 males and 7 females).The pre-trial period was 4 days,and the formal experiment period was 28 days.The results showed as follows:① Compared with the control group,expect the 10% mulberry leaf meal group,the semi-eviscerated percentage were significantly decreased in other experimental groups (P<0.05).Adding 10% mulberry leaf meal or 20% fermenterd mulberry leaf meal extremely significantly reduced the eviscerated percentage (P<0.01) and 10% fermenterd mulberry leaf meal reduced it significantly (P<0.05).The leg muscle percentage of chickens in 5% fermenterd mulberry leaf meal group and 20% mulberry leaf meal group reduced extremely significantly (P<0.01) and significantly (P<0.05),respectively.② Compared with the control group,the shear force of chest muscle in all experimental groups,and leg muscle in all mulberry leaf meal groups decreased,but only the shear force of chest muscle in 10% mulberry leaf meal group decreased significantly (P<0.05).Adding 20% mulberry leaf meal increased the pH_{45 min} of leg muscle significantly (P<0.05).The b^* value of leg muscle reduced in all experimental groups,and only that in the 20% mulberry leaf group reduced significantly (P<0.05).③ Compared with the control group,the number of E.coli of the cecum of chickens in 5% and 10% mulberry leaf meal groups reduced extremely significantly (P<0.05),and the ratio of Lactobacillus to E.coli of cecum in all experimental groups had an increasing trend (P>0.05).All over,the content of mulberry leaf meal in Huxu chickens'diet at the late growth stage could be up to 10%,and fermented mulberry leaf meal could be up to 20%.

This experiment was conducted to study the effects of different dietary crude protein levels on the nutrient absorbability in the small intestine of lambs.Three Kazakh lambs (30 kg body weight) fitted with permanent ruminal,duodenal and ileal fistula were selected.3×3 Latin square design was adopted.A dual-phase marker system with LiCr-EDTA and Yb-Ac as the liquid-phase or particulate-phase digesta flows maker was adopted to measure the true digesta flows at duodenal and ileal fistula,respectively.The results showed that nitrogen intake,total tract apparent digestibility,N deposition,the apparent digestibility of dry matter and organic matter were significantly increased with increasing levels of dietary crude protein (P<0.05),but had no significant effects on intestinal digesta flows (P>0.05).Increasing dietary crude protein levels had significant effects on most part of nutrients of duodenal digesta except fat and purine (P<0.05),while it only significantly effected CP,Ash,dry matter,neutral detergent fibre and acid detergent fiber of ileal digesta flows (P<0.05).In addition,intestinal absorbability of nutrients except for CP,Ash,dry matter,neutral detergent fibre and acid detergent fiber were significantly affected with increasing levels of dietary crude protein(P<0.05).The test provided basis for further study of the effects of dietary CP level on the digestibility and utilization of lamb.

This experiment was conducted to investigate the effects of energy level on the physical and chemical indicators,the composition and content of amino acids and fatty acids in muscle of house-fed yaks.A complete randomized design was adopted in this study,and sixty healthy and 3-year-old Maiwa yaks with an average body weight of (192.66±3.64)kg were randomly divided into 3 groups with 20 replicates per group and 1 yak per replicate.Diets for all groups were of the same crude protein content (14.81%) and different net energy levels for gain (NE_g) with 4.17(LE),4.48(ME) and 4.79 MJ/kg (HE),respectively for 97 days (7-day pretrial period,and 90-day feeding trial).The results showed as follows:The level of energy had no effect on the value of pH,cooking yield,drip loss,moisture,crude protein (CP),crude fat (EE),calcium (Ca) and phosphorus (P) (P>0.05).Except for the content of proline and histidine in muscle of group ME were significantly higher than the other groups (P<0.05),the content of total amino acids and other kinds of amino acids had no significant difference (P>0.05).The content of monounsaturated fatty acids,polyunsaturated fatty acids and total unsaturated fatty acids in ME group were significantly higher than the LE and HE groups,and the content of total saturated fatty acids in HE group higher than the LE and ME groups.For all kinds of fatty acid,the content of C15:1 n-3,C16:1 n-2 and C18:1 n-8 three monounsaturated fatty acids in ME group significantly increased than the other two groups (P<0.05);And the content of C17:0 in HE group had a significat rise trend.In conclusion,we recommend 4.48 MJ/kg (DM basis) as a diet appropriate NE_g level for the barn feeding of fattening yak under this experimental condition,appropriate NE_g level of diet could improve the fatty acids content in muscle of the yaks.

This experiment was conducted to investigate the effects of butyrin on the growth performance,nutrient apparent digestibility,slaughter performance,intestinal morphology and microbial flora of broilers.A total of 384 1-day-old Rose 308 broilers were selected and randomly divided into 4 groups,4 replicates per group and 24 broilers per replicate.The blank control group was fed on basal diet,the experimental groups were fed on basal diet supplemented with 250,500 and 750 g/t butyrin,respectively,which replaced 40% oil of the basal diet.The experiment lasted for 42 days.The result showed that compared with the blank control group,diets supplemented with butyrin had an promoting effect on the performance of broilers,and the middle dose group (500 g/t) was the best.The slaughter performance of supplemented groups showed a tendency to be improved,but there was no significant difference with blank control group (P>0.05);The height of duodenum and jejunum villus of middle supplemented group were significantly improved (P<0.05),and the crypt depth of supplemented groups tended to be reduced,but there was no significant difference (P>0.05);Compared with the blank control group,the apparent digestibility of crude fat and crude fiber of middle supplemented group were significantly improved (P<0.05);The amount of Escherichia coli of middle supplemented group was significantly decreased (P<0.05);And the amount of Lactobacillus was significantly improved (P<0.05).In conclusion,basal diet supplemented with butyrin could promote the growth performance,increase the nutrient digestibility,improve the intestinal morphology and microbial flora of broilers.The best supplemented level of butyrin for diets was 500 g/t.

The objective of this study was to evaluate the effects of 2-methylbutyrate on daily gain,dietary nutrient digestion and methane emissions in Simmental cattle.Thirty-six Simmental cattle (12-month-old) consuming a corn straw diet were divided randomly into four groups (control group,experimental groups 1,2 and 3) and supplemented with four levels of 2-methylbutyrate as 0,1.0,2.0 and 3.0 g/(kg·BW),respectively.Dietary nutrient digestibilities,average daily gain and methane emissions were determined.The results showed that OM digestibilities of the cattles in experimental group 2 and 3,CP digestibilities of the cattles in experimental groups 2 were significantly higher than that in control group (P<0.05).NFE digestibilities were not affected by adding 2-methylbutyrate (P>0.05).NDF digestibilities of the cattles in experimental group 2 were significantly higher than that in control group and experimental group 1 (P<0.05),while their ADF digestibility were significantly higher than that in control and other experimental groups (P<0.05).Compared with control group,the average daily gains of cattles increased significantly in experimental groups 2 and 3 (P<0.05),the dry matter intakes of cattles decreased significantly in experimental group 2 (P<0.05),and the feed gain ratio decreased significantly in experimental groups 2 and 3 (P<0.05).In addition,the methane emissions were decreased significantly in experimental groups 2 and 3 (P<0.05).The results indicated that the average daily gain,nutrient digestion could be improved,and methane emissions could be restrained by supplementing 2-methylbutyrate in Simmental beef cattle,the optimal dose of 2-methylbutyrate supplementation was 2.0 g/(kg·BW).

The test was conducted to study the effect of different culture ways on health and development of Japanese Black calves in cold area,20 healthy and newborn calves were divided into 2 groups using single factor randomized block design.Calves of control group were fed using traditional feeding method while calves of experimental group were fed using artificial feeding method.The test period was 180 d,body weight and body steep size indexes of calves were measured,and the health condition of calves were observed.The results showed that when weaned at 3 months old,body weight and average daily gain of calves in experimental group were significantly higher than that of control group (P<0.05);Body weight and average daily gain of calves with 6 months old in experimental group were significantly higher than that of control group (P<0.05);The fecal index,diarrhea rate,diarrhea frequency and death rate of experimental group were lower than that of control group,and the difference of diarrhea rate and diarrhea frequency were significant (P<0.05).And the respiratory disease incidence,incidence frequency and death rate of experimental group were lower than that of control group,the difference of the three indexes were significant (P<0.05).The results suggested that artificial feeding method could improve the Japanese Black calves survival rate,strengthen the prevention of diarrhea and respiratory disease and sufficiently play the potential to improve calf weaning weight.It not only could reduce the economic costs of farms,and also significantly improve the economic efficiency of domestic high-quality beef cattle.

In order to improve the stability of super oxide dismutase (SOD) with some modification,we used thermal denaturation extraction method to extract Cu/Zn-SOD from newborn bovine blood,and used layer-by-layer self-assembly technique,heparin and chitosan as material to modificate the SOD which separated and purified from newborn bovine blood.The results showed that compared with the natural SOD,the modified SOD increased 3.3 times in the thermal stability;In acid and alkaline,under the condition of pH 3.0 and pH 11.0,the acid-base resistance property of the modified SOD raised 2.0 times;On the antitrypsin hydrolysis ability,the relative enzyme activity of modified SOD had increased 2.7 times than natural SOD.The modified SOD with shell outside made of heparin and chitosan by physical modification could get better effect,and had the strong mechanical strength,better biocompatible,outer surface of chitosan was helpful to cross the cell membrane transport,and it could be applied to the development of oral dosage forms of SOD.

The culture conditions of Trichoderma reesei solid-state fermentation to produce α-galactosidase were optimized by response surface method. First of all, according to the single factors,the experiments were designed as Plackett-Burman and three main factorswere selected out:The ratio of corn cob and bagasse carbon source, the ratio of beef extract and ammonium nitrogen source and the fermentation initial pH. On the basis of using the method of the steepest uphill path approximation maximum response area, the response surface method was used to determine the main factors of the interaction between the main factors and the optimal culture conditions. The results showed that the optimized culture conditions in Trichoderma reesei solid-state fermentation producing α-galactosidase were:the ratio of carbon source (namely corn cobsand to bagasse ratio) was 3.86, the ratio of nitrogen source (beef paste to ammonium sulfate) was 1.17, and the initial pH was 9.12. After optimization, the α-galactosidase activity produced in Trichoderma reesei solid-state fermentation could be up to 342.98 U/g and were close to 353.43 U/g, the model prediction. It was increased by 113.91% than before optimization. In conclusion,the most important factors in Trichoderma reesei solid-state fermentation were carbon source, nitrogen source and pH value,which interacted each other.The activity of α- galactosidase was maximum when the ratio of carbon source was up to 3.86, the ratio of nitrogen source was up to 1.17 and pH value reach to 9.12.

Signalling lymphocyte activation molecule (SLAM,also called CD150) serves as a main cell receptor for PPRV (peste des petits ruminants virus).This study was aimed to establish a cell line,using Vero cells as the parental cell,to express goat SLAM stably,which could be used to isolate and propagate PPRV.The gene encoding goat SLAM in vitro was synthesized and cloned into eukaryotic expression vector pIRES2-GFP,and the recombinant expression plasmid pIRES2-gSLAM was obtained.The positive stably transfectant Vero-gSLAM cells were screened by G418 and identified by immunofluorescence(IF) and RT-PCR.The result of virus titration by Vero-gSLAM cell line showed that PPRV strain N75/1 had a titre of 10^-4.65 TCID₅₀ per 0.1 mL in Vero cell at 5 day after infection and the titre of PPRV N75/1 strain was 10^-5.75 TCID₅₀ per 0.1 mL in Vero-gSLAM cells.The cell line would play an active role in virus isolation,biological characteristics study and vaccine virus production of PPRV.

This study was aimed to establish a fetal fibroblast cell line of double transgenic (pGH/IGF-Ⅰ) pigs,and reserve fibroblast cells of double transgenic pigs for the follow-up study.A method of trypsin digestion was adopted to isolate and culture body tissues from pregnant porcine,and porcine fetal fibroblasts were isolated successfully through primary culture,subculturing and cryopreservation.The morphological observation,determination of viability before cryopreservation and after recovery,dynamic growth analysis,vimentin immunohistochemistry and microbial contamination detection were all done to study the biological characteristics of the cell line.The results showed that the fibroblasts were cultured and isolated successfully by trypsin digestion and differential centrifugation.The cell viability before cryopreservation and after recovery were 94.3% and 91.2%,respectively.The growth curve was sigmoidal,and experienced the incubation period,exponential growth period and platform three stages.The vimentin immunohistochemistry was positive,the microbial contamination detection were all negative.The results indicated that a fibroblast cell line of double transgenic porcine was successfully established.

In order to explore the influence of temperature changes on animal digestive tract smooth muscle,the contractility of isolated rabbit small intestine (duodenum,jejunum and ileum) were measured at different temperatures (39→32℃,32→39℃,39→42℃ and 42→39℃) using the biological signal processing system.The results showed that the contraction frequency of duodenum and jejunum was positively correlated with temperature changes between 39 and 32℃,while the amplitude and tension of duodenum were negatively correlated with temperature changes.Only the contraction frequency of ileum was positively correlated with temperature changes between 39 and 32℃.Changes of frequency,amplitude and tension of duodenum between 39 and 42℃ were similar with those changes between 39 and 32℃.However,the contraction of jejunum and ileum was irregular.When the temperature was decreased from 42 to 39℃,the contraction of small intestine could not return to the condition in 39℃.The results suggested that the optimal bath temperature was 37℃ in contraction test of rabbit small intestine in vitro,and the present results could be referenced to explore the disorder of gastrointestinal movement due to the change of environment temperature in animals.

The test was aimed at determining the effects of compound Codonopsis oral liquids with Codonopsis pilosula as monarch drug on levels of IgG,IL-2 and ACTH of serum with immunological stress in layer chickens.One hundred and fifty healthy chickens at one day old with the same body weight were divided into five groups of 30 chickens per group:High dose group,medium dose group,low dose group,model control group and base control group,respectively.From the 3 days old,the high,medium and low dose groups were respectively administered 8,4 and 2 mg/(kg·BW) compound Codonopsis oral liquids for 10 days;The chickens of 3 different dose groups and model control group were intramuscularly injected Newcastle disease (ND) vaccines Ⅰ to make immunosuppressive models on 7th day.The samples were collected and IgG,IL-2 and ACTH contents in serum were determined on the 8th,16th,24th,32th and 40th day.The results showed that on the 16th,24th and 32th day,the IgG content of 3 different dose groups were extremely significantly higher than that of model control group (P<0.01),and high dose group>middle dose group>low dose group.On the 8th and 16th day,the IL-2 level of high and middle dose groups were extremely significantly or significantly higher than that of model control group (P<0.05;P<0.01),and on the 24th and 40th day,3 different dose groups were extremely significantly higher than that of model control group (P<0.01),and that of high dose group was extremely significantly higher (P<0.01)while middle dose group was significantly higher (P<0.05)than the model control group on the 32th day;On the 8th,16th,24th and 40th day,the ACTH content of high and middle dose groups were extremely significantly or significantly lower than that of model control group (P<0.05;P<0.01),and that of high dose group was significantly lower than model control group on the 32th day (P<0.05).At the late experiment period,the ACTH content of 3 different dose groups were gradually became similar to base control group,indicating that the effect of immunosuppressant on ACTH was transient.In conclusion,the compound Codonopsis oral liquids could increase the level of levels of IgG,IL-2 and reduce the level of ACTH of serum,but it could not decrease to the level of base control group,and high dose had better effects.

The determination of major histocompatibility complex (MHC) classⅠcomplex structure can elucidate the antigen-binding mechanism,and then effectively utilize the specific CTL response.Currently,several MHCⅠstructures of the livestock and poultry are reported,containing cattle MHCⅠ allele (N*01301,N*01801),pig (SLA-1*0401) and chicken (BF2*2101,BF2*0401,BF2*0201 and BF2*1401).All above MHCⅠstructures are composed of α1,α2,α3 and β₂m chains;And the antigen groove is consisted of six pockets (A,B,C,D,E and F).Generally speaking,how different MHCⅠ binds miscellaneous peptides depends on the B and F pockets.In addition,the MHCⅠsequences have intraspecific and interspecific differences,so the peptides are various for different MHCⅠmolecules.But,different MHCⅠmolecules also can present same peptide,implementing the peptides of cross-presentation.The determination of animal MHCⅠstructures will promote the immunology and vaccine application research.

This study was aimed to investigate the distribution of genetic polymorphism of taste receptor family 1 member (T1R) gene between Ujimqin sheep and Hu sheep.DNA pools direct sequencing method and MALDI-TOFMS method were used to analyze genetic variation of T1R genes in 172 sheep of two Chinese sheep strains of Mongolian,and bioinformatics software predicted what impact polymorphic loci had to mRNA and protein secondary structure of T1R gene.The results showed that 9 SNPs were screened in T1R gene of two groups.Chi-square test for independence was taken to find the genotypes of the 5 SNPs which were significantly different between two sheep population(P<0.05),SNP2 located in TAS1R1 gene,SNP4,SNP7 and SNP8 located in TAS1R2 gene,SNP10 located in TAS1R3 gene.SNP2,SNP7 and SNP10 were silent mutations.SNP2 and SNP10 lead to corresponding gene mRNA secondary structure and the minimum free energy change,while the SNP7 only lead to the minimum free energy changes.SNP4 and SNP8 were missense mutations,the two missense mutations respectively led to asparagine(Asn) into serine (Ser),threonine (Thr) into methionine (Met),and according to online software forecast,the protein secondary structure of TAS1R2 gene all changed in mutations before and after.

To develop the potential function of dairy cow mammary stem cells (DCMECs) in regulation of lactation,we identify putative DCMECs which were BrdU label retaining epithelial cells,at the same time,analysis the location of two new mammary stem cells molecular marks FNDC3B and PROCR to verify the feasibility of them to indicate DCMECs.The mRNA levels of prolactin,growth hormone,insulin-like growth factor-1 and their receptors were detected along with cell passage by Real-time quantitative PCR.The results showed that the proportion of BrdU label-retaining epithelial cells was nearly 0.4% after 25 d continuous culture (passaged 4 times) and few cells were positive for FNDC3B or PROCR.Moreover,we observed the BrdU labelled epithelial cells by asymmetric division.The mRNA levels of prolactin,growth hormone,insulin-like growth factor-Ⅰ and their receptors in primary and passage cells were extremely significant difference(P<0.01).DCMECs would rapidly lose some physiological characteristics and the ability of milk synthesis when not under the condition of induction of lactation differentiation,but a certain percentage of mammary stem/progenitor cells will be retained,whose potential effects on the regulation of lactation and mammary acinar remodeling were worthy of attention.

The experiment was aimed to explore the expression of CD9 in the follicles at different developmental stages, and investigate the effect of in vitro maturation and cryopreservation on CD9. The expression of CD9 protein in the follicle was detected by immunohistochemistry technique, the expression of CD9 mRNA was detected by Real-time quantitative PCR, CD9 protein was detected by Western blotting. The results showed that the CD9 fluorescence signal was detected by immunohistochemistry technique, and the fluorescence signal gradually increased with the maturation of the follicle, and fluorescence signal was the strongest in mature follicle;Real-time quantitative PCR showed that the expression of CD9 mRNA in fresh MⅡ oocytes was significantly increase (P<0.05), and was the least in frozen GV oocytes;The result of Western blotting indicated that CD9 protein was the most in fresh MⅡ oocytes and the expression of frozen GV oocytes was the least, consistent with the results of Real-time quantitative PCR. Cryopreservation of oocytes was more extensive than embryo cryopreservation. Tetraspanins CD9 played a very important role in sperm egg fusion. The above experimental results showed that the cryopreservation made damage to oocytes, decrease content of tetraspanins CD9 protein, and CD9 injury was one of the important reasons for the decline of fertilization rate.

The experiment was aimed to study the expression and localization of NFκB1 in bovine mammary epithelial cell (BMEC),and to clarify the regulation of transcription for milk synthesis in BMEC by NFκB1.Primary cells were cultured using the tissue pieces culture method,and the BMEC was purified and passaged according to the different sensitivity of fibroblasts and BMEC to trypsin.Western blotting and immunofluorescence (IF) method were used to detect cell keratin 18 and β-casein expression to identify the cell purity and lactogenesis function.Furthermore,0.6 mmol/L methionine (Met) was added to BMEC culture to establish amino acids-stimulating BMEC model in vitro.Western blotting and IF were used to detect the expression and localization of NFκB1 and p-NFκB1 after adding Met.These results showed that BMEC was successfully purified.NFκB1 had two molecular forms in cytoplasm,the molecular weights were about 141 and 105 ku,and in nucleus only 105 ku form exists.The p-NFκB1 (50 ku) mainly existed in the nucleus.After Met stimulation,the protein level of NFκB1 141 ku form was slightly upregulated,the protein level of NFκB1 105 ku did not changed in the cytoplasm while obviously increased in the nucleus,the protein level and nuclear localization of p-NFκB1 were both obviously increased.These results suggested that NFκB1 was involved in the transcriptional regulation of lactogenesis in BMEC,and amino acids promoted lactogenesis by triggering NFκB1 phosphorylation in the nucleus.

In order to breed a beef line in Chinese Simmental cattle,this research defined breeding objective traits and selected traits according to real breeding condition of Chinese Simmental beef cattle.Using the balance method to calculate the marginal benefit of each objective trait and getting the corresponding economic weight of breeding objective traits through the standard value of the error corrected marginal benefit of breeding objective traits.The results showed that breeding objective traits mainly included growth traits (including weaning weight,fattening daily gain,18th month weight),carcass traits (including carcass quality,dressing percentage,pure meat percentage) as well as reproductive traits (including age at first calving,calving interval,stay group time).In the current market and production conditions,the marginal benefit of the breeding objective traits were 17.93 yuan/kg,16.2 yuan/kg,7.17 yuan/kg,297.99 yuan/grade,497.82 yuan/%,594.46 yuan/%,-3.62 yuan/d,-26.55 yuan/d and 232.75 yuan/y,respectively,and the ratio of relative economic weights of three kinds of traits was 31.49%:15.86%:52.65%,similar to 2:1:3,and reproductive traits occupied larger economic weight,was better than growth traits and carcass traits.The results indicated that reproductive traits played an important role in the process of breeding in Chinese Simmental beef cattle,and we needed to strengthen the selection of reproductive traits.

In order to investigate the effect of different concentrations of Ca²⁺ on in vitro capacitation of wapiti sperm,frozen-thawed sperm of Tarim Red deer (Cervus elaphus yarkandensis) was used as experimental materials in this study.Sperm capacitation status were assessed by chlortetracycline (CTC) staining,the expression levels of tyrosine phosphorylated protein were detected with Western blotting analysis of sperm membrane protein separated by SDS-PAGE,following the sperm were suspended in sp-TALP liquids,which contained different concentrations of Ca²⁺(0,1.1,2.2,3.5 and 5.0 mmol/L),and cultured for 0,2,4 h.The results showed that the low concentration of Ca²⁺ (1.1,2.2 mmol/L) was conducive to the maintenance of sperm motility (P<0.05),sperm capacitation rate was extremely significant higher than that of control group and high concentration groups (3.5,5.0 mmol/L;P<0.01),sperm survival time was the longest (P<0.01),but high concentration of Ca²⁺ (5.0 mmol/L) significantly inhibited sperm motility (P<0.05),sperm capacitation rate was extremely significantly lower than that in low concentration group (P<0.01),sperm survival time was the shortest (P<0.01). In addition,the expression levels of tyrosine phosphorylated proteins were different,and the levels of sperm protein phosphorylation in low concentration group (1.1 mmol/L) were extremely significant higher than those in other groups (P<0.01),and the expression levels of tyrosine phosphorylated proteins in high concentration groups of Ca²⁺ (3.5,5.0 mmol/L) were extremely significantly decreased (P<0.01). These results suggested that the appropriate Ca²⁺ concentration required for Tarim Red deer sperm in vitro capacitation was 1.1 mmol/L,and the presence of Ca²⁺ in process of capacitation was necessary.

To evaluate the safety of the total flavonoids of Limonium aureum (L.) Hill.and lay an experimental basis to study the pharmacological effects and clinical safety in the future,80 Wistarrats with same age and body weight were randomly divided into two groups (group Ⅰ,Ⅱ),and every group was divided into the high dose group (HG),medium dose group (MG),low dose group (LG) and control group (CG),respectively,of which the mice were given to the total flavonoids of Limonium aureum (L.) Hill.by gavage at the doses of 15,10 and 5 g/kg while the mice of CG were not given any medicine.The blood sample were collected and experimental indexes were measured at 10 (group Ⅰ) and 21 d (group Ⅱ).The results showed that comparing with CG,the weight gain of rats in MG and LG of group Ⅰhad no significant difference (P>0.05),and that of HG were significantly decreased (P<0.05);The weight gain of rats in LG of group Ⅱ had no significant difference (P>0.05),and that of MG and HG were significantly decreased (P<0.05);Organ indexes of experimental groups had no significant difference with CG (P>0.05);With increasing concentration of the total flavonoids of Limonium aureum (L.) Hill.,there was pathological damage to major organs and white lumps in lung and red bleeder existed in liver of rats in HG when given total flavonoids of Limonium aureum (L.) Hill.for 21 d.The results indicated that total flavonoids of Limonium aureum (L.) Hill.was low toxicity and could be safely used for animals with minding the dose was not too high.

The purpose of this paper was to study the acute toxicity in mice and long-term toxicity in rats of Gongjing perfusion to evaluate the clinical safety of medication and provide theoretical basis for clinical application.In the acute toxicity experiment,mice were given once the approximate lethal dose of Gongjing perfusion to measure the acute toxicity and the maximal tolerance dose (MTD).In the long-term toxicity observation,80 rats were divided into four groups:low-dose,middle-dose,high-dose test groups of Gongjing perfusion (2.5,5.0,10.0 g/(kg·d)) and control group with distilled water intragastric administration for 30 days.During the experimental period,the appearance,behavior and feces condition of rats were observed and recorded.Each rat was weighted every week,and the average daily gain were calculated.After 30 days,rats were executed and taken blood in their hearts,hematology and blood chemistry were detected.The results showed that the mice were all survived and LD₅₀ was not measured,the MTD was 40 g/kg.In the long-term toxicity test,compared with the control group,there were no significant difference in various index except individual index and the pathological examination revealed that no obvious pathological changes related to drug toxicity.So Gongjing perfusion had not long-term toxicity and acute toxicity under this experiment condition,which suggested Gongjing perfusion had a good clinical safety.

In order to understand the resistance of E.coli to clinical antimicrobial drugs from different farms in Kuitun area of Xinjiang,the bovine fecal samples were collected using the anal swab and E.coli was isolated from 123 (30),127 (30) and 128 (30) farms,respectively.The minimum inhibitory concentrations of E coli isolated from fecal samples to 10 antimicrobial drugs were determined by agar dilution method and the difference of resistance rate of E.coli from different farms was compared by chi square test.The results showed that the resistance rates of 128 farm to amoxicillin/clavulanic acid (33.3%) and apramycin (23.3%) were relatively high;The resistance rates of 127 farm to ampicillin (20.0%),amoxicillin/clavulanic acid (16.7%) and florfenicol (16.7%) were relatively high;The resistance rates of 123 farm to ampicillin (13.3%) and the apramycin (13.3%) were relatively high.The chi square test results showed that resistance rates of E.coli to florfenicol and enrofloxacin from 127 farm were significantly higher than that from 123 and 128 farms (P<0.05);The resistance rates of E.coli to amoxicillin/clavulanic acid and apramycin from 128 farm were significantly higher than that from 123 and 127 farms (P<0.05),respectively.The most of the antibiotics resistance pattern of E.coli from different farms was 1 to 2 resistant.The results showed that the drug resistance rates of bovine E.coli in Kuitun was not high,while the resistance rates to commonly used drugs such as amoxicillin/clavulanic acid and apramycin were high.It suggested that this kinds of antimicrobial drugs should be replaced or avoid using to treat bacterial diseases.In addition,the antimicrobial resistance between farms was different,indicating the farms medicine would impact the drug resistance in bacteria.

In this study, an indirect ELISA method was established to detect inhibin hormone (INH) epitope peptide vaccine antibody, it would provide oretical reference for the determination of Fine-wool sheep after active immune body INH epitope peptide vaccine antibody. On the basis of the predecessors, using indirect ELISA method to determinate serum INH epitope peptide antibody levels of sheep, and through control different experimental conditions to look for the best experimental conditions. Through explorating the experimental conditions, finally, the testing experiment conditions were determined, which was blocked solution with skimmed milk powder, INH and GnIH synthetic peptides dilution degrees for 20 000 times, the optimum reaction time was 60 min, the best color action time was 15 min. In this experiment, a kind of method to detection antibody in the body after INH active immune sheep was built, it would provide a reference for future research.

To investigate the anti-IBDV activity of IFN-α and its effects on chicken lymphocyte signaling molecule PI3K and NF-κB activity,forty 10-day-old non-immune chickens were randomly separated into two groups:Blank control group and IFN-α group.The chicken of two groups were inoculated with IFN-α and saline via intramscular injection for three days,respectively.Then they were inoculated with IBDV.From 13 to 18-day-old,the lymphocytes were isolated from peripheral blood of chicken.The lymphocyte proliferation of chicken were detected by the MTT method,the PI3K and NF-κB p65 concentration in lymphocyte and the IBDV-Ag of serum were detected at different time with ELISA.The results showed that compared with the blank control group,IFN-α could stimulate chicken lymphocyte proliferation and the PI3K and NF-κB p65 concentration in chicken lymphocyte were extremely significantly increased before infection (P<0.01).The lymphocyte proliferation and the expression of PI3K and NF-κB p65 in cell after IBDV infection were extremely significantly higher than before (P<0.01).IFN-α could extremely significantly inhabit the proliferation of IBDV-Ag (P<0.01).IFN-α could significantly promote lymphocyte transformation by increasing PI3K concentration and the biological activity of lymphocyte transformation associated with the PI3K and NF-κB p65 expression,activation and nucleo-cytoplasmic transport.It also could reduce IBDV-Ag proliferation in chickens.The study built foundation for IFN-α protein immuno enhancement and anti-viral activity.

The experiment was done to investigate the antibacterial effect of aqueous extract from Chrysanthemum indicum L. combined with antibiotics on extended spectrum β-lactamases (ESBLs)-producing E. coli,and aimed to offer theoretical basis for its further development.The ESBLs-producing E.coli was induced to passage by combining Chinese herbal medicine with antibacterial drugs.Then the minimal inhibitory concentration (MIC) of medicines were determined with double microdilution method.The results showed that the MIC of Chrysanthemum indicum L.aqueous extract was 250 mg/mL.And the MIC of ceftriaxone,ceftiofur sodium,cefotaxime,florfenicol,mequindox,amikacin and lincomycin decreased significantly after combining with 1/2 MIC of Chrysanthemum indicum L.aqueous extract.This research showed that aqueous extract Chrysanthemum indicum L.could increase the antibiotic sensitivity of the ESBLs-producing E.coli.

This study was to investigate the ribosome genotyping and antimicrobial resistance in Enterococcus faecalis strains isolated from 2 large-scale farms in Shanghai.40 isolates were evaluated for their sensitivity to 12 antimicrobial agents by broth microdilution,and the ribosome genotyping (ribotype) was characterized by the Riboprinter^® Microbial Characterization System.The resistance rates of most antimicrobial drug were relatively high and multidrug resistant strains were detected with more than 80%.4 strains of Enterococcus faecalis isolated from pigs were resistant to vancomycin,including 2 strains of vancomycin highly resistant (MIC>64 mg/L) with the same time highly resistant to gentamicin and streptomycin (MIC>2 048 mg/L).The results showed that multidrug resistance of Enterococcus faecalis was a serious issue,and the resistant phenotypes of the same type of ribotype was not entirely consistent.

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease that spreads all over the world.It is caused by porcine reproductive and respiratory syndrome virus (PRRSV).Once infected with PRRSV,NSPs (nonstructural proteins) were expressed firstly.At least 14 kinds of NSPs,including NSP1α,NSP1β,NSP2,NSP3,NSP4,NSP5,NSP6,NSP7a,NSP7b,NSP8,NSP9,NSP10,NSP11 and NSP12,were produced during PRRSV life,which plays an important role in virus proliferation,virus virulence,immunological characteristics and so on.In this paper,we make a brief overview in terms of the research progress on NSPs to make a basis for the prevention of PRRS.

This experiment was conducted to study the effects of propolis flavone (PF) on antiviral ability of host cell.First,the maximal safe concentration of PF to CEF and PK-15 was determined by MTT method.Then PF was diluted to five concentrations (from 250 to 15.6 μg/mL) with maintenance medium and acceded to foster system of CEF or PK-15 with NDV,IBDV,TGEV and PPV,respectively,and the cell viability was observed by MTT method at 24,48 and 72 h after infection as the index of high antiviral ability of host cell.The results showed that PF could promote the antiviral ability of host cell.And the effects were related to PF concentration,as the concentration increased,the effects were better.The antiviral activity of PF to NDV and TGEV with envelope in host cell existed in infectious earlier stage,while for IBDV and PPV without envelope,the activity appeared in infectious later stage.The results indicated that the antiviral effect period and mechanism of propolis flavone was different for different viruses,propolis flavone could be use for the prevention of NDV and TGEV,and for the treatment of IBDV and PPV.

The experiment was designed to investigate growth and development pattern and reveal the correlation of each traits of Xinjiang Brown cattle according to analysis body index data.2 794 Xinjiang Brown bulls were selected to measure body index and draw the growth curve of body weight during year 2010 to 2015 in Yili Zhaosu region.The body index was composed by body weight,body height,body length,chest circumference and canon circumference.All measured data were analyzed and validated underwent correlation and regression analysis.The results showed that body length of Xinjiang Brown bulls were grow fastely at the age 14 to 15 months;Body weight was extremely significantly positive correlated with other four measured body index (P<0.01).Of them,the correlation coefficient with chest circumference was the highest (r=0.796),and the lowest was with body length (r=0.163).The body weight regression estimate formula of Xinjiang Brown bulls was Y=-564.607-0.174X₁+2.441X₂+3.497X₃-1.086X₄,and the validation result implied this formula was statistical significance.Our work offers a good data basic for genetics selection and breeding of Xinjiang Brown cattle.

Nitrofurans are broad-spectrum antibiotics which have been widely used in food animals to the prevention and treatment of gastrointestinal infections.Due to abuse of drugs by criminals,animals could produce drug-resistant strains,which reduces the role of antibiotics.Meanwhile,excessive drugs residues in food has carcinogenic,teratogenic and mutagenic effects to the human body.In this paper,the damage and the detection methods of the residues of the nitrofurans in food are surveyed.The main detection methods include high performance liquid chromatography,liquid chromatography-mass spectrometry,liquid chromatography tandem mass spectrometry,enzyme-linked immunoassay,colloidal gold immunoassay and fluorescence immunity analysis method.The research status of various detection methods are summarized and the suggestions for further research are put forward.It is expected to provide reference for future research.

Soybean isoflavone is a kind of faint estrogen-like mixture of polyphenols which derived from the leguminous plants.Its active components include genistein,daidzein and glycitein.It is used in animal husbandry for its physiological functions that it can enhances performance,immune function,antioxidant and improves bone metabolism.But some following reports about safety and side effects of soybean isoflavones are also appeared.That is a worthy further research topic how to use it effectively.The article summarizes the types of soybean isoflavones,nutrient metabolism characteristics,physiological characteristics,the promoting effect of livestock and poultry production and security problems about animal and human.